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doi: 10.1097/SHK.0b013e318173e2cc
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IFSS Award

1 DIETARY LIPIDS REDUCE INFLAMMATION VIA PERIPHERAL ACTIVATION OF THE AUTONOMIC NERVOUS SYSTEM

T. Lubbers, J. De Haan, M. Hadfoune, M. D. Luyer, C. H. Dejong, W. A. Buurman, and J. W. Greve. Maastricht University Medical Center, Maastricht, the Netherlands

Lipid-rich nutrition has been shown to inhibit systemic inflammation and prevent gut barrier failure by stimulation of the autonomic nervous system (ANS) via cholecystokinin (CCK) receptors. This study investigates whether enteral lipids activate the ANS peripheral via receptors on the afferent vagus or central by receptors in the ANS via circulating CCK. Prior to hemorrhagic shock, rats were fasted or fed a lipid-rich nutrition, containing 50.4 % of lipid, of which 30% phospholipids. Peripheral activation of the ANS via the afferent vagus was investigated by perineural application of capsaicin (deafferentation) prior to shock. Central activation was studied by infusion of CCK in fasted rats. Plasma and tissue samples were collected 90 minutes after shock to assess inflammatory status and intestinal integrity. Deafferentation reduces the inhibitory effect of dietary lipid on TNF-α (134 ± 32 pg/ml vs 45 ± 13 pg/ml [sham]; p<0.001) and IL-6 (168 ± 14 pg/ml vs 69 ± 9 pg/ml [sham]; p<0.001). Preservation of gut barrier function by enteral lipid-rich nutrition was blunted by deafferentation, expressed as leakage of horseradish peroxidase in ileal segments (6.1 ± 0.3 μg/ml vs 2.7 ± 0.3 μg/ml [sham]; p<0.001) and bacterial translocation (113 ± 20 CFU/g tissue vs 33 ± 4 CFU/g tissue [sham]; p<0.001). The protective effects of lipid-rich nutrition could not be observed by systemic infusion of high levels of CCK. Our study shows that lipid-enriched nutrition inhibits systemic inflammation and gut barrier failure via peripheral activation of the afferent vagus. Enteral administration of lipid-rich nutrition could provide a therapeutic option in inflammatory disease.

2 α7 NICOTINIC ACETYLCHOLINE RECEPTOR STIMULATION INHIBITS CYTOKINE RELEASE IN HUMAN WHOLE BLOOD STIMULATED WITH VARIOUS TOLL-LIKE RECEPTOR AGONISTS

Matthijs Kox, Jan C. Pompe, Peter Pickkers, Cornelia W. Hoedemaekers, Arie B. van Vugt, and Johannes G. van der Hoeven. Department of Intensive Care Medicine, Radboud University Nijmegen Medical Centre, The Netherlands

The efferent vagus nerve can limit the inflammatory response in a reflex-like fashion via the α7 nicotinic acetylcholine receptor (α7nAChR). To date, in vitro studies investigating the cholinergic anti-inflammatory pathway almost exclusively used the principal Toll-like receptor 4 (TLR4) agonist LPS. However, pattern recognition of diverse classes of microbial products involves various TLRs that modulate the subsequent immune response and cytokine profiles.

We investigated whether the immunomodulating effect of α7nAChR stimulation is dependent on the TLR agonist used to induce cytokine release in human whole blood. We compared the effects of both a selective and a non-selective α7nAChR agonist, GTS-21 and nicotine respectively.

Stimulation of the α7nAChR by both GTS-21 and nicotine resulted in a dramatic dose-dependent inhibition of pro-inflammatory cytokine release induced by stimulation of various Toll-like receptors in human whole blood. This effect was not specific for the TLR-agonist used. GTS-21 inhibited pro-inflammatory cytokine production stronger than nicotine at equimolar concentrations. The production of the anti-inflammatory cytokine IL-10 was generally not inhibited, but even significantly stimulated.

In conclusion, stimulation of the α7nAChR has a profound anti-inflammatory effect which is not restricted to a specific TLR. Therefore, this pathway appears to be a general mechanism that can modulate the inflammatory response to any kind of infection. Targeting of the α7nAChR holds much promise for future treatment options to modulate the innate immune response.

ESS Award

3 PLATELET FACTOR 4 (PF4) GENE EXPRESSION IS HIGHLY UPREGULATED IN DENDRITIC CELLS AFTER SEVERE TRAUMA

Emanuel V. Geiger, Marcus Maier, Sebastian Wutzler, Dirk Henrich, and Ingo Marzi. Department of Trauma, Hand, and Reconstructive Surgery, Johann Wolfgang Goethe University, D-60590 Frankfurt/Main, Germany

Objective:

Trauma causes a marked alteration of many immune functions, including T-cell activation, proliferation and cytokine release as well as macrophage antigen presentation and cytokine release. Dendritic cells (DC) are potent antigen-presenting cells, reflecting an important linkage between the innate and adoptive immune system, within the systemic inflammatory response. Stimulating DC leads to distinct transcriptional changes in genes relevant for signal transduction, apoptosis and mediator synthesis. Hence, we performed this study to analyse early gene expression pattern of peripheral DC in multiple trauma.

Material and Methods:

The study was approved by the local ethics committee. Repeated blood samples were obtained from 10 multiple trauma patients (ISS 30± 9.7) and 5 healthy volunteers (controls) on day 0 to 5 after admission. Messenger RNA was isolated from highly purified peripheral DC and whole blood, respectively. Target cDNA and reference samples (monocytic cell line SIGM5) were cohybridized on a thematic medium-density microarray assessing 5331 inflammation-related transcripts. The results were confirmed by qRT-PCR. Platelet factor 4 (PF4), an important chemokine to activate inflammation, cell adhesion and oxidative burst, was detected by intracellular staining and FACS-analysis. Data were normalized and transformed employing arcus sinushyperbolikus. Statistical analysis was performed using the principle componence test. A p value < 0.05 was considered statistically significant. Ultimately, genes were clustered by means of the "average linkage" method to describe related expression pattern between subjects.

Results:

In addition to PF4, 24 different transcripts were significantly altered (e.g. CXCL5, CCL5, CCR1, GUCY1B3, and TNFSF11), i.e. the transcripts of 23 genes increased, whereas expression of C9ORF68 continuously decreased during the observation period. Age, ISS, and SOFA-score had no impact on the temporal gene expression. PF4 significantly increased with a maximum on day 4, and correlated with serum CRP (p= 0.01) as well as the total DC number (p= 0.01). Moreover, intracellular concentration of PF4 in DC cells parallels the results of microarray analysis and PCR.

Conclusion:

The central role of PF4 within the innate immune system emphasizes the importance of the DC for the posttraumatic activation of the inflammatory response in multiple trauma. This mechanism will be further elucidated.

4 INSULIN DECREASES SYSTEMIC BACTERIAL DISSEMINATION AND IMPROVES SURVIVAL AFTER BURN WOUND INFECTION

Gerd G. Gauglitz, Tracy E. Toliver-Kinsky, Felicia N. Williams, Gabriela Kulp, Juquan Song, Geoffrew Lerew, David N. Herndon, and Marc G. Jeschke.

Objective:

Severe burn injury is frequently associated with burn wound infections that lead to sepsis, multi organ failure, and increased mortality. Whereas insulin administration has been shown to improve morbidity and mortality in critically ill patients, the effects of insulin on the host response to severe burn-associated infection are not known. This study was designed to determine the effects of insulin on the host and inflammatory response to a lethal burn wound infection.

Methods:

Sprague Dawley rats were subjected to a 60% TBSA burn and wounds were topically inoculated with 5 × 106 cfu of Pseudomonas aeruginosa six days later. Randomized groups were treated daily for 28 days with saline, insulin (1U, 2.5 U, 5U/kg BW) or Metformin (12 mg/kg BW), n = 10 for each group. Survival was monitored and bacterial cultures were performed with various tissues. Serum was harvested at various time points and inflammatory cytokines were measured by ELISA.

Results:

Insulin affected survival in a dose-dependent manner. One unit of insulin significantly increased overall survival, as well as the mean time to mortality compared to saline or 5U insulin treatment, (p<0.05). 2.5U of insulin also significantly increased survival compared to control saline treatment (p<0.05), whereas 5U of insulin had no effect on survival. There was a significant correlation between the dose of insulin administered and the mean survival time after wound infection. Metformin, which also decreased blood glucose, did not increase survival after burn wound infection. 1 U of insulin also decreased systemic bacterial dissemination after burn wound infection. Nine days following wound inoculation, fewer insulin-treated rats had positive blood and spleen cultures compared to the saline-treated rats. However, local bacterial clearance from the burn wound was not improved with insulin treatments. We suggest that increased resistance to infections after insulin treatments in part due to an altered inflammatory response as we found that insulin administration significantly decreased early production of IL-6, MCP-1, CINC-1 and CINC-2 in response to burn injury and IL-6 in response to a burn wound infection.

Conclusion:

Insulin increases the resistance of burned rats to a highly lethal burn wound infection in a dose-dependent manner. Attenuated systemic inflammation and bacterial dissemination suggest that insulin may have beneficial effects on the immune system that are independent of glucose control.

US-SS Awards

5 ESTROGEN SUPPRESSES CARDIAC IL-6 AFTER TRAUMA-HEMORRHAGE VIA HIF-1α PATHWAY

E.A. Nickel*, C.H. Hsieh*, J.G. Chen*, M.G. Schwacha, and I.H. Chaudry. Center for Surgical Research, University of Alabama at Birmingham, AL 35294, USA

Cardiac dysfunction is a major concern after trauma-hemorrhage (T-H) and increased IL-6 is an underlying cause. Studies have shown that estrogen (E2) treatment following T-H normalized cardiac IL-6 levels and restored cardiac functions under those conditions. Since HIF-1α is expressed during hypoxia and cellular stress and regulates the expression of IL-6, we hypothesized that HIF-1α induces cardiac IL-6 after T-H and that E2 suppresses its induction. Mice (5-8/group) were subjected to T-H (2-cm midline laparotomy; mean arterial pressure of 35 mmHg for 90 min; resuscitation with 4x shed blood volume of Ringer's lactate) or sham (S)-operation (laparotomy, no hemorrhage or resuscitation). Vehicle, YC-1 (an HIF-1α inhibitor) or E2 was administered to T-H and S groups. Mice were sacrificed after 2h; whole heart proteins were extracted and IL-6, HIF-1α levels and binding activity were measured. IL-6 and HIF-1α levels and HIF-1α binding activity were markedly elevated after T-H; all of these parameters were normalized by E2 as well as YC-1 administration after T-H. Since elevated IL-6 levels following T-H were decreased with YC-1 treatment, it indicates that IL-6 expression in cardiomyocytes is induced via HIF-1α. Also, E2 decreased elevated HIF-1α andIL-6 after T-H. These results indicate that the beneficial effects of E2 on cardiac function following T-H appear to be mediated by the inhibition of HIF-1α expression and activity. (NIH RO1 GM37127).

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Figure

6 TRAUMA-SHOCK LYMPH-INDUCED LUNG INJURY OCCURS VIA THE TLR-4 PATHWAY

Vadim Pisarenko, Danielle Doucet, David Palange, Qi Lu, Da-Zhong Xu, and Edwin A. Deitch. UMDNJ-NJMS, Newark, NJ 07103

Objective:

We tested the hypothesis that gut-derived factors in trauma/hemorrhagic shock (T/HS) lymph directly produce lung injury that involves toll-like receptor 4 (TLR-4) signaling.

Methods:

Mesenteric lymph was collected from adult male Yorkshire minipigs subjected to T/HS (laparotomy+MAP 40-60 mm Hg for 3 hours) or trauma/sham shock (T/SS). Then, T/HS or T/SS lymph samples were injected IV into C3H/HeJ (TLR-4 knockout [KO]) and C3H/HeOuJ wild-type (WT) mice over 3 hours (0.01 ml/g/hour IV), after which the animals were sacrificed and lung injur determined by histology (lung score and # PMN/hpf) as well as, myeloperoxidase measurement (MPO, specific measure of PMN infiltration).

Results:

Lung injury occurs in WT mice infused with T/HS, but not T/SS lymph. Conversely, the TLR-4 KO mice injected with T/HS lymph did not show lung injury.

Table
Table

Conclusion:

These results support the hypothesis that T/HS lymph is sufficient to cause lung injury in otherwise normal mice and its injury-inducing capacity requires TLR-4 signaling.

7 LACTATE CLEARANCE AS A DETERMINANT OF SURVIVAL IN PATIENTS WITH SEVERE SEPSIS

R.C. Arnold, N.I. Shapiro, A.E. Jones, C. Schorr, J. Pope, E. Casner, R.P. Dellinger, S. Trzeciak, and on behalf of the EMSHOCKNET. UMDNJ-RWJMS, Cooper University Hospital, NJ, USA 08103

Objective:

Previous studies have shown the prognostic significance of lactate measurement in sepsis patients. We hypothesized that 1) early lactate clearance predicts survival in Emergency Department (ED) patients with severe sepsis and 2) failure to clear lactate would be identified by concomitant failure to achieve central venous oxygen saturation (ScvO2) ≥70%.

Methods:

A collection of 3 prospective registries of consecutive ED patients treated with goal-directed resuscitation for sepsis from a 3-hospital network (2003-07). Inclusion: (1) age >17, (2) suspected infection with ≥2 SIRS criteria and either (a) systolic blood pressure <90 mmHg after ≥20cc/kg fluid or (b)initial serum lactate ≥4 mM/L, (3) repeat serum lactate <6 hours from initial, and (4) ICU admission. Subjects were classified into 2 groups: 1) lactate clearance (CLR) defined as a repeat lactate that (a) decreased by ≥10% from initial or (b) both initial and repeat levels ≤2.0 mM/L, and 2) non-clearance (NO-CLR). We analyzed the difference in proportions of death between the two groups using 95% confidence intervals and the associated z statistic.

Results:

There were 159 subjects. Mean initial lactate was 4.5 ± 2.6 mM/L. NO-CLR occurred in 11/159 (7%). Mortality was 45% in NO-CLR vs. 18% in the CLR group, proportion difference 27% [2 to 55%], p=0.02. The majority (7/11) of NO-CLR cases achieved ScvO2 ≥70%.

Conclusion:

In this sample of ED patients with severe sepsis, early lactate clearance during initial resuscitation predicted survival and achievement of ScvO2 ≥70% did not reliably exclude non-clearance. These data support the rationale for a clinical trial of lactate clearance as a distinct endpoint of early sepsis resuscitation.

8 CAFFEINE RESTORES MYOCARDIAL CYTOCHROME OXIDASE ACTIVITY AND CARDIAC FUNCTION DURING SEPSIS

R. Verma*, Z. Huang*, and R.J. Levy. New York Medical College, Valhalla, NY 10595 USA

Impaired mitochondrial function is a potential cause of sepsis-associated myocardial depression. Cytochrome oxidase (CcOX), the terminal oxidase of the electron transport chain, is inhibited in the septic heart. Oxidative phosphorylation is regulated by cAMP and PKA. Caffeine increases CcOX activity by increasing cAMP and PKA activity. We hypothesize that caffeine will increase myocardial CcOX activity, restore cardiac function, and improve survival during sepsis. Sprague-Dawley male rats underwent cecal ligation and double puncture (CLP) or sham operation. At 24 and 48 hours, rats underwent i.p. injection of either caffeine (7.5 mg/kg) or equal volume of saline. One hour following the 48-hour injection, animals were sacrificed, cardiac ventricles harvested and mitochondria were isolated. Steady state CcOx kinetic activity was measured and normalized to citrate synthase activity. Cardiac function was assessed at 48 hours using an isolated rat heart preparation and survival was tracked. Significance was determined with student's t-test. CLP significantly decreased myocardial CcOX activity, O2 consumption, left ventricular pressure (LVP), and pressure developed during isovolumic contraction (+dP/dt) and relaxation (-dP/dt). Caffeine restored these parameters to sham values following CLP and increased ±dP/dt by >40%. Survival was 72% at 24 hours and 50% at 48 hours in saline injected CLP rats compared to 100% at 24 hours and 93% at 48 hours in caffeine injected CLP rats. Therefore, caffeine may be a novel therapy to abrogate sepsis-associated myocardial depression.

9 MODULATION OF BCL-2 FAMILY BLOCKS SEPSIS-INDUCED DEPLETION OF DENDRITIC CELLS AND MACROPHAGES

O. Peck-Palmer, J. Unsinger, K. Chang, J.E. McDunn, C. Coopersmith, and R. Hotchkiss. Departments of Anesthesiology/Surgery, Washington University School of Medicine, St. Louis, MO 63110

Introduction:

It is well established that sepsis induces extensive apoptosis in cells of the adaptive immune system. However, whether sepsis induces apoptosis in the innate immune system has not been clearly defined. Our previous in vitro studies demonstrated that both primary and cultured monocytes undergo E. coli-induced apoptosis, which was prevented by the delivery of anti-apoptotic peptides. The purpose of this study was to determine whether sepsis induces apoptosis in dendritic cells (DC) and macrophages (MΦ) in vivo. Secondly, we examined the effects of overexpression of Bcl-2 or knockout of Bim on sepsis-induced cell depletion.

Methods:

Male, 6-8 week old, C57BL/6 mice, Vav-Bcl2 mice (Bcl-2 overexpression in hematopoietic cells), and Bim−/−mice underwent sham or CLP injury. Twenty hours post injury the absolute numbers of splenic DC and MΦ were determined via flow cytometry. Two macrophage populations were defined, i.e., a well differentiated "mature" MΦ (CD11binterF480hi) and a less differentiated 'monocyte-like' population (CD11bhiF480inter). DC were defined as CDllbhiMHCIIhi.

Results:

Compared to sham, sepsis induced an 64+7% and 77+3% decrease in absolute cell numbers of splenic DC and 'less differentiated' MΦ, respectively (n=7, p<0.05). Importantly, there was no decrease in either of these two cell populations in Bcl-2 transgenic or Bim−/−mice. Sepsis did not decrease the more 'well-differentiated' MΦ.

Conclusion:

Sepsis causes extensive loss of innate immune cells. Prevention of apoptosis by the modulation of Bcl-2 family members was exhibited in DC and the less differentiated tissue MΦ. These results strongly implicate apoptosis as the mechanism of innate immune cell depletion by sepsis.

Minisymposium

10 IN VIVO GENE SILENCING OF PEROXISOME PROLIFERATOR ACTIVATED RECEPTOR-γ ENHANCES MYOCARDIAL REPERFUSION INJURY

B. Zingarelli, P.W. Hake*, M. O'Connor*, and A. Denenberg*. Critical Care Med., Cincinnati Children's Hosp. Med. Ctr., University of Cincinnati, Cincinnati, OH 45229, USA

Peroxisome proliferator activated receptor-γ (PPARγ) is a nuclear factor with pleiotropic effects on lipid and glucose homeostasis, and inflammation. PPARγ ligands have been reported to exert cardioprotective effects in experimental myocardial reperfusion injury. In this study we tested the hypothesis that reduced activation of PPARγ enhances reperfusion injury. With "loss-of-function" experiments, we inhibited PPARγ function using an in vivo model of post-transcriptional gene silencing by small interfering RNA (siRNA). Specifically, Wistar rats were administered with siRNA for PPARγ or scrambled siRNA (80 nmol/rat via jugular vein). At 48h after siRNA injection, animals were subjected to myocardial ischemia and reperfusion. In rats treated with scrambled siRNA, ischemia and reperfusion resulted in development of myocardial contraction bands and interstitial edema. Tissue damage was confirmed by elevation of plasma levels of troponin-1 (54.4 ±25.1 ng/ml). Increase in cardiac myeloperoxidase activity (20.6±2.1 U/100 mg tissue) indicated tissue neutrophil infiltration. In rats treated with siRNA for PPARγ, myocardial damage appeared massive with derangement of myofibrils, hemorrhage and infiltration of inflammatory cells. PPARγ siRNA-treated rats also exhibited higher levels of plasma troponin-1 (135.4±20.3 ng/ml) and increased activity of tissue myeloperoxidase (135.4±20.3 U/100 mg tissue) when compared with scrambled-treated rats (P<0.05). Our data suggest that PPARγ is an important cardioprotective regulator during reperfusion injury.

Supported by National Institutes of Health (R01 GM-067202; R01 AG-27990).

11 EARLY NOREPINEPHRINE INFUSION DELAYS CARDIAC ARREST AFTER SEVERE HEMORRHAGIC SHOCK IN RATS

Jae Hyuk Lee, Kyuseok Kima, You Hwan Jo, Kyung Su Kim, Woon Yong Kwon, Joong Eui Rheea, and Gil Joon Suh. Department of Emergency Medicine, Seoul National University College of Medicine, Seoul, Korea, aDepartment of Emergency Medicine, Seoul National University Bundang Hospital, Seongnam-si, Kyung-gi do, Korea

Severe hemorrhagic shock often results in cardiac arrest due to vital organ hypoperfusion, especially of the heart. Although fluid resuscitation is the mainstay of management in hemorrhagic shock, treatment of cardiac arrest in association with severe hemorrhagic shock is unclear. This study was designed to determine the effect of early infusion of norepinephrine on the hemodynamics and survival in severe hemorrhagic shock. Twelve Sprague-Dawley rats were bled to about 35% of estimated blood volume for 30 minutes and randomized to norepinephrine (10mcg/kg/min) in 5% dextrose solution (n=6) or the same volume of 5% dextrose (n=6), concurrently with Lactated Ringer's solution. After 30 minutes of resuscitation, half of the shed blood was transfused. Time to cardiac arrest and mean arterial pressure (MAP) were compared between the two groups. MAP during the resuscitation period was higher in the norepinephrine group than in the control group. Five of 6 rats in the norepinephrine group but none of the control group survived until the transfusion period (83.3% vs. 0.0%; P=0.003). Median time to cardiac arrest was significantly longer in the norepinephrine group (67.0 minutes [IQR, 60.0-77.0]) than in controls (41.0 minutes [IQR, 40.0-47.0]; P=0.002). Early use of norepinephrine in a rat model of severe hemorrhagic shock increased mean arterial pressure during the resuscitation period and delayed the onset of cardiac arrest.

13 HUMANIZED MICE AS A POWERFUL TOOL TO STUDY THE EFFECTS OF SEPSIS ON HUMAN HEMATOPOIETIC CELLS

Jacqueline Unsinger, Jacquelyn S. McDonough, Katherine C. Chang, Richard S. Hotchkiss. Department of Anesthesiology, Washington University School of Medicine, Saint Louis, MO 63110

Sepsis is a complex systemic disease. Hallmarks of sepsis are massive apoptosis of immune effector cells and profound immunosuppression. An important limitation in the study of sepsis has been the major differences in the mouse versus the human immune system. The purpose of this study was to generate mice with a "humanized" immune system and to investigate the effects of sepsis in a more clinically relevant model.

Methods:

To analyze the effects of sepsis on the human hematopoietic system in vivo, NOD/SCID/IL2rγnull mice were purchased from Jackson Laboratory. These mice lack mature murine T and B cells and display extremely low levels of NK cell activity. 1x105 purified human CD34+cord blood cells purchased from Cambrex Lonza were transplanted into these neonatal mice via the superficial temporal vein 24 hours post 100 rads whole body irradiation.

Results:

Six weeks post-injection mice display high level of chimerism of human hematopoiesis in the myeloid as well as in the lymphoid compartment. High level of engraftment could be detected in peripheral blood, spleen, thymus and bone marrow. Human cord blood cells differentiated into mature cells including T and B lymphocytes, NK cells, monocytes, granulocytes and dendritic cells. The bone marrow also displayed high engraftment of hCD34+hematopoietic progenitor cells. To investigate the effects of sepsis directly on the human hematopoietic system, these mice were subjected to CLP surgery to induce peritonitis. Human lymphocytes in septic mice had a marked increase in apoptosis compared to Sham operated mice Additionally, 24 hours post CLP, human cytokines could be detected in the septic mouse serum but were undetectable in Sham operated mice.

Conclusion:

"Humanized" mice provide a powerful tool to study the effects of sepsis on the human hematopoietic system and to investigate the effects and efficacy of drug therapy on the outcome of sepsis.

14 TRANSLATION OF THE TREATMENT OF HEMORRHAGIC SHOCK FROM THE BENCH TO THE BEDSIDE IN CIVILIAN AS WELL AS MILITARY PATIENTS

Michael M. Krausz. Rambam Health Care Campus, Technion-Israel Institute of Technology' Haifa Israel 31096

The classic treatment of hemorrhagic shock in trauma casualties was introduced by G. Tom Shires in the 1960s and 1970s to replace the large extracellular fluid (ECF) deficit in hemorrhagic shock by massive infusion of both shed blood and lactated Ringer's solution. This was the basis of the well known dogma "3 to 1 rule" for treatment of hemorrhagic shock which was adopted by the ATLS guidelines for treatment of trauma casualties. This practice was challenged by experimental laboratory studies in rats (Gross et al. 1988) and swine (Bickell et al. 1989). It was observed that attempting to increase blood pressure to normal by aggressive fluid resuscitation in uncontrolled hemorrhagic shock resulted in increased blood loss from injured vessels, hemodynamic decompensation and increased mortality, when compared to no resuscitation. This concept was later adopted by Bickell et al. in 1994 to treat hypotesive patients with penetrating torso injuries. It was observed that the hemodynamic response and survival were better in patients when resuscitation was delayed until surgery. Further experimental animal studies in swine by Kowalenco et al. in 1992 showed improved outcome with hypotensive resuscitation and this practice has recently become the preferred treatment of civilian as well military trauma casualties in uncontrolled hemorrhagic shock. The civilian guidelines of prehospital care in Israel, and the instructions for fluid treatment of hemorrhagic shock in the Israeli army at present, are to increase systolic blood pressure only up to 80 mmHg, and maintain this blood pressure until the bleeding source is controlled at surgery.

15 THE NON-GENOMIC PATHWAY MEDIATES SOME OF THE EFFECTS OF ESTROGEN ON CYTOKINE PRODUCTION BY T CELLS FOLLOWING TRAUMA-HEMORRHAGE

Takao Suzuki, Huang-Ping Yu, Ya-Ching Hsieh, Mashkoor A. Choudhry, Kirby I. Bland, and Irshad H. Chaudry. Center for Surgical Research and Department of Surgery, University of Alabama at Birmingham, AL 35294

Studies have shown that administration of 17β-estradiol (E2) following trauma-hemorrhage (T-H) attenuates the T-H-induced suppression in T cell cytokine production. However, it remains unknown whether E2 mediates its effects via genomic or non-genomic pathway. To determine the role of non-genomic pathway and the role of MAPK in mediating the non-genomic effects of E2, E2 conjugated to BSA (E2-BSA) which acts on only cell surface membranes was used. Male rats underwent T-H (mean BP 40mmHg for 90min, then resuscitation). E2, E2-BSA with or without an estrogen receptor antagonist (ICI 182,780), or vehicle was injected intravenously during resuscitation. IL-2 and IFN-γ productive capacity and the activation of MAPK (p38, ERK1/2, JNK) in response to anti-CD3 in T cells decreased at 2 h after T-H (Fig). However, E2 administration restored the values to normal. Although E2-BSA administration also significantly attenuated the suppression in T cell cytokine production, the values were still lower than sham. In contrast, administration of E2-BSA prevented T-H-mediated changes in MAPK activation in T cells to the same extent as E2 treatment. Co-administration of ICI 182,780 abolished the effects of E2-BSA. Moreover, IL-2 and IFN-γ production was further suppressed in vitro by selective inhibitors of ERK1/2 (PD98059) or JNK (SP600125) pathway in T cells, while p38 inhibitor (SB203580) had no significant effects. Thus the salutary effects of E2 on T cell cytokine production are mediated at least in part via the non-genomic pathway and these non-genomic effects are likely mediated via MAPK.

Figure
Figure

16 INCREASED ENDOTHELIAL PROGENITOR CELLS IN SEPSIS

Asako Matsushima, Hiroshi Ogura, Taichin Toh, Naoyuki Matsumoto, Hideo Hosotsubo, and Hisashi Sugimoto. Department of Traumatology and Acute Critical Medicine, Osaka University Graduate School of Medicine, Osaka, Japan

Introduction:

Endothelial progenitor cells (EPC) are reported to contribute to tissue repair. In septic condition, severe endothelial damage deteriorates micro-vascular circulation and causes multiple organ failure. Endothelial repair with EPC could be a key to recover from sepsis. In this study, we evaluated EPC and hematopoietic progenitor cell (HPC) and EPC related growth factor in peripheral blood of septic patients.

Methods:

Eighteen patients with sepsis (S) and 17 healthy volunteers (H) were included. Flow cytometric analysis was performed to count EPC and HPC. EPC was defined as the triple positive cell with CD117, vascular endothelial growth factor receptor 2 (VEGFR-2) and CD34. HPC was defined as the positive cell with CD34. Serum VEGF, E-selectin and IL-6 were measured by ELISA. Serial evaluation of these variables was also performed for the period of 14 days.

Results:

EPC and HPC increased significantly in sepsis (EPC; S 4192±329 /mL vs H 2780±108 /mL, p < 0.05, HPC; S 30032±2428 /mL vs H 24330±838 /mL, p < 0.05). Serum VEGF, E-selectin and IL-6 were all elevated in sepsis. In the serial study, EPC and HPC counts were elevated during the observation period, whereas serum IL-6 decreased gradually. Serum concentration of VEGF was increased during this period.

Conclusions:

EPC and HPC increased significantly in sepsis even after the decrease of pro-inflammatory cytokines, which suggests the pivotal role of these cells for the neovascularization and tissue repair in septic condition.

Novo Nordisk Award

17 PROSPECTIVE MULTICENTER STUDY OF THE RELATIONSHIP BETWEEN GLOBAL TISSUE HYPOXIA AND COAGULATION ACTIVATION IN EARLY SEPSIS RESUSCITATION

S. Trzeciak, A.E. Jones, N.I. Shapiro, A.E. Pusateri, R.C. Arnold, M. Rizzuto, J.E. Parrillo, and R.P. Dellinger, and for the Emergency Medicine Shock Research Network (EMSHOCKNET)

Experimental data suggests that endothelial exposure to hypoxia can activate coagulation. We hypothesized that the quantity of exposure to global tissue hypoxia would be associated with the degree of coagulation activation in early sepsis resuscitation. This is an interim analysis of a prospective multicenter study. Inclusion criteria: age >17, acute infection, hypotension despite fluid challenge or serum lactate >4 mM/L, and use of a continuous oxymetric central line during resuscitation as part of early goal-directed therapy (EGDT). We measured central venous oxygen saturation (ScvO2) every 10 sec for 6 hours and calculated the 0-6 hour cumulative area under the curve for ScvO2 <70% (Hypoxia Index). We measured coagulation biomarkers at 0 and 6 hours. A priori, we set a cut-off for clinically significant exposure as a Hypoxia Index >3000. We compared mean change in coagulation biomarkers over 0-6 hours between exposures and non-exposures. Of 23 subjects, 52% had exposure to hypoxia. Of all biomarkers [plasminogen activator inhibitor-1, antithrombin III, tissue plasminogen activator, thrombin-antithrombin and plasmin-antiplasmin complexes] only Protein C (PC) activity significantly differed between the groups, decreasing over 0-6hours in hypoxia exposures (−4.2% vs. a rise of 1.4% in non-exposures, p=0.045). In this interim analysis, exposure to global tissue hypoxia during early sepsis resuscitation was associated with significantly diminished PC activity, suggesting PC pathway activation. This ongoing research aims to determine if amelioration of global tissue hypoxia can attenuate sepsis-induced coagulation activation.

18 ROLE OF HEME OXYGENASE INDUCTION IN THE EARLY PHASE OF REPERFUSION AFTER TRAUMA/ HEMORRHAGIC SHOCK IN RATS

J. C. Duvigneau1, A. Postl1,2, R. T. Hartl1, S. Haindl2, T. Behling2, C. Zifko2, I. Miller1, K. Staniek1, W. Gregor1, L. Gille1, M. Gemeiner1, S. Bahrami2, A. V. Kozlov2, and H. Redl2. 1Veterinary University Vienna; 2Ludwig Boltzmann Institute for Traumatology, AUVA Research Center, Vienna, Austria

It is known that reperfusion after trauma/hemorrhagic shock (Trauma / Hemorrhage / Resuscitation - THR) is associated with the induction of heme oxygenase-1 (HO-1), a stress response protein. Catalytic activity of HO yields carbon monoxide, biliverdin (BV), which is rapidly metabolized to bilirubin (BR), and iron ions (Fe2+). While BV/BR were shown to have anti-oxidative properties, in-creased levels of Fe2+ mediate oxidative damage. Therefore, the role of HO-1 is unclear, since its induction can have both cytoprotective and deleterious effects.

Our project aimed at investigating the anti-/pro-oxidative potential of HO-1 in various organs in the early phase of THR. HO-1 induction can impart cytoprotection by simultaneously increasing production of proteins responsible for iron processing and sequestration, which would decrease the risk of iron-mediated oxidative stress. Therefore we analyzed the effect of THR on the expression of relevant proteins affecting iron homeostasis.

However, as even temporarily increased levels of the "free" iron pool are potentially dangerous, we investigated whether THR leads to critically elevated levels of "free" iron and consequently oxidative damage (protein nitration and lipid peroxidation).

Since delayed organ damage, which is possibly associated with increased oxidative stress, often develops after THR, we also analyzed the functions of essential cellular structures, such as mitochondria (respiration) and ER (hepatic cytochrome P450 1A1 activity), as well as markers for increased cell death (apoptosis/necrosis).

19 TOWARDS A MOUSE MODEL FOR ACUTE COAGULOPATHY OF TRAUMA/SHOCK

Daniel Frith1, Brian Chesebro2, Nimesh Patel1, Chris Thiemermann1, Karim Brohi1, Mitchell Cohen2, and Rupert Pearse1. 1William Harvey Research Institute, Barts and the London School of Medicine & Dentistry, Queen Mary, University of London; 2San Francisco General Hospital, University of California San Francisco

Background:

Recent clinical studies have identified an Acute Coagulopathy of Trauma-Shock (ACoTS) that is established early after injury and appears to be driven by systemic shock in combination with tissue injury. Arrival in the emergency department with ACoTS is associated with a 4-fold increase in mortality. The biological mechanism resulting in ACoTS, and the initiators of this pathway isunknown. We describe our experience developing a murine model of minimal resuscitation haemorrhagic shock for mechanistic investigation of ACoTS.

Methods:

The model was based on a previous murine model ofresuscitated haemorrhagic shock. There were significant obstacles to the development of a robust non-resuscitative model. These centred around 1) a method for reliably withdrawing enough blood for analysis at the end of resuscitation; 2) producing a reproducible and consistent degree of hypoperfusion as measured by the base deficit and lactate; 3) the inter-mouse variability in ventilatory response to anaesthesia regimens resulting in a respiratory acidosis and 4) determining a sufficient degree of trauma and shock to consistently reproduce ACoTS.

Results:

In the current model, C57 mice are anaesthetised with isoflurane (0.8-1.5%) carried in oxygen and breathe spontaneously throughout the study. One common carotid artery is catheterised with polyethylene tubing (internal diameter 0.28mm; non-heparinised) connected to a digital pressure transducer. Mice are randomly assigned to sham control (C); haemorrhagic shock (HS); trauma (T) and haemorrhagic shock + trauma (HS+T) groups. In the HS groups, blood is withdrawn to reduce the mean arterial pressure to 40 +/- 5mmHg and maintained for 60 minutes. 15% circulating volume of Ringers' Lactate is used to flush the line and core temperature is maintained at 37°C. Mice in trauma groups receive a 2cm paramedian laparotomy prior to haemorrhage. After 60 minutes blood is aspirated from the carotid line for analysis and the mouse is sacrificed under deep anaesthesia. Mice in all 4 groups exhibit normal respiratory physiology. HS mice have on average 40%±5% of their blood volume withdrawn. Mice subject to haemorrhage display a consistent metabolic acidosis with significantly higher base deficit (BD) (Fig 1). Control (C) and Hypoperfused (HYPO - BD >4mmol/l)) mice did not exhibit ACoTS, while HYPO+T mice had prolonged Activated Partial Thromboplastin Times (Fig 2). End aspirate yields a mean of between 443±74μl for C+T mice and 290±40μl for HS groups.

Figure
Figure
Figure
Figure

Conclusion:

We have developed a robust minimal resuscitation murine model of trauma/haemorrhagic shock that may be used to investigate the pathophysiology of ACoTS.

20 Interaction of the Complement and Coagulation System during Hemorrhagic Shock after Severe Trauma

Markus Huber-Lang1, Florian Gebhard1, and Uwe B. Brückner2. 1Dept. of Traumatology, Hand-, Plastic-, and Reconstructive Surgery; 2Division of Surgical Research; University of Ulm; Germany

Severe tissue trauma and hemorrhagic shock are associated with extensive activation of the coagulation cascade. In previous studies on polytraumatized patients suffering additional hemorrhagic shock an early development of disseminated intravascular coagulopathy (DIC) was found with secondary hyperfibrinolysis within the first 60 min after the injury. There is rising experimental and clinical evidence that the rapid activation of the coagulation system is accompanied by a very early onset of a progressive and uncontrolled innate immune response with often lethal consequences.

When analyzing plasma and sera obtained early after severe injury, we found that almost simultaneously to the activation of the coagulation system, there is also a massive and uncontrolled activation of the complement system with excessive generation of complement activation products (C5a, C3a, MAC), loss of complement hemolytic activity (CH50) and specific changes of the complement-regulatory protein (CReg) profiles on blood leucocytes. In analogy to the trauma-induced coagulopathy, this rapid impairment of the complement-associated immune defense may be termed "complementopathy". Since both cascades mainly house serine proteases, in vitro exposure of key complement components (C3, C5) with various key serine proteases of the coagulation system was performed, resulting in the robust generation of biologically active anaphylatoxins (C3a, C5a). Furthermore, investigation of C5-deficient plasma resulted in global impairment of the clotting function, which was almost completely reversible by adding C5, indicating a cross-talk between the coagulation and complement system.

In conclusion, the present data suggest a common serine protease cascade including both, the coagulation and complement system, as important players in the host's hemostatic and innate immune response (Supported by ESS/Novo Nordisk Award 2005).

Trauma/Multiple Injuries Basic Research

P1 TEMPARARY INTRAVASCULAR SHUNTS AS A DAMAGE CONTROL ADJUNCT TO MAINTAIN INTESTINE PERFUSION AFTER SUPERIOR MESENTERIC ARTERY INJURY IN SWINE MODEL

Weiwei Ding, Xingjiang Wu, Pengfei Wang, Wenbo Zhang, Guanwen Gong, Yousheng Li, and Jieshou Li. Research Institute of General Surgery, School of Medicine, Nanjing University, Jinling Hospital, Jiangsu Province, China

Background:

Trauma to the superior mesenteric artery (SMA) injury is rare but most critical. Attempts to repair the injured artery may be difficult and time-consuming because of limited medicine care or surgical experiences with massive casualty in military forward field and terrorist attacks in civil time. We extended use of temporary intravascular shunts (TIS) as a damage control adjunct in superior mesenteric artery injury swine model and hypothesized that temporary intravascular shunt could maintain hemodynamics more stable, shorten intestine ischemia time, maintain distal perfusion while resuscitation, and thus improve survival rate while comparing to primary anastomosis in the austere condition.

Methods:

Pigs (weight 22 ± 2.5 kg) were anesthetized and instrumented with arterial and a thermodilution cardiac output catheter, and splenectomized. SMA flow was interrupted and completely transected while animals were hemorrhaged to a mean arterial pressure (MAP) of 40 mmHg and maintained for 30 minutes. Cold lactated Ringer's was gradually infused to induce hypothermia. Prehospital resuscitation was simulated to maintain MAP 60 mmHg. Pigs were then randomized into control (C; n=6), primary anastomosis (PA; n=6), and temporary shunt (TS; n=6) groups. Abdomen was temporary closed and all pigs were closely monitored and resuscitated for 6 hours. After resuscitation, delayed anastomosis was performed in TS. The survival animals were executed on post operation day 2. Arterial and mixed venous blood gases and serum lactate were measured at intervals throughout the experiment. Systemic and intestine hemodynamic parameters were hourly recorded. Data were analyzed using analysis of variance with Dunnet post hoc test when appropriate. Significance was defined as P<0.05.

Results:

At the end of shock and hypothermia procedures all animals presented with disastrous conditions: low core temperature (34.7 ± 0.3 °C), severe acidosis (PH 7.18 ± 0.2), decreased blood pressure (40.9 ± 0.3 mmHg), depressed cardiac output (1.6 ± 0.4 L/min) and systemic oxygen delivery (7.8 ± 0.1 mL/kg/min). Without resuscitation it brought up to 100% mortality in control group. Animals in TS needed less resuscitation fluid than PA group throughout the resuscitation period (TS 159.0 ± 12.9 mL/kg; PA 206.7 ± 17.3 mL/kg; P<0.01). Systemic oxygen delivery in TS group was higher than that in PA at the according time point but it didn't reach statistical significant (P>0.05). Average SMA flow rates return to 42 ± 11 % of that in the baseline in TS at the first resuscitation hour (higher than PA 36 ± 8 %, P<0.05)and remained higher than PA in the following resuscitation hours. Serum lactate in TS decreased rapidly than that in PA from the fourth resuscitation hour (TS 5.7 ± 0.3; PA 6.6 ± 0.2; P<0.05) but remained higher at the end than baseline (4.1 ± 0.3 vs. 2.0 ± 0.2; P<0.01). Overall survival rate was significantly higher in TS than that in PA (66.7 % vs. 33.3 %, P<0.05).

Conclusion:

We built up a severe SMA injury animal model for damage control surgery investigations and extended TIS as an important damage control adjunct to maintain intestine perfusion. TIS is advocated when compared with primary artery anastomosis because it needs less volume of resuscitation fluid; accelerates the clearance of lactate acid; maintains hemodynamics stable and thus improves early survival rate. The relations between shunt time and vascular endothelium damage are deserved to be detailed in further experiments.

P2 ESTABLISHMENT OF AN ACUTE SUPERIOR MESENTERIC ARTERY INJURY MODEL FOR DAMAGE CONTROL SURGERY

Weiwei Ding, Xingjiang Wu, and Jieshou Li. From Research Institute of General Surgery, School of Medicine, Nanjing University, Jinling Hospital, Jiangsu Province, China

Background:

Managements of superior mesenteric artery (SMA) injuries are troublesome and often result in a disappointing outcome. Damage control surgery has been approved to be an effective and reliable strategy for severe trauma victims. We aimed to build up a severe trauma-shock-hypothermia model of superior mesenteric artery injuries for damage control study and determine the optimal time to institute damage control surgery.

Methods:

Pigs were anesthetized and instrumented with arterial and a thermodilution cardiac output catheter, and splenectomized. SMA flow was interrupted and completely transected while animals were hemorrhaged to 45% estimated blood volume. Pigs were maintained shock and intestine for three different durations: intestine ischemia for 30min (I-30; n=6), for 60min (I-60; n=6), and for 120min (I-90; n=6). Meanwhile, cold lactated Ringer's (10ml/kg) was gradually infused to induce hypothermia. SMA was then declamped and kept in reperfusion for 6 hours. Hemodynamic data were collected during shock and resuscitation. Complete bloodcounts, lactate, coagulation studies, and renal and liver function were measured throughout the experiment. Distal ileum was collected at theend of reperfusion. Significance was defined as P<0.05.

Results:

All animals presented with disastrous conditions at the end of shock: low core temperature, severe acidosis, decreased blood pressure, depressed cardiac output and oxygen delivery. I-90 animals suffered the lowest temperature (P<0.01), the most severeacidosis (P<0.01), lowest blood pressure (P<0.01), depressed cardiac output and oxygen delivery (P<0.01). Coagulopathy developed in I-90 while normal prothrombin time and thrombin timewere detected in I-30 and I-60. Aspartate aminotransferase, lactate dehydrogenase, creatine kinase and alkaline phosphatase were equally within groups (P>0.05). One hundred percent (6/6) of I-30, 83.3% (5/6) of I-60, and 16.7% (1/6) of I-90 pigs survived to at the end (P<0.01 overall). Base excess in I-90 was much lower than I-30 and I-60 animals (-10.4 ± 1.0 vs.-7.5 ± 0.5 vs.-8.8 ± 0.8, respectively).

Conclusions:

We firstly built up an acute SMA injury animal model for damage control surgery investigations and determined that the optimal institution time of damage control was before 60 minutes in the trauma-shock-hypothermia swine model.

P3 ELIMINATION OF C5aR REDUCED APOPTOTIC CELL DEATH AND CIRCULATING CYTOKINE LEVEL FROM INTESTINAL ISCHEMIA-REPERFUSION INJURY

Ruihua Chen, Harini Rajan, Xiaoman Yang, Da-Zhong Xu, Sergey B. Zaets, Nancy Bogdan, Qi Lu, Edwin A. Deitch, and Cindy Cao. Novo Nordisk US, North Brunswick, NJ 08902 and UMDNJ-NJ Medical School, Newark, NJ 07103

Objective:

C5a pathway has been demonstrated to be an important mediator in inflammation and ischemia/reperfusion. We aimed to examine several molecular events in addition to histological examination in order to reveal the downstream mechanism of C5a activation in ischemic reperfusion.

Methods:

Superior mesenteric artery occlusion (SMAO) model was adopted to create gut ischemia-reperfusion (I/R) injury. C5aR knock-out (KO) and wild type (WT) mice were subjected to SMAO or sham SMAO. Apoptotic cell death in intestine and circulating cytokine levels were determined after 3 hours of reperfusion.

Results:

Apoptotic cell death was induced 3hr after I/R injury in WT-SMAO, and was reduced by more than 50% in C5aR deficient mice compared to WT mice (see graph below). The mean plasma levels of TNFα was increased ~5.1 fold in WT subjected to SMAO compared to SHAM despite a wide range of circulating cytokine levels among individual animals within a given time point. In contrast, the mean TNFα level was only increased 1.5 fold in the C5aR KO mice subjected to SMAO (WT SMAO vs KO SMAO, P<0.05) compared to SHAM. A similar pattern was observed with plasma IL6 levels between WT and C5aR KO mice.

Figure
Figure

Conclusion:

Elimination of complement C5aR protects the gut from ischemia-reperfusion injury, and reduces local apoptotic cell death and systemic inflammation.

P4 MICROWAVE OVEN: HOW TO USE IT AS A CRYSTALLOID FLUID WARMER

Siriwasan Akanitthaphichat, and Kaweesak Chittawatanarat. Department of Surgery, Faculty of Medicine, Chiangmai University, Chiangmai

Background:

Hypothermia is a common complication in the hypovolemic patient. Warm intravenous fluids have been shown asa valuable adjunct in volume replacement to prevent this complication. The microwave oven is considered an applicable alternative method for warming fluids but the method to use have not been established.

Objective:

To evaluate the efficacy of the microwave oven in warming crystalloid fluids, while examining the variables influencing its operation, and determine the appropriate formula to calculate the safety temperature of warming fluid by this method.

Method:

We conducted the experimental study into 3 steps 1) to evaluate the variables influencing the operation, 2) determine the appropriate formula for calculating warming duration, and 3) apply the formula to the application. We underwent the study by using 4 type of microwave oven to evaluation the variables influencing the operation which including package shaking, type of container, variation of microwave irradiation and type of crystalloid fluid. In this study we calculate the duration of warming fluid for expected increase of temperature by theoretical formula:

Result:

The important variables influencing the operation of the microwave oven included the difference between crystalloid fluid and room temperature, microwave oven capability, variation of microwave irradiation, and fluid shaking. The median time achieve maximum temperature after warming fluid was 0.37 sec (0.43+/-0.24 sec). The maximum difference from variation of microwave irradiation between the maximum and the baseline temperature (ΔTmax) was 0.6 degree Celsius. The maximum temperature when we adjusted 0,5,10,15 and 20 percent to theoretical formula were 37.1, 37.3,37.8,38.1 and 38.9 degree Celsius respectively. In term of safety margin about 39 degree Celsius when we added the ΔTmax to the adjusted temperature. The appropriate formula for calculating warming duration was:

Conclusion:

The microwave oven is a safe and practical method for enabling the warming of crystalloid fluids especially in the area where lack of warmer equipment.

P5 POLOXAMER 188 IMPROVES SURVIVAL AFTER PROLONGED HYPOTENSIVE RESUSCITATION

E.A. Gonzalez, R. Zhang, R.A. Kozar, F.A. Moore, and R.L. Hunter. University of Texas and Methodist, Houston, TX 77030

Patients who develop decompensated shock require early interventions. The membrane sealant Poloxamer P188 is a unique and innovative pharmacologic adjunct to resuscitation. We hypothesized that P188 would improve early survival after prolonged hypotensive resuscitation of lethal traumatic shock.

Methods:

Male rats had femoral arterial and venous catheters placed. The rats were bled over 15 min to a MAP of 30 mm Hg for 30 min, then assigned to 40 min of hypotensive resuscitation (MAP 60) with a continuous infusion of a) Hextend at 1.4 ml/100 gm or b) Hextend at 1.4 ml/100 gm+P188 140 mg/kg. At 6 hr survivors where sacrificed.

Table
Table

Results:

Resuscitation with P188/Hex significantly reduced the amount of volume required during the first six hours of resuscitation to maintain a BP of 60 mmHg. Moreover, P188/Hex significantly extended survival time from onset of hemorrhage as compared to Hex resuscitation alone.

Conclusion:

P188 a novel resuscitative improved early survival after hemorrhagic shock. Patients who develop decompensated shock require early interventions for survival and may benefit from P188 administration.

P6 CHANGED EXPRESSION PROFILE OF COMPLEMENT REGULATORY PROTEINS (CREGS) FOLLOWING POLYTRAUMA IN HUMANS

U. Amara1, D. Rittirsch1, G.M. Anjum2, M. Schneider2, U.B. Bruckner3, M. Weiss2, F. Gebhard1, and M. Huber-Lang1. Departments of 1Traumatology and 2Anaesthesiology, 3Division of Surgical Research, University of Ulm, University Hospital, 89075 Ulm, Germany

In cases of severe injury and sepsis the complement cascade, a critical part of the innate immune system, becomes uncontrolled and excessively activated. However, protection against complement-mediated tissue destruction is provided by various soluble and membrane-bound complement regulatory proteins (Cregs). So far, the cellular profile of the inhibitory Cregs in patients with multiple injuries is not understood. Therefore, the expression profile of the most relevant Cregs was investigated using flowcytometry analysis of neutrophils, monocytes and lymphocytes obtained from either age-matched healthy volunteers (n=10) or polytraumatized patients (n=10; injury severity score ISS>18). Blood samples were harvested immediately at admission to emergency room and 4, 12, 24, 120 and 240 h after trauma, respectively, and analyzed immediately. Significantly altered expression profiles of CD35 (complement receptor 1, CR1), CD46 (membrane co-factor protein, MCP), CD55 (decay accelerating factor, DAF) and CD59 (MAC inhibitor) in neutrophils, monocytes and lymphocytes were found. Some of them showed a cell type-dependent reverse expression pattern, whereas others exhibited a similar cellular expression, indicating different regulation pathways of the Cregs.

The study evidently suggests that Cregs' expression profile on leukocytes from polytraumatized humans is significantly changed. Therapeutic intervention by Creg modulation might be an effective future strategy to beneficially modulate the inflammatory response after severe trauma.

P7 GENDER DIFFERENCES IN THE RESPONSE TO INCREASE OF INTRA-ABDOMINAL PRESSURE IN RATS

Michael M. Krausz, Evgeni Gleizerov, Luba Seminikhina, Dalit E. Dar, and Ahmad Mahajna. Rambam-Health Care Campus, Haifa Israel 31096

Abdominal compartment syndrome (ACS) is defined as increased intra-abdominal pressure (IAP>20mmHg) in combination with signs of organ dysfunction. It has been demonstrated that estradiol, has a protective effect on cardiovascular and hepatocellular functions after trauma-hemorrhage. The response to increases of IAP was therefore compared in male and female rats.

Methods:

After anesthesia and cannulations, a group male and female rats were randomly divided into 4 groups: Group 1 (Males n=12 females n=9) sham-operated including canulation and insertion of a sterile balloon into the abdominal cavity. Group 2 (males n=10 females n=8), IAP was increased to 10 mmHg. In group 3 (males n=9 females n=9), IAP was increased to 20 mmHg, and in group 4 (males n=10 females n=8), IAP was increased to 30 mmHg. The animals were observed for 4 hours.

Results:

Increase in IAP to 20 mmHg and 30 mmHg led to a fall in mean arterial pressure (MAP) to 51.8±7.1 mmHg (p<0.01) and 33.3±9.3 (p<0.01) mmHg respectively, in male rats. In female rats a similar increase of IAP led to a fall in MAP to 73.1±6.3 mmHg (p<0.05) and 52.0±3.4 (p<0.05) mmHg, respectively. The fall in MAP in males was significantly lower than in females (p<0.05). Increase in IAP to 20 or 30 mmHg resulted in reduction of serum glucose levels and increase of serum lactate in males, while in female rats blood glucose was reduced and lactate levels increased only when they were subjected to IAP of 30 mmHg. The pH levels were reduced in both males and females subjected to 20 or 30 mmHg compared to control rats. The survival of females that were subjected to IAP of 30 mmHg was significantly longer (p<0.05) survival than males subjected to the same IAP.

Conclusions:

Female rats better preserved their hemodynamic and metabolic parameters than male rats during increases of IAP, and may therefore be better protected against lethal abdominal compartment syndrome than males.

P8 RESUSCITATION WITH HEXTEND ALONE OR WITH A DRAG-REDUCING POLYMER (DRP) IN A SWINE HEMORRHAGE (HEM) MODEL

Michael Dubick, Jill Sondeen, Leticia Medina, Dale Prince, Eriko Yoshimaru, and Marina Kameneva*. US Army Institute of Surgical Research, San Antonio, TX and *University of Pittsburgh, Pittsburgh, PA, USA

Objective:

As part of our continuing investigation of adjuncts to fluids to improve outcomes in a hypotensive resuscitation strategy, the current study investigated 10 ppm DRP added to Hextend for the treatment of hemorrhagic shock in swine.

Methods:

Anesthetized, splenectomized and instrumented 40 kg swine (n=7−8/gp) were subjected to a controlled HEM of 20 ml/kg over 5 min that duplicated the blood loss profile of an uncontrolled HEM. At 10 min, pigs received 30 ml of Hextend or DRP. After 30 min, a second HEM of 8 ml/kg and limited resuscitation was given with up to 1 L of Hextend or DRP. Hemodynamics were monitored continuously and blood samples were drawn for CBC, blood gases, chemistries and coagulation function at baseline (BL) and at select times throughout the 4 hr experiment or until death.

Results:

DRP did not affect the viscosity of Hextend or plasma. Although MAP, heart rate and cardiac output (CO) were similar between groups, CO was 30% higher immediately after DRP than Hextend infusion. Lactate levels were 28% and 36% lower in the DRP than Hextend group at 2 h and 4 h, respectively. In addition, base excess and CVP were better maintained near BL values after DRP than Hextend infusion. DRP did not affect coagulation function as determined by thrombelastography. Survival was 8/8 in the DRP group and 5/7 in the Hextend group.

Conclusions:

Although DRP in Hextend did not exhibit general improvement in hemodynamics over Hextend infusion alone, it appeared to reduce signs of hypoxia associated with hemorrhagic shock and did not affect coagulation.

P9 BLUNT CHEST TRAUMA INDUCES MIGRATION OF PERIPHERAL BLOOD MONOCYTES TO THE LUNG ALONG A CHEMOTACTIC GRADIENT

U. Niesler, A. Palmer, M. Sulger, D.H. Seitz, M.S. Huber-Lang, and M.W. Knöferl. Dept. of Trauma Surgery, University of Ulm, 89075 Ulm, Germany

Previous studies in a rat model of blunt chest trauma showed a significantly higher number of alveolar macrophages (AM) after lung contusion. We hypothesized that the increase in cell number is initiated by chemokine release from resident AM which leads to the migration of monocytes to the lung along the chemotactic gradient. To study this, male Sprague Dawley rats were subjected to either sham procedure or blunt chest trauma, induced by a single blast wave. At 6h, 24h and 48h after injury, concentrations of CINC-1, MCP-1 and MIP-2 in bronchoalveolar lavage fluids (BAL) were determined by ELISA. Additionally, protein concentrations in BAL were measured. To analyze the capacity of monocytes to migrate to the inflammatory environment in the lung, blood monocytes from untreated male Sprague Dawley rats were used in a modified Boyden chamber chemotaxis assay with BAL obtained from trauma as well as sham animals as a chemoattractant. Concentrations of CINC-1 and MCP-1 in BAL were significantly increased at 6h, 24h and 48h after blunt chest trauma, when compared to sham; MIP-2 concentrations were significantly elevated at 24h and 48h after trauma. Protein concentrations in BAL were increased at all time points after trauma. Chemotactic activity of monocytes was significantly enhanced at 6h and 48h after trauma. These results suggest that blunt chest trauma causes a chemotactic gradient which, along with increased vessel permeability as shown by increased BAL protein concentration, facilitates the migration of circulating monocytes into the lung with two peaks in the early (6h) and late (48h) phase after trauma.

P10 FORMATION OF MICROPARTICLES IS CLOSELY RELATED TO ENDOTHELIAL DAMAGE IN VITRO

E. Feketeova*, R. Feinman*, Q. Lu, and E.A. Deitch. Surgery Department, UMDNJ, Newark, NJ 07103

We have previously shown that trauma/hemorrhagic shock (T/HS) mesenteric lymph leads to MODS. Since under conditions of stress, endothelial microparticles (MP) have been show to be increased and to both activate the endothelium and to have vasoactive properties, we tested the hypothesis that T/HS lymph induces MP formation from endothelial cells.

Methods:

Rats were subjected to T/HS n=7 or Trauma/Sham shock (T/SS) n=7 and Mesenteric lymph was collected for 3 hours post shock. The T/HS and T/SS lymph samples were ultra-centrifuged and the MP pellet labelled with AnnexinV/CD144 following which the the number of MP were counted by flow (sample gated on 1 u beads). In a second Exp, 5% vv T/HS or T/SS lymph was incubated with confluent Human Umbilical Vein Endothelial Cells (HUVEC) for 1 hour at 37°C following which the cell cultures were harvested and processed to form a MP pellet, that was stained with AnnexinV/CD144. Statistical analysis was done via Nonparametric ANOVA.

Results:

We have found 22 fold increase of MP in T/HS lymph (1122±341) MP/ul, when compared to T/SS lymph (50±20) MP/ul. MP formation was 10 fold higher in the HUVEC incubated with T/HS lymph (3299±1299 MP/ul) as compared to T/SS lymph (306±60). The number of MP in the HUVEC cultures incubated with T/SS lymph was similar to that seen in HUVECs culured in media alone (266±23 MP/ul).

Conclusion:

The number of MP is increased in T/HS lymph and T/HS lymph induces MP formation from HUVECs. Since MP may have significant biologic activity T/HS-induced MP formation may contribute to the biologic activity of T/HS lymph.

P11 DISRUPTION OF SKELETAL MYOCYTES INITIATES ENZYMATIC SUPEROXIDE GENERATION

Uta Kerkweg, and Herbert de Groot. Institute of Physiological Chemistry, University Hospital Essen, Hufelandstr. 55, 45122 Essen, Germany

Major muscle trauma is frequently succeeded by severe inflammatory reactions, which originate from early local incidents within the injured tissue. Since generation of reactive oxygen species (ROS) as an early local reaction to muscle crush injury has often been predicted, we studied ROS formation of cultured C2C12 skeletal myocytes after lethal mechanical damage using e.g. lucigenin chemilumi-nescence. Rupture of myocytes resulted in an immediate release of superoxide (O2) from the damaged cells; O2mainly originated from the membrane fraction and was enhanced by addition of NADH or NADPH. NAD(P)H oxidase inhibitors largely diminished this NAD(P)H-enhanced O2generation in isolated membrane fractions and cell lysates. In particular, the participation of mitochondria to O2formation was examined in C2C12 cells and mitochondria isolated from rat gastrocnemius muscle. Directly after exposure to injury-mimicking conditions (extracellular fluids), mitochondria took up extreme loads of calcium and swelled massively. Even though the mitochondrial respiratory chain completely lost its functionality, O2was produced by damaged mitochondria. Furthermore, O2formation was largely enhanced upon the addition of NADH. Thus, in response to muscle injury, O2is produced by membrane-bound electron-transferring enzymes, especially NAD(P)H oxidases, which are localized at the mitochondrial membranes and the plasma membrane. This suggests a decisive role of ROS and enzyme-catalyzed redox reactions in the pathogenesis of blunt tissue trauma, thus being an initiator of the signalling mechanism from injured myocytes to the surrounding tissue and, potentially, to the whole body.

P12 SOLUBLE FAS LIGAND RELEASED BY ALVEOLAR MACROPHAGES AFTER LUNG CONTUSION IS INVOLVED IN APOPTOSIS INDUCTION IN ALVEOLAR TYPE 2 CELLS

A. Palmer, U. Niesler, S. Mangold, M. Perl, M.S. Huber-Lang, F. Gebhard, M.W. Knöferl, and D.H. Seitz. Dept. of Trauma Surgery, University of Ulm, 89075 Ulm, Germany

Apoptosis of alveolar type 2 (AT2) cells after lung contusion has been previously described; however the mechanisms of apoptosis induction remain unclear. Potential inducers of apoptosis in the lungs are alveolar macrophages (AM) and soluble Fas ligand (sFasL). Therefore we hypothesized that sFasL secreted by AM induces apoptosis in AT2 cells after blunt chest trauma. To study this, AT2 cells were isolated from male Sprague Dawley rats and cultured for 66 h without stimulation. Thereafter, cell cultures were incubated for 18 h with sFasL in different concentrations (50-500 ng/ml) and apoptotic events were detected by Annexin V stain. Another set of animals was subjected to sham procedure or blunt chest trauma induced by a single blast wave. At 6, 24 or 48 h after trauma, bronchoalveolar lavage fluids (BAL) were collected and AM were isolated and cultured for 24 h without stimulation. sFasL concentrations of AM supernatants and BAL were determined by ELISA. In AT2 cell culture, apoptotic cell counts were significantly increased after addition of sFasL, compared to vehicle. sFasL concentration in BAL was significantly elevated 6 h after chest trauma and further increased at later time points. AM showed a markedly increased release of sFasL 6 h after lung contusion. These results indicate that sFasL is released by activated AM after lung contusion and appears in BAL. sFasL seems to be involved in AM-derived apoptosis of AT2 cells after chest trauma.

P13 DIVERGENT NEUTROPHIL REGULATION OF ASPECTS OF LUNG INJURY AND INFLAMMATION FOLLOWING BLUNT CHEST TRAUMA

M. Perl, M. Kieninger*, M.S. Huber-Lang, F. Gebhard, A. Ayala, and M.W. Knöferl. Department of Orthopedic Trauma, University of Ulm, 89075 Ulm, Germany

Extravasation of neutrophils (PMN) into the lungs is frequently seen in response to trauma and inflammation. However, so far it remains unclear what role activated PMN play in modulating trauma induced lung injury and/ or pulmonary inflammation. To study this, male C3H/ HeN mice intravenously received a PMN-depleting antibody (Gr-1) or control immunoglobulin (Ig) and were subjected to an isolated bilateral lung contusion (Tx) 24 hrs thereafter. Three or 30 hrs later bronchoalveolar lavage fluids (BALF) and lungs were harvested and the concentrations of inflammatory mediators were quantified. In addition, the degree of lung injury was assessed by quantifying BALF total protein concentration, evaluation of lung histology and differentiation of leukocytes infiltrating into the alveoli. PMN depletion resulted in a 95% reduction of PMNs in blood and BALF. Tx induced increase in lung myeloperoxidase activity was also markedly diminished after PMN depletion. IL-6, MIP-2, KC and MCP-1 were all substantially increased in BALF and lungs in response to Tx. Interestingly, BALF and lung chemokines but not IL-6 were even further increased in the absence of PMN. In contrast, Tx induced lung protein influx was substantially decreased and lung histology was markedly improved in PMN-depleted animals. Taken together, these data indicate that activated PMN aside from injuring lung tissue, appear to also possess antiinflammatory properties, which may be important in protecting lung tissue from injury. As such the ability to differential exploit either of these aspects of PMN function may be valuable in the therapeutic approach of Tx induced lung injury. (Supported by DFG-KN 2-2).

P14 RESUSCITATION OF HEMORRHAGED SEDATED MATURE MALE MINIATURE SWINE WITH 17β-ESTRADIOL (E2)

L.A. Baer, J.W. Burns, B.S. Jordan, J.L. Sondeen, M.A. Dubick, I.H. Chaudry, and C.E. Wade. US Army Institute of Surgical Research, San Antonio, TX USA 78234

In environments with limited resuscitation fluids, maintaining life after of hemorrhage is difficult. We hypothesized the use of E2 infusion would increase survival to 180 min. A miniature swine hemorrhage model was developed to test various resuscitation products. Anesthetized minipigs (43.4±1.3kg;14.2±0.4mths) were catheterized for hemorrhaging, infusions, sample collection and monitoring. Animals were placed in a sling with feet on the floor during recovery and sedated with midazolam to a brain bispectral index of 80-90. Swine were hemorrhaged 60% of estimated blood volume exponentially over 1 hr and assigned to no resuscitation (control;n=15), resuscitation with 1ml/kg/min of Hextend (Hex;n=5) to a systolic blood pressure (SBP) of 80±5 mm Hg, or a 1 mg/kg bolus (1.125mg/ml=45±1.2ml) of E2 (n=6). Animals were observed for 180 min or death. Baseline data were not different between treatments. At the end of hemorrhage there were reductions in MAP and base excess (BE), and an increase in lactate (Lac). Survival time for controls was 60±11.7 min after hemorrhage with a 7% survival rate at 180 min. Hex animals required 669±138ml, 38±8.2% replacement of the shed blood volume to attain the target SBP and 100% survival. E2 nearly doubled survival time to 117±23.6 min (p<0.05) and survival rate to 17%. In a model of severe hemorrhage in male swine, that could be resuscitated with fluids, E2 tended to increase survival time, but not to 180 min. Vascular reactivity in vitro indicates hyporesponsiveness of pig carotid artery to E2-induced vasorelaxation compared to rat aorta, suggesting higher doses of E2 may be required.

Table
Table

Mean ± SEM; * different from the control, p<0.05.

P15 THE ROLE OF NOREPINEPHRINE IN PROGENITOR CELL MOBILIZATION TO SITES OF INJURY

Jason P. Ulm, Alicia M. Mohr, Ziad C. Sifri, Salil G. Shah, and David H. Livingston. UMDNJ-NJMS, Department of Surgery, Newark, NJ 07103

Background:

Severe trauma leads to elevated levels of catecholamines as well as bone marrow (BM) dysfunction. Our previous work has shown that hematopoietic progenitor cells (HPCs) are sequestered in injured tissue following trauma. The aim of this study was to evaluate the role of norepinephrine (NE) in HPC mobilization to injured lung tissue.

Methods:

To deplete NE, male Sprague-Dawley rats (300-400gm) underwent chemical sympathectomy with injections of 6-hydroxydopamine (6-OH). Two groups underwent unilateral lung contusion (LC and LC+6-OH). HPC presence was assessed by BFU-E, CFU-E, and CFU-GEMM colony growth both in lung and BM tissue 3 hours after injury. *P values<0.5 by ANOVA and Tukey-Kramer tests (n=3-7/group).

Results:

6-OH maintains BM cellularity but leads to suppression of HPC colony growth both in BM and injured lung. Following LC and 6-OH+LC, HPC colony growth was 50% suppressed in BM. Growth of HPC was significantly increased in lung tissue following injury and the addition of 6-OH abolished this response (figure).

Conclusions:

Chemical sympathectomy abolished the growth of all HPCs in normal and injured lung tissue despite maintaining BM cellularity. NE appears to play a role in HPC mobilization to the site of tissue injury.

P16 AN ERYTHROCYTE SUBPOPULATION WITH DECREASED DEFORMABILITY FOLLOWING HEMORRHAGIC SHOCK

K. Caprio*, M.R. Condon*, D. Xu, E. Feketeova*, Z. Spolarics*, E.A. Deitch, and G.W. Machiedo*. *Surgical Services, DVA New Jersey HealthCare System, E. Orange, NJ 07018, and New Jersey Medical School, Newark 07103, USA

Using a trauma/hemorrhagic shock (T/HS) model in rats, this study tested the hypothesis that T/HS induces decreased erythrocyte deformability in specific RBC subpopulations.

Methods:

Following trauma-sham shock (T/SS, n=6) or T/HS (n=7), erythrocyte populations were isolated on the basis of size by centrifugal elutriation (subpopulations stratified by rate of elutriation in ml/min). The deformability of each subpopulation was analyzed using ektacytometry. Lineweaver-Burk conversion of deformability-response curves to shear stress was used to determine the shear stress at half-maximal cell elongation (KEI) and maximal cell elongation (EImax). A higher KEI signifies a decrease in deformability.

Results:

KEI values for cells eluted at 12 ml/min were significantly higher in T/HS (2.39+0.3) than T/SS animals (1.6+0.18, p<0.05). Also, the older and smaller subpopulations (eluted at 8 and 9 ml/min) showed a trend toward decreased deformability following shock. As expected, cell size, measured as mean corpuscular volume (MCV), increased linearly with flow rate from 8 mL/min to 13 mL/min. However, there was no difference in the MCV of cells collected from T/SS versus T/HS animals.

Figure
Figure

Conclusion:

Centrifugal elutriation successfully isolated and identified the subpopulation of T/HS RBC with decreased deformability (12% of total RBCs). It appears that the increased rigidity in this RBC subpopulation may contribute to the microcirculatory dysfunction seen after hemorrhagic shock.

P17 HEME OXYGENASE UPREGULATION DECREASES HEMORRHAGE SHOCK-INDUCED NITRIC OXIDE LEVELS IN THE RAT MESENTERIC MICROCIRCULATION

Michael Moncure, John G. Wood, Carla C. Braxton, Kendra Haydel, Jonathan Mahnken, Kahdi F. Udobi, and James H. Thomas.

Introduction:

Hemorrhagic shock (HS) results in microvascular damage as a result of leukocyte adherence (LA), emigration and eventual vascular permeability (VP) changes. Our lab has demonstrated increases in reactive oxygen species (ROS) generation, LA and VP following HS and hypoxia which are significantly reduced by heme oxygenase (HO) upregulation. We hypothesized that part of HO mediated microvascular protection may be the result of Nitric Oxide (NO) increases at the endothelial cell level.

Methods:

Sprague Dawley rats (300g) were administered Hemin (n=5) or vehicle (n=5) six hours prior to HS and resuscitation and a control group given vehicle (n=5). HO-1 expression was measured by RT-PCR in various tissues. Shock was induced in pentobarbital anesthetized animals by decreasing MAP to 40 mmHg for 60 minutes followed by standard resuscitation measures. NO levels were measured in the mesenteric venules of rats using intravital microscopy following administration of intravenous diaminoflurescein-2 diacetate (DAF-2).

Results:

Hemin administration significantly increased HO-1 expression. NO measurements were significantly less following resuscitation in the Hemin compared to the HS without Hemin group (152.05±6.36 vs. 190.79±6.36, p=0.0004) and were similar to controls (152.05±6.36 vs. 159.98±4.49, p=0.236). HS without Hemin significantly increased NO levels compared to controls. These differences were maintained throughout the entire length of the experiment.

Conclusions:

HO is upregulated systemically in the animals by hemin pretreatment and less so by HS. Protection of the microcirculation after HS as a result of HO upregulation appears to have no relation to increased microvascular NO levels. Our results showed that DAF-2 fluorescence, an index of venular NO levels, was significantly decreased in the face of HO upregulation.

P18 IMMEDIATE EFFECTS OF CHEST TRAUMA ON OXYGENATION IN SWINE

Andriy Batchinsky, Bryan Jordan, Jenifer Beck, David Martini, Corina Necsoiu, Belinda Meyers, Michael Lucas, and Leopoldo Cancio. U.S. Army Institute of Surgical Research, 3400 Rawley E. Chambers, Fort Sam Houston, Tx, 78234

Background:

Pulmonary contusion (PC) is a common cause of acute respiratory distress syndrome, but the pathophysiology of its immediate effects on oxygenation is unknown.

Methods:

Anesthetized pigs (GR1, n=8) received right chest blunt trauma with a captive-bolt device. Controls (GR2) received sham injury. Multiple inert gas elimination technique was used to define the cause of hypoxia pre-, and 5-, 30-, 60-and 120-min post PC, as a percentage of cardiac output (Q) to ventilation-perfusion compartments (V/Q). Volumes of normal (VOL norm), poorly (VOL poor), and non-aerated lung (VOL non) were assessed via CT scans.

Results:

GR1 data are in the Table.

Table
Table

Conclusions:

PC leads to immediate and sustained hypoxia, caused by sustained increases in true shunt. Transient increases in V/Q mismatch, and an increase in dead space ventilation, play lesser roles. CT scans reveal a gradual worsening of VOL non over 120 min. PC is a dynamic process over the initial minutes after injury, suggesting the importance of early intervention such as institution of positive end expiratory pressure.

P19 HEMORRHAGIC SHOCK AND RESUSCITATION DO NOT ALTER BAROREFLEX GAIN

Eric Irwin, Matt Byrnes, Kristine Mulier, and Greg Beilman. North Memorial Medical Center, University of Minnesota

Introduction:

Baroreflex function has been found to be affected by aging and disease processes. Little is known of the effects of hemorrhagic shock and resuscitation on baroreflex sensitivity. We used whole band average (WBA) cross-spectral analysis to test the hypothesis that baroreflex sensitivity (BRS) is not altered by reversible shock and resuscitation.

Methods:

Five rats under general anesthesia and mechanical ventilation underwent serial hemorrhages (shed volume of 30%), then 20 minutes of unresuscitated shock followed by resuscitation with shed blood and two equal volumes of lactated ringers (LR). Fast Fourier Transforms were performed on two minute continuous EKG and arterial pressure recordings (ADI Powerlabs) at each time point. The Low Frequency power (0.04-0.73 Hz) was calculated and BRS determined as the baroreflex gain calculated as the square root of the ratio LF content for RR intervals/arterial pressure using the WBA technique as described by Pinna et. al. ANOVA was used to determine if differences between time points were significant.

Results:

No significant differences were found for BRS values at any time point studied.

Conclusion:

Severe reversible hemorrhagic shock does not alter baroreflex sensitivity in this murine model. Further studies are ongoing to determine if BRS is altered in more severe irreversible forms of shock.

Table
Table

P20 HEART RATE VARIABILITY ANALYSIS MONITORS PROGRESSION OF SHOCK AND RESUSCITATION

Eric Irwin, Kristine Mulier, Joshua Huelster,and Greg Beilman. North Memorial Medical Center, University of Minnesota

Introduction:

Heart Rate Variability (HRV) analysis studies autonomic modulation of sinoatrial node function (SNF). We used HRV analysis to test the hypothesis that hemorrhage and resuscitation cause changes in both sympathetic (S) and parasympathetic (P) modulation of SNF.

Methods:

Six rats under general anesthesia and mechanical ventilation were hemorrhaged (shed volume=30%), unresuscitated shock (20 min), then resuscitation with shed blood and lactated ringers. HRV was analyzed at steady state for each condition using fast Fourier Transforms from continuous EKG recordings to obtain Low Frequency (LF)(0.04-0.73 Hz, increases as S tone increases) and High Frequency (HF)(0.73-2.4 Hz, increases as P tone increases) power. These were normalized (LFn and HFn) for total power 0.04-2.4Hz.

Results:

LFn increased with hemorrhage/shock, returning to baseline with resuscitation. HFn decreased during hemorrhage/shock, increasing to baseline with resuscitation.

Figure
Figure

Conclusions:

These findings show modulation of both P and S tone and indicate a possible role for HRV analaysis in monitoring the development of shock and the response to resuscitation.

P21 THE SELECTIVE V1A RECEPTOR AGONIST FE 202158 REVERSES PLATELET ACTIVATING FACTOR (PAF)-INDUCED HYPOTENSION, VASCULAR LEAK, IMPAIRED TISSUE PERFUSION, AND MORTALITY IN RATS

Régent Laporte1, Halina Wisniewska1, James Russell2, Donald Landry3, Pierre Rivière1. 1Ferring Research Institute, Inc., San Diego, United States, 2St-Paul's Hospital & University of British Columbia, Vancouver, Canada, 3Columbia University, New York City, United States

Introduction:

PAF is a phospholipid autacoid inducing peripheral vasodilation and vascular leak syndrome, both pathophysiological hallmarks of sepsis-induced cardiovascular dysfunction resulting in hypotension [1].

Indeed, PAF has been implicated in various animal models of sepsis and in septic humans [2]. A deficiency in the vasopressor hormone arginine vasopressin (AVP), a mixed V1a/V2 receptor agonist, also contributes to the cardiovascular dysfunction of septic shock, leading to clinical use of AVP for this condition [3,4]. These various findings led us to hypothesize that the selective V1a receptor agonist FE 202158 would be effective in a rat model of PAF-induced hypotension.

Methods:

Male Wistar rats were anesthetized with thiobutabarbital, surgically instrumented, and put on assisted ventilation. After obtaining baseline data, mean arterial pressure (MAP) was reduced to 40 mmHg by titration of an intravenous infusion of PAF. Once this level of hypotension was reached, test animals were given an intravenous infusion of FE 202158 or AVP, titrated to raise and maintain MAP at 70 mmHg for 3 h, while the PAF infusion was continued. Control rats received only an equal infusion of vehicle in addition to the continued PAF infusion.

Results:

FE 202158 dramatically reduced mortality in PAF-treated animals compared with PAF+vehicle controls. Survival was 80% in the FE 202158-treated group (8/10) versus 21% in the vehicle-treated group (3/14). Improvements were also observed in other parameters related to vascular leak syndrome and impaired tissue perfusion. In contrast, AVP was unable to reverse PAF-induced mortality (22% survival; 2/9), only delay it.

Conclusions:

This rat model of PAF-induced hypotension reproduced the pathophysiological hallmarks of sepsis-induced cardiovascular dysfunction. The selective V1a receptor agonist FE 202158 was not only able to reverse the associated mortality, but also other manifestations of this dysfunction including vascular leak syndrome. AVP could not reverse mortality but only delay it.

References

1. Montrucchio et al.: Physiol Rev 2000;80:1669-99.

2. Horkheimer et al.: Advances in Sepsis 2002;2:2-7.

3. Holmes et al.: Crit Care 2004;8:15-23.

4. Russell: Curr Opin Crit Care 2007;13:383-91.

P22 COMPARISON OF LYOPHILIZED SWINE PLASMA (LSP) TO FRESH FROZEN PLASMA (FFP) PLUS TWO RATIOS OF PACKED RED BLOOD CELLS (RBC) IN A COLD, COAGULOPATHIC, POLY TRAUMA SEVERE HEMORRHAGIC SHOCK SWINE MODEL

Jill L. Sondeen, M. Dale Prince, Leticia Medina, I. Amy Polykratis, Michael A. Dubick, John B. Holcomb. US Army Institute of Surgical Research, San Antonio, TX

The purpose of this study was to determine if LSP was as effective as FFP in reversing the coagulopathy in our multi-trauma model (anesthetized, instrumented female pigs, n=3-4/gp, femur fracture, 60% hemorrhage [HEM], 30-min shock period, hypothermia, Grade V liver injury). LSP was made from type matched pooled swine FFP using a Lyogard form (gift from Hemcon, Inc.). Type-matched RBC was made from donor pigs. Normal saline (NS) resuscitation was given (3:1 NS: HEM volume) after the shock period. Treatment consisted of liver packing and randomization to one of six groups of fluids (see Table), with the volume=HEM volume. Samples were collected at baseline (BL), post shock (PS), pre infusion (PI), and hourly to 4 h. LSP was as effective as FFP in restoring various indices of coagulation (PT, PTT, fibrinogen, ACT, and thrombelastogram (TEG, citrated, 15min incubation, kaolin, measured at pig's body temperature), including the maximum amplitude of the TEG shown in the Table (mm, x±sd) only 1 h shown). The lyophilisation process preserves the beneficial effects of plasma and LSP can be stored at room temperature and dissolves within 10 minutes.

Table
Table

P23 PRE-HOSPITAL RECOMBINANT FACTOR VIIA (RFVIIA) IMPROVED OUTCOME IN UNCONTROLLED HEMORRHAGIC SHOCK IN SWINE

Anke H. Scultetus, Francoise Arnaud, Ashraful Haque, Kohsuke Teranishi, Richard McCarron, and Daniel Freilich. Naval Medical Research Center, Silver Spring, MD 20910, U.S.A.

The objective of this study was to compare the effects of pre-hospital resuscitation with hemoglobin based oxygen carrier-201 (HBOC-201) alone (Group 1, n=14, 10 ml/kg/10 min infusion) or in combination with recombinant Factor VIIa (rFVIIa) (Group 2, n=18, 90 μg/kg/infusion) in a swine model of uncontrolled hemorrhage as part of an effort to optimize pre-hospital care in the critically injured. Fluids were administered at 15, 30 and 45 min after grade III liver crush/laceration. After simulated hospital arrival (60 min), animals received saline (for hypotension or tachycardia) and whole blood (for anemia), liver injury was repaired, and animals were followed for up to 72 hours. The reported data was obtained from an interim analysis focusing on physiological parameters. Baseline factors were not significantly different. Physiologic parameters were similar in the two treatment groups except lactate was significantly lower in Group 2 compared to Group 1 animals (p<0.002). There were trends to improved SaO2, pre-hospital blood loss (24.6 vs. 31.2 ml/kg), and survival (72.2 vs. 42.8%) in Group 2 vs. Group 1 animals. We conclude that in comparison with HBOC-201 alone, pre-hospital infusion of HBOC-201 with rFVIIa improved clinical outcome in a shock model of uncontrolled hemorrhage in swine. That blood loss was similar with addition of rFVIIa despite improved markers of shock suggests clinical benefit that may be independent of hemostatic activity.

P24 HEMORRHAGIC SHOCK AND RESUSCITATION DO NOT ALTER BAROREFLEX GAIN

Eric Irwin, Matt Byrnes, Kristine Mulier, and Greg Beilman. North Memorial Medical Center, University of Minnesota

Introduction:

Baroreflex function has been found to be affected by aging and disease processes. Little is known of the effects of hemorrhagic shock and resuscitation on baro-reflex sensitivity. We used whole band average (WBA) cross-spectral analysis to test the hypothesis that baroreflex sensitivity (BRS) is not altered by reversible shock and resuscitation.

Methods:

Five rats under general anesthesia and mechanical ventilation underwent serial hemorrhage (shed volume of 30%), 20 minutes of unresuscitated shock, then resuscitation with shed blood and lactated ringers (LR). Fast Fourier Transforms were performed on continuous EKG and arterial pressure recordings at each time point. Low Frequency power (0.04-0.73 Hz) was calculated and BRS determined using the WBA technique as described by Pinna et. al. ANOVA was used to determine if differences between time points were significant.

Results:

No significant differences were found for BRS values at any time point studied.

Figure
Figure

Conclusion:

Severe reversible hemorrhagic shock does not alter baroreflex sensitivity in this murine model. Further studies are ongoing to determine if BRS is altered in more severe irreversible forms of shock.

P25 HEMATOLOGICAL EFFECTS OF PREHOSPITAL ADMINISTRATION OF HBOC-201 WITHOUT OR WITH RFVIIA IN UNCONTROLLED HEMORRHAGE DUE TO LIVER INJURY IN SWINE

Francoise Arnaud, Anke Scultetus, Richard McCarron, and Daniel Freilich.

Patients with traumatic hemorrhagic shock may benefit from rapid bleeding control and increased tissue oxygenation. The goal of this study was to compare the effects of an oxygen-carrying resuscitation fluid (HBOC-201) with a hemostatic agent (rFVIIa). Yorkshire swine were hemorrhaged by liver injury andresuscitated during the prehospital phase with up to 3 doses of HBOC-201 infused at 10 ml/kg without (Grp 1) or with an admixed 90 μg/kg rFVIIa (Grp 2). After 75 min (hospital phase), animals received saline (for hypotension) and blood transfusion (for anemia), and were followed for up to 72 hrs. Hematology profile (CBC) and coagulation parameters (PT, TEG, PFA, and TAT) were measured. Significance (p<0.05) was assessed by Student's t-test. Dilutional coagulopathy occurred in both groups, but PT and TEG-R were less adversely affected in Grp 2. Other hematologic and hemostasis parameters and post-treatment blood loss volumes were not different. There was a trend to improved survival in Grp 2 compared with Grp 1 animals (Table). We conclude that in comparison with HBOC-201 alone, prehospital treatment with a resuscitation fluid containing HBOC-201 and rFVIIa diminishes dilutional coagulopathy and may improve survival in swine with uncontrolled hemorrhage due to liver injury.

Table
Table

P26A MINOCYCLINE DECREASES LIVER INJURY AFTER HEMORRHAGIC SHOCK AND RESUSCITATION IN MICE

Christoph Czerny1,2, Tom P. Theruvath1, Eduardo Maldonado1, Mark Lehnert2, Ingo Marzi2, and John J. Lemasters1,3. 1Center for Cell Death, Injury and Regeneration, Departments of 1Pharmaceutical and Biomedical Sciences and 3Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, and 2Department of Trauma Surgery, J.W. Goethe University, Frankfurt am Main, Germany

Background:

Patients that initially survive hemorrhage and resuscitation may develop a systemic inflammatory response syndrome (SIRS) that leads to injury and dysfunction of vital organs (multiple organ dysfunction syndrome, MODS). SIRS and MODS may involve mitochondrial dysfunction.

Aim:

We investigated whether minocycline, an inhibitor of mitochondrial permeability transition (MPT), can prevent mitochondrial dysfunction and decrease liver injury after hemorrhage and resuscitation.

Methods:

Under pentobarbital anesthesia, C57BL6 mice were hemorrhaged to 30 mm Hg for 3 h and then resuscitated with shed blood plus half the volume of lactated Ringer's solution containing minocycline (10 mg/kg body weight), tetracycline or vehicle. Serum alanine aminotransferase (ALT) and caspase-3 activity were assessed 6 h after resuscitation. Mitochondrial polarization was assessed by intravital multiphoton microscopy of rhodamine 123, a potential-indicating fluorophore.

Results:

After resuscitation with vehicle and tetracycline, ALT increased to 4538 U/L and 3999 U/L, respectively. Minocycline treatment decreased ALT to 1763 U/L (p<0.01). Minocycline also decreased caspase-3 activity by 69% (p<0.01) and preserved mitochondrial polarization (p<0.05) after hemorrhagic shock and resuscitation.

Conclusion:

Minocycline decreases liver injury after hemorrhage and resuscitation by preventing MPT-dependent mitochondrial dysfunction. Minocycline might be useful clinically after hemorrhage shock and resuscitation to prevent SIRS and MODS.

P26B NECROTIC AND APOPTOTIC CELL DEATH IS DIMINISHED IN JNK2-/-KNOCKOUT MICE AFTER HEMORRHAGE AND RESUSCITATION

Christoph Czerny1,2, Tom P. Theruvath1, Eduardo Maldonado1, Mark Lehnert2, Ingo Marzi2, and John J. Lemasters1,3. 1Center for Cell Death, Injury and Regeneration, Departments of 1Pharmaceutical and Biomedical Sciences and 3Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, and 2Department of Trauma Surgery, J.W. Goethe University, Frankfurt am Main, Germany

Background:

Hemorrhage and resuscitation (H/R) lead to phosphorylation of mitogen-activated stress kinases (MAPKs), an event that is associated with organ damage. Phosphorylation of JNK2 promotes the mitochondrial permeability transition, which causes cell death.

Aim:

was to determine whether necrosis and apoptosis after hemorrhage and resuscitation were decreased in JNK2 deficient mice.

Methods:

Under pentobarbital anaesthesia, wild-type and JNK2 deficient mice were hemorrhaged to 30 mm Hg for 3 h and then resuscitated with shed blood plus half the volume of lactated Ringer's solution. Serum alanine aminotransferase (ALT) and caspase-3 activity were assessed 6 h after resuscitation.

Results:

ALT in wild-type mice increased 4538 IU/L at 6 h after resuscitation. In JNK2-/-mice, this increase of ALT after H/R was blunted to 1785 IU/l (p<0.05). Caspase-3 activity in livers of JNK2-/-mice after H/R was also decreased by 62% compared to wildtype mice 6 h after H/R.

Conclusion:

JNK2 contributes to hepatic injury after H/R. Thus, pharmacological interventions aimed at inhibiting JNK2 activity may be beneficial in H/R and protect against multi-organ-failure.

P27 A COMPARISON BETWEEN VASOPRESSIN AND TERLIPRESSIN IN THE RESUSCITATION OF UNCONTROLLED HEMORRHAGIC SHOCK

Chien-Chang Lee1,2, I-Jing Chang3, Po-Chu Lee4, Tzung-Hsin Chou1, Wei-Kuo Chou1, and Shyr-Chyr Chen1. 1Department of Emergency Medicine, National Taiwan University Hospital, Taipei, Taiwan; 2Graduate Institute of Epidemiology, College of Public Health, National Taiwan University Hospital, Taipei, Taiwan; 3Taipei City Hospital, Taipei, Taiwan; 4Department of Surgery, National Taiwan University Hospital Yun-Lin Branch, Taiwan

The aim of this study was to evaluate the effect of vasopressin or terlipressin on a rodent model of uncontrolled hemorrhagic shock (UHS). UHS was produced by inducing 3 mL/100 g hemorrhage followed by tail amputation 30 min before fluid resuscitation to simulate a prehospital setting. Triple the volume of shed blood lactate ringer (LR) resuscitation was given from 75 to 105 mins after the start of the experiment to simulate the hospital phase. Forty-eight rats were randomized into four groups: the LR group, who were given LR resuscitation equal to the volume of shed blood in the pre-hospital phase; the Vaso group, who were given bolus vasopressin (0.8 U/kg) in the pre-hospital phase; the Terli group, who were given bolus terlipressin (15mcg/kg) in the pre-hospital phase; and the Sham group, who were underwent the same operative procedure without induction of UHS. The animals were observed for three days. The mortality rates of the Vaso and Terli groups III was were both 25% (3/12), lower than the LR group (33.3%, 4/12). In the pre-hospital phase, the MAP of the terlipressin-treated rats was maintained at 80-110 mmHg; while that of vasopressin-treated rats rose in the initial five minutes and declined gradually. The MAP of the LR group was kept at around 40 mmHg. Vasopressin-and terlipressin-treated rats were associated with less proinflammatory and more anti-inflammatory cytokine production. Statistical significance was detected between the Terli and LR groups or Vaso group for IL-6, TNF-alpha and IL-10. Histopathological examination showed significantly less lung injury change in the Vaso and Terli groups, compared with the LR group. Early optimization of hemodynamics with terlipressin or vasopressin in an animal model of UHS was associated with improved hemodynamics and inflammatory cytokine profile. Compared with vasopressin, terlipressin has the advantage of ease of use and sustained effects.

P28 PROTEASOME DEPENDENT PROTEOLYSIS AFTER MECHANICALLY INDUCED SKELETAL MUSCLE TRAUMA

N. Ponelies1, M. Seiffert1, N. Ising1, D. Gosenca1, M. Schoen2, B. Vollmar2, and U. Obertacke1. 1Dept. Trauma Surg., University Clinic Mannheim; 68167 Mannheim, Germany; 2Inst. Exp. Surg.; 18055 Rostock, Germany

The ubiquitin proteasome pathway (upp) is the main proteolytic mechanism in normal but also pathological processes. The upp involvement and metabolic consequences of local soft tissue trauma are rarely defined. Thus, we investigated upp activities after skeletal muscle injury in human and animal system.

Methods:

Standardized closed soft tissue trauma of Sprague-Dawley rats hind limb (EDL) and contused human skeletal muscle extracts were examined concerning the intracellular ubiquitin content, the ubiquitin ligation (UL) and proteasome activities (PA). Additional information about systemic effects were gained by examine the animals lung (upp) and blood (CK/LDH).

Results:

For the animal model immediately (10 min) after contusion upp activities (muscle) drop down (UL - 49 %, PA - 44%, p < 0,05). The same progression is observed in human tissue (UL - 58 %, p < 0,05; PA - 77 %, p = 0,125). The turn over of free and conjugated ubiquitin is blocked 6 h after impact in rat tissue whereas no effect is detected for ubiquitin levels in contused human muscles. The examined lung tissue revealed as well a reduced upp activity (UL - p = 0,069, PA - p < 0,05).

Conclusions:

The upp is actively suppressed in the early phase (48 h) after muscle contusion different from severe trauma and other systemic diseases. Otherwise the traumatic impact is sufficient to generate a systemic reaction.

P29 FUNCTIONALIZED HYBRIDS ON QUARTZ SENSOR ARRAY FOR ENHANCED GAS/BIO SENSING

Shabbir Moochhala*1, Palaniappan2, Francis E.H. Tay2, and Melissa A.L. Teo1. 1DSO National Laboratories, 27 Medical Drive, Singapore 117510 2Department of Mechanical Engineering, National University of Singapore. *Corresponding author: Tel: (65)64857201; Email: [email protected]

Analysis of breath is feasible and could provide a non-invasive method for early detection, continuous monitoring and management of lung injury/diseases caused by battlefield trauma and infectious/chronic illness. Currently, clinical measurement of nitric oxide NO in exhaled air is proving to be a reliable marker of lung inflammation and oxidative stress. However, current detector systems are expensive, benchtop system and only detect a single analyte. Recently, several other volatile gases, such as carbon monoxide CO and ammonia NH3 have been detected and correlated with various types of pulmonary injury/disease.

Functionalized hybrids matrix deposited on quartz sensor array been tested as receptors for gaseous bio-markers detection. Bio-marker such as Nitric Oxide (NOx), Carbon monoxide (COx), etc., is identified and quantified by functionalized hybrids deposited on Quartz Crystal Microbalance (QCM) electrodes. The receptor hybrids are anchored chemically on to a mesoporous silica network which is deposited on QCM. Scanning Electron Microscopy (SEM), Fourier Transform Infra-red spectroscopy (FTIR), Time of Flight Secondary Ion Mass Spectroscopy (Tof-SIMS) is used to characterize the hybrids. The sensor response towards the bio markers is tested in a sealed gas chamber. The sensor response of QCM functionalized with non-modified QCM is also recorded. The developed sensors are tested as breath analyzer using animal models. Experiment results show that sensitivities up to ppb/Hz could be achieved by using the developed hybrids deposited on QCM electrodes.

Keywords:

QCM, Gas/bio sensor, Breath Analyzer, mesoporous silica

P30 TRAUMA, HEMORRHAGE AND RESUSCITATION (THR) RELATED FREE IRON RELEASE ASSOCIATED WITH HEMEOXYGENASE (HO-1) INDUCTION IN RATS

A. Postl1,2, C. Zifko1, H. Redl1, S. Bahrami1, A. Kozlov1,2, M. Gemeiner2, and J.C. Duvigneau2. 1Ludwig Boltzmann Institute for Traumatology, AUVA Research Center, Vienna, Austria; 2University of Veterinary Medicine, Vienna, Austria

Background:

Free iron is a potent inducer of oxidative stress leading to the formation of reactive oxygen species (ROS) and causing lipid peroxidation, protein and DNA damage. The Hemeoxygenase (HO-1) releases ferrous iron, but its pathophysiological potential in THR is unclear. The aim of the present study was to determine whether the induction of HO-1 in different organs is associated with changes of free iron level under THR. Anesthetized rats (n=6) were subjected to a THR-model, including laparotomy, bleeding and resuscitation (adequate/ inadequate phase) followed by a 2h observation. HO-1 expression was determined in heart and liver by real time PCR and its activity was measured in microsomal liver fraction. Free iron concentration was measured by means of electron paramagnetic resonance spectroscopy (EPR) in non-homogenized liver and heart tissue. The gene expression of HO-1 was significantly increased in heart and liver at 2h after THR. In heart, the free iron level in THR group increased by 26% compared to the sham. In liver no significant difference in free iron levels were found between the groups irrespective of the significant increase in HO-1 expression and microsomal HO-1 activity. Our data showed that free iron increase coincided with the up-regulation of the HO-1 in heart but not in liver, suggesting that the increase of free iron levels in heart was potentially mediated by HO-1.

P31 ORGAN SPECIFIC ROS FORMATION AFTER TRAUMA, HEMORRHAGE, AND RESUSCITATION (THR) IN RATS: PATHOPHYSIOLOGICAL RELEVANCE?

C. Zifko, A. Postl, M. Jafarmadar, H. Redl, A. Kozlov, and S. Bahrami. Ludwig Boltzmann Institute for Traumatology and the Research Center of AUVA, Vienna, Austria

THR-induced multiple organ failure is partly ascribed to the presence of reactive oxygen species (ROS).

We characterized THR-induced ROS formation in different organs and determined the effect of ROS scavenging on THR-induced early organ damage. Anesthetized rats (n=6) were subjected to THR-model, including adequate/ inadequate resuscitation phase followed by a 2h observation. 1-hydroxy-3-carboxy-2,2,5,5-tetramethyl-pyrrolidine hydrochloride (CPH), a non-toxic spin trap, was infused, starting at the onset of resuscitation up to the end of the experiment. Electron paramagnetic resonance spectroscopy was used to quantify the amount of ROS produced in individual organs. ROS formation was lower in THR than in sham animals. ROS levels varied in different organs as follows: liver<heart<colon<jejunum<duodenum<kidney<lung<ileum. At the end of experiment, THR-induced histopathological changes were observed only in the intestine. ROS scavenging ameliorated THR-induced edema in intestine.

Our data show organ specific changes in ROS formation early after trauma, hemorrhage and resuscitation in rats. The histological abnormalities observed in the intestinal tract only suggest susceptibility of this organ to THR, which, at least in part, is ROS dependent.

P32 ROLE OF PULMONARY RESISTANCE IN RESPONSE TO RESUSCITATION OF PORCINE SHOCKED TRAUMA AND CIRCULATORY COLLAPSE

S. Zenker, H. Gomez, P. Polanco, R. Namas, A. Torres, G. Clermont, Y. Vodovotz, M. Pinsky, and J. Puyana. University of Pittsburgh, PA 15261

Trauma and severe hemorrhagic shock (HS) nearing exsanguination may lead to circulatory collapse (CC) despite hemorrhage control and resuscitation. The underlying physiological mechanisms are only partially understood.

Objective:

We investigated the relationship between acute mean arterial pressure response to volume resuscitation (ΔMAP) and pulmonary and systemic hemodynamics in swine subjected to severe trauma/CC.

Methods:

12 Yorkshire-Durock pigs were subjected to trauma/HS as previously described (Crit Care Med 2007;12(Suppl):A28). Hemodynamic parameters, left atrial pressure (LAP-7/12 animals) and aortic and pulmonary flow were measured. Characteristic aortic impedance (Zc), pulmonary input resistance (PIR), and pulmonary vascular resistance (PVR) were calculated, as well as their changes under resuscitation (Δ). Survival was assessed at 2hafter resuscitation. Data were analyzed using non-parametric methods.

Results:

8 animals survived (SG) and 4 died (NSG) after resuscitation. ΔMAP ranged from-7 to 23 mmHg (median 6.4 mmHg). ΔMAP was negatively correlated with ΔPIR (n=12, Spearman's ρ=−0.72, p=0.01). A similar trend was observed for ΔMAP vs. ΔPVR (n = 7, ρ=−0.68, p = 0.11). ΔZc under resuscitation differed significantly between SG and NSG (median-0.046 vs. 0.020, p = 0.012), and was negatively correlated with ΔMAP (ρ=−0.71, p=0.02).

Conclusion:

The relationship between ΔPIR/ΔPVR and ΔMAP supports a role for pulmonary streambed obstruction in impairing response to resuscitation, with ΔZc difference reflecting downstream effects. Causality of this association remains to be studied.

P34 EVIDENCE FOR ENDOTHELIAL CELL ADHERENS JUNCTION DAMAGE IN HEMORRHAGIC SHOCK INDUCED HYPERPERMEABILITY

Ed W. Childs, Binu Tharakan, and Felicia A. Hunter. Department of Surgery, Texas A and M University Health Science Center-College of Medicine and Scott and White Memorial Hospital, Temple, Texas 76508 USA

Objective:

Hemorrhagic shock (HS) activates apoptotic signaling and disrupts the microvascular endothelial cell barrier leading to hyperpermeability. We hypothesized that caspase-3 mediated cleavage of the endothelial cell adherens junction protein beta catenin and subsequent cell-cell detachment is a key route to vascular hyperpermeability following HS.

Methods:

In rats, HS was induced by withdrawing blood to reduce the MAP to 40 mmHg for 60 minutes followed by resuscitation. The rats were injected intravenously with FITC-albumin (50 mg/kg) and hyperpermeability was observed in mesenteric post-capillary venules using intravital microscopy. The mesenteric microvasculature was analyzed for caspase-3 activity. The serum collected from HS rats and active caspase-3 peptides were used to induce hyperpermeability in rat lung microvascular endothelial cell (RLMEC) monolayers. RLMEC were used for immunofluorescence of beta catenin and VE cadherin or f-actin staining.

Results:

In rats, HS induced vascular hyperpermeability in vivo and resulted in caspase-3 activation (p<0.05). Serum from HS rats or transfection of active caspase-3 induced hyperpermeability in RLMEC monolayer. In RLMEC, HS serum and active caspase-3 transfection lead to disruption of beta catenin and formation of f-actin stretch fibers. A decrease in VE cadherin immunofluorescence was also observed.

Conclusion:

These findings demonstrate that caspase-3 mediated cleavage of the cell adherens junction protein beta catenin is a key event that leads to hyperpermeability. The separation of beta catenin from VE cadherin following the caspase-3 mediated cleavage may be the underlying mechanism that leads to the cell-cell detachment and hyperpermeability.

P35 HYPERTENSION INCREASES VULNERABILITY TO HEMORRHAGIC SHOCK IN HYPERTENSIVE VS NORMOTENSIVE RATS

S. Song, P.S. Reynolds, F. Tamariz, and R.W. Barbee. Virginia Commonwealth University Medical Center and VCU Reanimation and Shock Center, Richmond VA 23298 USA

Hypertension is the most common co-morbid condition in aged trauma patients, and is associated with poorer outcomes. We hypothesized that hemodynamic changes associated with hypertension should result in greater vulnerability to shock relative to normotensive controls. We examined oxygen delivery (DO2) dynamics as a function of blood loss in a rodent model of controlled hemorrhage; we compared hypertensive (SHR) to 2 normotensive (Sprague Dawley SD, Wistar-Kyoto WKY) rat strains.

Methods:

Male rats from 3 strains were anaesthetized, intubated, ventilated, and thoractomized. Cardiovascular (cardiac output CO, MAP mean arterial pressure, SVR systemic vascular resistance) and respiratory (VO2) variables were measured or calculated. DO2 was estimated from CO and arterial oxygen content. DO2crit was estimated by nonlinear regression of both VO2 and surrogate markers on DO2.

Results:

CO and DO2, were lower, and SVR higher, for SHR compared to normotensive rats (P < 0.001). While initial MAP of SHR rats was much higher (176 vs 130 mm Hg for normotensives; P < 0.001), MAP was equal to normotensives within 10% TBV (total blood volume) hemorrhage. DO2crit and delivery-dependent VO2 rates did not differ between strains. However, SHR rats tolerated less blood loss before reaching DO2crit compared to normotensive strains (24% TBV vs 35% TBV, P < 0.001).

Conclusions:

The greater vulnerability to hemorrhage exhibited by SHR rats can be partially attributed to differences in vascular compliance. These data suggest that triage criteria for trauma patients with known hypertensive medical histories need to be re-evaluated.

P36 RAT WHOLE BLOOD ASSAY DETECTS NEUTROPHIL PRIMING FOR SUPEROXIDE ANION

P. Eckels, E. Moore, E. Peltz, J. Jordan, A. Banerjee, S. Kashuk, and C. Silliman. Denver Health Medical Center, University of Colorado Denver and Bonfils Blood Center, Denver, CO, 80204, U.S.A.

Priming is a pro-inflammatory event that induces adhesion and maximizes O2production to a subsequent stimulus and may lead to neutrophil (PMN)-mediated mediated tissue damage. Animal modelling for PMN-induced organ injury in rats is constrained by xenobiologic interactions and a dearth of available PMN. Our purpose was to develop a species-specific whole blood assay to measure priming in rat PMN.

Methods:

Rats underwent a controlled hemorrhagic-shock model where mesenteric lymph was collected. 100μL of blood from healthy rats underwent chemi-luminescent measurement of O2with Diogenes in a luminometer. O2was measured post incubation (37°C) with 10% (v/v) lymph fractions prior to stimulation with fMLP. All samples were standardized to PMN number and platelet-activating factor (PAF) served as positive priming control.

Results:

O2generation from blood treated with post-shock mesenteric lymph (PSML) had a 3-fold increase over naïve blood response to fMLP (p<0.05; n=7) and pre-shock levels (p<0.05). Pre-shock samples were not statistically significant from controls. Superoxide dismutase treated PMN did not produce O2.

Figure
Figure

Conclusions:

This assay employs a selective, non-toxic reagent (Diogenes) to measure O2directly in smaller sample size available from rodents, and allows for evaluation of PMN priming of the NADPH oxidasein whole rat blood.

P37 POST-SHOCK MESENTERIC LYMPH IS A MAJOR SOURCE OF SYSTEMIC IL-6 FOLLOWING TRAUMA

E. Peltz, E. Moore, S. Damle, J. Jordan, P. Eckels, S. Kashuk, and A. Banerjee. University of Colorado Denver and Denver Health Medical Center, Denver, CO 80262

Circulating IL-6 is a robust predictor of ARDS/MOF following trauma, but its tissue origin remains unclear. We hypothesize that hemorrhagic shock elaborates the release of IL-6 from the ischemic gut into mesenteric lymph thereby potentiating systemic elevations.

Methods:

Trauma/hemorrhagic shock (T/HS) was induced in SD rats by controlled hemorrhage (30mmHg x 40min) and the mesenteric duct was cannulated for lymph collection/diversion. Resuscitation was performed with NS and 1/2 shed blood. Lymph and plasma samples from pre-shock and for 3 hours post-shock were evaluated for IL-6 using bead array technology. Students T-test was used for statistical analysis.

Results:

Levels of IL-6 in post-shock mesenteric lymph increased progressively over time with a 6 fold increase seen during the 3rd hour post-shock (p<0.05). Adjusting for the increase in post-shock lymph production reveals a 24 fold increase in the hourly delivery of IL-6 to circulation by the 3rd hour (p<0.05). Mesenteric diversion attenuated the increase seen in plasma IL-6 levels following T/HS.

Figure
Figure

Conclusion:

IL-6 is markedly elevated in mesenteric lymph following T/HS and a gradient exists from lymph to plasma. These results suggest the gut is amajor source of circulating IL-6following T/HS, which may be amarker for the magnitude ofsplanchnic ischemia associated with injury.

P38 DOES 17β-ESTRADIOL PROTECT THE BRAIN INFLAMMATORY RESPONSE FOLLOWING TRAUMA-HEMORRHAGE?

H. Akabori*, M.A. Choudhry, K.I. Bland*, I.H. Chaudry. Center for Surgical Research and Dept of Surgery, University of Alabama at Birmingham, AL 35294

Although 17β-estradiol (E2) improves the depressed immune functions following trauma-hemorrhage (T-H), it remains unknown whether E2 has any salutary effects in the brain under those conditions. Adult male Sprague-Dawley rats underwent a midline laparotomy (i.e., induction of soft-tissue trauma) and were then bled to and maintained at a mean arterial blood pressure of 35-40 mmHg for ~ 90 min. The rats were then resuscitated with 4 x the shed blood volume in the form of Ringer's lactate over 60 min. E2 (1 mg/kg BW i.v.) or vehicle was administered at the onset of resuscitation. Immediately after and 2 hr after resuscitation, IL-1β and dopamine levels in the hypothalamus were measured. Circulating levels of IL-1β, dopamine, and prolactin (PRL) were also assayed. T-H significantly increased dopamine and IL-1β levels both in the hypothalamus and plasma after resuscitation (p<0.01 compared to sham). However, these parameters were normalized by E2 treatment. Furthermore, E2 significantly increased circulating levels of PRL after resuscitation (p<0.05 vs. vehicle). Thus, administration of E2, which decreases dopamine secretion and upregulates PRL secretion, appears to be a useful adjunct for downregulating inflammatory cytokine release following T-H even in the hypothalamus. (NIH R37 GM39519).

*p < 0.05 vs. T-H + vehicle, **p < 0.01 vs. other groups. n = 6 rats/group; ANOVA and Tukey's test.

Figure
Figure

P39 INHIBITION OF INOS PATHWAY LIMITS MOLECULAR EVENTS ASSOCIATED WITH HEMORRHAGE OR RADIATION INJURY

Juliann G. Kiang. Armed Forces Radiobiology Research Institute, Uniformed Services University, Bethesda, MD 20889-5603, USA

Exposure to ionizing radiation results in DNA breaks that activate the ATM and CHK2 pathways. NF-kB/p53/CDC25 are then activated, which arrests the cell cycle. This in turn leads to acute radiation syndrome (ARS) followed by multiple organ dysfunction (MOD) and multiple organ failure (MOF). iNOS expression and NO production increase after either hemorrhage or radiation exposure. We previously showed that inhibiting iNOS expression prevents hemorrhage injury. We therefore investigated whether inhibiting iNOS expression also limits radiation injury. We exposed human Jurkat T cells to gamma radiation (4 Gy). Cell lysates were collected 4 hr later. We found radiation exposure increased iNOS expression by increasing levels of its transcription factors NF-kB-p50 and KLF6. Cells collected 24 hr after irradiation showed that cell viability was reduced by 35%. In these cells, NO production, lipid peroxidation, ATP levels, and caspase-3 activity were significantly elevated, suggesting the iNOS pathway had been activated. Treatment with17-DMAG, an iNOS inhibitor, 24 h prior to radiation limited these changes. These results suggest radiation and hemorrhage injury both involve the iNOS pathway, and agents that prevent hemorrhage injury should be useful for treating radiation injury. (Supported by AFRRI RAB2CF)

P40 SYSTEMATIC ANALYSIS OF THE SALUTARY EFFECT OF ESTROGEN ON CARDIAC PERFORMANCE FOLLOWING TRAUMA-HEMORRHAGE

Z.F. Ba*, J.T. Hsu*, J. Chen*, W.H. Kan*, M.G. Schwacha, and I.H. Chaudry. Center for Surgical Research, University of Alabama at Birmingham, Birmingham, AL 35294

Although 17β-estradiol (E2) as well as estrogen receptor (ER) agonist administration following trauma-hemorrhage (T-H) improves cardiac function, it remains unknown what the optimal estrogen or ER agonist dosage is to elicit these beneficial effects. To study this, the dose-dependent effects of E2, propylpyrazole triol (PPT, ER-α agonist) and diarylpropionitrile (DPN, ER-β agonist) on heart performance (+dP/dt) were determined in sham rats, at the time ofmaximal bleedout (MBO) or at 2 h following T-H. The results ofthe maximal dose (MD) and the 50% effective dose (ED50) of E2,DPN and PPT in sham and following MBO and T-H are shown below:

Table
Table

E2 and DPN induced dose-dependent increases in+dP/dt in all groups, whereas PPT was ineffective at all doses. MD and ED50 of DPN were approximately 100-fold lower than those of E2. The half-life of E2 in plasma was approximately 25 min in sham and MBO groups. A positive correlation between the estrogen-induced increase in+dP/dt and survival in MBO rats was observed. These results collectively suggest that the salutary effects of estrogen on cardiac performance are dose-dependent and mediated via ER-β. (Supported by NIH R37 GM39519)

P41 TEMPORAL TRANSCRIPTOMIC RESPONSE OF MURINE LIVER TO SEVERE INJURY AND HEMORRHAGIC SHOCK

Rebecca D. Edmonds, Claudio E. Lagoa, Jose M. Prince, Ryan M. Levy, George Tseng, Yoram Vodovotz, and Timothy R. Billiar.

Survivors of trauma-hemorrhagic shock (T-HS) frequently develop organ dysfunction and infectious complications as a result of imbalance between pro-inflammatory and anti-inflammatory pathways. We employed a murine model of T-HS to study the specific gene expression patterns and molecular pathways that may provide insight into the pathophysiology of trauma-hemorrhagic shock. C57BL/6 mice were divided into the following experimental groups: 1) Anesthesia only controls; 2) 1.5 hours of femoral artery cannulation; 3) 1.5 hours HS, bilateral femur fracture (BFF), 4.5 hours of resuscitation with shed blood and lactated ringers. RNA was extracted from the liver and hybridized to Codelink and Affymetrix mouse whole genome microarray chips. The microarray data sets were subjected to standard statistical analysis, hierarchical clustering, and pathway analysis. Evaluation of both Codelink and Affymetrix platforms by statistical analysis for micrarrays (SAM) revealed significant differential expression of genes when comparing groups 1 and 2 suggesting that surgical trauma results in a significant insult. Comparison between groups 2 and 3 demonstrated differential expression of 5960 genes on the Affymetrix array and 2979 genes on the Codelink array. Ingenuity Pathway Analysis (IPA) of differentially expressed genes between groups 2 and 3 indicates that the main signaling pathways involved are IL-10, SAPK/JNK, p38 MAPK, PPAR, IL-6, NF-κβ, apotosis, and ERK/MAPK signaling.

P42 HIBERNATION-BASED THERAPY IN A LARGE ANIMAL MODEL OF HEMORRHAGIC SHOCK

Julie Gilkeson, Kristine Mulier, and Greg Beilman. University of Minnesota, Minneapolis, MN

Introduction:

Levels of the neuroprotective ketone beta-hydroxybutyrate (BHB) and antioxidant melatonin are increased in hibernating mammals. The aim of our study was to assess potential benefits of these compounds in hemorrhagic shock. We compared small volume infusions of 4M D-BHB and melatonin (BHB/M) to 4M saline controls in a porcine model of hemorrhagic shock.

Methods:

We induced hemorrhagic shock to systolic blood pressure<60 mmHg for 60-90 minutes. Pigs treated with BHB/M or 4M NaCl (control) underwent 8 hours of targeted resuscitation using lactated Ringers and shed blood.

Results:

Both groups (total n=16) had similar hemorrhage volumes and mortality (BHB/M=20%, control=17%, p=1.0). BHB/M treated animals demonstrated significantly improved base excess (p=0.0004) when compared to controls. BHB/M treated animals also demonstrated trends toward earlier lactate normalization, higher peripheral tissue perfusion during shock, and less intravenous fluids given.

Conclusions:

BHB/M in resuscitation after hemorrhage results inmproved markers of end organ perfusion. Further study of molecularprotective mechanisms active in hibernation may contribute to strategies for treatment of hemorrhagic shock.

Figure
Figure

P43 ENDOTHELIAL CELL CD36 AND CD49 EXPRESSION IS UPREGULATED AFTER TRAUMA-HEMORRHAGIC SHOCK IN VITRO

K. Caprio, E. Feketeova, M. Condon, Q. Lu, Z. Spolarics, E.A. Deitch, and G.W. Machiedo. Surgical Services, VA New Jersey Health Care System, East Orange, NJ 07108, Surgery Dept. UMDNJ, Newark, NJ 07103

We have previously shown an increased adhesion of RBC to human umbilical vein endothelial cells (HUVEC) in trauma-hemorrhagic shock (T/HS), the majority of which are CD36+. The goal of the study was to investigate the HUVEC response to T/HS blood.

Methods:

Rats were subjected to T/HS (n=10) or trauma-sham shock (T/SS, n=6) and sacrificed at 3 hours post shock. T/HS or T/SS RBC were added to confluent HUVEC in tissue culture plates, incubated for 5minutes at 370 C, and washed 3 times. Then plates were labeled with α-CD36 FITC (thrombospondin) and α-CD49d (α4 β1 integrin) PE antibodies. The number of adherent CD36+and CD49d+RBC together with number of CD36+and CD49d+HUVEC was counted in 3 randomly selected fields for each study condition on fluorescent microscope Zeiss. Statistical analysis was done via unpaired t test.

Results:

In our in vitro model, the percentage of HUVEC expressing CD36 (84.4±4.7, n=10) is significantly increased after exposure to T/HS blood when compared to T/SS blood (3.5±2.2, n=6, p<0.0001). Similarly, HUVEC CD49 expression is increased when exposed to T/HS blood (72.4±7.7, n=10), but not seen with T/SS blood (n=6).

Conclusion:

In addition to increased adhesion of RBC to HUVEC after T/HS, we have shown a selective HUVEC response by the upregulation of CD36 and CD49. A factor unique to T/HS blood may play a role in the observed expression of these molecules and contribute to the microcirculatory dysfunction seen after trauma-hemorrhagic shock.

P44 DIFFERENTIAL HIF-1α-MEDIATED KUPFFER CELL PHAGOCYTOSIS FOLLOWING TRAUMA-HEMOR-RHAGE AND HYPOXIA

C.H. Hsieh*, E.A. Nickel*, J.G. Chen*, M.G. Schwacha, M.A. Choudhry, R. Raju*, K.I. Bland*, and I.H. Chaudry. Center for Surgical Research, University of Alabama at Birmingham, AL 35294

HIF-1 is a biological O2 sensor that enables adaption to hypoxia. Studies have shown that HIF-1α enhances macrophage (MΦ) phagocytosis in hypoxia. In contrast, although trauma-hemorrhage (T-H) produces a hypoxic insult, phagocytosis of MΦ is suppressed after T-H despite HIF-1α activation. We hypothesized that HIF-1α-mediated Kupffer cell (KC) phagocytosis is differentially regulated following hypoxia and T-H. Male C3H/HeN mice were randomly assigned to sham, T-H (laparotomy, 90 min hemorrhagic shock, BP 35 mmHg, resuscitation) or hypoxia (5% O2, 60 min). T-H and hypoxia groups were given Wortmannin (PI3K inhibitor), YC-1 (HIF-1α inhibitor) or vehicle at maximum bleed out time or while PaO2 was 30 mmHg during hypoxia. Mice were sacrificed 2 hr after T-H or hypoxia. The severity of hypoxia in KC, the p-Akt and activated HIF-1α as well as the KC phagocytosis were measured. KCs from hypoxia group were found to have more Akt activation and enhanced phagocytosis. Wortmannin decreased the activated HIF-1α levels in both T-H and hypoxia groups. Furthermore, administration of Wortmannin and YC-1 prevented the increased phagocytosis of KCs after hypoxia but not after T-H. Hence, activation of HIF-1α through PI3K pathway is crucial for HIF-1α in mediating KC phagocytosis after hypoxia but not following T-H (NIH R01GM37127).

Figure
Figure

P45 AUTONOMIC LINKAGE IS PRESERVED IN HEMORRHAGIC SHOCK

Eric Irwin, Matt Byrnes, Kristine Mulier, and Greg Beilman. North Memorial Medical Center, University of Minnesota

Introduction:

Cardiac stability requires balance between parasympathetic (P) and sympathetic (S) tone. Heart rate variability (HRV) was used to study the effects of resusci-tation and hemorrhage on S and P modulation of sinoatrial node function (SNF).

Methods:

Six rats under anesthesia and mechanical ventilation were hemorrhaged (shed vol=30%), monitored for 20 minutes then resuscitated with shed blood and ringers lactate. HRV was analyzed at each time point using: Standard deviation (SD) of RR intervals (varies inversely with S tone), and root mean square of successive RR intervals (rMSSD varies directly with P tone). rMSSD was plotted against SD to assess any linkage.

Results:

The correlation between SD and rMSSD was stronger during baseline, hemorrhage and shock (r2>0.85, p<0.001) than resuscitation (r2>0.604, p<0.008). The regression line slope was statistically less (p<0.01) during resuscitation, suggesting greater S tone, but increased to baseline after resuscitation suggesting a return to baseline autonomic linkage.

Conclusions:

S and P modulation of SNF are linked and shift towards greater change in S tone during resuscitation.

Figure
Figure

P46 EARLY PRESENCE OF NITROSATIVE STRESS USING AN OVINE MODEL OF ACUTE LUNG INJURY

Aimalohi Esechie1, Collette Jonkam2, Perenlei Enkbaatar2, Eszter Horvath3, Lillian Traber2, Csaba Szabo2,3, and Daniel Traber1,2. 1Departments of Neuroscience and Cell Biology, 2Anesthesiology, University of Texas Medical Branch, Galveston, Texas and 3Department of Surgery, University of Medicine and Dentistry of New Jersey, Newark, New Jersey

Trauma due to burn and smoke injury is commonly associated with elevated levels of free radicals such as nitric oxide and generation of reactive nitrogen species.

Objective:

Elucidate the changes involved in nitrosative stress using a clinically relevant model of ALI.

Method:

Sheep (n=5 per group) were surgically instrumented for chronic study. 5 days after surgery, the animals were randomly allocated as follows: Injury (instrumented; 3rd degree 40% total body surface area flame burn and insufflation with 48 breaths of cotton smoke under deep anesthesia); control (instrumented; no injury); non-injury (no instrumentation; no injury). Injured sheep were sacrificed at the following time points post-injury (hrs): T=4h, 8h, 12h, and 24h. Data is reported as mean ± SEM.

Results:

Pulmonary gas exchange was attenuated 24h post burn and smoke injury (p<0.05) and correlated with increased lung water content at 18h and 24 h (p<0.05). Inducible nitric oxide synthase (iNOS) expression increased significantly after injury compared with the non-injury and control groups; peak iNOS expression was at 12h post burn and smoke inhalation (p<0.05 vs. control and un-injured; n=5). Plasma nitrate/nitrite (NOx) correlated with protein expression: plasma NOx values were significantly increased 12h post injury (p=0.046). 3-nitrotyrosine, an index of reactive nitrogen species formation, significantly increased after burn and smoke inhalation compared with the control (p<0.05, n=5) and non-injury (n=2) groups. The extent of DNA damage was assessed by measuring repair enzyme, poly (ADP) ribose polymerase (PARP). PARP activity increased in the injury group (p<0.05 vs. control).

Conclusion:

Nitrosative stress occurs early after burn and smoke inhalation injury. Therapeutic interventions to mediate excess free radical production could ameliorate impaired pulmonary function associated with ALI.

Support:

R01GM60688; SBI 8450; SBI 8954; SBI 8820

P47 MECHANISM OF HEPATOPROTECTION IN PROESTRUS FEMALE RATS FOLLOWING TRAUMA-HEMORRHAGE: HEME OXYGENASE-1-DERIVED NORMALIZATION OF HEPATIC INFLAMMATORY RESPONSES

S. Hu*, S. Yang*, J. Chen*, M.A. Choudhry, K.I. Bland*, and I.H. Chaudry. Center for Surgical Research, University of Alabama at Birmingham, AL 35294

Although hepatic damage occurs in males and ovariectomized females (OVX) but not in proestrus (PE) females following trauma-hemorrhage (T-H), the mechanism responsible for this hepatoprotection remains unknown. We hypothesized the protection is due to downregulation of liver inflammatory responses via enhanced heme oxygenase-1 (HO-1). To study this, PE and OVX female rats underwent T-H (midline laparotomy and 60% blood loss followed by resuscitation). In another PE group, HO-1 inhibitor chromium mesoporphyrin-IX chloride (Cr-MP, 2.5 mg/kg) was administered intraperitoneally at the onset of resuscitation. Rats were sacrificed 2 hrs thereafter. Our results indicate that following T-H: 1) LV performance was maintained in PE rats but depressed in OVX and Cr-MP-treated PE groups; 2) liver damage was not evident in PE rats; however, Cr-MP abrogated the protection; 3) liver HO-1 expression increased significantly in PE rats; in contrast, liver ICAM-1, caspase-3, Bcl-2, P-IκB-α, and NF-κB expressions increased in OVX and Cr-MP-treated PE groups; 4) liver MPO activity, NF-κB DNA-binding activity, and CINCs increased in OVX and Cr-MP-treated PE groups; 5) plasma estradiol levels and hepatic estrogen receptor-α and-β expression were unaltered by Cr-MP administration. Thus, enhanced HO-1 in PE cycle modulates inflammatory responses and protects the liver following T-H. (NIH R37 GM39519)

Figure
Figure

P48 17β-ESTRADIOL IMPROVES BONE MARROW-DERIVED MACROPHAGE SURVIVAL AND ANTIGEN PRESENTING FUNCTION AFTER TRAUMA-HEMORRHAGE

J.G. Chen*, C.H. Hsieh*, E. Nickel*, M.A. Choudhry, M.G. Schwacha, K.I. Bland*, and I.H. Chaudry. Center for Surgical Research and Department of Surgery, University of Alabama at Birmingham, AL 35294

While trauma-hemorrhage (T-H) alters Kupffer cell, splenic and peritoneal macrophage functions, it remains unclear whether T-H also affects the function of bone marrow-derived macrophages (BM-MΦ). To study this, male C3H/HeN mice 6-8 wk and weighing 20-25 g were subjected to sham operation or T-H, receiving vehicle or 17β-estradiol (E2) (1 mg/kg). T-H was induced by midline laparotomy and ∼90 min of hemorrhagic shock followed by fluid resuscitation. The mice were sacrificed 24 hr after resuscitation. CD11b+BM cells were isolated and cell proliferation, apoptosis and antigen presentation were assessed in vitro. Apoptosis was measured using LPS-(1 μg/ml) simulated cells (16 hr) and proliferation was assessed following 48-hr stimulation with LPS. Adherent cells were cultured for 7 days prior to determining antigen presenting capacity of the cells to present conalbumin (cona) to T-cells. A decrease in the proliferation and increased apoptosis of BM-MΦ were observed following T-H. BM-MΦ antigen presentation was also significantly suppressed after T-H. E2 administration significantly reduced the BM-MΦ apoptosis and improved antigen presentation capacity. In conclusion, administration of E2 following T-H improves BM-MΦ functions, and thus is likely to decrease susceptibility to subsequent sepsis. (NIH RO1 GM37127)

Figure
Figure

P49 MECHANISM OF ESTRADIOL-MEDIATED CARDIO-PROTECTION FOLLOWING TRAUMA-HEMORRHAGE: UPREGULATION OF CARDIAC O-GLYCOSYLATION /PHOSPHORYLATION

S. Yang1*, S. Hu1*, L. Zou2*, M.A. Choudhry, J.C. Chatham2*, K.I. Bland1*, R.B. Marchase2*, and I.H. Chaudry1. 1Center for Surgical Research and Dept Surgery, 2Dept Cell Biology, University of Alabama at Birmingham, AL 35294 USA

Our studies have shown that estradiol (E2) and glucosamine prevent trauma-hemorrhage (T-H)-induced cardiac depression and cardioprotection by glucosamine was associated with increased cardiac N-acetyl-glucosamine (O-GlcNAc) levels. However, it is not known whether the E2-mediated cardioprotection is due to upregulation of cardiac O-glycosylation/phosphorylation. T-H was induced in adult male rats (removal of 60% of circulating blood volume followed E2 [1 mg/kg] or vehicle [cyclodextrin, CD, 20.7 mg/kg]) administration i.v. during resuscitation. In another E2-treated T-H group, the E2 receptor antagonist ICI 182,780 (3 mg/kg) was administered 30 min before E2, i.p. Two hrs after T-H, LV performance was determined, cardiomyocytes isolated, cardiac nuclei extracted andcardiac mitochondria isolated. Following T-H: 1) LV performance and cardiac CTD 110 decreased significantly (p<0.05); E2 restored LV performance and enhanced cardiac CTD110; 2) cardiac mitochondrial ATP levels andcytochrome-C activity significantly decreased; E2 restored mitochondrial ATP levels and cytochrome-C activity; 3) cardiac P-IκB-α, NF-κB and ICAM-1 expression and MPO activity increased significantly; E2 prevented those increases; 4) ICI 182,170 abolished protective effectof E2.

Figure
Figure

Thus, E2-mediated cardio-protection after T-H appears to be due to upregulation of cardiac O-glycosylation/phosphorylation. (NIH R37GM39519 and R01 HL076165)

P50 STIMULATING EFFECTS OF SERA FROM SEVERE TRAUMA PATIENTS ON NF-κB ACTIVITY IN MACROPHAGE AND THEIR RELATIONSHIP WITH PATIENTS PROGNOSIS

Liang Hua-Ping, Xu Xiang, Xia Hao, Fei Jun, Wu Dan, and Wang Zheng-Guo. State Key Laboratory of Trauma, Burns, and Combined Injury, Department 1, Research Institute of Surgery, Daping Hospital, Third Military Medical University, Chongqing 400042, China

Objective:

To investigate the stimulating effects of sera from severe multiple trauma patients within 24h post trauma on nuclear factor-κB(NF-κB) activity in macrophage and their relationship with patients prognosis.

Methods:

24h after transfection of the recombinant NF-κB plasmid containing luciferase reporter gene into the mouse macrophage line (RAW264.7), the cells were stimulated by different sera for 6h, then stimulating effects of sera on NF-κB were detected by luciferase action, The concentration of interleukin-1β, tumor necrosis factor α, interleukin-10, interleukin-1 receptor antagonist, and soluble tumor necrosis factor receptor I were detected with ELISA kits.

Results:

The stimulating activity of NF-κB was increased significantly in traumatic sera group, which is higher in MODS group, nonsurvivors group than that in non-MODS, survivors group respectively. These changes were correlated positively with APACHE II score, while had no relationship to the cytokine or endogenous antagonist levels. The area under the receiver operator characteristic (ROC) curve of NF-κB activity for predicting MODS and mortality was significantly higher than that of APACHE II score.

Conclusion:

Early measurement of NF-κB stimulating activity of sera from severe multiple trauma patients may show the predictive value for the late occurrence of MODS and mortality.

Keywords:

trauma; NF-κB; prognosis

P51 BLAST INJURY DECREASES HEMORRHAGE TOLERANCE IN RATS

Timothy B. Bentley, and Joseph B. Long. Walter Reed Army Institute of Research, 503 Robert Grant Ave., Silver Spring, MD 20910-7500

Primary blast, defined as the blast overpressure (BOP) wave generated by explosives, induces brain and systemic trauma that likely alters cardiovascular (CV) compensation to hemorrhage (HEM). To better understand the polytrauma interactions associated with blast, we investigated the injuries and altered CV responses to hemorrhage after graded BOP exposure. Under isoflurane anesthesia, male S-D rats (365g avg) received femoral arterial and venous cannulae and were then subjected to sham injury, BOP, HEM or combined BOP-HEM injuries. BOP was generated using a compressed air-driven shock tube yielding a single overpressure wave at peak pressures of 86, 97 or 118 kPa. Following air-blast, rats underwent a 15 min computer controlled hemorrhage to a target mean arterial pressure (MAP) of 30 mmHg, which was isobarically maintained by additional bleeding as necessary. Arterial pressures and shed volume (SBV) were recorded continuously until death or euthanasia at 5 hr, after which necropsies were performed. Number, severity and distribution of internal injuries increased with the severity of BOP and included contusions and hemorrhage in brain, ear, eye, lung, heart, kidney, cecum, bladder, skin and testicles and tearing in heart, liver and stomach. BOP caused apnea (10-17 sec) and hypotension (20-40 mmHg) pro-portional to BOP severity, but no bradycardia. BOP compromised CV tolerance to hemorrhage with a decrease in SBV (12 to 6.6 ml), time until onset of decompensatory shock and with more severe blast, a reduction in time until death. Thus, BOP disrupts cardiovascular compensation after hemorrhagic hypotension, and in so doing, likely alters fluid resuscitation requirements.

Trauma/Multiple Injuries Clinical Research

P52 BLOOD PRODUCT EFFECT ON SURVIVAL FOR COMBAT TRAUMA PATIENTS IN SHOCK

Philip C. Spinella, Jeremy G. Perkins, and John B. Holcomb. US Army Institute of Surgical Research, San Antonio, TX

Objective:

To determine independent effects of blood products on survival for trauma patients in shock.

Methods:

This retrospective study analyzed the following variables: admission vital signs and lab values, 24 hour blood product totals, ISS and hospital mortality for trauma patients with shock, defined as base deficit (BD) of =-6, admitted to one combat hospital in Iraq in a 1 year period (2004). Variables that were associated with survival on univariate analysis were evaluated by multivariate logistic regression to determine independent associations.

Results:

There were 360 patients included with a BD of =-6. Median (IQR) ISS was 16 (10-25) with a mortality of 20%. On univariate analysis survival was associated with SBP, HR, Temp, BD, GCS, hematocrit, ISS, recombinant VIIa use, and 24 hr total amount transfused of RBCs, FFP, and whole blood. Results of the regression analysis correctly classified 84% of outcomes with an area under the curve of 77%, (Table). When BD =-3 and-9 were both used to define shock, independent associations were still found for RBC and FFP amount transfused with death and survival, respectively.

Table
Table

Conclusion:

Trauma patients in shock may benefit from an increased ratio of plasma to RBCs. Prospective study is needed to determine if resuscitation with more plasma and less RBCs improves outcomes in trauma patients in shock.

P53 CEREBRAL FAT EMBOLISM (CFE): CLINICAL FEATURES, MRI FINDING AND EVOLUTION

N. Baffoun1, K. Baccar1, M. Magouri1, Z. Zaghdoudi1, S. Abassi1, M. Achour1, and C. Kaddour1. 1Institute Of Neurology, anesthesiology and critical care, La Rabta, Tunisia

Background:

Fat embolism syndrome (FES) is a serious complication of both long bone fracture and orthopaedic surgery with intramedullary manipulation. It was shown that fat emboli traverse pulmonary vasculature and cause brain embolisation, which result in neurological impairment.

Patients and methods:

We review over three year (2005-2007) the cases of patients who presents long bones fractures and/or fracture of pond in association or not with brain injury or other lesions and admitted in our ICU. We study the incidence of FES, clinical status and MRI finding.

Results:

27 patients were involved in this study. Seven of them have present isolated long bones and/or pond fracture. The 20 other patients were polytrauma. Seven young men have presented FES (3 patients with mild to moderate brain injury and one of them present thoracic trauma). Age: 33 ± 8 years, Apache III: 31 ± 15, ISS: 23 ± 22, ventilation duration: 7 ± 5 days, duration of stay: 16 ± 10 days.

Five patients were admitted in ICU for respiratory distress with neurological impairment 24 to 48 hours after trauma or after orthopaedic surgery. Three of them present an ARDS. One patient was admitted for persistent drowsiness with delirium. The last one was a poly trauma patient who presents a moderate brain injury (GCS 10) with frontal contusion on CT scan. He underwent orthopaedic surgery and present hypotension and oxygen desaturation. The days after, the neurological status worsens and the patient presents a cerebral death status. Two patients have a thrombocytopenia and only one have petechiae. The MRI of these patients reveals essentially multiple high intensity signals throughout white matter, corpus callosum and in periventricular on T2 and FLAIR sequences.

Five patients from the survivors were discharged without neurological sequela. The sixth remained drowsy at discharge. The control MRI shows partial or complete regression of lesions.

Discussion:

The FES can be made on the basis of clinical signs. Respiratory signs are seen in 75%, which may progress to respiratory failure. Half of our patients have presented an ARDS. Petechiae are seen in 50%. Neurological changes, seen in up to 86% of patients, may present as diffuse encephalopathy and/or with focal abnormalities. Criteria and scoring system were published to be used to diagnosis.

MRI has been shown to be more sensitive than CT in the identification of CFE with typically abnormalities appearing as hyper intense lesions on T2-weigted, proton density and FLAIR sequences. CFE can cause capillary inflammation and break-down of the blood barrier, without associated haemorrhage or permanent neurological deficit.

The outcome in patients with FES, which is not correlated to MRI finding, is generally favourable with mortality rates of less than 10%.

P54 THE INCLUSION OF SLOVENIAN MULTIPLE TRAUMA PATIENTS INTO THE DGU REGISTRY

D. Brilej, R. Lefering, and R. Komadina. *Dept. of Traumatology, General and Teaching Hospital Celje, Oblakova 5, Celje, Slovenia; **IFOM - Institute for Research in Operative Medicine, University of Witten/Herdecke, Faculty of Medicine, Cologne, Germany

Introduction:

Trauma registries are the basis for programs that aim to improve the quality of the entire course of treatment of severely injured patients, from the accident site to completion. In 1992 we started implementing a polytrauma registry in Celje General Hospital to collect the data for comparison of treatment efficacy with international standards for the most severely injured. A major drawback of the Celje Registry was the small number of patients. At the same time when the idea emerged for a Slovenian registry of severely injured, an initiative in the European Union aimed to standardise the protocols for data gathering. This would allow databases to interconnect and form the core of a European registry of injured patients. Given the difficulties posed by the small sample size in Slovenia, we considered the option to get included in the DGU Registry. This article reports the results of the first year of inclusion of Slovenian patients into the DGU Registry.

Materials And Methods:

In 2006 we collected data on 80 patients who met the inclusion criteria: polytrauma patients with ISS of at least 18, isolated injuries and AIS score of 5 in individual regions, severe injury (AIS 4) and affected vital signs who continued their treatment in the Intensive Care Unit, patients with at least 2 fractures of long tubular bones and affected vital signs.

Results in 2007:

The average age was 50.5 years, 87.5% were male and average ISS score was 28.2. 15 injured patients (18.75%) died in the hospital during the acute treatment phase (before transfer to a care ward). Comparison with the DGU register average data shows (in tables) some differences in prehospital and hospital treatment strategies between Slovenian and German trauma centres, which do not influence our results significantly.

Discussion:

Data was evaluated in the Documentation Centre of the DGU Registry in Cologne during regular annual data analysis for all participating hospitals. The analysis focused on the structure of our patient group in comparison with the total registry population (demographics, types and severity of injuries) and on quality of care (TRISS and Revised Injury Severity Classification (RISC)). With improvement of prehospital critical care and increased average life expectancy in Slovenia, the pattern of injuries suffered by patients admitted to our hospital began to change. Besides the predominant high-energy blunt force trauma, the percentages of polytrauma patients and severe head injuries increased over time.

P55 DAMAGE CONTROL ORTHOPEDICS - WHAT DO WE REALLY KNOW?

E. Steinhausen1, R. Lefering2, S. Sauerland2, M. Maegele1, S. Wutzler2, E. Neugebauer2, B. Bouillon1, and D. Rixen1. 1Department of Trauma/ Orthopedic Surgery, 2Institute for Research in Operative Medicine, University of Witten/ Herdecke, Cologne-Merheim, Germany

Objectives:

Today, there is a trend towards the concept of "Damage Control Orthopedics" (DCO) in the management of multiple trauma patients with long bone fractures. While this trend is reflected by a significant increase in primary external fixation of femur fractures in an analysis of the German Trauma Registry, current literature is insufficient.

Methods:

A systematic literature analysis on the clinical evidence of DCO was performed. Based on these results a new trial was designed.

Results:

Most of the 68 identified controlled trials had a retrospective design with evidence level (EL) 2b-4. Only four studies had a randomized design (EL 1b). Results are contradictory, a generalized management strategy is missing.

Conclusion:

Evidence from the current literature is insufficient. Thus, in the era of "evidence-based medicine", there is the need for a more specific, clarifying randomized controlled trial. Therefore a new trial was initiated which is entitled: Randomized controlled trial on risk adapted damage control orthopaedic surgery of femur shaft fractures in multiple trauma patients. Temporary fracture fixation with fixateur externe (DCO) is compared to primary reamed nailing. Primary objective is to reduce the extent of organ failure as measured by the maximum SOFA-Score within 4 weeks after trauma. The required number of patients was calculated to be 140 for intention-to-treat analysis. Patient recruitment started in July 2007. Up to date, 21 level I trauma centers participate, including all five regional CHIR-Net-centers. The trial is supported by the Deutsche Forschungsgemeinschaft (DFG).

P56 TRACKING CENTRAL HYPOVOLEMIA WITH LINEAR AND NONLINEAR INDICES OF HEART RATE VARIABILITY (HRV) IN HUMANS

K.L. Ryan1, C.A. Rickards1, W.H. Cooke1, and V.A. Convertino1. 1US Army Institute of Surgical Research, Fort Sam Houston, TX 78234; 2University of Texas at San Antonio, San Antonio, TX 78249

Objective:

Novel HRV analyses could provide a non-invasive method of tracking early reductions in blood volume with hemorrhage, promoting earlier intervention that might prevent hemorrhagic shock. We hypothesized that both linear and nonlinear indices of HRV could track reductions in central blood volume earlier than standard vital signs (e.g., blood pressure (BP)).

Methods:

Ninety-three (41F/52M) healthy subjects (age 28 ± 1 yrs; height 174 ± 1 cm; weight 76 ± 2 kg) completed a progressive lower body negative pressure (LBNP) protocol (0, -15, -30, -45, -60, -70, -80 mmHg for 5 min each) to presyncope, followed by a recovery period. ECG, BP and stroke volume (SV) were measured continuously. Linear (RRI HF, RRISD, pNN50) and nonlinear (sample entropy (SampEn), fractal dimensions by curve length (FD-L) and dispersion analysis, symbol dynamics (SymDyn)). HRV indices were calculated at each level of LBNP and recovery.

Results:

SV was reduced from baseline (84 ± 2 ml) by-30 mmHg LBNP (p<0.001), fell to ~60% of baseline at-80 mmHg (34 ± 1 ml) and was restored during recovery (93 ± 2 ml). SampEn, FD-L, SymDyn and pNN50 were also lower than baseline by-30 mmHg (p=0.025), while the remaining HRV indices and BP fell by-45 mmHg LBNP (p=0.012). All linear and non-linear HRV measures were highly correlated with the reduction and recovery of SV, with amalgamated R2 values =0.88.

Conclusion:

Both linear and nonlinear indices of HRV directly track the reduction and recovery of central blood volume during LBNP in healthy humans; some of these indices changed earlier than BP.

P58 C-JUN EXPRESSION IN MONOCYTES IN MULTIPLE TRAUMA PATIENTS: A COMPARATIVE PILOT-ANALYSIS IN MRNA GENE EXPRESSION AND NUCLEAR PROTEIN

Viktoria Bogner1, Veit Stöcklein1, Peter Richter1, Christian Suren1, Daniel Teupser1, Joachim Thiery1, Wolf Mutschler1, and Peter Biberthaler1. 1Chirurgische Klinik und Poliklinik-Innenstadt, Ludwig-Maximilians Universität, München, 2Institut für Laboratoriumsmedizin, Klinische Chemie und Molekulare Diagnostik, Universitätsklinikum Leipzig

Background:

Modulations of the monocyte function seem to advantage the development of SIRS (Systemic Inflammatory Response Syndrome) and consecutive multiple organ failure in multiple trauma patients and consequently has profound impact on the patients' clinical outcome. However, the thereby underlying initial intracellular mechanisms are not well understood yet. Our precedent genome-wide microarray study showed a significant influence of differential c-Jun mRNA expression on the patients' clinical development. Yet, it is unclear, if this differential regulation also dominates intracellular monocyte protein concentration.

Patients and Methods:

Seven multiple trauma patients exhibiting an injury severity score between 16 and 75 points have been enrolled in the study. Blood samples were drawn on admission (90min after trauma) and at 6h, 12h, 24h, 48 and 72h. Monocyte isolation from whole blood was followed by nuclear protein preparation. C-Jun protein expression was assessed using Phosphoprotein Assay (Biorad). The results have been correlated to the mRNA gene expression and the patients' clinical outcome.

Results:

Three of seven patients had an adverse outcome. Nuclear c-Jun protein expression shows an initial peak (0h) in all patients, followed by a decrease and a second peak at 72h post trauma. Deceased patients in general exhibit higher c-Jun protein levels at any time after trauma in comparison to patients who survived. This is supported by the microarray mRNA expression data. Though having a distinct tendency, the yet low sample size does not allow for a sufficient significance in non-parametric statistics.

Conclusion:

Our genome-wide mRNA Microarray Expression study revealed a significant correlation between monocyte c-Jun mRNA Expression and clinical outcome in multiple injured patients in the very early posttraumatic period. For the first time, this phenomenon can be confirmed in nuclear monocyte protein in a yet low patient collective. This further supports a superior role of c-Jun concerning the development of a post trauma immune dysfunction syndrome with adverse clinical outcome. Follow up studies to further increase the sample size are currently underway.

P59 HOSPITAL-BASED TRAUMA REGISTRY AND TRISS FOR IMPROVING INJURY SURVEILLANCE AND DEVELOPING THE RESOURCES

L. Fattori, G. Pesenti, C. Marradi, A. Intelisano, and A. Nespoli. Ospedale San Gerardo dei Tintori, Dipartimento di Chirurgia, via Pergolesi 33, 20052 Monza, Italy

Introduction:

In a teaching-hospital in urban area, referral centrefor severely traumatized patients, a trauma registry was implemented. A homogeneous treatment should be guaranteed by a favourable (70%) rotation of ATLS certified doctors in Emergency Department (ED).

The empirical criteria of hospitalization was defined to enrol the patients to the study. Type of trauma, mechanism of injury, age, gender, Injury Severity Score (ISS), Trauma Related Injury Severity Score (TRISS), diagnostic and surgical treatment, length of stay and discharge status (alive or dead) were recorded. Results:100.000/year admissions to the ED, 12.000 admissions to the hospital wards/year and among these, trauma patients are 900/year (7.5%). 18% of trauma patients are ISS>15. The 70% of trauma patients admitted the wards underwent to surgical procedures that is not significantly different from the 65% of patients with ISS>15. 70% of major injuries resulted in 15<ISS<25(Mortality Rate(MR) 0%), 11% in 26<ISS<40 group (MR 8%, in non-survivors predicted survival rate by TRISS:<20) and 18% in ISS>41% (MR 50%, predicted survival rate in non survivors by TRISS:<10%). In ISS>41 group, TRISS overestimate mortality risk in 20% of survivors. Conclusions: The empirical criteria of hospitalization correlates with an elevated index of surgical procedures, and this index is similar in ISS>15 patients. ISS should not be considered a relevant parameter to suggest admission to hospital wards, but only to stratify patients at discharge. Mortality rate in high risk patients is better than expected by TRISS: TRISS tends to over estimate potentially deaths in elevated ISS scores.

P60 PROGNOSTIC FACTORS OF DIFFERENT ASSESSMENTS IN MULTIPLE TRAUMA PATIENTS WITH PARAPLEGIA

Cornelia Putz, Thomas Grieser, Christian Schuld, Carl Hans Fürstenberg, Hans Jürgen Gerner, Rüdiger Rupp, and Bernd Wiedenhöfer. Stiftung Orthopädische Universitätsklinik Heidelberg, D-69118, Schlierbacher Landstr. 200a.

Background:

Injuries of the thoracic vertebrae in multiple trauma patients are often accompanied by severe thoracic injuries and sensorimotoric deficits. The neurological course can influence the success of rehabilitation. The aim of the study was to evaluate if the type of fracture, the Polytraumaschluessel (PTS), the AIS (ASIA Impairment Scale) and the SCIM (Spinal Cord Injury Measurement) are important tools for rehabilitation outcome in thoracic multiple trauma patients. A retrospective data analysis of 21 multiple trauma patients with thoracic vertebrae and spinal cord injury was performed in the course of rehabilitation.

Methods:

We selected 21 operated multiple Trauma patients with thoracic vertebrae injury and complete or incomplete paraplegia from the Heidelberger EMSCI database (n = 330, time interval: 2002-2007). In all patients the PTS, the AO-Classification, the AIS and the SCIM were collected. Data was statistically evaluated with SPSS 15.0®. T-test and Chi2-Test were performed (p < 0.05).

Results:

According to the AO-Classification 5 A-(23.8%), 10 B- (47.6%) and 6-C fractures (28.6%) of thoracic spine (T1-12) were found. After one year 14 (66.7%) patients (group 1) maintained complete paraplegia, whereas 7 (33.3%) patients (group 2) improved in the AIS. A conversion of complete to incomplete paraplegia was found in two patients of group 2. In group 1 all patients with C-fractures (n = 6) maintained their neurological status (AIS A). A relation between neurological improvement and the overall PTS (μgroup1= 32.07 ± 8.6 SD, μgroup2 = 23.86 ± 8.9 SD, p = 0.058) with statistical significance for the sub item PTST in the thoracic area (μgroup1 = 14.5 ± 4.3 SD, μgroup2 = 7.57 ± 5.3 SD, p = 0.005) was found. The SCIM (μgroup1=63.07 ± 15.2SD, μgroup2 = 78.71 ± 14.0 SD, p = 0.035) showed a significant improvement after one year in the group 2.

Conclusion:

Poor neurological outcome was found in all patients having C-fractures and an AIS of A. The PTS, the AO-Classification and the SCIM constitute important assessment criteria regarding neurological outcome and rehabilitation success in multiple trauma patients with paraplegia.

P61 SEX AS PROGNOSTIC MARKER AFTER SEVERE MULTIPLE TRAUMA

H. Trentzsch1, R. Lefering2, R. Kraft1, M. Burggraf1, S. Zedler1, E. Faist1, S. Piltz1, and the trauma registry of German Society for Traumasurgery (DGU). 1Dept of Surgery, Hospital of the University of Munich Großhadern, 2Institut for Research in operative medicine (IFOM), Campus Köln-Merheim

Several studies have evaluated sex as a risk factor for survival and complication after trauma. Female sex is considered beneficial, however, clinical data remains inconsistent. We have analyzed data from the DGU trauma registry with regard to gender-differences. From 29353 trauma cases prospectively recorded we selected patients being primarily treated with an ISS≥9 (n=20.288). Additionally, we stratified by age and performed a matched-pair-analysis including 3965 pairs. Matching considered age-group, injury severity according to abbreviated injury scale (AIS) and availability of data referring to shock in the field. Males (n=14720) had higher rates of multiple organ failure (MOF, 22% vs. 20%, Chi2-Test, p<0.001) and sepsis (11% vs. 7%, Chi2-Test, p<0.001) but females (n=5568) had higher mortality rates (20% vs. 17%, Chi2-Test, p<0.001). Stratified by age, we observed no difference in survival rates. The matched-pair-analysis, however, gave less mortality rates in females (16% vs. 18%, Chi2-Test, p=0.037). MOF and sepsis rates were unchanged penalizing male sex (MOF: 20% vs. 16%, Chi2-Test, p=0.007; sepsis: 9% vs. 7%, Chi2-Test, p<0.001). Data suggests higher mortality rates in females to be confounded by mean age, injury severity by AIS, incidence of shock and co-morbidities. This was controlled in the matched-pair-analysis. In conclusion, we find female sex to be beneficial in terms of survival rates and risk for developing sepsis or MOF, although epidemiologic effects may quench this advantage.

P62 THE PROGNOSTIC VALUE OF CYTOKINES AND LABORATORY PARAMETERS FOR ORGAN DYSFUNCTION AND MORTALITY IN PATIENTS WITH MULTIPLE INJURIES

Frank Hildebrand, Michael Frink, Christian Krettek, and Martijn van Griensven#. *Trauma Department, Hannover Medical School, Hannover; #Ludwig Boltzmann Institute, Vienna, Austria

Introduction:

Although therapeutic concepts of patients with major trauma have improved during recent years, organ dysfunction still remains a frequent complication during clinical course in intensive care units. It has previously been shown that cytokines are upregulated under stress conditions such as trauma or sepsis. However, it is still debatable if cytokines are adequate parameters to describe the current state of trauma patients. To elucidate the relevance of cytokines, we investigated if cytokines and other laboratory parameters (thrombocytes, lactate, CRP, base excess) predict development of multi organ dysfunction syndrome (MODS) or outcome.

Methods:

In this prospective study, patients with an Injury Severity Score (ISS)>16 and an age>16 years were included. Blood samples for determination of cytokines (IL-6, IL-8, IL-10, IL-1β und TNF-α) and laboratory parameters (CRP, thrombocytes, lactate, Base Excess) were drawn over a period of 14 days. Patients were divided into groups according to the development of MODS (+MODS vs. -MODS, Marshall Score) and outcome. To determine the association between levels of cytokines and laboratory parameters and development of MODS the Spearman rank correlation coefficient was calculated and logistic regression and analysis was performed.

Results:

143 patients were included in this study. IL-6 represented the most reliable parameter for predicting the development of MODS with an overall accuracy of 84.7% (specificity: 98.3%, sensitivity: 16.7%). The threshold value of IL-6 at admission for development of MODS was 761.7pg/ml and 2176.0pg/ml for mortality. Among the laboratory parameters lactate was the most reliable parameter for MODS-prediction (overall accuracy 82.9%, specificity 97.1%, sensitivity 7.7%). By combining IL-6, lactate and thrombocytes, sensitivity was improved to 27.3%.

At admission, only systemic IL-6 and lactate levels demonstrated a significant correlation with outcome (specificity: IL-6 100%, lactate 99.0%; sensitivity: IL-6 28.6%, lactate 5.3%). The threshold value of IL-6 for adverse outcome was 2176pg/ml.

Conclusion:

In this study, IL-6 represented the most reliable parameter for assessment of the clinical status. However, the low sensitivity of IL-6 and partly overlapping IL-6 plasma concentrations in patients with or without MODS demonstrate that no single marker alone is able to accurately predict the clinical course and outcome of multiple trauma patients. This is not surprising as these patients represent a highly diverse group (e.g. genetic-or gender-dependent differences). Therefore, laboratory markers (especially lactate), clinical assessment as well as inflammatory parameters(especially IL-6) should be combined for evaluation of the clinical status.

P63 INTESTINAL CELL DAMAGE PRECEDES INFLAMMATION IN MULTITRAUMA-PATIENTS

J. de Haan, J.P.M. Derikx, B. Relja, T. Lubbers, A. van Bijnen, M.D. Luyer, W.A. Buurman, J.W. Greve, and I. Marzi. Maastricht University Medical Center, Maastricht, Netherlands and JW Goethe University Hospital, Frankfurt am Main, Germany

Intestinal damage is considered to play an important role in the development of SIRS and sepsis after severe trauma. However clinical evidence remains scarce. This study investigates the early presence of intestinal cell damage in multi-trauma patients in relation to the inflammatory response. Trauma patients (n=95) admitted to the Emergency Room (ER) were classified according to the Injury Severity Score (ISS) and presence of abdominal trauma (AT). In plasma collected at ER, concentrations of Intestinal Fatty Acid Binding Protein (I-FABP), a cytosolic protein present in mature enterocytes and released after cellular damage, were measured. Circulating procalcitonin (PCT), a marker for bacterial infections, was determined at day 1 post-trauma. At ER, mean (±SEM) I-FABP concentrations were significantly elevated in trauma patients with AT compared to patients without AT (980±235 vs. 262±28 pg/ml; p<0.05 MWU). Interestingly, I-FABP was also increased in patients without AT compared to 76 healthy volunteers (164±12 pg/ml; p<0.01). I-FABP levels at ER were correlated positively with ISS (Spearman r=0.56; p<0.001). Furthermore, I-FABP values at ER correlated with PCT levels on day 1 (Spearman r=0.61; p<0.001). In conclusion, this is the first study to provide evidence for rapid development of intestinal epithelial cell damage in severe multi-trauma patients with and without abdominal trauma. The extent of early intestinal damage is associated with the subsequent inflammatory response. Further studies are needed to determine whether therapies aimed at reduction of intestinal damage improve clinical outcome in multi-trauma patients.

P64 ARTERIAL LACTATE MATCHES VO2 IN PREDICTING CRITICAL OXYGEN DELIVERY IN A RAT MODEL OF HEMORRHAGIC SHOCK

P.S. Reynolds, S. Song, F. Tamariz, and R.W. Barbee. Virginia Commonwealth University Medical Center and VCU Reanimation and Shock Center, Richmond VA 23298 USA

Systemic critical oxygen delivery (DO2crit) is a major indicator of vulnerability to tissue hypoxia. However DO2crit is difficult to measure in the acute trauma patient. We developed a rodent model to assess threshold DO2 obtained from both VO2 and lactate following severe controlled hemorrhage.

Methods:

Male rats were anesthetized, intubated, ventilated, and thoractomized. Cardiovascular variables were measured continuously; hematological variables were measured at baseline and after each 0.5 ml hemorrhage increment. DO2 was estimated from cardiac output and arterial oxygen content, and VO2 from respiratory gases. DO2crit was estimated by nonlinear regression of VO2 and surrogate markers on DO2.

Results:

DO2crit estimated from lactate did not differ from that estimated from VO2 (11.5 vs 11.6 mL/min/kg, P>0.2); lactate levels at DO2crit averaged 3.0 mmol/L (SD 0.8).

Conclusions:

There are few published measurements of the dynamics of the VO2:DO2 relationship during acute hemorrhage in rats. Our model represents an improvement in existing VO2:DO2 dynamical models for rodents because of continuous sampling of hemodynamic variables and rapidly sequenced measures of hematological variables. These features allow superior quantification of VO2:DO2 kinetic curve and higher resolution of the transition point. Values obtained for DO2crit in this study are similar to those previously reported followingprolonged hemorrhage (10.4 - 10.8 ml/min/kg) or anemic hypoxia (10.0 ml/min/kg). Data from this study further support the viability of lactate as a surrogate marker for DO2crit during acute hemorrhage.

P65 COMPARISON BETWEEN TWO POSTINJURY MULTIPLE ORGAN FAILURE (MOF) SCORES

Angela Sauaia, Ernest E. Moore, Jeffrey Johnson, David J. Ciesla, Walter Biffl, and Anirban Banerjee. University of Colorado Denver and Denver Health Medical Center, Denver, CO 80262

The use of different scores to define postinjury MOF have seriously hampered research on novel therapies. Thus, we propose to compare objective outcomes of patients identified as having MOF by two widely accepted scores: Marshall's (MS) and Denver's (DS).

Methods:

1389 consecutive trauma patients with ISS>15, admitted from 1992-2004, had MOF scored daily. The DS grades (from 0-3) only 4 organ dysfunctions (heart, lung, kidney, liver) and defines MOF as score >4. The MS grades, in addition, hematologic and CNS dysfunctions (total=6 organs, 0-4 scale) and defines MOF as score>6.

Results:

According to MS, 691(50%) had MOF, of whom 107(15%) died, 57% required mechanical ventilation (MV)>7 days and 63% had ICU stay>14 days. In contrast, DS identified only 308 (22%) patients with MOF, of whom 92(30%) died, 76% required MV>7 days and 73% had ICU stay>14 days. Comparisons between the scores showed:

Table
Table

Conclusion:

Compared to the Marshall MOF Score, the Denver MOF Score performed better at identifying trauma patients at risk for mortality and high resource utilization.

P66 POSTTRAUMATIC NEUTROPHIL FAS/FASL SIGNALLING

Adnana Paunel-Görgülü, Tim Lögters, Stefan Margraf, Jens Altrichter, Detlev Kindgen-Milles, Joachim Windolf, and Martin Scholz. Department of Trauma and Hand Surgery, University Hospital Duesseldorf, D-40225 Düsseldorf, Germany

Fas/FasL system is crucial for the regulation of innate immune responses. Intensive care patients frequently suffer from posttraumatic immune deregulation and organ failure, largely due to aberrant and prolonged neutrophil hyperactivity. However, the role of Fas/FasL signalling in the deregulation of immune responses in severely injured patients has not been studied. Therefore, in this study the expression of Fas/FasL in neutrophils from severely injured patients was determined over a time period of two weeks after trauma. Moreover, the ex vivo sensitivity of neutrophils to immobilized anti-Fas IgM was investigated. Neutrophils were obtained over time (I: days 1-3; II: days 4-6; III: days 7-11) from -15- multiple trauma patients with an injury severity score (ISS)>16. The gene expression of Fas and FasL was significantly up-regulated in neutrophils from severely injured patients compared with neutrophils from healthy controls. Whereas Fas protein expression was significantly up-regulated on the surface membrane of neutrophils over time, no difference was found for FasL. Interestingly, sFasL in patient plasma decreased over time. When neutrophils were challenged with immobilized anti-Fas IgM, caspase 3 and-8 activity significantly increased in patient neutrophils, however, to a minor degree compared with neutrophils from healthy donors. Evidence was found that the reduced ability to undergo Fas-mediated apoptosis may be due to increased anti-apoptotic MCL-1 expression in patient neutrophils.

P67 HMGB1 IS MARKEDLY ELEVATED WITHIN SIX HOURS OF MECHANICAL TRAUMA IN HUMANS

E. Peltz, E. Moore, S. Damle, P. Eckels, Y. Tsuruta, J. Johnson, A. Sauaia, A. Banerjee, and E. Abraham. University of Colorado Denver and Denver Health Medical Center, Denver, CO 80262

HMGB1 is a late mediator of the systemic inflammation and multiple organ failure (MOF) associated with sepsis. Recently, HMGB1 has been shown in animal studies to be a mediator of hemorrhage-induced organ dysfunction. We hypothesize that HMGB1 is elevated in human plasma following trauma.

Methods:

Trauma patients at risk for MOF (ISS 16 - 57; mean 30.75) were identified for inclusion (n=16) and HMGB1 levels in plasma samples from<6 hours through>72 hours after trauma were measured by ELISA. Statistics were done by ANOVA, with post-hoc Bonferroni test for multiple comparisons.

Results:

Plasma HMGB1 levels were greatest within 6 hours of injury (mean 713 ng/ml, p<0.01), decreased more than 50 percent by 24 hours (268 ng/ml), then remained elevated above normal levels (<2ng/ml) through 72 hours.

Figure
Figure

Conclusion:

Plasma HMGB1 is dramatically elevated within 6 hours of mechanical trauma and remains elevated above normal levels through 72 hours post injury. These results suggest that, in contrast to sepsis, HMGB1 release is an early event following traumatic injury. Thus, HMGB1 may be integral to the early inflammatory response to trauma.

P68 SPECIES-SPECIFIC INFLAMMATORY RESPONSE TO TRAUMA/HEMORRHAGE

Rajaie Namas, Andres Torres, Mazen Zenati, Juan Ochoa, Timothy R. Billiar, and Yoram Vodovotz. University of Pittsburgh, Pittsburgh, PA

Introduction:

Traumatic injury/hemorrhagic shock (T/HS) is the most common cause of death in people<44 years old in the US, in part due to the induction of an acute inflammatory response. Mice and rats are often used as pre-clinical models of T/HS, but no single study has documented the correspondence between inflammation in these animals and their human counterparts.

Methods:

Ten male T/HS patients, all survivors, 26-53 y.o., ISS 24-50 were analyzed. Patients were treated surgically and received 0-5 units of blood+fluids. Male C57Bl/6 mice (6-10 wk old; n=5) and male Sprague Dawley rats (10-12 wk old; n=10) were anesthetized, cannulated surgically, and subjected to HS (25 mmHg [M] and 35-40 mmHg [R]). Mice were resuscitated with shed blood+2X lactated Ringer's solution. Rats were resuscitated with shed blood+0.9% saline. Plasma blood samples were taken 24h post-trauma and assayed for IL-6, IL-10, TNF-α, and NO2/NO3. Results were analyzed by ANOVA followed by Holm-Sidak post-hoc test.

Results:

IL-6 (485±165 [H], 19±11[R], 200±22 [M]; P<0.009 vs. both animals) and IL-10 (267±100 [H], 13±4.1[R], 14±2.6 [M]; P=0.019 between H and R) were higher, and TNF (0±0[H], 9±0.4[R], 2±2[M]; P<0.001 vs. both animals) and NO2/NO3(31±3[H], 6±0.9[R], 33±2.8[M]; P<0.001 vs. both animals) were lower 24h post-T/HS in humans.

Conclusion:

Differences in the inflammatory responses of humans vs. mice and rats may suggest that care should be taken when extrapolating from pre-clinical to clinical studies of inflammation in T/HS.

P69 COPEPTIN LEVELS PREDICT THE SEVERITY OF SHOCK IN TRAUMA PATIENTS

R. Jaffe, L. Holmes, N. Martin, S. Sonnad, M. Bergey, N. Morgenthaler, J. Struck, A. Horan, Y. Guan, C. Deutschman, P. Reilly, C.W. Schwab, and C. Sims. University of Pennsylvania, Philadelphia, PA, USA

Arginine vasopressin (AVP) is a critical element in the endocrine response to hemorrhagic shock. Copeptin, a stable fragment of the AVP precursor, is a reliable surrogate for plasma AVP and a more stable biomarker.

Objective:

To determine if initial copeptin levels reflect the severity of hemorrhagic shock.

Methods:

Serial blood samples were drawn from trauma patients (n=22) during initial resuscitation/hospitalization. Copeptin was measured using a novel sandwich immunoassay. Kruskal Wallis, Mann-Whitney, and Friedman tests were used to compare groups.

Results:

Copeptin is markedly elevated following trauma and falls rapidly with resuscitation over time (p<0.001) (Fig 1). Patients receiving 10-20 units of blood product in 12 hrs had statistically higher levels of copeptin on arrival (p=0.01) (Fig 2). An ROC curve demonstrated copeptin adequately identified high unit transfusions (AUC=86%).

Figure
Figure
Figure
Figure

Conclusion:

Initial copeptin levels reflect severity of shock and predict the need for massive blood product resuscitation in trauma patients.

Neurotrauma Basic Research

P70 MODULATION OF TLR2 REDUCES BRAIN INJURY FOLLOWING FOCAL ISCHEMIA AND REPERFUSION

Fang Hua, Jiang Ma, Tuanzhu Ha, Jim Kelley, David Williams, Race Kao, John Kalbfleisch, and Chuanfu Li. Depts of Surgery, Internal Medicine, Biometry and Medical Computing, East Tennessee State University, Johnson City, TN 37614

Recent studies have shown that Toll-like receptors (TLRs) contribute to ischemia/reperfusion (I/R) injury. In this study we investigated the effect of modulation of TLR2 on focal cerebral I/R injury. Pam3CSK4, a TLR2 specific ligand, was administered to mice 24 hrs before the mice were subjected to focal cerebral ischemia (1 hr) and reperfusion (24 hrs). Cerebral infarct size, blood brain barrier (BBB) permeability and expression of tight-junction proteins was examined after cerebral I/R. Modulation of TLR2 by Pam3CSK4 significantly reduced brain infarct size (1.9 ± 0.5% vs 9.4 ± 2.2%, p<0.05) compared with the untreated I/R group. Pam3CSK4 administration preserved BBB function as demonstrated by decreased leakage of serum albumin (0.528 ± 0.026 vs 0.771 ± 0.059) and Evans Blue (9.23 ± 0.72 μg/mg vs 12.56 ± 0.65 μg/mg) into brain tissue. Pam3CSK4 also blunted the decrease in occluding protein expression when compared with the untreated I/R group. In addition, Pam3CSK4 treatment reduced the acute (24 hr) mortality (4.3% vs 24.2%, p<0.05), preserved neurological function (8.22 ± 0.64 vs 3.91 ± 0.57, p<0.05), and attenuated brain edema following focal cerebral I/R injury. Our results suggest that modulation of TLR2, with a TLR2 specific ligand, will reduce the morbidity and mortality associated with focal ischemic brain injury. In addition, preservation of blood brain barrier function by modulation of TLR2 may be one of the mechanisms of the neuroprotection.

P71 BENEFICIAL EFFECTS OF ETHYL PYRUVATE IN A MOUSE MODEL OF SPINAL CORD INJURY

Tiziana Genovesea, Emanuela Espositoa,b, Emanuela Mazzona, Rosanna Di Paolaa, Rosaria Melib, Placido Bramantia, Mitchell P. Finkd, and Salvatore Cuzzocreaa,c. aIRCCS Centro Neurolesi "Bonino-Pulejo", Messina, Italy; bDepartment of Experimental Pharmacology, University of Naples "Federico II", Via D. Montesano 49, Naples, Italy; cDepartment of Clinical and Experimental Medicine and Pharmacology, School of Medicine, University of Messina, Italy; dDepartments of Critical Care Medicine, Surgery and Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, USA

Despite advances in the field of medicine, injury to the spinal cord remains a devastating problem. Contemporary management of the spinal cord injury (SCI) currently consists of supportive care and stabilization of the spine. Numerous pharmacologic therapies have been evaluated for the treatment of SCI, but none have met with significant success. The aim of the present study was to evaluate in a mouse model of SCI the effect of the treatment with ethyl pyruvate (EP), a simple aliphatic ester, which has been shown to have anti-inflammatory effects in previous numerous cell culture and animal studies. SCI was induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy in mice. SCI in mice resulted in severe trauma characterized by edema, neutrophil infiltration, production of inflammatory mediators, tissue damage, and apoptosis. Treatment with EP (75, 25, or 8.5 mg/kg) 1 and 6 h after the SCI significantly reduced the deleterious effects of SCI in a dose-dependent manner. Specifically, treatment with EP decreased (1) the degree of spinal cord inflammation and tissue injury (histological score); (2) neutrophil infiltration (myeloperoxidase activity); (3) nitrotyrosine formation; (4) pro-inflammmaory cytokines expression; (5) NF-κB activation; (6) ERK1/2 mitogen activated protein kinase phosphorylation; and (6) apoptosis (TUNEL staining, FAS ligand, Bax and Bcl-2 expression). Moreover, EP (75, 25, or 8.5 mg/kg) significantly ameliorated in a dose-dependent manner the loss of limb function (evaluated by motor recovery score). Taken together, our results demonstrate that EP treatment reduces the development of inflammation and tissue injury associated with spinal cord trauma.

P72 GITR-FC FUSION PROTEIN INHIBITS GITR TRIGGERING AND PROTECTS FROM THE INFLAMMATORY RESPONSE FOLLOWING SPINAL CORD INJURY

Salvatore Cuzzocrea, Giuseppe Nocentini, Tiziana Genovese#, Rodolfo Bianchini, Emanuela Mazzon#, Simona Ronchetti, Emanuela Esposito#, Di Paola Rosanna#, Placido Bramanti#, and Carlo Riccardi. *Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia, Tossicologia e Chemioterapia, Università di Perugia, Italy. Dipartimento Clinico e Sperimentale di Medicina e Farmacologia, Torre Biologica, Policlinico Universitario, 98123 Messina, Italy; #IRCCS Centro Neurolesi "Bonino-Pulejo", Messina, Italy

Glucocorticoid-Induced Tumor necrosis factor receptor-Related (GITR) protein is a co-stimulatory molecule that plays a role in inflammation so that GITR-Fc fusion protein can exert an anti-inflammatory effect. To investigate the mechanism by which GITR-Fc exerts its effects, we first used GITR knock-out (GITR−/−) mice to verify whether GITRL/GITR system played a pro-inflammatory role in the spinal cord injury (SCI) model. Notably, a less pronounced disease was induced in GITR−/− as compared to GITR+/+mice. We then evaluated the effect of GITR-Fc fusion protein against SCI-induced injuries in GITR−/− and wild type (GITR+/+) mice. Administration of GITR-Fc ameliorated SCI-induced inflammation in GITR+/+ mice as evaluated through: 1) histological damage and apoptosis, 2) modulation of apoptosis-related transduction factors (Bax and Bcl-2), 3) expression of inflammatory markers (nitrotyrosine, iNOS, IL-2, IL-12 and TNF-α) and 4) T lymphocyte infiltration. GITR-Fc was effective in GITR+/+but not in GITR−/− suggesting that, in this experimental model, its anti-inflammatory action is due to inhibition of GITR triggering and not to GITRL activation. In conclusion, GITR plays a role in SCI and administration of GITR-Fc results in amelioration of SCI severity prompting further studies on the potential anti-inflammatory properties of GITR-Fc.

P73 REDUCTION OF PERIVASCULAR AQUAPORIN-4 EXPRESSION IN CORTEX AND STRIATUM BY AGMATINE IN AMELIORATION OF TRANSIENT MIDDLE CEREBRAL ARTERY OCCLUSION-INDUCED INFARCTION AND FUNCTIONAL DEFICITS

Ching-Ping Chang1,2, Che-Chuan Wang1,3, Yun-Chin Yao2, Jinn-Rung Kuo2,3,4, Bor-Chih Cheng1,5, and Mao-Tsun Lin1,2. 1Department of Biotechnology, Southern Taiwan University, Tainan 710, Taiwan; 2Department of Medical Research, Chimei Medical Center, Tainan 710, Taiwan; 3Department of Surgery, Chi-Mei Medical Center Tainan 710, Taiwan; 4Institute of Clinical Medicine, School of Medicine, National Cheng-Kung University, Tainan 710, Taiwan; 5Department of Cardiovascular Surgery, Chi-Mei Medical Center, Tainan, Taiwan

The aquaporin-4 (AQP4) pool in the perivascular astrocyte membrane has been shown to be involved in the pathogenesis of brain edema, which is a major cause of morbidity and mortality in stroke. Here we demonstrate by quantitative immunohistochemistry that the ischemia cortex and striatum show similar patterns of AQP4 expression in the reperfusion phase after 90 minutes of middle cerebral artery occlusion in the rat. The cortical and striatal cores display an increase of perivascular AQP4 expression at 1 hour of reperfusion with no sign of subsequent recovery. The cortical and striatal loss of AQP4 is accompanied by cerebral infarction and apoptosis as well as proprioception dysfunction. This increase of cerebral AQP4 expression cerebral infarction, and functional deficits show no recovery toward 72 hours of reperfusion. Agmatine (100mg/kg; intraperitoneally) administrated immediately after middle cerebral artery occlusion attenuated cerebral infarction and apoptosis, proprioceptive dysfunction, and striatal and cortical AQP4 expression evaluated 72 hours after middle cerebral artery occlusion. The present data indicate that agmatine may attenuate deficits by reducing increase of perivascular AQP4 in both the cortex and striatum.

Keywords:

aquaporin, astrocyte, brain infarction, apoptosis, stroke, agmatine

P74 HYPOTHERMIC RETROGRADE JUGULAR VEIN FLUSH MAY ATTENUATE TRAUMATIC BRAIN INJURY IN RATS BY REDUCING OXIDATIVE STRESS

Jinn-Rung Kuo1,2,3, Che-Chuan Wang1,2, Ching-Ping Chang2,4, and Mao-Tsun Lin2,4. 1Department of Surgery, Chi-Mei Medical Center Tainan 710, Taiwan; 2Department of Biotechnology, Southern Taiwan University, Tainan 710, Taiwan; 3Institute of Clinical Medicine, School of Medicine, National Cheng-Kung University, Tainan 710, Taiwan; 4Department of Medical Research, Chimei Medical Center, Tainan 710, Taiwan

The mechanisms underlying the protective effects of hypothermic retrograde jugular vein flush (HRJVF) therapy in traumatic brain injury (TBI) remain unclear.Here we attempted to ascertain whether the possible occurrence of oxidative stress exhibited during TBI could be reduced by HRJVF. In the present study, the fluid percussion technique was used to produce TBI. Experiments were conducted to determine the effect of TBI on the malondialdehyde (MDA), glutathione peroxidase (GPx) activity, glutathione reductase (GR) activity, superoxide dismutase (SOD) and catalase activity in different brain structures inn rats with or without HRJVF. HRJVF was accomplished by intravenous infusion of cold saline (5ml, 4) over 5 minutes. It was found that the MDA values obtained from different brain tissues were significantly greater than those of sham group. In contrast, the GR, GPx, SOD and catalase activity values in brain were significantly lower compared with sham group. The increased values of MDA and the decreased values of GR, GPx and SOD in different brain tissue were significantly attenuated by HRJVF adapted immediately after TBI. These findings demonstrate that brain cooling caused by HRJVF therapy may attenuate TBI-induced cerebral ischemia injury by reducing oxidative stress in brain.

Keywords:

traumatic brain injury, hypothermic retrograde jugular vein flush, malondialdehyde, glutathione peroxidase, glutathione reductase, superoxide dismutase, catalase

P75 EXPRESSION OF KV1.2 IN MICROGLIA AND ITS PUTATIVE ROLES IN MODULATING PRODUCTION OF PROINFLAMMATORY CYTOKINES AND REACTIVE OXYGEN SPECIES

Jia Lu1*, Fan Li2, Shabbir Moochhala1, and Eng-Ang Ling2. 1Defense Medical and Environmental Research Institute, DSO National Laboratories, 27 Medical Drive, Singapore 117510; 2Department of Anatomy, Block MD10, 4 Medical Drive, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597

Microglial cells are endowed with different potassium ion channels but their expression and involvement in specific functions has remained to be fully explored. This study has shown Kv1.2 expression in microglia both in vivo and in vitro. Kv1.2 immunoreactivity was first localized in the amoeboid microglia in the postnatal rat brain between 1 (P1) and 10 (P10) days. At P14, it was localized in the ramified microglia derived from the amoeboid microglia. Kv1.2 immunoexpression in microglia was progressively reduced with age and it was undetected at P21. In P1 rats exposed to hypoxia, Kv1.2 immunoreactivity in amoeboid microglia was markedly enhanced. RT-PCR analysis confirmed Kv1.2 mRNA expression in microglial cell culture and murine BV-2 cells. Furthermore, it was up-regulated when the cells were subjected to hypoxia exposure. Additionally, Kv1.2 mRNA expression coupled by that of IL-1ß and TNF-α was increased following ATP and LPS application. However, the intracellular potassium concentration in activated microglia as determined by PBFI fluorescence was decreased. Blockade of Kv1.2 channel TsTX resulted in partial recovery of intracellular potassium concentration accompanied by a reduced expression of IL-1ß and TNF-α as well as production of intracellular reactive oxygen species (ROS). We conclude that Kv1.2 in microglia is sensitive to hypoxia, ATP and LPS. Its up-regulated expression as elicited by the external stimuli may be linked to IL-1ß and TNF-α expression and ROS production. Hence, optimizing the dosage of TsTx in suppression of Kv1.2 expression may be potential therapeutic means in ameliorating neuroinflammation in which microglial activation is implicated.

P76 EXOGENOUS ADMINISTRATION OF GLIAL CELL LINE-DEVIVED NEUROTROPHIC FACTOR IMPROVES RECOVERY AFTER SPINAL CORD INJURY

Cheng-Kuei Chang. Department of Surgery, Mackay Memorial Hospital, Taipei, Taiwan and Graduate Institute of Injury Prevention and Control, Taipei Medical University and Municipal Wan-fan Hospital, Taipei, Taiwan

The aim of present study was to examine whether systemically delivered glial cell derived neurotrophic factor (GDNF) was beneficial in reversing the spinal cord injury (SCI) in a spinal cord compression model. Rats were divided into three major groups: (1) sham operation (laminectomy only); (2) laminectomy+SCI+normal saline (1 ml/kg body weight, i.v.); and (3) laminectomy+SCI+GDNF (50 ng/kg body weight, i.v.). Spinal cord injury was induced by compressing the spinal cord for 1 min with an aneurysm clip calibratedto a closing pressure of 55 g. GDNF or saline was administered immediately after SCI via the tail vein. Behavioral tests of motor function measured by maximal angle an animal could hold to the inclined plane was conducted at day 1 to 7 after SCI. The triphenyltetrazolium chloride staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end labeling assay were also conducted after SCI to evaluate spinal cord infarction and apoptosis respectively. Both GDNF and vascular endothelial growth factor (VEGF) in the injured spinal cord were assayed by immunafluorescence. It was found that systemically delivered GDNF, but not vehicle solution, significantly attenuated the SCI-induced hind limb dysfunction and spinal cord infarction and apoptosis. Both GDNF and VEGF could be detected in the injured spinal cord after GDNF, but not vehicle solution, therapy. The results indicate that GDNF treatment may be beneficial in reversing hind limb dysfunction by reducing spinal cord infarction and apoptosis in a spinal cord compression model.

Keywords:

Spinal cord injury, neurotrophic factor, apoptosis, vascular endothelial growth factor

P77A HYPERTONIC/HYPERONCOTIC TREATMENT (HHT) AND SURGICAL EVACUATION AFTER ACUTE SUBDURAL HEMATOMA (ASDH) IN RATS

B. Alessandri, D. Jussen, C. Papaioannou, A. Heimann, and O. Kempski. Johannes Gutenberg-University, Inst. For Neurosurg. Pathophysiology, D-55131 Mainz, Germany

Treatment of ASDH consists mainly of surgical evacuation of the hematoma in order to restore micro-circulation. Early preoperative neuroprotection with HHT may improve survival rates. The benefit of HHT on functional and histological outcome as supportive therapy ac-companying surgical intervention was investigated n a model of ASDH (400 μl subdural blood infusion). Thirty (30) minutes after ASDH, rats (n=50) received i.v. either HyperHAES (7.2% saline/ 6% hydroxyethyl starch) or vehicle (NaCl 0.9%), followed by surgical evacuation of the hematoma 1 hr after ASDH induction in those rats scheduled for surgical treatment. We explored acute effects of HHT on blood variables, changes of intracranial pressure (ICP) and investigated the chronic effects of HHT, surgical blood clot evacuation, andthe combination of both on functional and histological outcome following ASDH (12 days). ASDH increased ICP and decreased CPP in all groups. All treated groups showed a better recovery with respect to neurological function and neuronal cell death compared with the vehicletreated ASDH group. HHT with surgical evacuation or HHT alone improved functional and histological outcome slightly more thansurgical evacuation alone. This significantly improved functional andhistological outcome by HHT was comparable to the effects after blood evacuation alone. The combination of evacuation of subdural blood and early HHT improved outcome further but not significantly, which was due to the strong effects of single treatments and a ceiling effect of the combined treatment in this model.

P77B HIGH BRAIN TISSUE CONCENTRATIONS OF THE HEMOPOIETIC CYTOKINE ERYTHROPOIETIN AMELIORATES NEUROPOTECTIVE PROPERTIES AFTER ACUTE SUBDURAL HEMATOMA IN RATS

B. Alessandri, M. Wähmann, M. Rahimi, A. Raabe, H. Vatter, A. Heimann, and O. Kempski. Johannes Gutenberg-University, Inst. for Neurosurg. Pathophysiology, D-55131 Mainz, Germany

The hemopoetic cytokine Erythropoietin (EPO) has been shown to be neuroprotective in animal model of traumatic brain injury. The necessary brain tissue concentration for neuro-protection is not thoroughly investigated. The present study investigates acute effects of EPO on intracranial pre-ssure, cerebral perfusion pressure, tissue oxygen concent-ration (ptiO2) and cerebral blood flow as well as the neuro-protective potential of systemically applied EPO in a rat model of ASDH (n>5/group, subdural infusion of 400μl blood). 20) minutes after subdural blood infusion, rats re-ceived i.v. either NaCl, 200, 2000 or 20'000 IU EPO and brain tissue EPO was measure 1 hour and histological da-mage 48 hours after ASDH. Ipsilateral brain tissue con-centration of EPO rose to: 1.7±0.2, 11.7±0.2. 88.4±4 and 672±63 mIU/ml tissue in the NaCl, 200, 2000 and 20'000 IU group, respectively. EPO at 20'000 IU increased ICP significantly during the last 10 mn of monitoring. Measure-ment of lesion volume 48 hours after ASDH revealed a significant neuroprotective effect of200 and 2000 IU EPO (12.5±0.4; 9.6±0.5 mm3) when compared to the NaCl group (16.6±0.3), whereas 20'000 IU was neurotoxic (20.2±0.7). EPO was neuroprotective in a model of ASDH. However, high tissue EPO concentration received by i.v. in-jection of 20'000 IU led to ICP increase and produced ad-verse effects on lesion growth. Thus, save concentration ranges for acute treatment with EPO should be evaluated for the use of EPO in TBI patients.

P78 THE FIRST EXPERIENCE OF INVESTIGATION OF STEM CELLS ACTION IN PATIENTS WITH PROLONGED UNCONSCIOUS STATE AFTER CRANIOCEREBRAL INJURIES

A.N. Kondratiev, and R.V. Nazarov.

Mesenchymal stem cells are pluripotential cells that could be selected from the stroma of bone marrow. In vitro isolated stem cells display great proliferative activity. They are able to increase in quantity about 10000 times by reproducing themselves without loosing multivalent differentiation potential. Selected and cultivated cells are capable of differentiation (both in vivo and in vitro) in neural, vascular tissues, osteocytes, chondrocytes, myocytes and bone marrow cells. The most exploited ways of differentiation are neurogenesis and angiogenesis. The restoration of vessels and neural tissue of central nervous system is contemporary and worth-while method of treatment for patients with long time unconscious state? Aim: to investigate the safety and efficiency of application autological stem cells in treatment of patients in prolonged unconscious state after craniocerebral injures. Materials and methods: in our investigation we included 20 randomized patients in unconscious state that persisted longer then 4 weeks. Control group was formed of 10 patients who received the standard therapy. Another 10 patients besides the same therapy came through stem cells transplantation. In patients from the experimental group we took the bone marrow from the ischiadic bone (about 50 ml) and then, after the selection and cultivation of stem cells till they reached the quantity of 5 mln in cubic centimeter, they were transplanted into patients. Our investigation was approved by the ethical committee of the institute and the informed agreements of the closest relatives of the patients were received. Results: we noted no complications related with investigation in experimental group throughout 6 month after the stem cells transplantation. Within 2 weeks after transplantation all the patients demonstrated positive effects in general status: improving the condition of nails, hair, and skin. After 6 month period 4 patients from the control group and 6 patients from the experimental group restored to consciousness. But due to the small size of patients group and short observing period, it is difficult to define clearly the direct effects of stem cells on neural tissue. Discussion: The results we had got made it possible to affirm the expediency of using stem cells in complex therapy of the patients with severe damages of central nervous system. We noted such effects as stimulation of angiogenesis in damaged areas of the brain, removal of the deficiency of macroergical substances, improving of general status. But in the same time we did not get such important effect as restoration of neuronal connections which was demonstrated in experiments on animals. That is why it is necessary to care out further investigations of the stem cells transplantation.

P79 HEMATOLOGIC EFFECTS OF PREHOSPITAL ADMINISTRATION OF RECOMBINANT ACTIVATED FACTOR VII (rFVIIA) IN TRAUMATIC BRAIN INJURY (TBI) IN SWINE

Francoise Arnaud, Anke Scultetus, Richard McCarron, and Daniel Freilich.

Brain injury is an important cause of morbidity (including disability) and mortality after trauma. The purpose of this study was to assess the hemostatic effects of rFVIIa administered after TBI during the prehospital phase to diminish intracranial hemorrhage. Yorkshire pigs were subjected to TBI (frontal-parietal fluid percussion; 2.8±0.6 atm) (T0) and then administered rFVIIa (90 μg/kg, n=10) or nothing (n=10) at T15. All animals received standard supportive care. At T75, simulated hospital phase treatment also included infusion of mannitol for elevated intracerebral pressure (ICP). Hemodynamic monitoring continued up to 6 hours after which animals were euthanized. Serial blood samples were analyzed for CBC, chemistry, and coagulation indices (PT, TEG, PFA, thrombin-antithrombin [TAT], and ATIII). Significance (p<0.05) was assessed by Student's t-test. All animals lived to 6 hours in both groups without clinical evidence of clinical thrombotic complications. There were no time or group differences for hemoglobin, hematocrit, white cell count, platelets, and ATIII. In both groups, TAT was reduced from T0 to T15 (p<0.001) due to brain injury. At T60, PT and TEG-R were lower with rFVIIa compared to controls (p<0.05); other TEG indices and PFA were similar. pH was significantly higher, and HCO3and lactate trended to be improved with rFVIIa. Overall, this mild TBI model resulted in increased thrombin generation but not coagulopathy. We conclude that in swine, prehospital administration of rFVIIa tend to improve indirect global measures of tissue perfusion without significant effects on hematologic/hemostatic parameters.

P80 A NOVEL APPROACH OF SELECTIVE BRAIN COOLING AFTER EXPERIMENTAL TRAUMATIC BRAIN INJURY IN THE RAT

Hinnerk Doll, Florian Kipfmüller, Hubert Truebel, and Marc Maegele. Institute for Research in Operative Medicine (IFOM), Department of Pediatrics, HELIOS Klinikum Wuppertal, Cologne, University of Witten-Herdecke (Germany)

To assess a novel approach of pharyngeal selective brain cooling (pSBC) for improved neurofunctional outcome in a model of experimental traumatic brain injury (TBI). Male Sprague-Dawley rats were subjected to lateral fluid-percussion (LFP) brain injury and received either pSBC for 3 hours post-injury or no treatment. In the pSBC group brain temperature was selectively reduced to 33°C±1°C at 15 min post-injury and maintained for 3 hours. Whole body temperature was kept stable at normothermic levels. Neuromotor and sensorimotor dysfunction and coordination immediately after injury and recovery on days post-injury 7 and 14 (DPI) were assessed. All animals subjected to TBI showed an identical pattern of severe neurofunctional impairment at 24 hours after injury. In the time course of the experiment pSBC animals showed superior neurofunctional recovery at both time points, i.e. on DPI 7 (pSBC: 19,21 points ±2,24; TBI only: 14,33 points ±2,60; p<0001) and DPI 14 (pSBC: 20,54 points ±1,56; TBI only: 16,40 points ±2,88; p<0,001). Similarly, distances walked on the rotating rod (Rotarod test) were substantially longer in animals that had received pSBC after injury versus their nontreated counterparts on both DPI 7 (pSBC: 1,74m ± 0,41; TBI only: 0,98m ± 0,28; p<0,001) and DPI 14 (pSBC: 2,04m ± 0,23; TBI only: 0,98m ± 0,33: p<0,001). pSBC was associated with improved neuromotor and sensorimotor outcome in a model of experimental TBI but additional work is necessary to further characterize its effects.

P81 KETAMINE DOES NOT EXACERBATE TRAUMATIC BRAIN INJURY INDUCED INFLAMMATION IN THE RAT

Matthew T. Harting, Jeremy L. Ward, Kenneth S. Helmer, Charles S. Cox Jr., and David W. Mercer. University of Texas Medical School, Houston, TX

Traumatic brain injury (TBI) induces an acute intracerebral (IC) inflammatory response. Ketamine is an anesthetic with potent anti-inflammatory actions. However, its use may be limited in TBI patients as controversy exists as to whether ketamine aggravates or attenuates TBI. We hypothesized that ketamine would not exacerbate TBI induced inflammation. Male rats were given intraperitoneal ketamine (7mg/kg) or saline 1h before sham injury or induction of TBI using a moderate controlled cortical impact injury model. Six hours after injury, the brain was extracted and the direct area of injury and penumbral areas isolated. Cytokine production (Bioplex) and cerebral edema (tissue water percentage) were measured. As shown in the representative figures(*), TBI caused intracerebral edema and induced significant production of IC IL-1α*, IL-1β, IL-6, IL-10, and TNF-α* in both the direct injury and penumbral areas when compared to sham, effects not attenuated or exacerbated by ketamine. As a positive control, this same dose of ketamine attenuated serum cytokine production in the rat in response to endotoxin at 6 hours (not shown). These data indicate that the anti-inflammatory effects of ketamine are absent in the brain using a rat model of TBI. However, the data also indicate that ketamine does not exacerbate TBI induced inflammation and further studies are warranted to determine whether it may be used safely in patients sustaining TBI.

Figure
Figure

Neurotruama Clinical Research

P82 PROGNOSTIC FACTORS BETWEEN YOUNG AND ELDERLY GROUPS IN A SEVERE HEAD INJURY-SIGNIFICANCE OF CPP MANAGEMENT IN GERIATRIC HEAD INJURED PATIENTS

Shoji Yokobori, Tomohiko Masuno, Takao Suzuki, Hidetaka Sato, Akira Fuse, Takashi Araki, Takahiro Atsumi, Masatoshi Kitazono, and Hiroyuki Yokota. Department of emergency and critical care medicine, Nippon medical school, Tokyo, Japan

There has been a rapid expansion of aged head injury patients and it becomes a social problem in Japan. It is important to examine prognostic factors for improvement of prognosis in elderly patients. We analyzed the prognostic factors of elderly head injured patients, especially in terms of cerebral perfusion pressure and cerebral pressure autoregulation. We divided the 68 severe head-injured (GCS=8) patients into three groups, the "Young group (group-Y; 16-30 y.o.)", "Middle-aged group (group-M; 31-65 y.o.)", and "Elderly group (group-E;>65 y.o.)" and compared vital signs, ICP, CPP, and the outcome on discharge in three groups. In addition, we calculated the pressure reactivity index: PRx (Czosnyka et al.1997) for evaluation of cerebral pressure autoregulation, and compared them among the three groups. The ratio of unfavourable outcome in group-E was 86% and the worst in three groups. There were no significant differences in heart rate, respiratory rate, and arterial blood gas data on admission. However the systolic and diastolic blood pressure was higher and the body temperature was significantly lower in group-E. Moreover, the average of CPP was lower and the PRx was higher in group-E. These results suggested that the cerebral pressure autoregulation was impaired with aging, and the decreasing of CPP strongly affected the prognosis in geriatric head injured patients. Therefore, it appears that the maintenance of sufficient CPP is more significant in elderly than younger head injured patients.

P83 FLUCTUATIONS IN CORTICAL SYNCHRONIZATION IN PAEDIATRIC TBI

Vera Nenadovic1,2,3, James S. Hutchison1,3,4, Luis Garcia Dominguez3, Hiroshi Otsubo5, Martin P. Gray1, Roy Sharma5, Jason Belkas3, and Jose Luis Perez Velazquez*2,3,5. 1Critical Care Medicine Program, Hospital for Sick Children, Toronto; 2Department of Paediatrics and Institute of Medical Science, University of Toronto, Toronto; 3Neuroscience and Mental Health Program, Hospital For Sick Children Research Institute, Toronto; 4Interdepartment Division of Critical Care, Faculty of Medicine, University of Toronto, Toronto; 5Division of Neurology, Department of Paediatrics, Hospital for Sick Children, Toronto

Traumatic brain injury (TBI) is the leading cause of death and acquired disability in the paediatric population worldwide. We hypothesized that electroencephalography synchrony and its temporal variability, analysed during the acute phase following TBI, would be altered from that of normal children and as such would offer insights into TBI pathophysiology. Seventeen paediatric patients with mild to severe head injury admitted to a paediatric critical care unit were recruited along with ten age and gender matched controls. Patients had two electroencephalographs performed three days apart. Outcome was measured at one year post TBI utilizing the Pediatric Cerebral Performance Category score (PCPC). Maximal synchrony between EEG channels correlated to areas of primary injury as seen on CT scan. The temporal variability of phase synchronization among EEG electrodes increased as patients recovered and emerged from coma (p<0.001). This temporal variability correlated with outcome (Pearson coefficient of 0.74) better than worst Glasgow Coma Score, length of coma or extent of injury on CT scan. This represents a novel approach in the evaluation of TBI in children.

Burn Injury Basic Research

P84 SUBSTANCE P IS A KEY MEDIATOR IN SYSTEMIC INFLAMMATORY RESPONSE SYNDROME AND LUNG DAMAGE FOLLOWING BURN INJURY

Madhav Bhatia1, Selena Sio1, Manoj Puthia2, and Shabbir Moochhala2. 1Department of Pharmacology, National University of Singapore, Singapore; and 2Defence Medical and Environmental Research Institute, DSO National Laboratories, Singapore

Substance P (SP) is a neuropeptide, known for its pro-inflammatory effects on numerous respiratory functions. Pulmonary complications in burn individuals have been reported even when the lungs have not sustained direct thermal damage. However, the involvement of neuropeptides in lungs following thermal injury remains unknown. Therefore, this study aimed to investigate the effect of local thermal injury on inducing distant organ pulmonary SP release and its relevance to lung injury. Male BALB/c mice, 6-8 weeks old, were anesthetized, shaved and received a 30% total body surface area full thickness burn after exposure to 95°C water for 8 seconds. Sham mice, served as controls, were anesthetized, shaved and exposed to 24°C water. Mice were subsequently resuscitated with 1 ml saline (i.p.). Subgroups were treated with specific neurokinin-1 receptor (NK1R) antagonist, L703606 (12mg/kg, i.p.) or vehicle 1h before burn. Lung and blood sample were collected 1h and 8h post-burn. Our results show that burn Injury augments significant expression of SP and biological activity of SP-NK1R signaling early after SIRS had set in. Administration of L703606, significantly suppressed PPT-A mRNA, which encodes for SP, NK-1R mRNA levels and protein levels of SP in both plasma and lung. The enhanced SP-NK-1R response correlates with exacerbated lung damage after burn characterized by increased microvascular permeability, edema and neutrophil accumulation. However, disruption of SP-NK1R signaling by L703606 significantly reverses this effect. The development of heightened inflammation and lung damage associated with leukocyte sequestration in the lungs and blood was made worst along with increased pro-inflammatory interleukin (IL)-1β, tumor necrosis factor α (TNF-α) and IL-6 mRNA and protein production as early as 1h after injury in lung homogenates. Chemokines, macrophage inflammatory protein-2 (MIP-2) and MIP-1 α were markedly increased suggesting the active role of SP induced chemoattractant production in mediating trafficking of inflammatory cells. Likewise, these effects of cytokines and chemokines were markedly suppressed upon administration of L703606. Additionally, increased lymphocyte and monocyte production in the blood was reduced by L703606. Our findings provide for the first time, important information on SP neural activity in exacerbating pulmonary inflammatory events and injury via NK1R signaling after burn. Additionally, we show that SP can modulate circulatory population of immune cells in thermal injury. Furthermore, the inhibition of tachykinin actions may provide a possible form of therapeutic intervention for the prevention of an acute pulmonary inflammatory cascade following severe injury.

P85 ADMINISTRATION OF PEROXYNITRITE DECOMPOSITION CATALYST INTO BRONCHIAL ARTERY ATTENUATES PULMONARY PATHOPHYSIOLOGY AFTER SMOKE INHALATION AND BURN INJURY IN OVINE MODEL

Hamahata Atsumori.

Introduction:

The bronchial circulation plays a critical role in the pathophysiology of burn and smoke inhalation-induced acute lung injury. There is ~20-fold increase in bronchial blood flow associated with excessive production of peroxynitrite following combined smoke inhalation and cutaneous burn. We hypostasized that direct delivery of low dose WW-85 (peroxynitrite decomposition catalysts) into the bronchial artery would attenuate smoke/burn-induced acute lung injury.

Methods:

Sheep were surgically prepared before the injury. Through 4th intercostals thoracotomy bronchial artery was cannulated preserving the blood flow. Acute lung injury was induced 7-10 days later by 40% total body surface area 3rd degree cutaneous burn and smoke inhalation (48 breaths of cotton smoke,<40°C) under deep anesthesia. Adult female sheep (n=10, 35.4 ± 1.0 kg) were divided into two groups following the injury: 1) WW-85 group: 1 h post-injury, WW-85 (0.002 mg/kg/h, 2ml/h) was continuously injected into bronchial artery, n=5. 2) Control group: 1h post-injury, equal amounts of saline was injected into bronchial artery, n=5. After the injury, all animals were placed on ventilator, and fluid resuscitated equally. Studies were conducted in awake state for 24 h.

Results:

Pulmonary function evaluated by measurement of blood gas analysis, pulmonary mechanics, and pulmonary transvascular fluid flux was severely deteriorated in control group. However, the above changes were markedly attenuated by WW-85 infusion into bronchial artery (Table 1).

Table 1
Table 1:
(at 24h)

Conclusions:

We demonstrated that low dose (50 times lower than i.v. dose) of WW-85 directly administered into bronchial artery attenuated the pulmonary dysfunction in same extent. Our data strongly suggest that local airway production of peroxynitrite contributes to pulmonary dysfunction following smoke inhalation and burn. Regional delivery of the pharmaceutical compounds might be both beneficial and cost efficient in treatment of burn victims, especially those who have concomitant smoke inhalation.

P86 INSULIN THERAPY IMPROVES GLUCOSE DISPOSAL IN BURNED, HINDLIMB UNLOADED RATS

H.F. Pidcoke, C.E. Wade, X. Wu, L.A. Baer, and S.E. Wolf. Univ of Texas Health Science Center, San Antonio and US Army Institute of Surgical Research, Texas, USA

Introduction:

Severe trauma results in profound hypermetabolism and immunocompromise. Insulin treatment improves muscle protein accretion and immune function in patients; however efforts to study the mechanism have been hampered by an inadequate preclinical burn model. Large burn alone in rats does not reproduce the severity of metabolic insults seen in human subjects; a new model combining burn and disuse in the form of hindlimb unloading (BHU) more closely resembles the human condition and produces greater metabolic derangements. We hypothesized that insulin therapy after injury and disuse decreases weight loss and improves glucose disposal in BHU rats.

Methods:

Sprague-Dawley rats were randomised to daily treatment with insulin (INS: 5 units/kg SQ insulin), or vehicle (NS). Rats from both groups were scalded (40% BSA) and hindlimb unloaded for 14 days. Glucose tolerance testing (GTT) was performed on Day 14.

Results:

The INS group retained more mass compared to the NS group (287±11 v 272±12 g, p=0.023). Fasting glucose levels were lower for the INS group compared to NS (42±21 v 105±9 mg/dL, p=0.005), and natural log regression demonstrated the slope of the glucose disposal curve was steeper in the INS group (-0.018 v-0.007, p=0.014). The area under the curve adjusted for starting glucose was lower in the INS group (6985±1260 v 10865±2003 mg*min/dL, p=0.003).

Conclusions:

Data demonstrate that daily insulin therapy in BHU rats decreases weight loss and results in faster glucose disposal. The BHU preclinical model is adequate for studying insulin therapy after severe injury, and further research examining protein turnover and immune function is planned.

P87 CARDIOMYOCYTES LACKING CD14 DO NOT MOUNT A CYTOKINE RESPONSE TO LPS

R.C. Barber*, D.L. Maass, and J.W. Horton. UT Southwestern Medical Center, Dallas, TX 75390

Objective:

We have shown previously that blockade of CD14 signaling by molecular (CD14 KO) or pharmacologic (geldanamycin Tx) means abrogated cardiomyocyte inflammation after burn injury. Herein, we investigated the role of CD14 in the cardiomyocyte response to a sterile inflammatory stimulus.

Methods:

Dose-response data were generated by challenge of CD14 KO mice and wild-type littermates with 0 to 10 μg of LPS and measurement of inflammatory cytokine secretion by cultured cardiomyocytes. Statistical analyses were performed by ANOVA, with by post-hoc analysis with SNK.

Results:

Supernatants from CD14 KO cardiomyocytes held lower levels of cytokines, relative to wild-type cells (Figure). Interestingly, cytokine levels were also significantly lower in supernatants from unstimulated CD14 KO cardiomyocytes, relative to wild-type cells, suggesting that stimulation induced by cardiomyocyte isolation also signalled through CD14.

Figure
Figure

Conclusion:

CD14 signalling is critical to the cardiomyocyte response to a sterile inflammatory challenge. Study support: NIH/NIGMS P50-GM21681-43 [JWH].

Cytokine concentrations in supernatants from wild-type or CD14 KO cardiomyocytes after LPS challenge. Values are mean +/− SEM.

*Significant difference from 0 LPS (P < 0.05). Significant difference from wild-type at equivalent LPS level (P < 0.05).

P88 GEMCITABINE AMELIORATES POST BURN INFLAMMATION AND IMMUNOSUPPRESION

J. Noel, X. Guo*, L. Haar, Q. Wang*, and C. Ogle. Shriners Hospital for Children, Cincinnati Ohio 45209

Previous data in our lab have indicated that thermal injury induces an increase in monocyte progenitors and immature monocytes, and these latter are a major source of post burn inflammation. This suggested that thermal injury induced a burst of proliferation in the myeloid system, leading to increased production of monocytes and their progenitors. We reasoned that drugs that target proliferation cells would be effective in vivo in reducing post burn alterations I immunity. Mice were burned or sham burned, and on post burn day 6, injected with 120 mg/kg gemcitabine, a ribonucleotide reductase inhibitor. On post burn day 8, the mice were injected with 5 mg/kg LPS and mortality monitored, or the splenocytes isolated, stained with CFSE, then and stimulated with Concanavalin A to measure the proliferative abilities of T cells. Sham mice, and burn mice treated with gemcitabine were not killed injection of LPS, but burn mice had 100% mortality. Sham mice and sham mice treated with gemcitabine had robust proliferation in their CD4 T cells, while burn mice had significantly reduced proliferation of these cells. Gemcitabine treatment restored proliferation of CD4+T cells to normal level in burn mice. These data suggest that intervening in the process of hematopoiesis may be a fruitful area to ameliorate post burn immune dysfunction.

Figure
Figure

P89 BURN-INDUCED GUT BARRIER INJURY IS ATTENUATED BY PHOSPHODIESTERASE INHIBITION: EFFECTS ON TIGHT JUNCTION STRUCTURAL PROTEINS

T.W. Costantini, W. Loomis, J. Deree, S.H. Choi, J.G. Putnam, B.P. Eliceiri, A. Baird, V. Bansal, and R. Coimbra. Division of Trauma, University of California-San Diego, San Diego, CA 92103

Background:

Loss of intestinal barrier function after burn injury allows movement of intraluminal bacteria across the mucosa, which can lead to the development of distant organ injury and multiple organ failure. Tight junction function is highly regulated by membrane associated proteins including occludin and ZO-1, which can be modulated by systemic inflammation. We hypothesized that (1) burn injury leads to decreased expression of the tight junction proteins occludin and ZO-1, and (2) phosphodiesterase inhibition attenuates these burn induced changes.

Methods:

Male balb/c mice undergoing a 30% total body surface area steam burn were randomized to resuscitation with normal saline (NS) or NS+Pentoxifylline (PTX, 12.5mg/kg). Intestinal injury was assessed by histology and by plasma TNF-α levels using ELISA. Occludin and ZO-1 levels in gut extracts were obtained by immunoblotting.

Results:

Burn injury resulted in a marked decrease in occludin and ZO-1 levels. Compared to animals receiving NS alone, treatment with PTX resulted in an improvement in occludin protein levels of 24% (p<0.05) at 6 hours, which remained significant at 24 hours (25%; p<0.05). In PTX treated animals, ZO-1 protein levels increased 12% at 6 hours. Plasma TNF-α levels in PTX treated animals decreased by 70% (p<0.01) compared to NS alone at 2 hours. Histologic analysis showed improved intestinal villous height and decreased inflammatory cell infiltrate in animals treated with PTX.

Figure
Figure

Conclusion:

Treatment with PTX prevented the burn-induced decrease in intestinal occludin and ZO-1 protein levels. Therefore, phosphodiesterase inhibition may be a useful adjunct strategy in the attenuation of burn-induced gut barrier injury.

P90 AGING AND SARCOPLASMIC RETICULUM CALCIUM ATPASE DYSFUNCTION AFTER BURN COMPLICATED BY SEPSIS

L. Wang, J. Quan, D.L. Maass, D. Kianpour, J.W. Horton, and W. Tao. Univ. Texas Southwestern Medical Ctr, Dallas, TX 75390

Aging is associated with poor outcome in burn victims. Dysfunction in sarcoplasmic reticulum calcium ATPase (SERCA 2a) has been previously shown to contribute to post-burn myocardial depression. To evaluate differences in SERCA 2a function between young and old subjects, we induced 40% total body surface area burn followed by S. pneumoniae sepsis (B+S) in male C56/BL7 mice at 2 and 14 months (MO) of age. Hemodynamic and cardiac function was measured with left ventricular pressure-volume analysis. SERCA 2a protein and mRNA were measured with Western blot and real-time RT-PCR, respectively. Aging per se was not associated with poor cardiac function after sham injury. After B+S injury, however, there was worsened cardiac dysfunction in the 14 MO mice than in the 2 MO mice. Compared with 2 MO mice, there was a significant decrease in SERCA 2a gene expression and protein production in 14 MO mice after sham, and such a decrease was worsened after B+S injury.

Figure
Figure

These data suggest differences in SERCA 2a production associated with aging contributes to the more pronounced cardiac dysfunction after burn and sepsis in older subjects.

P91 AGE-RELATED CARDIAC CYTOKINE EXPRESSIONS IN RESPONSE TO IL-6 DEFICIENCY AFTER BURN COMPLICATED BY SEPSIS

J. Quan, L. Wang, D.L. Maass, J.W. Horton, and W. Tao. Univ. Texas Southwestern Medical Ctr, Dallas, TX 75390

Interleukin-6 is closely related to aging. We aim to compare the effect of IL-6 deficiency on cardiac inflammation and function in wild type (WT) and IL-6 knockout (KO) mice at 2 months (MO) and 14 MO of age after 40% total body surface area burn complicated by S. pneumoniae sepsis (B+S). Global hemodynamic and cardiac contractile function was measured by left ventricular pressure-volume analysis, and cardiac inflammation was measured by quantitative RT-PCR of TNF-α and IL-1β. In 2 MO KO mice, hemodynamic and cardiac function was improved compared to 2 MO WT mice after B+S; in 14 MO KO mice, however, it was significantly worsened compared to 14 MO WT mice. Cytokine gene expressions showed aging itself was associated with an increase in TNF-α and IL-1β expressions, which was further exacerbated by B+S injury. After B+S injury, there was a significant reduction in IL-1β in 2MO KO mice, but not in 14 MO KO mice.

Figure
Figure

These data suggest IL-6 knockout is partially protective of 2 MO mice, but not 14 MO mice after B+S injury. The associated changes in IL-1β is closely related to such age-dependent discrepancy.

P92 EARLY USE OF SELECTIVE DIGESTIVE DECONTAMINATION ATTENUATES MYOCARDIAL DEPRESSION FOLLOWING BURN INJURY

T. O'Keeffe, J. White, D.L. Maass, SM. Lemaire, and J.W. Horton. UT Southwestern, Dallas, TX. 75390

Background:

Selective Digestive Decontamination (SDD) has been shown to reduce early myocardial depression following burn injury in animal models. We hypothesized that use of a new and simplified antibiotic regimen would reduce burn-induced myocardial depression.

Methods:

Adult male Sprague-Dawley rats were divided into fourgroups; burn or sham burn with or without the addition of SDDantibiotics (Neomycin, Metronidazole, Nystatin) given orally in weight-based doses at four and sixteen hours post insult. Hearts were harvested 24hrs post insult and myocardial function studied invitro.

Results:

Hearts from burned rats treated with SDD had significantly higher systolic (+dP/dt) and diastolic (-dP/dt) responses to fluid challenges compared to vehicle treated rats (1844 mmHg/s ± 80 vs. 1115 ± 99 and 1588 ± 97 vs. 917 ± 105). Mean Arterial Pressure (MAP) was higher in burned rats receiving SDD; 154.8 ± 6.4 vs. 111.6 ± 8.3 (p<0.01). Mean hemoglobin and hematocrit werealso significantly higher in the burned rats that received SDD(14.4g/dL ± 0.5 vs. 10.5 ± 0.8 and 44.3% ± 1.6 vs. 31.7 ± 2.5). Intracellular calcium and sodium levels were reduced in the heartsof burned rats that received SDD compared to no SDD (Ca2+157nMol/L ± 11 vs. 293 ± 17, and Na+15mMol/L ± 3 vs. 38 ± 2.4). There were no differences between sham burn, and sham burn+SDD in any of the above comparisons.

Conclusions:

Early use of this simplified SDD regimen can attenuate burn-induced myocardial depression. Reduced intra-cellular concentrations of Sodium and Calcium suggest that this effect may be mediated by cell membrane stabilization. Further research should focus on possible clinical application of these results in human burn patients.

P93 CASPASE-1 MEDIATED CARDIAC DYSFUNCTION AND INFLAMMATORY ALTERATIONS ASSOCIATED WITH BURN TRAUMA

D.L. Carlson, J. Hue, and D.J. White. UT Southwestern Medical Center at Dallas, Dallas, TX 75390-9063

Previously we have shown that broad spectrum caspase activity is directly associated with murine cardiac dysfunction following burn trauma. Caspase-1(Cas1) induces apoptosis, and cleaves the precursor forms of IL-1β and IL-18 into active peptides, suggesting that Cas1 is a key player in the inflammatory response. The purpose of this study was to investigate the involvement of Cas1 in cardiac dysfunction and the inflammatory response following burn trauma. NOD.129S2(B6)-Casp1tm1Sesh/LtJ (Cas1−/−) and NOD/ShiLtJ (Cas1+/+) mice were divided into sham and burn (40%TBSA) groups. Following burn, Langendorff (18hrs) and echocardiograms (4, 8, 12, 24, 48hrs) were used to asses systolic function. Both cardiac tissue and circulating cytokines were measured (IL-6, IL-10, TNF-α, IL-1β, and MIF). TUNEL analysis of cardiac sections was performed. Cas−/−animals demonstrated no expression of cardiac IL-6, and IL-1β expression was decreased 22-fold from control at the highest level of expression (p<0.05). Cardiac TNF-α was also significantly depressed in Cas−/−animals (437±21 vs.1497±312, p<0.02). Cas1 deficiency significantly preserved cardiac function at 18hrs (Langendorff) post thermal injury (LVP [mmHg]: 98.4±5.6 Cas−/−sham, 94.6±1.3 Cas−/−burn, 95.6±3.4 Cas+/+sham, 80.1±2.7 Cas+/+burn). These results were confirmed by fractional shortening data. No differences in TUNEL were detected between Cas−/−and Cas+/+groups. Taken together, these results suggest that Cas1 plays a central role in theinflammatory response following thermal injury, and affects cardiacfunction either directly, or as a result of the alterations in inflammatory pathways.

P94 SERCA2 OVEREXPRESSION RESCUES CARDIAC CALCIUM AND SODIUM HANDLING AND REDUCES MYOCARDIAL INFLAMMATION AFTER BURN INJURY

D.L. Maass*, R.C. Barber, and J.W. Horton. UT Southwestern Medical Center, Dallas, TX 75390-9160

Objective:

Burn injury is known to produce myocardial dysfunction and inflammation. We have shown previously that burn injury alters calcium handling in the heart by reducing expression of SERCA2, a key calcium handling protein in cardiac tissue. Herein, we further investigated the mechanism underlying altered calcium/sodium handling and inflammation in cardiomyocytes isolated from SERCA2 overexpressing mice.

Methods:

Transgenic mice designed to overexpress SERCA2 (ona FVB/N background) and FVB/N controls were given a 40%TBSA cutaneous burn. Cardiac myocytes were isolated and cultured. Intracellular levels of cardiomyocyte calcium and sodium were measured along with cytokine concentrations in cell-culture supernatants.

Results:

SERCA2 overexpression significantly reduced burn-induced alterations in cardiomyocyte calcium and sodium as well as supernatant cytokine concentrations, relative to FVB/N controls.

Conclusion:

Overexpression of SERCA2 moderated calcium and sodium handling in cardiac myocytes and reduced myocardial inflammation following burn injury.

Study support:

NIH/NIGMS R01-GM057054-08 [JWH].

Figure
Figure

Intracellular calcium and sodium concentrations in FVB/N or SERCA2 overexpressor cardiomyocytes. Values are mean +/− SEM.

P95 ENVIRONMENTAL ACIDIFICATION OF MACROPHAGES IN CULTURE REDUCES THE INFLAMMATORY RESPONSE AND ENHANCES PHAGOCYTOSIS OF OPSONIZED PARTICLES

Antonio De Maio, Julia Grabowski, and Mark Talamini. UCSD, Department of Surgery, La Jolla, CA 92093

Previous studies have demonstrated that the advantages of laparoscopic surgery are not simply the product of reduced tissue trauma, but rather a direct biological effect of CO2 insufflation, which produces a small reduction in peritoneum pH. We hypothesize that extracellular acidification modulates the inflammatory response. To test this hypothesis, a murine macrophage line (J744) was pre-incubated in culture medium at a pH of 6.0 or 7.4 for 3 hours. Cells were returned to neutral pH and stimulated with LPS. Macrophages that were pre-incubated at a pH of 6.0 displayed a decrease in the levels of LPS-induced TNF-alpha (53%) or IL-6 (40%) as compared to cells pre-incubated at a pH of 7.4 (p<0.05, Student's t-test). This reduction in cytokine production could be correlated with a decrease in the translocation of NF-kB into the nucleus after LPS stimulation. In contrast, an increase in phagocytosis of opsonized particles (2 fold) was observed in macrophages that were pretreated at pH 6 as opposed to cells incubated at pH 7.4 (p<0.05, Student's t-test). These results suggest that, indeed, macrophage function is modulated by a short and transient exposure to an acidic environment, such as that induced during CO2 insufflation. These data shed light upon the molecular mechanism involved in the beneficial effect observed in patients undergoing laparoscopy surgery.

P96 RELATIONSHIP BETWEEN HIGH MOBILITY GROUP BOX-1 RELEASE AND IMMUNOSUPPRESSION FUNCTION OF REGULATORY T CELLS IN RATS AFTER THERMAL INJURY

Yong-Ming Yao, Li-Feng Huang, and Zhi-Yong Sheng. Burns Institute, First Hospital Affiliated to the Chinese PLA General Hospital, Beijing 100037, P.R.China

The present study was to investigate in vivo the effect of high mobility group box 1 protein (HMGB1) on immunosuppression function of regulatory T cells (Tregs) and the influence on T cell-mediated immunity after thermal injury. Male Wistar rats were randomly divided into four groups as follows: sham burn group, burn group, burn with ethyl pyruvate (EP) treatment group, and burn with anti-receptor for advanced glycation end products (RAGE) antibody treatment group, and they were sacrificed on postburn days (PBD) 1, 3, 5 and 7 respectively with 8 animals at each time point. Magnetic cell sorting microbeads were used to isolate splenic Tregs and a column of nylon wool to obtain T cells. Phenotypes were analyzed by flow cytometry and cytokines were determined by ELISA kits. Serum HMGB1 levels were significantly elevated during PBD 1-7. In the burn group, CD152 (cytotoxic T-lymphocyte-associated antigen 4, CTLA-4) and forkhead/winged helix transcription factor p3 (Foxp3) expression levels of Tregs were strongly enhanced in comparison to the sham-injured group, and the capacity of Tregs to product IL-10 was markedly increased. Administration of EP to inhibit HMGB1 could significantly decrease expression levels of CTLA-4, Foxp3 on Tregs and interleukin IL-10 production after burns. Simultaneously, proliferative activity and expression levels of IL-2 as well as IL-2Rα of Tcell were restored. The excessively released HMGB1 might stimulate CD4+CD25+Treg activity via binding RAGE on the surface of Tregs, and trigger shift of Th1 to Th2 with suppression of Tlymphocyte immune function following burn injury.

P97 INDUCTION OF HEMEOXYGENASE-1 INHIBITS NEUTROPHIL O2 PRODUCTION BY DOWN-REGULATING p47phox AND p67phox ACTIVATION AND PREVENTS INTESTINE TISSUE DAMAGE IN A TWO-HIT MODEL OF ALCOHOL INTOXICATION AND BURN INJURY

X Li, MG Schwacha, IH Chaudry, and MA Choudhry. Univ. of Alabama at Birmingham, AL.

The mechanism by which hemeoxygenase-1 (HO-1) protects against oxidant-mediated tissue injury remains unknown. Recently, we have shown a role for neutrophils in intestinal damage following alcohol (EtOH) intoxication and burn injury. Since neutrophil-mediated tissue injury involves the release of superoxide anions (O2), we examined whether HO-1 modulates neutrophil O2 production following EtOH and burn injury. We also investigated whether HO-1 antioxidant properties are mediated via p47phox and/or p67phox proteins. Male rats (~250g) were gavaged with EtOH to achieve a blood EtOH level of~100 mg/dL prior to ~12.5% TBSA burn or sham injury. Some rats were treated with HO-1 activator Cobalt protoporphyrin IX chloride (Copp, 25 mg/Kg BW) at the time of injury. One day after injury, EtOH combined with burn injury significantly increased neutrophil O2 production and p47phox/p67phox activation. In contrast, it decreased caspase-3 activity, apoptosis and HO-1 levels. Copp treatment normalized neutrophil HO-1, O2 (Fig), p47phox and p67phox following EtOH and burn injury. Caspase-3 expression; however, was further decreased in Copp-treated rats. Moreover, administration of Copp prevented the increase in intestinal edema and permeability following EtOH and burn injury. These findings provide new insight into the mechanism by which HO-1 regulates neutrophil O2 production and protects intestine from damage following EtOH and burn injury. (R01AA015731& R21AA015979)

Figure
Figure

P98 BURN INJURY ACTIVATES INTRINSIC APOPTOTIC SIGNALING AND INDUCES VASCULAR HYPERPERMEABILITY

Benjamin Smith, J. Greg Whaley, Binu Tharakan, Felicia A. Hunter, and Ed. W. Childs. Department of Surgery, Texas A and M University Health Science Center-College of Medicine and Scott and White Memorial Hospital, Temple, Texas 76508, USA

Objective:

Vascular permeability is increased following burn injury. Activation of the intrinsic apoptotic pathway has been shown to promote vascular hyperpermeability. We hypothesized that the intrinsic apoptotic pathway is activated following burn injury and contributes to vascular hyperpermeability.

Methods:

Sprague-Dawley rats were subjected to a 40% TBSA full-thickness burn of the dorsal skin. Sections of normal, sham, and burned skin were examined with routine H and E staining as well as Masson's trichrome stain to confirm depth and completeness of burn. All rats were given 50 mg/kg FITC-albumin intravenously, and mesenteric post-capillary venules were examined with intravital microscopy. The flux of albumin from the intravascular space to the interstitial space was determined by the change in fluorescent intensity over time. Mesenteric tissue from a second set of experimental and sham rats was harvested at three hours post-burn; cytosolic cytochrome c levels were determined by ELISA, and caspase-3 activity was determined by a fluorometric assay.

Results:

Vascular permeability was significantly increased in the experimental group at 140, 160, and 180 minutes post-burn (p<0.05). Release of cytochrome c from the mitochondria into the cytosol was increased in the burned rats (p<0.05), and activation of caspase-3 was demonstrated in this group (p<0.05).

Conclusions:

Burn Injury induces vascular hyperpermeability and increases mitochondrial release of cytochrome c and of caspase-3 activation. The activation of the intrinsic apoptotic signaling cascade may be one of the factors responsible for the disruption of endothelial cell-cell junctions and resulting vascular hyperpermeability.

P99 CD4+FOXP3+TREGS ARE ACTIVATED BY INJURY

Goro Tajima, Malcolm P. MacConmara, Fionnuala O'Leary, Adam J. Delisle, John A. Mannick, Vijay K. Kuchroo, and James A. Lederer

Enhanced CD4+CD25+regulatory T cell (Treg) potency occurs following severe injury suggesting that Tregs may play a natural role in controlling the host response to injury. This study addresses whether injury can activate Tregs. To accomplish this, we used a FoxP3-GFP knock-in mouse as a tool to accurately identify and measure phenotypic changes in FoxP3-expressing Tregs in sham versus injured mice. Groups of FoxP3-GFP mice were anesthetized and underwent sham or burn injury. At days 1, 3, and 7 after injury, mice were sacrificed and cells were prepared from the injury site, lymph nodes draining the injury site, and spleen. Cell suspension were stained to detect CD4 T cells and counterstained to detect changes in CD25, CD69, CD62L, ICOS, PD-1, and CTLA-4 expression levels. We found that injury caused a significant increase in the percentage of FoxP3+Tregs in the lymph nodes draining the injury site at all time points after injury. In contrast, significant increases in Tregs were not detected until 7 days after injury at the site of injury and in the spleen. The results of experiments testing for phenotypic changes in Tregs after injury showed that injury induced increased expression of CD25, CD62L-low, ICOS, PD-1, and CTLA-4 on lymph node and spleen FoxP3+Tregs as early as 1 day after injury. At 7 days after injury, FoxP3+CD4 T cells showed further signs of activation and expressed significantly higher levels of ICOS and CTLA4. We confirmed that injury activates Tregs by performing an adoptive transfer of FoxP3-GFP+cells into CD4-/-mice and showing that Tregs from burn mice expressed an activated phenotype. Thus, Tregs are activated by injury and injury can increase the percentage of Tregs at the site of injury, the draining lymph nodes and spleen. Therefore, the enhanced Treg function that occurs after injury might be due to the injury-induced activation of FoxP3+Tregs.

P100 LIGHT EMITTING DIODES AND COX-2 INHIBITOR AS NOVEL NON-INVASIVE THERAPEUTIC APPROACH FOR THE MANAGEMENT OF BURN WOUNDS

Manoj Kumar Puthia1, Mui Hong Tan1, Steven Thng2, Ng Wee Tong2, Pierce Chow3, Shabbir M. Moochhala1, and Lu Jia1. 1Defence Medical and Environmental Research Institute, DSO National Laboratories, 27 Medical Drive, DSO (Kent Ridge) Singapore 117510, 2HQMC (Headquarters Medical Corps), Singapore, 3Department of Experimental Surgery, Singapore General Hospital

Burn injuries have always been a significant problem and their burden on medical services is overwhelming especially during mass casualties. Burns are a leading cause of accidental deaths resulting in considerable morbidity and disfigurement causing significant functional and social impairment. Despite major advances, burn treatment remains a highly specialized area that requires modern facilities and there is a need to develop novel therapeutic strategies. In this study, our aim was to evaluate the efficacy of Light-Emitting Diodes (LEDs) and topical COX-2 inhibitor in the management of burn wounds. We used a pig model of partial thickness burn injury and treated with near infrared LED exposure and COX-2 inhibitor topical application. Efficacy of various treatment modalities were studied by a) wound contraction, b) Laser Doppler imaging, c) histology, and d) immunohistochemistry for Ki-67, proliferating cell nuclear antigen. Our findings showed that LED and cox-2 inhibitor combined treatment is effective with best wound healing profile among all treatment groups. Even when used alone, LED treatment of burn wounds showed significant healing. Overall, results suggest that treatment of burn wounds with LED alone or in combination with celecoxib has potential to be used as a novel non-invasive therapeutic intervention.

P101 EFFECT OF SPHINGOSINE 1-PHOSPHATE ON HYPER-PERMEABILITY RESPONSE IN SCALDING INJURY

LIU Xianglan, WANG Zhanhua, LI Qiang, HUANG Xuliang, and HUANG Qiaobing. Department of Pathophysiology, Key Lab of Shock and Microcirculation Research of Guangdong Province, Southern Medical University, Guangzhou 510515, P.R. China

Objective:

To study the effect of sphingosine 1-phosphate (S1P) on morphological alteration and hyper-permeability response in endothelial cells (ECs) and intact vessels after burn injury.

Methods:

The distribution of VE-cadherin and F-actin were double stained in primary cultured HUVEC with immunofluorescence and fluorescent probes respectively. Permeability changes were measured by a fluorescence-ratio-technique in isolated skin micro-vessels as well as in mesentery venules of mice. Burn injury was induced by burn serum in cultured and isolated vessel or a third-degree scald burn in mice on the dorsal skin covering about 30% total body surface area.

Results:

The intervention of cultured ECs with burn serum could cause a noticeable alteration of VE-cadherin spreading at cellular border, displaying a disruption and diffusion on the location of per se smooth lining without burn serum stimulation, accompanying with the formation of F-actin stress fibers. Appropriate amount of S1P prevented these burn-induced morphological alterations as well as the hyper-permeability response in cultured endothelial monolayer and isolated and intact vessels. The inhibition of S1P synthesis with N-Dimethylsphingosine (DMS) significantly increased the mesentery venular permeability, while the exogenous S1P did not altered the basal vascular barrier function.

Conclusion:

S1P might be essential in maintaining the basal vascular barrier function and could be protective in burn insult by preventing the increase of venular permeability. (This work is sponsored by the National Key Foundation for Basic Science Research of China (grant G2005CB522601)).

P102 A NEW THERAPEUTIC WAY FOR BURN SHOCK: EFFICACY OF LACTOSYL DERIVATIVES AND UNDERLYING MECHANISMS

Zhihui Zhao*, Qing Li§*, Zhongjun Li§, Jinghua Liu, Qiaobing Huang, Lin Zhang, Peng Deng, Xiaowei Gong, Jian Chu, Kesen Zhao, Shuangquan Zhang, and Yong Jiang. Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, College of Life Science, Nanjing Normal University, Nanjing 210097, China; Key Laboratory for Functional Proteomics of Guangdong Province, Department of Pathophysiology, Southern Medical University, Guangzhou 510515, China; §State Key Laboratory of Natural and Biomimetic Drugs, Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing 100083, China

Rationale:

Severe burn shock remains an unsolved clinical problem with an urgent need to explore novel therapeutic approaches.

Objectives:

To evaluate the therapeutic effect of a series of synthetic lactosyl derivatives on rats with burn shock and elucidate the underlying mechanisms.

Methods:

The bioactivities of synthetic lactosyl derivatives were determined on a burn shock model of rats. Histology was used to check the morphological damage to vital organs. A flow chamber assay was used to assess the adhesion of leukocytes. The binding and localization of the oligosaccharides on cells were observed by fluorescent or confocal microscopy. Flow cytometry was employed on the binding assay of the oligosaccharides.

Main Results:

An-2 and Gu-4, two lactosyl derivatives with bi-and tetravalent β-D-galactopyranosyl-(1,4)-β-D-glucopyranosyl ligands, significantly prolonged the survival time (P < 0.05 vs. saline), stabilized blood pressure and ameliorated the injuries to vital organs after burn in a dose-dependent manner. An-2 and Gu-4 markedly decreased the adhesion of leukocytes to primary rat lung microvessel endothelial cells. A CD11b antibody significantly interrupted the interaction of them with leukocytes from rats with burn shock in a dose-dependent manner. The oligosaccharides selectively bound to leukocytes and with a colocalization of CD11b on the cell membrane.

Conclusions:

Both An-2 and Gu-4 significantly inhibit the adhesion of leukocytes to endothelial cells by binding to CD11b on the membrane of leukocytes and thereby exert protective effects on severe burn shock; They are promising candidates for the treatment of burn shock and may be applied also to other CD11b-dependent diseases.

Keywords:

leukocyte-endothelial cell interaction; lactosyl derivatives; integrin; burn shock

Burn Injury Clinical Research

P103 MITOCHONDRIAL VARIANT (4216C) ASSOCIATED WITH INCREASED SEVERE SEPSIS RISK AFTER BURN INJURY

R.C. Barber*, D. McGee, L.Y. Chang, J.E. Bender, T.S. O'Keeffe, A. Burris, S.M. Lemaire, G.F. Purdue, J.L. Hunt, B.D. Arnoldo, and J.W. Horton, UT Southwestern, Dallas, TX 75390-9160

Objective:

Studies have linked impaired mitochondrial activity with increased risk for clinical complications after injury. A polymorphism (T4216C) in the NADH DH1 gene is known to cause decreased cellular respiration.

Methods:

We evaluated the association between T4216C and outcome after burn injury in a cohort of 310 patients from Parkland Hospital, Dallas, TX with burns ≥15% TBSA without significant non-burn related trauma (ISS ≤16), traumatic or anoxic brain injury or spinal cord injury, who survived >48 hours under an IRB-approved protocol. Clinical data were collected prospectively and T4216C was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results: After adjustment for full-thickness burn size, presence of inhalation injury, age, gender and race/ethnicity, carriage of the 4216 C-allele was associated (P = 0.020)with an odds ratio of 2.99 (95% CI; 1.19-7.57) for complicated sepsis (Figure). Full-thickness burn size was the only other risk factor investigated that was associated with risk for complicated sepsis. Conclusions: Carriage of the C-allele at nucleotide 4216 of the mitochondrial genome appears to decrease mitochondrial function and increase risk for infectious complications after burn injury. This study was supported by NIH/NIGMS R01-GM057054-08 [JWH].

Figure
Figure

P104 PROPRANOLOL TREATMENT IMPROVES MORBIDITY AND MORTALITY IN SEVERELY BURNED PATIENTS

Marc Jeschke, Felicia Williams, Celeste Finnerty, Gaby Kulp, Ludwik Branski, Gerd Gauglitz, and David Herndon. Shriners Hospital for Children, Galveston, TX 77550

Background:

The aim of the present study was to determine the effect of propranolol administration on clinical outcomes in severely burned pediatric patients during acute hospitalization in a large randomized double-blinded single center study.

Patients and Methods:

Burned children (n = 344) with >40% total body surface area burn (TBSA) were randomized to receive standard burn care (control, n = 242) or standard burn care plus propranolol (n = 102). Clinical parameters, mortality, infection, sepsis, insulin resistance, serum hormones, and cytokines were measured throughout acute hospitalization. Statistical analysis was performed by Student's t-test, or ANOVA.

Results:

Demographics, burn size, and clinical data were similar in both groups. Propranolol restored the acute phase and hormonal response by decreasing α2-macroglobulin and free fatty acids, while it increased IGF-I, IGFBP-3, and GH, P < 0.05. Propranolol significantly decreased MCP-1, GM-CSF, and increased IL-10 which is associated with a decreased number of infections (control: 2.5 ± 0.2 vs. propranolol: 1.8 ± 0.2), sepsis (control: 17% vs. propranolol: 6%) and pneumonia (control: 11% vs. propranolol: 4%), P < 0.05. 49% of control patients required insulin administration but only 23% of propranolol treated patients required insulin administration, P < 0.01. The amount of insulin in the control group was on average 5550 ± 60 IU per patient while it was 400 ± 40 IU in the propranolol group, P < 0.01. While 19 patients died in the control group (8%) only two patients died in the propranolol group (2%), P < 0.05.

Conclusion:

In this large prospective, double-blinded, randomized single center study propranolol markedly improved inflammation, metabolism, incidence of infection and sepsis, morbidity and mortality during acute hospitalization.

P105 A NEW HORMONAL REGULATOR FOR POSTBURN INSULIN RESISTANCE: OSTEOCALCIN

Marc Jeschke, Gerd Gauglitz, Gabriela Kulp, and David Herndon. Shriners Hospitals for Children, Galveston, TX 77550

Introduction:

Using a genetic manipulated mouse model it was recently shown that osteocalcin increases β-cell proliferation, insulin secretion and insulin sensitivity. Our group has demonstrated that severely burned patients are insulin resistant for over 2 years postburn. The aim of this study was to determine osteocalcin levels postburn and correlate these levels to markers of insulin resistance in a large pediatric population post burn up to three years.

Methods:

Severely burned pediatric patients with burns>40% of their total body surface area (n = 138) were included in the present study. A standard 75 g oral glucose tolerance test was performed during patient hospital stay and at 3, 6, 9, 12, 24, and 36 months post burn. Glucose uptake and insulin secretion were assessed by measuring serum glucose, insulin and C-peptide following the glucose load using standard methodologies. Serum osteocalcin levels were determined at the same time. Statistical analysis was performed by ANOVA with Bonferroni's correction.

Results:

During acute hospitalization and up to 3 years patients demonstrated a pathologic OGTT, implying persistent insulin resistance. Serum glucose, insulin and C-peptide levels remained elevated over the three year study period when compared to controls. Serum osteocalcin levels drastically decreased immediately postburn and remained decreased over the entire 3 year study period, P < 0.05. Osteocalcin levels show a strong correlation with pathologic OGTT levels.

Conclusion:

Pathologic response to insulin is a hallmark of the acute phase in patients post burn. In this study, we have demonstrated that alterations in insulin sensitivity persist up to 3 years after the burn injury, although the patients do not display clinical diabetes.

P106 BURNS AS THE MODEL OF SIRS AND MOD/F

T. Spiridonova, and S. Smirnov. Burn Center of the Sklifosovsky Institute for Emergency Medicine, 3 B. Sukharevskaya Sq. Moscow 129010, Russia

The term "burn disease" is used in Russia to define a complex of pathophysiologic manifestations developing over time in a patient with burn. The purpose of our study was to investigate the pathogenesis of burn disease and multiple organ dysfunction / failure (MOD/F) syndrome to improve the diagnosis and treatment. Two hundred patients were studied for SIRS-typical symptoms and laboratory parameters. SIRS was revealed in all patients with Frank Index more than 30 units. SIRS duration and scored severity correlated to injury severity in uncomplicated course. On the basis of SOFA Score, we have developed a Score for MOD/F clinical analysis that incorporates the SIRS-adapted scoring of dysfunction symptoms in respiratory, cardiovascular and coagulation systems. A stomach tissue biopsy performed in 109 burn patients in the period of 1-21 days post-burn revealed acute erosive-ulcerous lesions in 90.4% of patients, gastroduodenal haemorrage in 2/3 of them. The analysis of these results urged to supplement our Score with symptoms of gastroduodenal tract erosive-ulcerous lesions. We have found that MOD/F in burn patients develops either as a result of SIRS without infectious complications, or as a result of infectious complications and sepsis developed secondary to SIRS. The study results suggest that burn disease may serve the model of SIRS and MOD/F. SIRS makes a pathogenetic ground of burn disease and MOD/F. Obtained results helped to improve MOD/F diagnosis, organ-protective therapy, immunotherapy, and surgical tactics.

P107 DECREASED DIETARY FAT IS CRITICAL IN THE MANAGEMENT OF PEDIATRIC PATIENTS POST BURN

Gerd G. Gauglitz, MD, Jong O. Lee, MD, David N. Herndon, MD, Hal K. Hawkins, MD, PhD, Stefanie C. Halder, MS, Arthur P. Sanford, MD, and Marc G. Jeschke, MD, PhD

Background:

Most nutritional supplements used for critically ill patients throughout the world include a high proportion of fat. However, high fat administration has been linked to increased hepatic steatosis, infection, and mortality. During a small prospective study, we found that a low fat diet was associated with decreased morbidity in severely burned children. The purpose of this study was therefore to asses in a large cohort of severely burned children the effects of a low fat/high carbohydrate diet (Vivonex® T.E.N.) on clinical outcomes when compared to a high fat diet (milk).

Materials and Methods:

Nine hundred forty-four children with burns over more than 40% of their total body surface area (TBSA) were divided into two groups: patients receiving Vivonex®T.E.N. (low fat, 1996 to 2006; n = 518) were compared to those receiving milk (high fat, 1985 to 1995; n = 426). We determined patient demographics, caloric intake, length of hospital stay, incidence of sepsis, incidence of fatty metamorphosis of the liver and organomegaly at autopsy and mortality. Significance was accepted at P < 0.05.

Results:

Demographics and caloric intake were similar in both groups. Patients receiving Vivonex®T.E.N. had shorter ICU stays (Vivonex®T.E.N.: 31 ± 2 days; milk: 47 ± 2 days; P < 0.01), shorter LOS per % TBSA burn (Vivonex®T.E.N.: 0.5 ± 0.02 days/%; milk: 0.7 ± 0.03 days/%; P < 0.01) and lower incidence of sepsis (Vivonex®T.E.N.: 11%; milk: 20%; P < 0.01). Patients receiving Vivonex®T.E.N. lived significantly longer until death than those receiving milk (Vivonex®T.E.N.: 20 ± 3 days; milk: 10 ± 2 days; P < 0.01). Autopsies revealed decreased hepatic steatosis and decreased enlargement of the kidney and spleen.

Conclusion:

We found that a low fat/high carbohydrate diet is associated with shorter LOS, prolonged survival, and decreased incidence of organomegaly, infection and fatty metamorphosis of the liver in burned pediatric patients compared to a high fat diet. Based on these findings, we recommend a low fat/high carbohydrate diet in severely burned patients.

P108 BIOLOGICAL ALLOFIBROBLAST-BASED DRESSING DECREASES THE DURATION OF SUPERFICIAL BURN HEALING

E. Zhirkova, S. Smirnov, V. Khvatov, M. Sychevsky, and K. Smirnov. Burn Center of the Sklifosovsky Institute for Emergency Medicine, 3 B. Sukharevskaya Sq. Moscow 129010, Russia

Two thirds of patients admitted to a burn center have superficial burns. Application of traditional treatment methods ensures epithelialization of such burn wounds average on the 14th-21st day post-burn. We applied biological dressings based on cultivated live allofibroblasts (AFs) to treat burns in these patients. The purpose of the study was to define the optimal timing for the AF-based dressing application. Materials and methods. The study included patients having burns of 20-70% TBSA who were divided into 2 comparable groups. Cultivated live AFs on the silicon -organic substrate were applied on the 1st-2nd day post-burn in 21 patients (group I), and on the 3rd-7th day post-burn in 19 patients (group II). Observation showed that epithelialization of the whole burn wound surface in 20 patients of group I occurred at 5-7 day of the biological dressing application. In patients of group II, the application of these dressings at that time of eschar formation was less efficient. That may be explained by necrosis formation on the burn wound surface that loosened the contact of the dressing with the wound. Thus, in the treatment of superficial burns, the application of AF-based dressings on the 1st-2nd day of burn injury reduces the time needed for burn wound epithelialization.

Organ Dysfunction Basic Research

P110A HYPERTONIC SALINE INCREASES ACUTE LUNG INJURY BY UP-REGULATING NEUTROPHIL A3 ADENOSINE RECEPTOR EXPRESSION

Yoshiaki Inoue, Yu Chen, Reinhard Pauzenberger, Mark Hirsh, and Wolfgang G. Junger. Department of Surgery, Trauma and Surgical Critical Care, BIDMC, Harvard Medical School, Boston, MA 02215

Hypertonic saline (HS) modulates neutrophil (PMN) functions via ATP release and formation of adenosine. Negative feedback through A2a adenosine receptors inhibits PMN, while positive feedback via A3 adenosine receptors enhances PMN functions. Here we tested the role of A3 receptors in mediating lung tissue damage in a mouse sepsis model.

Sepsis was induced by cecal ligation and puncture (CLP) in wild-type (WT) and A3 receptor knockout (A3KO) mice before or after treatment with HS (7.5% NaCl, 4 ml/kg). Neutrophil elastase accumulation in lung tissues was measured as marker of inflammation. The tissue damage was assessed by histology.

HS treatment of PMN before cell activation significantly suppressed A3 receptor expression (P < 0.05), while delayed HS treatment enhanced A3 receptor expression. When administered before CLP, HS significantly reduced PMN accumulation and lung tissue damage. HS administered after CLP significantly increased PMN accumulation and tissue damage in WT but not in A3KO mice (n = 6, P < 0.05). Correspondingly, mortality after CLP and delayed HS treatment was significantly greater in WT (88%) than in A3KO mice (50%) and was similar to mortality in mice treated with HS before CLP (50%).

We conclude that HS treatment controls A3 receptor expression of PMN and that A3 receptors mediate aggravated tissue damage in response to delayed HS administration. (Funding: Novo Nordisk, NIH, and DoD).

P110B NEUTROPHIL ACCUMULATION IN THE LUNGS REQUIRES P2Y2 AND A3 ADENOSINE RECEPTORS

Yu Chen, Yoshiaki Inoue, Reinhard Pauzenberger, and Wolfgang G. Junger. Department of Surgery, Trauma and Surgical Critical Care, BIDMC, Harvard Medical School, Boston, MA 02215

Excessive neutrophil (PMN) infiltration into the lungs causes acute lung injury and organ damage that are responsible for high mortality among trauma patients. Our previous work has shown that PMN chemotaxis requires ATP release, adenosine formation, and feedback through A3 adenosine and P2Y2 receptors. Here we studied those mechanisms in a mouse model of acute lung injury.

We found that inhibitors of A3 (MRS-1191) and of P2Y2 receptors (suramin) suppressed chemotaxis of human PMN in vitro. Using a trans-well assay system, we studied chemotaxis of mouse PMN isolated from the bone marrow of wild-type (WT) mice or knockout mice lacking P2Y2 receptors (P2Y2 KO) or A3 receptors (A3 KO). Chemotaxis of PMN from P2Y2 KO and A3 KO mice was significantly lower than that of PMN from WT mice. To study the physiological relevance of these receptors in vivo, we assessed PMN accumulation in a model of acute lung injury (ALI) using WT and KO mice. ALI was induced using a cecal ligation and puncture (CLP) sepsis model. Elastase activities in lung tissue samples were used as markers of PMN accumulation. Lung tissue damage was assessed by histological examination. PMN infiltration into the lungs was significantly greater in WT mice than in A3 KO or P2Y2 KO mice (n = 6, P < 0.05). Lung tissue damage was more sever and mortality was significantly higher in the WT group compared to A3 KO or P2Y2 KO mice.

We conclude that A3 and P2Y2 receptors are involved in PMN induced ALI and inhibition of these two receptors may be useful to prevent ALI (Funding: Novo Nordisk, NIH, and DoD).

P111 CARDIAC SPECIFIC EXPRESSION OF CONSTITUTIVELY ACTIVE PI3K ISOFORM P110α ATTENUATES CARDIAC DYSFUNCTION AND INCREASES SURVIVAL OUTCOME IN CLP SEPSIS

David Williams, Fang Hua, Race Kao, Tuanzhu Ha, Tammy Ozment-Skelton, John Kalbfleisch, William Browder, and Chuanfu Li. Dept. of Surgery, East Tennessee State University, Johnson City, TN 37614

We have reported that activation of the PI3K/Akt signaling pathway positively correlates with improved outcome in a murine model of CLP induced septic shock. We hypothesized that activation of the PI3K/Akt signaling pathway prevents or blunts cardiac dysfunction during sepsis/septic shock. To evaluate this hypothesis, we examined cardiac function and survival in transgenic mice with cardiac myocyte specific expression of a constitutively active PI3K isoform p110α (caPI3K Tg). caPI3K Tg mice and age-matched wild type (WT) control mice were subjected to CLP. Sham operated mice served as the control. Mice that were not subjected to surgery or anesthesia served as the normal controls. Six hrs after CLP, control mice showed significant (P < 0.05) depression of cardiac function, i.e. stroke work (↓73.7%), cardiac output (↓78.5%), dp/dt max (↓47.6%), and ejection fraction (↓45.7%). However, cardiac function was maintained at control levels in caPI3K Tg mice subjected to CLP. Myocardial levels of phosphorylated Akt were significantly higher in caPI3K mice versus WT mice. The median survival time in WT CLP mice was ~28 hrs and time to 100% mortality was 46 hrs. In contrast, 90% of the caPI3K CLP mice were alive at ~28 hrs and 80% at 46 hrs after CLP (P < 0.01). Most importantly, 50% of the caPI3K CLP mice went on to survive indefinitely (P < 0.01), i.e.>30 days. This is the first demonstration of a causal relationship between PI3K/p110α, the cardiac myocyte, cardiac function and survival outcome insepsis. We conclude that activation ofa single isoform of PI3K, i.e. p110α, in a single cell type (the cardiac myocyte), in a single organ (heart), has a profound effect on the morbidity and mortality associated with fulminating sepsis/septic shock.

P112 DIFFERENTIAL EFFECT OF TLR2 AND TLR4 ON CARDIAC FUNCTION DURING SEPSIS/SEPTIC SHOCK

Chuanfu Li, Fang Hua, Race Kao, Tuanzhu Ha, John Kalbfleisch, Tammy Ozment-Skelton, William Browder, and David Williams. Dept. of Surgery, East Tennessee State University, Johnson City, TN 37614

Recent studies suggest that TLR2 and TLR4 may play important roles in sepsis/septic shock, however, the contribution of TLR2/4 to cardiac dysfunction in polymicrobial sepsis/septic shock has not been thoroughly investigated. We examined the role of TLR2 and 4 on cardiac function 6 hrs after CLP sepsis. TLR2 deficient mice showed decreased cardiac function and increased mortality in response to CLP sepsis. When wild type (WT) mice were treated with the TLR2 ligand, peptidoglycan (PDG), there was an attenuation of sepsis induced cardiac dysfunction. This positively correlated with improved survival outcome (50% vs 0%). These data suggest that TLR2 plays a protective role in sepsis. In contrast, CLP-induced cardiac dysfunction was attenuated in TLR4−/−mice. When compared to the control group, cardiac output in CLP TLR4−/−mice was maintained at control levels (Fig. 1), while ejection fraction (↑21.2%), dp/dt max (↑42%) and stroke work (↑61%) were increased, respectively. CLP sepsis increased cardiac myocyte apoptosis and caspase-3 activity in control mice, but not in TLR4−/−CLP mice. These data suggest that TLR4 has a role in mediating sepsis induced cardiac pathology. When taken together, our data indicate that TLR2 and TLR4 have opposing, i.e. differential, effects, on cardiac function during acute sepsis/septic shock. These data also suggest that differential modulation of TLR2/4 may be useful in preventing cardiac dysfunction during sepsis/septic shock.

Fig. 1
Fig. 1:
Differential effect of TLR2 and 4 on

P113 TLR2 MODULATION OF SEPSIS INDUCED CARDIAC DYSFUNCTION IS MEDIATED VIA A β-ARRESTIN 2 DEPENDENT MECHANISM

David Williams, Fang Hua, Race Kao, Tuanhzu Ha, John Kalbfleisch, Tammy Ozment-Skelton, Peter Rice, William Browder, and Chuanfu Li. Dept. of Surgery, East Tennessee State University, Johnson City, TN 37614

Modulation of TLR2 dependent signaling can prevent cardiac dysfunction and improve survival outcome in CLP-induced sepsis/septic shock, however, the mechanisms are unknown. β-arrestins are important in recruitment, activation, and scaffolding of cytoplasmic signaling complexes for receptor-mediated cellular signaling pathways. Thus, β-arrestins serve as multifunctional adapters that recruit a broad spectrum of signaling molecules. We hypothesized that β-arrestins may play a role in TLR2 induced cardioprotection in sepsis/septic shock. We administered the TLR2 ligand, peptidoglycan (PDG), at 50 μg/25g body weight to β-arrestin 2−/−and age-matched WT control mice. One hr after PDG administration, the mice were subjected to CLP. Cardiac function was examined 6 hrs post-CLP. TLR2 modulation with PDG attenuated CLP induced cardiac dysfunction. However, sepsis induced cardiac dysfunction was not ameliorated in β-arrestin 2−/−mice treated with PDG. There was no difference in cardiac output, dp/dt max (mm Hg/sec) or ejection fraction between CLP control and β-arrestin 2−/−CLP mice treated with PDG. To investigate how TLR2 and β-arrestin 2 mediate cardioprotection during sepsis/septic shock, we examined whether TLR2 and β-arrestin 2 associated in the myocardium following PDG administration. We found that TLR2 co-localized with (β-arrestin 2 in the myocardium following PDG priming. The data suggest that (β-arrestin 2 plays an important role in mediating TLR2-induced myocardial protection during sepsis/septic shock. These data also suggest that modulation of TLR2/β-arrestin 2 dependent signaling pathways may prevent morbidity and mortality associated with CLP induced sepsis/septic shock.

P114 LIPOPOLYSACCHARIDE INDUCED MYOCARDIAL PROTECTION AGAINST ISCHEMIA/REPERFUSION INJURY IS MEDIATED THROUGH A PI3K/AKT-DEPENDENT MECHANISM

Chuanfu Li, Tuanzhu Ha, Fang Hua, X. Liu, Jiang Ma, Julie McMullen, Tetsu Shioi, Seigo Izumo, Jim Kelley, Xiag Gao, William Browder, Race Kao, and David Williams. Dept. of Surgery, East Tennessee State University, Johnson City, TN 37614, Beth Israel Deaconess Medical Center and Dept. of Medicine, Harvard Medical School, Boston MA, 02215, Animal Model Research Center, Nanjing University, Nanjing, China, 210093, Baker Heart Research Institute, Melbourne, Victoria, Australia, 8008

The ability of lipopolysaccharide (LPS) pretreatment to induce cardioprotection following ischemia/reperfusion (I/R) has been well documented, however, the mechanisms have not been elucidated. LPS is a TLR4 ligand. Evidence indicates that there is cross-talk between the TLR and phosphoinositide-3-kinase/Akt (PI3K/Akt) signaling pathways. We hypothesized that PI3K/Akt signaling plays a critical role in LPS induced cardioprotection. To evaluate this hypothesis, we pretreated mice with LPS, 24 hrs before the hearts were subjected to ischemia (45 min) and reperfusion (4 hrs). Activation of the PI3K/Akt and NFκB signaling pathways were evaluated. The effect of PI3K/Akt inhibition in LPS-induced cardioprotection was also evaluated. LPS pretreatment reduced infarct size by 66.5% compared to untreated controls (11.7 ± 1.45% vs 34.9 ± 5.39%, P < 0.01). Cardiac myocyte apoptosis and caspase-3 activity were also lower following LPS + I/R. LPS increased the levels of phospho-Akt, phospho-GSK-3β, heat shock protein 27, and IκBα in the myocardium, respectively. Pharmacological inhibition of PI3K by LY294002 or genetic modulation employing kinase defective Akt (kdAkt) transgenic mice abolished the cardioprotection induced by LPS. These data indicate that LPS induced cardioprotection in I/R injury is mediated through a PI3K/Akt-dependent mechanism.

P115 BLOCKING OF COMPLEMENT ACTIVATION PROTECTS THE GUT FROM ISCHEMIA-REPERFUSION INJURY AND ATTENUATES LUNG ALTERATIONS

Sergey B. Zaets, Da-Zhong Xu, Qi Lu, John Zhang, Eleonora Feketova, Edwin A. Deitch, and Cindy Cao. Novo Nordisk US, North Brunswick, NJ 08902 and UMDNJ-NJ Medical School, Newark, NJ 07103

Objective:

Activation of complement system plays an important role in the mediation of ischemia-reperfusion injury. We hypothesized that elimination of C5a receptor (C5aR) protects the gut from ischemia-reperfusion injury and thus diminishes subsequent alterations in remote organs.

Methods:

Superior mesenteric artery occlusion (SMAO) model was used to re-create gut ischemia-reperfusion injury. C5aR knock-out (KO) and wild type (WT) mice were subjected to SMAO or sham SMAO. Gut histology as well as ileum, lung and liver myeloperoxidase (MPO) activity were assessed after a 3 hour reperfusion period.

Results:

The percentage of injured ileal villi as well as the number of neutrophils per 100 villi, which reflects the severity of the gut injury, was significantly higher in WT mice subjected to SMAO, compared to that in C5aR KO mice (see table). Similarly, ileum and lung MPO activity was significantly higher in WT mice subjected to SMAO.

Table
Table

Conclusions:

Elimination of complement C5a pathway protects the gut from ischemia-reperfusion injury and diminishes gut-derived lung alterations.

P116 RECOMBINANT FACTOR XIII DIMINISHES MULTIPLE ORGAN DYSFUNCTION (MOD) CAUSED BY GUT ISCHEMIA-REPERFUSION INJURY IN RATS

Sergey B. Zaets, Da-Zhong Xu, Qi Lu, Eleonora Feketova, Tamara L. Berezina, Maryann Gruda, Edwin A. Deitch, and Eva H.N. Olsen. Novo Nordisk US, North Brunswick, NJ 08902 and UMDNJ-NJ Medical School, Newark, NJ 07103

Objective:

Plasma factor XIII (FXIII) is responsible for stabilization of fibrin clot at the final stage of blood coagulation. FXIII has also been shown to modulate inflammation and endothelial permeability. We hypothesized that FXIII diminishes MOD caused by gut ischemia-reperfusion injury.

Methods:

A model of superior mesenteric artery occlusion (SMAO) model was used to induce gut ischemia-reperfusion injury. Rats were subjected to 45 min SMAO or sham SMAO and treated with recombinant FXIII (rFXIII) or placebo at the beginning of reperfusion period. Lung permeability, lung and gut myeloperoxidase (MPO) activity), gut histology, neutrophil respiratory burst, and liver microvascular blood flow were measured after a 3 hour reperfusion period. The effect of activated rFXIII on transendothelial resistance of human umbilical vein endothelial cells (HUVEC) was tested in vitro.

Results:

Lung permeability as well as lung and gut MPO activity were significantly lower in treated versus untreated animals (see table). Similarly, rFXIII treated rats had lower neutrophil respiratory burst activity and ileal mucosal injury. rFXIII treated rats also had higher liver microvascular blood flow compared to the placebo group. Invitro, rFXIII caused a dose-dependent increase in HUVEC monolayer resistance to injury.

Table
Table

Conclusion:

Administration of rFXIII diminishes SMAO-induced MOD in rats, presumably by preservation of endothelial barrier function.

P117 SELECTIVE ACTIVATION OF MELATONIN RECEPTORS WITH RAMELTEON IMPROVES LIVER FUNCTION AFTER HEMORRHAGIC SHOCK IN RAT

Alexander Mathes, Darius Kubulus, and Hauke Rensing. Department of Anesthesiology, Saarland University Hospital, D-66421 Homburg (Saar), Germany

Pineal hormone melatonin has been demonstrated to improve organ dysfunction in a variety of stress models by direct antioxidant mechanisms. Recent evidence suggests that melatonin receptor dependent effects may contribute to the beneficial action of the hormone. However, it has not been investigated whether isolated activation of melatonin receptors, without concomitant antioxidant effects, may result in organ protection. Therefore, we determined whether the novel hypnotic substance ramelteon, a selective melatonin receptor agonist without direct antioxidant capacity, may improve liver function after hemorrhagic shock in rat. After approval of the institutional review board, animals underwent hemorrhagic shock (MAP 35 ± 5 mmHg) for 90 minutes and resuscitation with shed blood and ringers (2 hours). At the end of shock, animals were treated with ramelteon (1.0 mg/kg), ramelteon plus melatonin receptor antagonist luzindole (each 1.0 mg/kg) or vehicle. At the end of the experiment, liver function was measured by plasma disappearance rate of indocyanine green (PDRICG). Compared to vehicle controls, ramelteon therapy significantly improved PDRICG (7.89 ± 2.12 % vs. 13.67 ± 2.51 %; P = 0.006). Antagonist luzindole abolished this effect completely. This demonstrates that selective activation of melatonin receptors may improve liver function after hemorrhagic shock in rat. It may therefore be speculated that direct antioxidant effects are contributory, but not mandatory for the beneficial effects of melatonin. Hypnotic ramelteon could thus play an interesting role in future sedation concepts for the critical care patient. [This study was supported by a grant of the ESA]

P118 ETHYL PYRUVATE REDUCES THE DEVELOPMENT OF NON-SEPTIC SHOCK INDUCED BY ZYMOSAN IN MICE

Rosanna Di Paolaa, Emanuela Mazzona, Tiziana Genovesea, Concetta Crisafullib, Placido Bramantia, Emanuela Espositoa,d*, Mitchell P. Finke, and Salvatore Cuzzocreaa,b. aIRCCS Centro Neurolesi "Bonino-Pulejo", Messina, Italy; bDepartment of Clinical and Experimental Medicine and Pharmacology, School of Medicine, University of Messina, Italy. dDepartment of Experimental Pharmacology, University of Naples "Federico II", Via D. Montesano 49, Naples, Italy. eDepartments of Critical Care Medicine and Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, USA

Ehyl pyruvate (EP) is a simple aliphatic ester, which has been shown to have anti-inflammatory effects in previous numerous cell culture and animal studies. In the present study, we investigated the effects of EP (75 mg/kg i.p.) on the development of non-septic shock caused by zymosan.

Mice received either intraperitoneally zymosan (500 mg/kg, administered i.p. as a suspension in saline) or vehicle (0.25 ml/mouse saline). EP (75 mg/kg s.c.) was administered 1 and 6 hours after zymosan administration. Organ failure and systemic inflammation in mice was assessed 18 hours after administration of zymosan and/or EP. Treatment of mice with EP attenuated the peritoneal exudation and the migration of polymorphonuclear cells caused by zymosan. EP also attenuated the lung, liver and pancreatic injury and renal dysfunction caused by zymosan as well as the increase in myeloperoxidase activity in the lung and intestine caused by zymosan. Immunohistochemical analysis for inducible nitric oxide synthase (iNOS), nitrotyrosine, poly (ADP-ribose) (PAR), TNF-α and IL-1β revealed positive staining in pancreatic and intestinal tissue obtained from zymosan-injected mice. The degree of staining for nitrotyrosine, iNOS, PAR, TNF-α and IL-1β were markedly reduced in tissue sections obtained from zymosan-injected mice, which had received EP. In addition, administration of zymosan caused a severe illness in the mice characterized by a systemic toxicity, significant loss of body weight and a 60% of mortality at the end of observation period (7 days). Treatment with EP significantly reduced the development of systemic toxicity, the loss in body weight and the mortality caused by zymosan.

P119 TRAUMA/HEMORRHAGIC SHOCK INDUCED GUT INJURY IS ASSOCIATED WITH THE DISRUPTION OF MUCUS LAYER UTILIZING A STABILIZED MUCUS LAYER TECHNIQUE

Qi Lu, Da-Zhong Xu, Susan Sharpe, Danielle Doecet, Vadim Pisarenko, Marlon Lee, and Edwin A. Deitch. UMDNJ-NJ Medical School, Newark, NJ 07103

The intestinal mucus layer is an important but understudied component of the intestinal barrier and we have recently reported that its disruption is associated with trauma-shock-induced loss of gut barrier function utilizing Periodic Acid Schiff staining of cryostat (frozen) tissue samples. However, mucus layer analysis of frozen section intestinal sections has many limitations (sub-optimal histologic quality as compared to fixed tissues, limited ability to perform immuno-histochemistry, and increased technical difficulties). Yet the study of frozen sections appeared necessary, since standard fixation techniques dissolve the mucus layer. To avoid the limitations of using frozen tissue samples, we tested whether a modified processing technique employing Carnoy's fixative solution would preserve the mucus layer yet allow tissue fixation in paraffin blocks.

Methods:

Male SD rats were subjected to a laparotomy (trauma) and 90 min of sham shock (T/SS) or actual shock (35 mmHg × 90 min) (T/HS group). At 3 hr after resuscitation the rats were sacrificed and samples of the terminal ileum were processed by frozen section or by fixation in Carnoy's solution. Gut injury was evaluated by determining the percentage of villi injured and disruption of the intestinal mucus layer was quantified by determining the percentage of the villi covered by mucus.

Results:

Histologic analysis of gut injury could not be reliably quantified on the frozen tissue samples but could be accurately assessed in the Carnoy fixed samples (T/SS = 2.5 + 0.5 % vs T/HS = 22.4 + 0.5% of villi injured; P < 0.01). T/SS rats had 98% of the ileal mucosa covered with a mucus layer and this was decreased after T/HS to 63 + 3% in the Carnoy fixative processed samples and 49 + 15% in the frozen sections (T/HS vs T/SS P < 0.001).

Conclusion:

This study shows that T/HS significantly reduces the intestinal mucosal layer and that the Carnoy method of tissue fixation has advantages over the standard approach of using frozen section techniques to assess the gut mucus layer.

P120 EFFECTS OF ALBUMIN ON PERITONITIS-INDUCED SEPTIC SHOCK WITH MULTIPLE ORGAN DYSFUNCTION SYNDROME IN RATS

Z.F. Chen1, S.J. Chen2, R.L. Chen1, C.T. Chiu1, and C.C. Wu1. Departments of 1Pharmacology and 2Physiology, National Defense Medical Center, Taipei; 1Department of Nursing, Kang-Ning Junior College of Medical Care and Management, Taipei; R.O.C., Taiwan

The main cause of death in patients with sepsis is multiple organ dysfunction syndrome (MODS). The development of MODS correlates with over-expressed inflammatory response (e.g. cytokines) and global tissue hypoperfusion (e.g. hypotension). In this study, we tried to examine effects of albumin (ALB) in Wistar rats with MODS induced by cecal ligation and puncture (CLP) surgery. Rats were divided into four groups, i.e. (1) sham operation (SOP), (2) SOP + ALB, (3) CLP and (4) CLP + ALB. It is noted that at 3 h after CLP, animals received ALB (25 % albumin 3 ml/kg, 25 μl/min). The changes of hemodynamics, blood sugar, rectal temperature, hepatic (GPT and GOT) and renal (BUN and CRE) function, blood gas (pH, Pco2, Po2, base excess, HCO3), plasma NO and interleukin-1β levels and electrolytes were monitored over 18 h. At 18 h after CLP, animals were sacrificed and most of organs were excised to perform the pathological studies, iNOS expression and superoxide measurement, whereas thoracic aortas were used to analyze vascular reactivity. Our results showed that ALB not only attenuated the hypotension but also ameliorated lung, hepatic and renal dysfunction in rats with CLP. Thus, ALB may serve beneficial effects in animals with MODS and these beneficial effects of ALB on sepsis animals may be attributed to the reduction of plasma NO level and organ superoxide level, and increases of organ blood flow, which led to decreasing the mortality in CLP-treated animals.

P121 CARDIOTROPIC EFFECTS OF RELAXIN ON MYOCARDIAL INFARCTION AND ADVERSE POST-ISCHEMIC REMODELLING AND HEART FAILURE

Silvia Nistri1, Lucia Formigli1, Daniele Bani1, and Emanuela Masini2. Depts.: 1Anatomy, Histology and Forensic Medicine, and 2Preclinical and Clinical Pharmacology, University of Florence, viale G.Pieraccini 6, I-50139 Florence, Italy

The hormone relaxin has been shown to cause coronary vasodilation and to prevent ischemia/reperfusion-induced cardiac injury in rodents. This study provides evidence that relaxin, used as an adjunctive drug to coronary reperfusion, reduces the functional, biochemical and histopathological signs of myocardial injury in an in vivo swine model of heart ischemia/reperfusion, currently used to test cardiotropic drugs for myocardial infarction. Human recombinant relaxin, given at reperfusion at doses of 1.25, 2.5 and 5 μg/kg b.wt. upon a 30-min ischemia, caused a dose-related reduction of key markers of inflammation, myocardial damage and cell apoptosis, by a mechanism involving reduction of oxygen free radical-induced tissue injury. Moreover, relaxin reduces the inflammatory activation of resident mast cells, the main source of cardiac histamine and prostanoids, thereby preventing the onset of severe arrhythmias, cardiodepressive effects and coronary spasm. Overall, relaxin reduced the extension of necrotic myocardium and leads to a substantial improvement of heart contractility.

Then we explored whether relaxin, which is able to promote extracellular matrix turn-over and neo-angiogenesis, could beneficially influence post-infarction heart remodelling and dysfunction using a swine model of stem cell therapy with C2C12 myoblasts genetically engineered to express relaxin. One month after cell engraftment, histological analysis showed that C2C12/RLX myoblasts selectively settled in the ischemic scar, where they induce extracellular matrix remodeling by the secretion of matrix metalloproteases (MMP) and increase microvessel density through the expression of VEGF. By echocardiography, C2C12/RLX-engrafted swine showed improved heart contractility compared with the ungrafted controls. In conclusion, our studies offer additional evidence for the potential therapeutic effects of relaxin in acute myocardial infarction and in preventing deleterious cardiac remodeling in the post-infarcted heart.

P122 IRON(II) COMPLEXES INCREASE IN MESENTERIC LYMPH AFTER RESUSCITATION FROM HEMORRHAGE

J.L. Atkins*, N.V. Gorbunov*, R. Van DuyneΦ, F. KashanchiΦ, and A.M. KomarovΦ. *Walter Reed Army Institute of Research, Silver Spring, MD, 20910, ΦThe George Washington University, Washington, DC, 20037

Most extracellular iron is in the form of Fe(III), however some physiological conditions with lower redox state may favor the presence of extracellular Fe(II). During hemorrhage/resuscitation (H/R) an Fe(II) pool could play an important role in exacerbating oxidative stress. We have previously shown that the electron paramagnetic resonance (EPR) spectrum of ceruloplasmin (CP,) the principal ferroxidase in plasma, is decreased during H (J. Appl. Physiol., 2006). Based on these considerations we examined mesenteric lymph for the presence of Fe(II). Anesthetized male rats were hemorrhaged to 40mmHg over 15 minutes, held at this pressure by blood withdrawal until decompensation and then resuscitated with 3 times the shed blood volume with Lactated Ringers. EPR of mesenteric lymph after H/R showed no detectable spectrum for CP. Fe(II) measured by ferrozine assay, was 4.4-15.4 (nmol Fe/mg protein) during control and hemorrhage and increased significantly to 27.4-170 during resuscitation. The iron was protein bound and was concentrated in two size fractions 1000-2000 kDa and 150-200 kDa. These Fe(II) complexes can participate in the Fenton reaction generating hydroxyl radicals as detected by EPR and spin trapping with DMPO in the presence of hydrogen peroxide. We conclude that H/R lymph has increased Fe(II) after H/R which may contribute to oxidative stress when thoracic duct lymph reaches the high oxygen environment of the lung.

(Dept of Army, PRMRP grant no. PR033201)

P123 AMPK-INDEPENDENT CARDIOPROTECTION OF ADIPONECTIN IN THE ISCHEMIC/REPERFUSED MOUSE HEART

T. Christopher, Y.J. Wang, L. Tao, B. Goldstein, E. Gao, W. Koch, R. Tian, and X. Ma. Thomas Jefferson University, Philadelphia, PA 19107, USA, and *Brigham and Women's Hospital, Boston, MA 02115, USA

Adiponectin (ADN) is an adipocytes-derived cytokine that regulates cellular metabolism largely via AMP activated protein kinase (AMPK). It has recently been demonstrated that ADN protects the heart against myocardial ischemia/ reperfusion (MI/R) injury. However, the role of AMPK in ADN's cardioprotective signaling is not well understood. Adult male mice with cardiac-specific overexpression of a dominant negative mutant of the AMPK α2 catalytic subunit (TG) or their littermates (WT) were subjected to 30 min of MI followed by 3 (apoptosis) or 24 (infarct size and cardiac function) hours of R and treated with vehicle or ADN (2 μg/g bw, 10 min before R, ip.). Treatment with ADN significantly reduced cardiomyocyte apoptosis (P < 0.01), decreased infarct size (69 ± 3.8% vs. 33 ± 4.5%, P < 0.01), and improved cardiac function (P < 0.01) in WT mice. The cardioprotective effects of ADN are blunted in TG mice (P < 0.05), indicating that AMPK plays an important role in ADN's cardioprotective signaling. However, a significant portion of ADN cardioprotection was retained in the TG mice as evidenced by reduced cardiomyocytes apoptosis (TUNEL positive cells: 24.5 ± 1.6% in vehicle treated TG mice vs. 16.4 ± 2.0% in ADN treated TG mice, P < 0.05; Caspase-3 activity: 1.18 ± 0.06 μM/mg protein/h vs. 0.89 ± 0.06 μM/mg protein/h, P < 0.05), decreased infarct size (81 ± 3.6% vs. 54 ± 4.7%, P < 0.01) and improved left ventricular ejection fraction (33 ± 2.4% vs. 52 ± 2.3%, P < 0.01). These novel results demonstrated that ADN protects ischemic/reperfused hearts through multiple signaling pathways, not exclusively through its known AMPK-mediated metabolic activity. Identifying these alternative signaling pathways will not only shed light on ADN intracellular signaling but will also potentially form the basis for future therapeutic interventions.

P124 FAILED SEPSIS-INDUCED ACTIVATION OF HEPATIC GLYCOPROTEIN (GP) 130 IS NOT DUE TO IMPAIRED PHOSPHORYLATION OF JANUS KINASE (JAK)-1

A. Abcejo, K. Andrejko, N. Raj, and C.S. Deutschman

Objective:

Sepsis is a highly lethal disease in surgical intensivecare units. One effector of sepsis during the acute phase response isInterleukin-6 (IL-6). IL-6 signals are mediated in part by activation ofsignal transducer and activator of transcription (Stat)-3. IL-6 binds toits cell surface receptor gp 130. This catalyzes phosphorylation of thegp130-associated protein JAK-1. JAK-1 inturn phosphorylates anumber of tyrosine residues on the cytoplasmic tail of gp130. Thesep-TYRs in turn phosphorylate Stat-3 monomers, which dimerize,migrate to the nucleus and induce organ specific gene expression.Previous work in a lethal sepsis model (cecal ligation and doublepuncture, 2CLP) has demonstrated failed Stat-3 DNA binding thatparallels mortality and decreased gp130 phosphorylation. Wehypothesize that this finding results from impaired JAK-1 activitythat is caused by a defect in JAK-1 phosphorylation.

Methods:

Liver homgenates and serum was collected from 6-8week old C57Bl/6 mice prior to operation (T0) and at 3, 6, 16,24, 48, and 72 hours after sham operation (SO), CLP (non-lethalsepsis) and 2CLP. Gp130 and Jak-1 were immunoprecipitated fromcytosolic protein extracts following surgery. Western blotting wasused to determine protein levels of gp130, phosphorylated (p) gp130,Jak-1 and p-Jak-1. IL-6 serum levels were detected by flow cytometry.

Results:

IL-6 serum levels were elevated in both CLP and 2CLPrelative to T0 and SO controls. The abundance of p-gp130 waselevated following CLP but decrease over time following 2CLP.There was no change in gp130 protein levels following CLP, 2CLPor SO. Jak-1 and p-Jak-1 protein levels show little variation after anytype of surgical intervention.

Conclusions:

Decreased Stat-3 DNA binding is not a resultof inappropriate IL-6 serum levels or a down regulation of its receptorprotein gp130. Loss of p-gp130 levels is reflective of failed Stat-3DNA binding. Therefore, failure of the Stat-3 signaling pathwaycannot be explained by a failure to phosphorylate Jak-1.

P126 EFFECTS OF β-ADRENOCEPTOR AGONISTS ON PERITONITIS-INDUCED SEPSIS

S.J. Chen1, C.M. Tsao2, M.T. Shih3, and W.J. Liaw4 and C.C. Wu3. 1Department of Nursing, Kang-Ning Junior College of Medical Care and Management, Taipei; 2Department of Anesthesiology, Taipei Veterans General Hospital, Taipei; Departments of 1Physiology, 3Pharmacology, and 4Anesthesiology, National Defense Medical Center, Taipei; R.O.C., Taiwan

The multiple organ dysfunction syndrome (MODS) in septic shock is mainly caused by the maldistribution of tissue perfusion and amplification of inflammatory responses, which are modulated by β-adrenoceptor agonists. To evaluate and compare the effects of terbutaline and dobutamine on MODS in cecal ligation and puncture (CLP) model of sepsis. Wistar rats received CLP or sham operation (SOP) followed by saline vehicle, dobutamine (0.3 μg/kg/min i.v. infusion from 3 h to 18 h after CLP) or terbutaline (0.3 mg/kg i.v. at 3 and 9 h after CLP). At 0, 9 and 18 h after CLP, the changes of hemodynamics, organ function indexes, as well as the plasma interleukin-1β (IL-1β) and NO levels were examined. At 18 h after CLP, animals were sacrificed and aortas, lungs, livers and kidneys wereimmediately exercised to analyze O2.−levels and inducible NO synthase (iNOS) expression. These organs were also evaluated by the pathological study. Results demonstrated that terbutaline, but not dobutamine, significantly attenuated vascular hyporeactivity to norepinephrine and the delayed hypotension, reduced hepatic and renal dysfunction, diminished plasma IL-1β and NO, lung iNOS expression, tissue O2.−level and the infiltration of polymorphonuclear neutrophils in the lung and the liver, and improved the 18-h survival rate. Terbutaline or β2-adrenoceptor agonist can be as an alternative treatment for severe sepsis-induced MODS. The protective effect of terbutaline is mediated by inhibition of proinflammatory mediators and attenuation of oxidant production.

P127 MECHANISM OF INTESTINAL TIGHT JUNCTION LOSS FOLLOWING HEMORRHAGIC SHOCK

G. Thuijls, J. de Haan, J.P.M. Derikx, I. Daissormont, E. Heineman, W.A. Buurman. Maastricht University Medical Center, Maastricht, Netherlands

Tight junctions (TJ) are an essential component of the intestinal epithelial barrier. Shock-induced loss of TJ integrity results in translocation of bacteria and bacterial products which is associated with an increased risk of SIRS. The integrity of TJ is based on an intact actin cytoskeleton, in which actin depolymerizing factor/cofilin (AC) plays a central role. The aim of this study is to investigate the sequence of events following hemorrhagic shock (HS) and proposed intestinal hypoperfusion, with special emphasis on AC activation, actin cytoskeleton severing and TJ loss. Rats were subjected to HS and sacrificed, along with controls, at 15, 30, 60 and 90 minutes after induction of shock. Intestinal hypoperfusion, activation of AC, actin cytoskeleton severing and TJ integrity loss (Claudin-3, ZO-1) were assessed. One-way ANOVA, with post-hoc Dunnett's multiple comparison test, was used for between group comparisons. HS caused intestinal hypoperfusion (−71.7% bloodflow) and a significant activation of AC from 15 min after HS (by 21%, P < 0.05 versus control). This was accompanied by actin cytoskeleton severing, followed by a significant intestinal TJ loss from 60 min after HS (Claudin-3; 100 ± 15 versus 20 ± 8 % and ZO-1; 100 ± 14 versus 56 ± 4%, P < 0.05 versus controls). This study shows for the first time that HS rapidly results in intestinal hypoperfusion, AC activation, actin depolymerization and subsequent TJ loss. Elucidation of the etiology of intestinal barrier failure increases our understanding of the physiological response to shock. Further steps are directed at the development of therapeutic interventions to reduce barrier function loss.

P128 ENDOTHELIAL MICROVASCULAR ACTIVATION IN HEMORRHAGIC SHOCK: THE ROLE OF THE NEUTROPHIL

Francis M. Wulfert1,2, Matijs van Meurs2,3, Ann de Haes1,2, Ageeth J. Knol1,2, Martin Houwertjes1, Jan G. Zijlstra3, Leon P.H.J. Aarts4, and Grietje Molema2. 1Department of Anesthesiology, 2Department of Pathology and Medical Biology, 3Department of Critical Care, University Medical Center Groningen, University of Groningen, 4Department of Anesthesiology, Leiden University Medical Center, Leiden. The Netherlands

Multiple organ dysfunction syndrome is a complication of hemorrhagic shock (HS). Interaction of activated neutrophils and endothelial cells, is considered to play a prominent role in the pathophysiology. Our hypothesis was that hemorrhagic shock rapidly activates endothelial cells toward a pro-inflammatory status due to neutrophil - endothelial cell interaction, and hence that depletion of neutrophils would prevent endothelial activation. In selected animals, neutrophil depletion was established by i.p.injection of anti-NIMP antibody 24 hour prior to HS induction. Male anesthetized mice were subjected to controlled HS with a MAP of 30 mmHg during 90 minutes followed by resuscitation with 6% hydroxyethyl starch. Mice were sacrificed before HS induction (control), after 90 minutes of HS, and 24 hours after resuscitation. mRNA and protein expression of adhesion molecules (E-selectin, P-selectin, VCAM-1 and ICAM-1) were measured in lungs, kidney, and liver by real time RT-PCR respectively immunohistochemistry, and compared to expression in non-treated counterparts and controls. Induction of inflammatory genes, presented by upregulation of mRNA and protein, was seen early during HS, immunohistochemistry showed a microvascular bed specific activation pattern. Only in the kidney, neutrophil depletion decreased E-selectin expression during the shock period, while at 24hrs after resuscitation ICAM-1 and VCAM-1 mRNAs was increased in all organs (table). Neutrophil depletion affected HS related microvascular endothelial cell activation during the early shock period. Further studies will focus on effects of neutrophil depletion on endothelial cell activation in the early post resuscitation phase.

Table
Table

Table:

Expression levels of P-selectin, E-selectin, VCAM-1, and ICAM-1 mRNA in lung, liver and kidney, analysed by quantitative RT-PCR using GAPDH as housekeeping gene. RNA levels were normalized to their respective levels in RNA isolates of healthy organs obtained from untreated control mice.

P129 NORMAL RAT GUT MUCUS LAYER PROTECTS AGAINST PROTEASE-INDUCED GUT INJURY

Susan M. Sharpe, Xiaofa Qin, and Edwin A. Deitch. UMDNJ-NJMS Newark, NJ 07103

Objective:

This study tested the hypothesis that the intestinal mucus layer of the normal rat gut prevents mucosal injury from intra-luminal proteases, and removal of the mucus layer via a mucolytic will cause an increase in gut injury in the presence of pancreatic proteases.

Methods:

Using a 30-cm ligated segment of normal rat distal ileum, four groups were tested: 1) saline (control for gut handling); 2) NAC (removal of mucus layer); 3) saline + proteases (control) and 4) NAC + proteases. After 3 hours, the gut segment was harvested and permeability was assessed using an everted gut sac method with an FD-4 probe. The physiologic properties of the mucus layer were tested by measuring the hydrophobicity of the gut segment; the larger the angle, the greater the hydrophobicity and the greater the physiologic barrier to luminal contents.

Results:

Table I-Gut permeability is increased and hydrophobicity is decreased after addition of NAC followed by proteases

Table
Table

Conclusions:

The gut mucus layer is protective against proteolytic damage. When the mucus layer is removed via a mucolytic, the hydrophobicity of the mucus layer is decreased, allowing luminal proteases to reach the intestinal mucosa and cause increased gut permeability.

P130 LUNG INFLAMMATION DURING INDIRECT ACUTE LUNG INJURY (ALI) IS REGULATED THROUGH FAS INDUCED APOPTOSIS - POTENTIAL CROSSTALK?

M. Perl, C.S. Chung, U. Perl, J. Lomas-Neira, and A. Ayala. Department of Surgery, Rhode Island Hospital and Brown University, Providence, RI 02903

Lung apoptosis and inflammation appear to be two key mechanisms in the development of ALI, however, potential crosstalk between these two mechanisms remains elusive. In particular it is unclear whether inhibition of apoptosis in the lung at the Fas receptor or at the executioner caspase level can differentially effect lung inflammation. To study this, male Fas receptor mutant animals (lpr) were subjected to hemorrhagic shock (HEM) and sepsis (cecal ligation and puncture [CLP]) 24hrs thereafter. Separately, wild type mice were treated alike but received control-or caspase-3-siRNA intratracheally after HEM to silence lung caspase-3 activity. 12 or 24 hours after CLP, lung apoptosis (active caspase-3 by western blotting), the degree of lung injury (lung protein influx and histology), lung neutrophil influx (myeloperoxidase activity) and pulmonary inflammation (TNF-alpha, IL-6, MCP-1, KC, MIP-2) were assessed. Pulmonary apoptosis, neutrophil influx and the degree of lung injury were all markedly reduced in the absence of Fas signaling as well as in response to caspase-3 silencing. Interestingly, while lung inflammation was markedly abrogated in both scenarios, in the absence of Fas signaling both, lung cytokines and chemokines were substantially decreased whereas caspase-3 silencing inhibited lung cytokines only. These data indicate, that a reduction of the severity of ALI can result from modulating apoptosis at the Fas receptor as well as at the caspase-3 level. However, inhibition of lung apoptosis under these circumstances diminishes lung inflammation, through a reduction of proinflammatory cytokines, while chemokines may be differentially regulated. (Supported by NIH-HL73525).

P131 MECHANISMS OF EXTRA-PULMONARY ACUTE LUNG INJURY: THE DENDRITIC CELL AS A REGULATOR OF THE MACROPHAGE'S INFLAMMATORY RESPONSE

Fabienne Venet, Joanne Lomas-Neira, Xin Huang, Yaping Chen, Chun-Shiang Chung, and A. Ayala. Div Surg Res,/Surgery, Rhode Island Hospital / Brown University, Providence, RI, USA

Acute Lung injury (ALI) remains the most common organ dysfunction seen in critically ill patients, however, the cellular interactions in the lung contributing to its pathophysiology remain unclear. The aim of this study was to determine the contribution of dendritic cells (DCs) to lung apoptosis, inflammation and neutrophil recruitment during ALI resultant from hemorrhagic shock (Hem) /sepsis (CLP). To assess this, we depleted DCs in CD11c-Diphteria Toxin (DT) Receptor conjugated mice by the respective delivery of DT either systemically (intraperitoneally [IP] injection) or locally in the lung (intratracheally [IT] instillation). Subsequently, extra-pulmonary lung injury was produced using a dual insult model of Hem followed 24h later by CLP. 24h after CLP, caspase 3 activity, lung myeloperoxidase (MPO) and pro-inflammatory cytokine production in the lung and plasma were measured. Systemic DC depletion (IP DT) was associated with a marked rise in IL-6, TNF-α and MCP-1 concentrations after Hem/CLP, whereas lung apoptosis and neutrophil recruitment (MPO) were not modified in comparison with background mice. Similar results were observed after local lung DCs depletion (IT DT). Flow cytometric phenotyping suggested that this DC effect was mediated through the recruitment of pro-inflammatory macrophages to the lung. Our results with macrophage depletion through IT clodronate administration support this assertion. Thus the resident and/or recruited DCs appear to play a here-to-for unanticipated role in suppressing lung inflammation and promoting the resolution of ALI. (NIH-HL73525)

P132 SMALL VOLUME RESUSCITATION IN A RAT MODEL OF HEATSTROKE

Mao-Tsun Lin and Ching-Ping Chang. Department of Medical Research, Chi Mei Medical Center, Tainan, Taiwan 710; and Department of Biotechnology, Southern Taiwan University, Tainan, Taiwan 710

We compared the effectiveness of different small volume fluid resuscitation in a rat model of heatstroke. Anesthetized rats, immediately after the onset of heatstroke, were randomizedly divided into five major groups and given the following: a) nothing; b) 0.9% NaCl solution (1-10 ml/kg body weight, i.v.); c) hydroxyethyl starch (HES) (6%, 1-10 ml/kg body weight, i.v.); d) 7.2% NaCl solution (1-10 ml/kg body weight, i.v.); and e) hyper-HES (6% HES plus 7.2% NaCl solution, 1-10 ml/kg body weight, i.v.). Another group of rats were exposed to room temperature (24°C) and used as normothermic controls. The instant at which the mean arterial pressure dropped to a value of 25 mmHg from the peak level and core temperature reached over 42°C was taken as the time point for onset of heatstroke. When the untreated or 0.9% NaCl solution-(1 or 5 ml/kg) treated rats underwent heat stress, their survival time (interval between the heatstroke onset and animal death) values were found to be 20-22 min. Resuscitation with 10 ml per kg of body weight of 0.9% NaCl solution, 6% HES, 7.2% NaCl solution, or hyper-HES their survival time values respectively are 93 ± 6, 101 ± 12, 154 ± 18, or 286 ± 21 min. Apparently, the order of effectiveness in resuscitation of heatstroke in a rat model is: hyper-HES>7.2% NaCl solution >0.9% NaCl solution or 6% HES. As compared with values for normothermic controls, the untreated heatstroke rats had lower mean arterial pressure, cerebral perfusion pressure, cerebral blood flow, brain partial pressure of oxygen, and plasma levels of protein C. In contrast, the untreated heatstroke rats had higher values of intracranial pressure, core and brain temperatures, brain levels of glutamate, glycerol, lactate-to-pyruvate ratio, neuronal damage scores, plasma levels of prothrombin time, partial thromboplastin time, D-dimer, and tumor necrosis factor-α, blood urea nitrogen, aspartate and alanine aminotransferase, and alkaline phosphatase. The hypotension, intracranial hypertension, cerebral hypoperfusion and hypoxia, increment of cerebral ischemia and damage markers, hypercoagulable state, and tumor necrosis factor-α overproduction were all significantly attenuated by hyper-HES therapy. The benefit of hyper-HES during heatstroke is related to small-volume effect.

Keywords:

Heatstroke, hyper-HES, hypotension, hypercoagulable state, inflammation, multiorgan dysfunction

P133 Heatstroke-specific peptidomics analysis of rat cerebrospinal fluid

Yong-Qi Zhao, Jun-Tao Gao, Shu-Hong Liu, Haitao Wu, and Ming Fan. Beijing Institute of Basic Medical Sciences, 27# Taiping Road, Beijing, PR China, 100850

The present study was performed to find the different peptides in rat cerebrospinal fluid (CSF) when rats suffering heatstroke. Pentobarbital anesthetized Wistar rats (200g ± 20g weighted) were exposed to ambient temperature of 42.5 ± 0.5°C to induce heatstroke. Another group of anesthetized rats was exposed to room temperature (23 ± 1°C) and used as normothermic controls. Femoral arterial pressure (FAP) was monitored by a pressure probe (Bio-Pac) inserting in right femoral arterial. The moment at which FAP dropped by a value of 25 mmHg from its peak levels was taken as a reference point for the onset of heatstroke. CSF of rats were taken suction by hollow needles punctured into 3-ventricle without blood contamination just at that time. CSF was analyzed with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). MS spectrum comparison showed that several peptides disappeared at the moment of heatstroke, instead of new peptides appeared. Most of these peptides were low molecular weighted (<10kDa), and following sequence characterization would be useful for identify their source and function. This finding has important implication for future peptide biomarker discovery about heatstroke.

Keywords:

heatstroke, cerebrospinal fluid, peptidomics

P134 MITOCHONDRIAL RESPIRATION IN PERMEABILISED MUSCLE FIBERS OF PIGS DURING AND AFTER HEMORRHAGIC SHOCK

B. Hauser1, C.D. Nguyen1, A. Söll2, F. Simon2, P. Radermacher1, and E. Calzia1. Sektion Anästhesiologische Pathophysiologie und Verfahrensentwicklung1 and Thorax-und Gefäβchirurgie2, Universitätsklinikum, 89070 Ulm

The aim of our study was to measure mitochondrial respiratory function in biopsies from the breast muscle of 10 pigs undergoing hemorrhagic shock induced by extracting 40% of the estimated intravascular blood volume which was subsequently re-transfused after 4h at the beginning of the reperfusion period. The biopsies were sampled before and 3h after shock induction, as well as at 12h of resuscitation. Mitochondrial respiration was measured in the saponin-permeabilised muscle biopsies by means of high-resolution respirometry (Oxygraph-2k, Oroboros, Austria) and quantified as the oxygen flux during state 3 respiration with complex I substrates (Malate, Glutamate, and Pyruvate) and with combined complex I and II substrates after further addition of succinate. The ratio between these two conditions was quantified as succinate control ratio (SCR)1. Data are presented as mean ± SD, oxygen flux unit is [pmol O2 / s * mg]

Table
Table

Mitochondrial respiratory function was markedly suppressed during shock but almost completely recovered after resuscitation. These findings potentially indicate adaptive mechanisms related to shock.

Reference

1. Lemieux et al, Mitochondrial Physiology Network (2008);12.13, 38-42.

P135 MITOCHONDRIAL RESPIRATION IS NOT IMPAIRED IN RENAL BIOPSIES OF PIGS AFTER AORTIC CLAMPING

F. Simon2, B. Hauser1, C.D. Nguyen1, A. Söll2, H. Schelzig2, P. Radermacher1, and E. Calzia1. Sektion Anästhesiologische Pathophysiologie und Verfahrensentwicklung1 and Thorax-und Gefäβchirurgie2, Universitätsklinikum, 89070 Ulm

Our goal was to study the effects of ischemia/reperfusion (I/R)-injury on renal mitochondrial respiration. Therefore, in two groups of anesthetized pigs (n = 7) we induced renal ischemia by 2h of aortic clamping followed by 8h of reperfusion. Mitochondrial respiration was measured in mechanically permeabilized1 small kidney biopsies taken from the renal cortex before clamping and after reperfusion respectively by means of high-resolution respirometry (Oxygraph-2k, Oroboros, Austria). We sequentially measured state 3 respiration (O2-flux per mg tissue) for complex I after addition of malate, glutamate and ADP, and for complex II after further adding rotenone and succinate. The respiratory control ratio (RCR) was determined for complex I. Complex IV activity was measured with ascorbate/TMPD as substrates and corrected for chemical background determined afteraddition of KCN2. Data are presented as mean ± SD, unit is [pmol O2 / s * mg]

Table
Table

Normalized O2-flux for all complexes and RCR were almost equal before clamping and after reperfusion. Therefore, we conclude that in our experiment ischemia/reperfusion did not impair mitochondrial respiratory function in the renal cortex.

References

1. Kuznetsov et al: Anal Biochem (2002) 305:186 - 194

2. Kuznetsov et al: Mitochondrial Physiology Network 6.6:1-4

P136 HEMODYNAMIC AND METABOLIC EFFECTS OF H2S DURING PORCINE ISCHEMIA/REPERFUSION INJURY

F. Simon1,2, R. Giudici1, C.D. Nguyen1, H. Schelzig2, S. Öter1, M. Gröger1, U. Wachter1, J. Vogt1, G. Speit3, C. Szabó3, P. Radermacher1, and E. Calzia1. 1Anästhesiologische Pathophysiologie und Verfahrensentwicklung, 2Thorax-und Gefäβchirurgie, 3Humangenetik, Universitätsklinikum, 89070 Ulm, Germany; 3Ikaria, Seattle WA 98102, USA

In awake mice inhaled H2S produced a "suspended animation-like" metabolic status with hypothermia and reduced O2 demand, thus protecting from lethal hypoxia [1]. Murine models may be questioned, however, since due to their large surface area/mass ratio rodents can rapidly drop their core temperature. Therefore, we investigated whether intravenous H2S (sulphide) would induce a comparable metabolic response in anesthetised and mechanically ventilated pigs. Since H2S was also reported to improve post-ischemic heart function, we also investigated whether sulfide would influence the norepinephrine (NE) responsiveness during reperfusion after aortic occlusion. After 2 hours of i.v. sulphide or vehicle, animals underwent 30 minutes of aortic occlusion with nitroglycerine, esmolol and ATP adjusted to maintain mean arterial pressure (MAP) at 80-120% of baseline. During reperfusion NE was titrated to keep MAP≥80% of this level. sulfide reduced heart rate and cardiac output without affecting stroke volume, markedly the NE needs required to maintain hemodynamic targets, and caused a drop in core temperature concomitant with lower VO2 and VCO2. Sulfide attenuated the reperfusion-related hyperlactatemia, while glycemia was higher at the end of the experiment. Intravenous sulfide allowed reducing energy expenditure in an anesthetised large animal model, improved the noradrenaline responsiveness during reperfusion after aortic occlusion, and thus may be organ-protective after ischemia/reperfusion injury.

Reference

1. Szabó C: Hydrogen sulfide and its therapeutic potential. Nat Rev Drug Disc 2007;6:917-935

P137 SIMVASTATIN REDUCES THE LIVER INJURY INDUCED BY ISCHEMIA-REPERFUSION IN THE RAT

João Rocha, Bruno Sepodes, Rui Pinto, Christoph Thiemermann, and Helder Mota-Filipe. Faculty of Pharmacy - University of Lisbon, 1649-019 Portugal

Simvastatin is a HMG-CoA reductase inhibitor and member of the therapeutic family of statins. Recent experimental studies demonstrated that pre-treatment with statins reduces the injury caused by ischemia-reperfusion of the heart, brain and kidney. There is evidence that statins can reduce cytokine-induced endothelial iNOS expression by interfering with the nuclear translocation of nuclear factor-κB (NF-κB). Studies have also demonstrated that statins are ligands of peroxisome proliferator-activated receptors (PPAR)-α and PPAR-γ, members of a hormone receptor family of ligand-activated transcription factors, known to have an important role in the modulation of inflammatory process in various animal models of inflammation. Recent developments have also shown that statins could induce heme-oxygenase activity, which is known to a have protective role in ischemic injury. Here we investigate the effects of simvastatin, on the liver injury caused by ischemia-reperfusion injury, in the rat. Male Wistar rats were randomly allocated into 4 groups as described: (1) Sham Group; (2) I/R Group: rats which were subjected to liver ischemia for 45 minutes interrupting blood supply to 3/4 of the liver, followed by reperfusion for 2 h; (3) Simvastatin + I/R Group: rats which received simvastatin (1 mg/kg i.v.) 30 minutes prior to liver I/R; and (4) Simvastatin Control Groups. Blood samples were obtained at the end of reperfusion for determination of biochemical markers of liver injury and tissue samples were also collected for histology. Our results show that simvastatin reduced the increase on the level of biochemical markers for liver injury when compared to I/R group: AST (965 ± 52 vs. 740 ± 27 IU/L) and ALT (1000 ± 63 IU/L vs. 535 ± 35 IU/L), but also LDH (8243 ± 873 vs. 4495 ± 432 IU/L) and γ-GT (3.67 ± 0.6 vs. 1.30 ± 0.3IU/L). We demonstrate here that simvastatin, an inhibitor of HMG-CoA reductase and a ligand of PPAR-α and PPAR-γ, causes a substantial reduction in the ischemia-reperfusion-induced increase in the serum levels of hepatic injury markers, providing the evidence that statin administration reduces the liver injury induced by ischemia-reperfusion, in the rat. We propose that statins, such as simvastatin, might be useful pharmacological tools in liver injury associated with ischemia-reperfusion of the organ.

First Author: [email protected]

P138 INSULIN AND TDZD-8, A GLYCOGEN SYNTHASE KINASE-3β INHIBITOR, REDUCE THE LIVER INJURY CAUSED BY ISCHEMIA-REPERFUSION IN THE RAT

Bruno Sepodes, Joao Rocha, Rui Pinto, Christoph Thiemermann, and Helder Mota-Filipe. Faculty of Pharmacy - University of Lisbon, 1600-083 Portugal

Glycogen synthase kinase 3β (GSK-3β) is a serine/threonine protein kinase involved in the modulation of the inflammatory response. GSK-3β plays a pivotal role in the regulation of the activation of NF-κB, activation of which has been implicated in the pathogenesis of several diseases. Here we investigate the effects of two GSK-3β inhibitors on the liver injury caused by ischemia-reperfusion injury, in the rat, using a synthetic inhibitor, TDZD-8, previously reported by our team to reduce liver and neuromuscular injury caused by skin burn; and the endogenous inhibitor, insulin, which as an important role in the therapy of critically ill patients (sepsis, trauma). Male Wistar rats were randomly allocated into 6 groups as described: (1) Sham Group; (2) I/R Group: rats which were subjected to liver ischemia for 45 minutes interrupting blood supply to 3/4 of the liver, followed by reperfusion for 2 h; (3) TDZD-8 + I/R Group: rats which received TDZD-8 (1 mg/kg i.v.) 30 minutes prior to liver I/R; (4) Insulin + I/R Group: rats which received insulin (1,4 UI/kg i.v.) 30 minutes prior to liver I/R; (5) TDZD-8 and (6) Insulin Control Groups. Blood samples were obtained at the end of reperfusion for determination of biochemical markers of liver injury and tissue samples were also collected for histology. Our results show that both TDZD-8 and insulin administration reduced the increase on the level of biochemical markers for liver injury when compared to I/R group: AST (1132 ± 61 vs. 541 ± 254 and 555 ± 10 IU/l, for TDZD-8 and insulin, respectively) and ALT (1941 ± 92 vs. 583 ± 90 and 174 ± 27 IU/l, for TDZD-8 and insulin, respectively), and the same was observed for LDH and γ-GT, and light microscopy examination We demonstrate here that TDZD-8 and insulin cause a substantial reduction in the ischemia-reperfusion-induced increase in the serum levels of hepatic injury markers, providing, to our knowledge for the first time, the evidence that inhibition of GSK-3β reduces the liver injury induced by ischemia-reperfusion, in the rat. We propose that GSK-3β inhibitors, such as TDZD-8 and insulin, may be useful in the therapy of liver injury associated with ischemia-reperfusion of the organ.

First Author: [email protected]

P139 Cariporide, a Na+/H+ exchange (NHE) inhibitor, blocks leukocyte endothelium interaction

M. Buerke, U. Buerke, D. Pruefer, and K. Werdan. Department of Medicine III, Martin-Luther-University, Halle/Saale, Germany; *Department of Cardiothoracic and Vascular Surgery, Johannes Gutenberg-University, Mainz, Germany

Background:

Cariporide (HOE 642) a Na+/H+exchange (NHE) inhibitor has been shown to ameliorate ischemia/reperfusion (I/R) injury by reduction of cytosolic Ca2+-overload. In this regard intracellular Ca2+-rise has been demonstrated to mediate cellular activation, inflammatory mediator release, gene activation and inflamatory injury.

Methods:

The effects of Cariporide on leukocyte-endothelial cell interactions were observed by intravital microscopy in the rat mesenteric microcirculation in vivo. Further, its effect on P-selectin expression on the vasculature was analysed by immunhistochemical analyses. Inflammatory activation (i.e., leukocyte rolling, firm adherence and transmigration) were studied in the microcirculation after thrombin superfusion (0.5 U/ml) or after hemorrhage (60 min) and reperfusion (90 min). Cariporide (5 or 10 mg/kg) was administrated as an i.v. bolus injection 5 minutes after thrombin superfusion or 5 minutes before reperfusion.

Results:

Treatment with Cariporide significantly reduced the leukocyte rolling and adhesion in the rat mesenteric microcirculation superfused with thrombin from 55 ± 8 to 4 ± 2 rolling cells/min and 12 ± 2 to 3.2 ± 1.6 adhering cells/100 μm (P < 0.01). After hemorrhage followed by reperfusion leukocyte rolling was decreased from 54 ± 6.2 to 2.4 ± 1 cells/min (P < 0.01) and leukocyte adherance was decreased from 6.3 ± 1.9 to 1.2 ± 0.4 cells/100 μm (P < 0.01) following Cariporide treatment. Leukocyte extravasation was decreased by approximately 90 min after reperfusion. Immunhistochemical detection of endothelial cell adhesion molecule P-selectin showed a profound decrease in endothelial cell surface expression within the internal vasculature following Cariporide administration compared to vehicle treatment (35.4 ± 3.2 vs 14.2 ± 4,1 % (P < 0.01)).

Conclusions:

These data provide evidence, that Cariporide is a potent and effective vasculoprotective compound that inhibits leukocyte-endothelial cell interactions after inflammatory activation. Thus, NHE inhibitor Cariporide seems to have important anti-inflammatory effects beside the lowering of cytosolic Ca2+levels. Reduction of endothelial P-selectin expression may present a mechanism underlying this Na+/H+exchange inhibitor effect.

P140 Sirolimus exerts anti-inflammatory potency by inhibition of leukocyte endothelium interaction

U. Buerke, D. Pruefer, K. Werdan, and M. Buerke. Department of Medicine III, Martin-Luther-University, Halle/Saale, Germany; *Department of Cardiothoracic and Vascular Surgery, Johannes Gutenberg-University, Mainz, Germany

Sirolimus, a mTOR inhibitor, has been shown to inhibit rejection after transplantation, inhibits SMC prolipferation, and ameliorate inflammation. In our study we observed the ability of Sirolimus to modulate leukocyte-endothelium interactions under acute inflammation.

The effects of Sirolimus on leukocyte-endothelial cell interactions were observed by intravital microscopy in the rat mesenteric microcirculation in vivo and P-selectin expression by immunohistochemical analyses in the microvasculature. Inflammatory effects (i.e., leukocyte rolling, adherence and transmigration) were studied after mesenteric superfusion with thrombin (0.5 U/ml) or after hemorrhage (60 min) and reperfusion (90 min). Sirolimus (2 μg/kg) was administrated by an i.v. bolus injection 5 minutes after thrombin superfusion or 5 min prior to reperfusion.

Treatment with Sirolimus significandly reduced leukocyte rolling and adhesion in the rat mesenteric microcirculation superfused with thrombin. Similar, following hemorrhage and reinfusion of the shed blood, Sirolimus reduced significantly leukocyte rolling and adherence. Further, leukocyte extravasation was decreased by Sirolimus approximately 90 min after reperfusion. Immunhistochemical analysis of endothelial cell adhesion molecule P-selectin expression showed a significant decrease within the intestinal vasculature after Sirolimus treatment.

These data provide evidence, that Sirolimus inhibits leukocyte-endothelium interaction after ischemia and reperfusion or thrombin activation. Thus, mTOR inhibitor Sirolimus seems to have important anti-inflammatory effects beside the anti-prolifarative effect. Reduction of endothelial P-selectin expression may present a mechanism underlying this effect.

P141 TNF-ALPHA DECREASES PSEUDOMONAS AERUGINOSA-INDUCED MORTALITY IN BURN THROUGH NEGATIVELY REGULATING TOLL-LIKE RECEPTORS

L.W. Chen, P.S. Chen, and C.M. Hsu. Institute of Emergency and Critical Care Medicine, National Yang-Ming University, Taipei; Taiwan

Tumor necrosis factor (TNF-α) is a potent proinflammatory cytokine and important in inducing the acute-phase response, leading to physiological changes that serve to eliminate the infecting organism. Toll-like receptors (TLRs) comprise a family of pattern-recognition receptors that detect conserved molecular products of microorganisms. P. aeruginosa is extremely efficient at colonizing burn wounds, spreading systemically, and causing sepsis, which often results in a systemic inflammatory response and death. To evaluate the role of TNF-α and TLRs in P. aeruginosa-induced mortality in thermal injury, we injected 109 P. aeruginosa (109/ml in PBS) in the back of the wild type (WT) mice, Tnfrsf1a-/- mice, and TLR4-/- mice at 8 hr after 30% total body surface area (TBSA) burn. At 24 hr after burn, MPO activity and TLR4 mRNA expression of lung of animals were measured. P. aeruginosa induced an increase of TLR4 mRNA as well as protein expression and increase of MPO activity and of lung in Tnfrsf1a-/- mice compared with WT mice. On the contrary, P. aeruginosa induced a less MPO activity and TLRs expression in lung of TLR4-/- mice compared with WT mice and Tnfrsf1a-/- mice. Taken together with the increase of mortality in Tnfrsf1a-/- mice and decrease mortality in TLR4-/- mice after P. aeruginosa infection in burn, we concluded that TNFα decreased P. aeruginosa-induced lung damage through negatively regulating TLRs expression.

P142 BLOOD FLOW REGISTRATION IN PORCINE LIVER ISCHEMIA/REPERFUSION INJURY MODEL - COMPARISON OF UREA AND ETHANOL CLEARANCE USING MICRODIALYSIS

S. Farnebo, E. Zettersten, A. Samuelsson, and F. Sjöberg. Department of Plastic Surgery, Hand Surgery and Burns, Institution of clinical and experimental medicine, University Hospital, Linköping, Sweden

We have in a previous study introduced urea as a possible marker for blood flow registration in rat skeletal muscle, using microdialysis. In this paper we compare this novel technique to the established and validated microdialysis ethanol technique in order to further strengthen its use as an important tool in the understanding of blood flow dependent recovery during microdialysis. A novel porcine liver ischemia/reperfusion injury model was used. A vast and significant increase of urea was seen promptly after induction of the ischemic phase (peak 128% ± 4,96% (mean, SEM), p<0,005) and an even greater rise was seen in ethanol (peak 162% ± 9,29% (mean, SEM), p<0,005). Metabolic markers including lactate, pyruvate and glucose varied accordingly in a manner well recognized as during tissue ischemia. As reperfusion was allowed, all parameters normalised with no significant signs of hyperperfusion, as might have been expected. The ethanol technique appears to be more sensitive than urea in this type of research setting, with a high perfusion rate and large changes in blood flow although the technique has many inherent drawbacks. In conclusion, the urea clearance technique seems highly reliable, easy to execute and widely applicable in various research settings, including the liver.

P143 T CELL SUBSETS DIFFERENTIALLY MEDIATE LIVER TISSUE DAMAGE AND NEUTROPHIL RECRUITMENT FOLLOWING ISCHEMIA-REPERFUSION

S. Kuboki, A.B. Lentsch, and C.C. Caldwell. The Laboratory of Trauma, Sepsis & Inflammation Research, Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio

The Laboratory of Trauma, Sepsis & Inflammation Research, Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio.Liver ischemia-reperfusion results in an acute inflammatory response culminating in the recruitment of activated neutrophils that directly injure hepatocytes. Recent evidence broadly suggests that T cells may mediate this neutrophil-dependent injury, however, the types of T cells involved has not been fully elucidated. Here, we induced partial hepatic ischemia on mice with altered or depleted T cell subsets. After 90 minutes of ischemia and 8 hours of reperfusion, serum ALT and hepatic MPO was determined.

As shown by the above table, depletion of NK1.1-expressing cells, which includes NK and NK T cells, resulted in a 25% decrease in serum ALT. Specific blockade of NK T cell function similarly reduced liver damage by 32%. Neutralization of regulatory T cells did not significantly change liver damage or neutrophil recruitment. Liver MPO, but not serum ALT, was significantly decreased in mice lacking γδ T cells. Mice with an invariant CD4 T cell receptor had a 20% decrease in serum ALT. Thus, the data show that T lymphocytes can mediate tissue damage and neutrophil recruitment following liver ischemia reperfusion. Further, the T cell actions are at least, in part, mediated by antigen specificity and T cell function. (Funded by grants from NIH-NIGMS and Shriners of NA).

Table
Table

P144 ACCURATE AND CONTINUOUS MEASUREMENT OF OXYGEN DEFICIT DURING HEMORRHAGE IN PIGS

J.P. Roesner1, P. Petzelbauer2, A. Koch3, T. Iber1, T.W.L. Scheeren1, B. Vollmar4, R. Zander5, G.E.F. Nöldge-Schomburg1, and K. Zacharowski3. 1Dept of Anesthesia & Intensive Care Medicine, University Hospital Rostock, Germany; 2Dept of Dermatology, Medical University of Vienna, Austria; 3Molecular Cardioprotection & Inflammation Group, Dept of Anesthesia, Bristol Royal Infirmary, University of Bristol, UK; 4Institute of Experimental Surgery, University of Rostock, Germany; 5Physioklin, Mainz, Germany

Hemorrhagic shock (HS)/reperfusion (R) are associated with organ failure and high mortality. Uncontrolled bleeding or prefixed bleeding volume or blood pressure controlled bleeding are traditional models of experimental HS. The latter models are influenced by compensatory mechanisms preventing accurate knowledge about the severity of cellular insult. We have improved an existing method (Rixen et al., 2001; Shock) using oxygen deficit (OD) as an endpoint of hemorrhage severity. 11 male pigs (weight: 28-32 kg) were anesthetized and subjected to an OD (approximately 110 mL/kg/h) using a metabolic monitor (DeltatracTm II, Datex, Ohmeda, Germany). It detects continuously O2 consumption and CO2 production allowing 'online' determination of OD/min. Overall, 4 pigs died in our study (n=2 during HS; n=2 during early R (<2h)). Pig were reperfused with shed blood and allowed to recover for 3 days. Endpoints: liver, kidney and heart damage. OD did not correlate with amount of blood withdrawn, arterial blood pressure or heart rate confirming the original concept of OD as a superior endpoint of severity of HS. An OD of 110 mL/kg/h caused significant liver, kidney and heart damage determined by plasma levels of AST, ALT, creatinine and cardiac TnT.

P145 Bβ15-42REDUCES ORGAN DAMAGE IN A PIG MODEL OF HEMORRHAGIC SHOCK AND REPERFUSION

J.P. Roesner1, P. Petzelbauer2, A. Koch3, T. Iber1, D. Vagts1, T.W.L. Scheeren1, B. Vollmar4, G.E.F. Nöldge-Schomburg1, and K. Zacharowski3. 1Dept of Anesthesia & Intensive Care Medicine, University Hospital Rostock, Germany; 2Dept of Dermatology, Medical University of Vienna, Austria; 3Molecular Cardioprotection & Inflammation Group, Dept of Anesthesia, Bristol Royal Infirmary, University of Bristol, UK; 4Institute of Experimental Surgery, University of Rostock, Germany

The fibrin-derived peptide Bβ15-42 has been shown to reduce myocardial infarct size following ischemia/reperfusion (R). Hemorrhagic shock (HS) followed by volume resuscitation represents a similar scenario, whereby a whole organism is vulnerable to R injury. We subjected male pigs (~30 kg) to HS by withdrawing blood to a mean arterial pressure of 40 mmHg for 60 min. Pigs were then resuscitated with shed blood and crystalloids for 60 min and at this time, Bβ15-42 (2.4 mg/kg, n=8) or vehicle control (PBS; 2.4 mg/kg, n=7) was injected as an iv bolus. Data are presented as mean±SEM. Five hours after resuscitation, controls presented acute lung injury (paO2/FiO2-ratio <300 mmHg; extra vascular lung water index (EVLWI; marker for lung injury): 9.0±0.5 ml/kg) and myocardial dysfunction/damage (cardiac index: 4.3±0.1 l/min/m2; cardiac TnT levels: 0.58±0.15 ng/ml). In contrast, Bβ15-42-treated pigs showed significantly improved pulmonary and circulatory function (paO2/FiO2-ratio >*400 mmHg; EVLWI: *5.2±0.6 ml/kg, cardiac index: *6.3±0.3 l/min/m2; cardiac TnT levels: *0.11±0.03 ng/ml; *p<0.05). We conclude that in a pig model of HS/R, administration of Bβ15-42 during R protects shock susceptible organs.

P146 EFFECT OF INHALED CARBON MONOXIDE ON MICROVASCULAR OXYGENATION OF THE GASTRIC MUCOSA AFTER HEMORRHAGIC SHOCK

I. Bauer, I. Schwartges, C. Beck, B. Berke, L.A. Schwarte, and O. Picker. Dept. of Anesthesiology, Heinrich Heine University Duesseldorf, 40225 Duesseldorf, Germany

Objective:

Maintenance of an adequate microvascular oxygenation (μHbO2) of the gastric mucosa seems to play a major role in the prevention and therapy of sepsis and MOF. Compensatory vasoconstriction under compromised hemodynamic conditions might be prevented by carbon monoxide (CO) through its vasodilatory effects. We investigated the effects of inhaled CO on μ HbO2 in hemorrhagic shock.

Methods:

Anesthetised (1.5MAC sevoflurane, FiO2=0.25; etCO2=35mmHg) dogs (26-28kg.) were randomly assigned to control or CO (100ppm); n=6. Hemorrhagic shock was induced by withdrawal of 20% of the estimated blood volume. We measured systemic hemodynamics, oxygen delivery (DO2), and μHbO2 (tissue spectrophotometry). Data are mean±SEM, ANOVA, Fishers PLSD, p<0.05.

Results:

Under physiological conditions, inhaled CO had no effect on systemic (DO2: 12.1±1.4 vs. 12.2±1.2 ml/kg/min) and local (μHbO2: 76±6 vs. 74±5%) oxygenation, and on systemic vascular resistance (SVR; 26.5±1.9 vs. 26.9±1.6 mmHg x min/l). Hemorrhagic shock led to a significant reduction of DO2 (8.0±0.7 ml/kg/h) and μHbO2 (55±9%), which was not significantly affected by inhalation of CO (DO2: 7.7±1.1; μHbO2: 55±9%). SVR increased with hemorrhagic shock independent of the presence of CO (con: 31.9±2.8; CO: 31.9±2.8 mmHg x min/l).

Conclusion:

In our animal model, low concentrations of inhaled CO had no effect on systemic and local oxygenation under physiological conditions and in hemorrhagic shock. This lack of protection may be explained by the lack of a vasodilatory effect observed with the used concentration of CO.

p147A REPERFUSION AFTER SEVERE HEMORRHAGIC SHOCK IS NOT ACCOMPANIED BY INCREASED OXIDATIVE DAMAGE AT ORGAN LEVEL

W. Gregor1, K. Staniek1, R. T. Hartl1, C. Zifko1, A. Postl1, A. Kozlov1, S. Bahrami1, H. Redl1, and J. C. Duvigneau1. 1Veterinary University Vienna, Austria 2Ludwig Boltzmann Institute for Traumatology, AUVA Research Center, Vienna, Austria

Hemorrhagic-traumatic shock (HTS) is believed to induce oxidative stress, mediating organ damage and multiorgan failure. Here we analyzed the oxidative damage at two different levels: lipid peroxidation (thiobarbituric acid-reactive substances = TBARS) and protein nitration (nitrotyrosine = NT) in various organs in a clinically relevant rat model of HTS [Shock 2006,25:283]. Since oxidative stress can occur at different sites within the cell, we investigated subcellular fractions of the liver (mitochondrial, microsomal fractions and cytoplasm). We did not find differences between sham and shock for both, TBARS and NT, in any of the fractions, but we found up-regulation of anti-apoptotic marker, Bcl-XL (mRNA) in HTS, and a positive correlation (r=0.5; p=0.016) of NT with the Bax/Bcl ratio. Additionally, in the heart we found increased levels of free iron in HTS and a positive correlation between TBARS and NT (r=0.64; p=0.001), as well as between TBARS and levels of nitrosyl-heme complexes (NHC), measured by electron spin resonance spectroscopy, (r=0.473; p=0.023) suggesting a moderate increase of oxidative stress. However, shock did not increase the tissue levels of NHC, and the application of a membrane-permeable scavenger of O2·− radicals during resuscitation (see abstract by C. Zifko) could not decrease the levels of TBARS and NT. Thus, in our shock model reperfusion does not induce serious oxidative damage, possibly because of the induction of potent antioxidative defence mechanisms mediated by Bcl-XL.

P147B EFFECT OF REPERFUSION ON CELLULAR FUNCTIONS IN RATS AFTER HEMORRHAGIC SHOCK

J. C. Duvigneau1, S. Haindl2, T. Behling2, R. T. Hartl1, C. Zifko2, A. Postl1,2, I. Miller1, W. Gregor1, L. Gille1, M. Gemeiner1, S. Bahrami2, H. Redl2, and A. V. Kozlov2. 1Veterinary University Vienna, Austria 2Ludwig Boltzmann Institute for Traumatology, AUVA Research Center, Vienna, Austria

Inadequate delivery of oxygen during severe hemorrhage and trauma (HTS) followed by reperfusion is supposed to induce oxidative stress resulting in the impairment of cellular functions. Therefore it was our aim to investigate markers of oxidative tissue damage together with the function of important cellular structures after HTS in different organs (liver, heart, kidney). Anesthetized rats (n=6) were subjected to HTS (laparotomy, bleeding and resuscitation (inadequate/adequate phase)) followed by a 2h observation period. In animals of the shock group mRNA (real-time PCR) of heme oxygenase (HO)-1 was significantly upregulated in all organs, resulting in an elevated HO activity (liver microsomes). The mitochondrial function (MF), estimated by respirometry, was unchanged in the heart, tended to decrease in the liver, and was significantly decreased in kidney of HTS vs. sham animals. In all organs MF negatively correlated with the mRNA of inducible NO synthase (iNOS) and in kidney additionally with the mRNA of HO-1, suggesting modulation by NO / CO. Activities of cytochrome P450 1A1 (liver microsomal fractions) were not considerably decreased by HTS. Our data show that the investigated cellular functions were not significantly affected under HTS, except in the kidney. Histological examination and markers of oxidative damage revealed no differences between sham and shock. Thus, oxidative stress-induced cellular dysfunction, supposed to cause mortality, is of minor relevance in our model of HTS.

P148 ARACHIDONIC ACID IN POSTSHOCK MESENTERIC LYMPH PROVOKES LUNG INJURY VIA LTB4

J. Jordan, E. Moore, E. Sarin, S. Damle, S. Kashuk, C. Silliman, and A. Banerjee. Denver Health Medical Center; University of Colorado Denver, Denver, CO 80262

Background:

Mesenteric lymph is the mechanistic link between splanchnic hypoperfusion and acute lung injury (ALI), but the culprit mediator(s) remain elusive. Systemic administration of a phospholipase A2 (PLA2) inhibitor attenuated neutrophil (PMN) mediated ALI. Consequently, we hypothesized that gut derived arachidonic acid (AA) is a key mediator in the pathogenesis of ALI via LTB4.

Methods:

Trauma/hemorrhagic shock (T/HS) was induced in SD rats via laparotomy and hemorrhage. The mesenteric duct was cannulated for lymph collection and diversion. Following T/HS, resuscitation included NS and ½ shed blood. AA and LTB4 were measured in the lymph and lungs by LC/MS/MS. AA and a LTB4 receptor antagonist were added in vitro to PMNs and O2 production was measured. Significance (p<0.05) was determined by ANOVA.

Results:

Postshock mesenteric lymph (PSML) increased PMN O2 with a Vmax of 11.56±1.25 vs. 3.95±0.29nmol O2/min (3.75×107 cells/mL); (n=5; p<0.01). PMNs pretreated with a LTB4 receptor antagonist had attenuated priming (2.64±0.58). LTB4 increased in the lungs following T/HS compared to control animals 104.5pmol±18.7 vs. 13.4pmol±4.7 (n=4; p<0.05). Lymph diversion reduced lung LTB4 by 75% and PMN accumulation by 40%. AA increased in PSML following T/HS. AA delivery to the lungs increased from 129.5μg/hr to 749.8μg/hr postshock.

Conclusion:

Splanchnic I/R activates gut PLA2 to release AA intoPSML where it is conveyed to the lungs and generates LTB4 which attracts PMNs, amplifies further LTB4 production and subsequent ALI.

P149 HYPOXIC MACROPHAGES DECREASE CARDIAC MYOCYTE SURVIVAL

V.P. Good, J.E. Baumgardner, K. Takahashi, E.J. Miller, K.B. Margulies, and C.M. Otto. University of Pennsylvania, Philadelphia, PA 19104

Introduction:

Hypoxia triggers a series of cellular responses by limiting substrate for metabolic and synthetic pathways and by activating transcription factors. Products of hypoxic cells can initiate signaling in nonhypoxic cells. We tested the effect of soluble factors released from hypoxic macrophages on cardiac myocytes cultured under standard conditions.

Methods:

A murine macrophage cell line (RAW264.7) was cultured for up to 20 hours in a forced convection cell culture system. Cells were randomly assigned to either 40 Torr O2 (normoxia) or 8 Torr O2 (hypoxia) in DMEM/5%FBS equilibrated with 5% CO2. Conditioned media was collected for 10 min following 6 and 18 hours of culture (flow rate 0.256ml/min). Media from neonatal rat cardiac myocytes grown to confluence on fibronectin coated wells was replaced with normoxic or hypoxic conditioned media or fresh DMEM/5%FBS. Adherent cells were lysed after 6 or24 hours of culture in a standard incubator. Cellular lysates wereassayed for protein and by western blot for inducible nitric oxide synthase (iNOS) and macrophage migration inhibitory factor (MIF).

Results:

Conditioned media from hypoxic macrophages significantly decreased adherent cardiac myocytes at 24 hours (p<0.001). At 6 hours, prior to any change in cardiac myocyte survival, exposure to hypoxic conditioned media increased MIF (p=0.046). iNOS was not expressed in any group.

Conclusions:

Hypoxic macrophages release soluble factors that alter cardiac myocyte protein expression and survival, a signaling pathway that may contribute to myocardial injury in diseases with regional tissue hypoxia.

Supported by NIH GM64486

P150 MANNAN-BINDING LECTIN DEFICIENCY PREVENTS ACUTE LUNG INJURY AFTER RUPTURED ABDOMINAL AORTIC ANEURYSM

William S. Johnson, Barry B. Rubin, and Thomas F. Lindsay. Toronto General Hospital, Ontario, Canada, M5G 2C4

Background:

Ruptured abdominal aortic aneurysm (RAAA) is associated with a systemic inflammatory response and multi-organ injury that is dependent on complement activation. We hypothesized that Mannan-Binding Lectin (MBL) was a principle mechanism for complement activation and multi-organ injury after RAAA.

Methods:

Male C57/BL6 wild type and MBL-A/C knockout mice modelled RAAA with 60 min. of hemorrhagic shock, 30 min. of supramesenteric aortic clamping, and 120 min. of reperfusion. Permeability to I-125 albumin quantified local gut and remote lung injury. Tissue levels of TNF and myeloperoxidase (MPO) assessed cytokine response and neutrophil sequestration.

Results:

Table
Table

*P<.05 vs. other groups

Conclusions:

MBL deficiency protects against remote lung injury after RAAA despite increased neutrophil sequestration. Deficiency does not protect against local gut injury, suggesting differential mechanisms of complement activation between tissues or the influence of other inflammatory pathways independent of TNF or neutrophils. Targeted intervention against MBL may hold value in protection against acute lung injury after RAAA.

P151 SPHINGOSINE 1-PHOSHATE HAS DUAL REGULATORY FUNCTIONS IN ENDOTHELIAL CELL (EC) PERMEABILITY AND CA2+ METABOLISM

Kiyoshi Itagaki1, Atsuko Yatani, Carl J. Hauser1, Zoltan Spolarics, and Edwin A. Deitch. 1Department of Surgery, Beth Israel Deaconess Medical Center, Boston, Massachusetts and Department of Surgery, UMDNJ, Newark, New Jersey USA

Objective:

Increased EC permeability is one of the major underlying causative mechanism of ALI and ARDS. Spingosine 1-phosphate (SIP) is a lysolipid that can mobilize cell Ca2+ either through receptor-based mechanisms or as an intracellular 2nd messenger. We studied the role of endogenously generated S1P in EC Ca2+ flux and barrier function.

Methods:

Human umbilical vein endothelial cells (HUVECs) were stimulated by thrombin (T), histamine (H) or other agonists with or without the cell S1P synthesis inhibitor Sphingosine Kinase Inhibitor 2 (SKI-2). Ca2+ flux was measured using a fluoroprobe and HUVEC permeability was assessed by dextran diffusion through monolayers.

Results:

Both T or H caused Ca2+ release from cell Ca2+ stores and then caused Ca2+ entry through store-operated channels (SOC). This was accompanied by an increase in EC permeability. SKI-2 decreased T and H induced Ca2+ entry and protected against associated permeability increases. In contrast to the effects of internally generated S1P, the external administration of S1P initiates Ca2+ release similar to that of thrombin or histamine but conversely, decreases EC permeability.

Conclusion:

These observations indicate that after stimulation by T or H, cellular generation of S1P serves as a positive modulator of HUVEC Ca2+ entry via SOC and mediates increased permeability. In contrast, extracellular exposure to S1P acts through different receptors that have opposite effects. Our data cautions that a potential dual role of endogenous and exogenous S1P in EC function needs to be considered in pharmacological studies targeting sphingosine metabolism in the treatment of ALI and ARDS.

Figure
Figure:
Thrombin or histamine-induced EC permeability increase was reduced by SKI-2 treatment.

P152 A NOVEL EVALUATION OF SHOCK ASSOCIATED LUNG INJURY BY ACTIVATED NEUTROPHIL COLLAGENASE 2 IN RATS

Bozena Antoniu, Kiyoshi Itagaki, Qin Zhang, Mustafa Raoof, and Carl J. Hauser. Department of Surgery, Beth Israel Deaconess Medical Center, Boston, MA 02215

Background:

Trauma and shock are commonly followed by inflammation that can cause acute lung injury (ALI). This can eventually progress to the Acute Respiratory Distress Syndrome or multiple organ failure. The pathogenesis of these processes is in part attributable to migration of activated neutrophils (PMN) through the endothelial basement membrane, which is required for organ attack. Migration of PMN through the extra-cellular matrix is facilitated by enzymes like the matrix methalloproteinases (MMPs) that can degrade extra-cellular matrix. MMP-8 / Collagenase2 is stored in PMN secondary granules and released on stimulation by proinflammatory cytokines or other immune stimuli. MMP-8 cleaves collagen I, II, and III and infiltrates connective tissues with high capacity resulting in breakdown of the extra cellular matrix.

Objectives:

1) Determine the role of activated MMP-8 in the genesis of shock related ALI; 2) assess whether the presence of MMP-8 can be used as a probe to elucidate extra-cellular matrix disruption in early, mild lung injury, and 3) since MMP-8 is exclusively expressed in PMN, assess whether it can be used to isolate and study the role of the PMN in the mechanisms of ALI.

Methods:

Traumatic / hemorrhagic shock (T/HS) was induced in rats by the performance of a laparotomy followed by 90 minutes of hemorrhage to MAP 30-40. Rats were then resuscitated with their own shed blood and allowed to recover for 3, 6, and 24 hours at which time the lungs were harvested to evaluate injury. Broncho-alveolar lavage fluids (BALF) and lung homogenates were assessed for MMP-8 by zymography followed by densitometry and MMP-8 specific fluorogenic substrate cleavage. MMP-8 was assayed by ELISA and MMP-8 expression was assessed by western blotting. Pulmonary PMN infiltration was assessed by MPO and capillary leakage was assessed by BALF albumin content.

Results:

MMP-8 activity by zymography /densitometry and kinetic measurement increased 4-fold from 1909(se±266) IDV in naive rat comparing to 7461(se ±1076) IDV in T/HS rats recovering for 3 and 7121(se ±6780) IVD for 6 hours Vmax from 2101 to 10261 mU/min, respectively. MMP-8 infiltration into lung tissue by ELISA showed increase at 3 and 6 hours (OD-450) from 0.07(se±0.017) to 0.44 (se±0.038) and 0.48 (se±0.044) respectively. MPO assay showed PMN presence peaked in tissue homogenates at 3 hours (120mU) and in BALF at 24 hours (48mU). Albumin leak was maximum (980 ng/ml) 3 hours after T/HS.

Conclusion:

MMP-8 is expressed by activated PMN and likely plays a key role upregulation of MMP-8 from PMN is associated with acute lung injury and may contribute to the pathogenesis of ARDS after T/HS. Further studies of the involvement of specific protease inhibitors may help elucidate the role on MMP secretion in lung injury after trauma and shock. MMP assays are a sensitive and potent tool for the study of early lung injury after trauma and shock.

P153 NMR METABOLOMIC INVESTIGATION IN POST-HEMORRHAGIC SHOCK MESENTERIC LYMPH

Tomohiko Masuno, Tetsutaka Sano, Hiroyuki Yokota, Norio Sato, Takao Suzuki, Keiko Hirakawa, Yokichi Ohno, Junichi Aiboshi, and Kaoru Koike. Dept. of Emergency and Critical Care Medicine Nippon Medical School, Tokyo, Japan 113-8603

Post-hemorrhagic shock mesenteric lymph (PSML) has been shown to contain proinflammatory mediators elaborated from the ischemic gut and cause distant organ injury. However, identification of bioactive mediators which trigger organ injury remains elusive. Metabolomics is the comprehensive analysis of metabolites which may allow discovery of biomarkers. The purpose of this study was to determine that metabolomic profiling employing nuclear magnetic resonance (NMR) spectroscopy and multivariate pattern recognition techniques could identify the different metabolite profiles of PSML.

Methods:

Rats underwent hemorrhagic shock (MAP 40 mm Hg × 30 min) and then resuscitation (shed blood + normal saline in 2x shed blood volume) over 2 hours. Mesenteric lymph was collected hourly up to 2 hours after shock. The spectra of metabolites in mesenteric lymph from each time point were obtained using NMR spectroscopy with different pulse programs, and the data were analyzed by multivariate analysis (principle component analysis and SIMCA method).

Results:

The analysis showed that the metabolite patterns of mesenteric lymph obtained during shock, first resuscitation hour (R1), and second resuscitation hour (R2) were distributed to each different cluster area. NMR spectroscopy using different pulse programs identified the changes in macromolecules (lipids and proteins) as well.

Conclusion:

NMR metabolomics was useful to identify the changes in metabolites, lipids, and proteins in mesenteric lymph following hemorrhagic shock. Metabolomics may help to discover the biomarkers and provide insight into the pathogenesis of post-hemorrhagic shock organ injury.

P154 TRANSPERITONEAL OXYGENATION WITH HYPERBARICALLY OXYGENATED PERFLUOROCARBONS IN A RAT HYPOXIC MODEL

Motohiro Nishiyama, Naoki Unno, Kazunori Inuzuka, Naoto Yamamoto, Daisuke Sagera, Hiroki Tanaka, and Hiroyuki Konno. Second Dept. of Surgery, Hamamatsu Univ. School of Medicine, Hamamatsu, Japan

Backgrounds:

Perfluorocarbons (PFC) have a high solubility for oxygen. The aim of this study is to investigate the effect of intraperitoneal administration of hyperbaricaly oxygenated PFC (oxy-PFC) on transperitoneal oxygenation in hypoxic condition.

Methods:

Rats were intubated and rendered hypoxic by ventilating them with 15 % oxygen and 85 % nitrogen to cause hypoxemia. Intraperitoneal administration of 5 atm HBO-PFC was performed at 10 minutes after the start of ventilation in six rats (Group A) and saline solution was administrated similarly in seven rats (Group C)(Time = 0). Arterial blood samples were drawn from the carotid artery every 15 minutes until 60 minutes (Time = 60). The blood gas was analyzed every 15 minutes.

Figure
Figure

Results:

In both groups, mean arterial blood pressure was stable being around 100 mmHg until 60 minutes (Time = 60). PaO2 gradually decreased, however, the reduction was significantly ameliorated in Group A.

Conclusion:

This study has demonstrated that some degree of transperitoneal oxygenation can be obtained by intraperitoneal administration of HBO-PFC. This technique may have potential for assisting patients with respitratory failure.

Organ Dysfunction Clinical Research

P155 RELATIVE ADRENAL INSUFFICIENCY IS ASSOCIATED WITH POSTINJURY MULTIPLE ORGAN FAILURE (MOF)

Walter L. Biffl, Jeffrey Johnson, Ernest E. Moore, David Ciesla, Anirban Banerjee, and Angela Sauaia. Denver Health Medical Center & University of Colorado, Denver, Colorado, USA

Adrenal insufficiency (AI) is common in critical illness, and appears to have prognostic implications in sepsis. Its significance in trauma, however, is unclear. We hypothesized that AI was common among trauma patients, and that treatment with cortisol (CORT) would improve survival.

Methods:

Over a two-year period, trauma patients at risk for MOF (Injury Severity Score [ISS])>15, >6U blood) had measurement of baseline CORT and a cosyntropin stimulation test (STIM) when AI was clinically suspected. Those with confirmed AI (baseline CORT <25μg/dL or response to STIM <10μg/dL) were treated with CORT.

Results:

Of 262 patients at risk for MOF, 55 (21%) had relative AI. Of those with AI, 24 (44%) developed MOF, compared with 23 (11%) of those without AI (p<.05). Mortality among AI patients was 5%, versus 6% among non-AI patients. Of those who were tested, nonsurvivors had significantly higher baseline and postinjury day 1 CORT levels than survivors. The effects of AI were independent of ISS and transfusion.

Conclusions:

Patients with relative AI have a higher incidence of postinjury MOF than similar-risk patients with normal adrenal function. However, mortality is not increased by AI. Further investigation is warranted to determine whether a) AI is a risk factor for, or a symptom of, postinjury MOF; and b) timely administration of CORT impacts mortality.

P156 TREATMENT OF MODS IN ACUTE PANCREATITIS

I. Bihalskyy, S. Chooklin, and O. Granat. Medical University, Lviv, Ukraine

System complications are most important in occurrence multiple organ dysfunctions and are a principal lethality cause in early terms of severe acute pancreatitis. Development of the multiple-organ dysfunction syndrome (MODS) often determined the unfavorable outcome of necrotizing pancreatitis. Among them proinflammatory cytokines and oxidative stress accept critical participation in development of local and system complications in severe acute pancreatitis.

Levels of some interleukins (IL), such as IL-1β, IL-8, and IL-18, tumor necrosis factor α (TNF-α), adhesion molecules (ICAM-1, E-selectin), malondialdehide (MDA), myeloperoxidase (MPO), lipid peroxide (LPO) were measured in 45 patients with acute pancreatitis. In 30 patients in complex treatment included dexamethasone (16 mg/day), ascorbic acid (4 g/day), pentoxifylline (400-600 mg), geptral (800 mg), N-acetylcystein (1200 mg).

MODS is the typical for the early stages of severe acute pancreatitis. Increased levels of all mediators were noted in all patients with acute pancreatitis already at the time of admission. Concentration of IL-1β, IL-18, ICAM-1, and E-selectin clear correlated with the severity of MODS and development of local complications (necrosis and fluid collections). The LPO level correlated with CRP concentration and index of pancreas damage, determined in computed tomography (Balthazar criterion). Concentration of TNF-α correlated with MDA level. After therapy researched parameters were faster normalized. Incidence of organ dysfunctions decreased.

Thus, the necrotizing pancreatitis characterized by the elevated levels of proinflammatory cytokines and adhesion molecules, oxidative stress activated, which determined the MODS development. The blocking of the excessive synthesis of inflammatory mediators, oxidative stress improves the clinical course of necrotizing pancreatitis.

P157 EFFECTS OF TIGHT GLYCEMIC CONTROL ON CHOLESTASIS AND BILIARY SLUDGE IN MEDICAL CRITICALLY ILL PATIENTS

YM Vanwijngaerden, J Wauters, D Mesotten, P Wouters, I Milants, A Wilmer, and G Van den Berghe. Department of Intensive Care Medicine, University Hospital Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium

Background:

Cholestatic liver dysfunction has been linked to increased risk of mortality in the ICU population. In a large prospective randomized controlled trial, the incidence of hyperbilirubinaemia was shown to decrease with tight glycaemic control via intensive insulin therapy (IIT) as compared with conventional insulin therapy (CIT). Our aim was to further examine cholestatic liver dysfunction and biliary sludge (BS) prospectively in a large medical ICU population.

Methods:

This study was a pre-planned subanalysis of a large (n=1200) trial on the effects of IIT on the outcome of medical critical illness. To estimate the prevalence of BS, patients (n=658) with a stay ≥ 5 days were selected. Of these 658 patients, 119 had an interpretable sonography (IIT group) on day 5 and 137 in the CIT group. A second sonography was performed on day 10 with only 68 patients in each therapy group. In all patients (n=658) morning blood samples, from admission to either discharge or death, were examined for total bilirubin (bili), alkaline phosphatase (AP) and gamma-glutamyl transferase (GGT). Cholestasis was defined as AP > 400 IU/L and gGT > 80 UI/L or bili > 3 mg/dL. The effect of IIT on the incidence of cholestasis was assessed using Kaplan-Meier cumulative hazard analysis. p<0.05 was considered significant.

Results:

IIT reduced the prevalence of BS from 66.4% in the CIT to 50.4% on day 5 (p=0.01). However, the difference in BS prevalence was not significant on day 10 (CIT: 66.2% vs IIT 57.4%, p=0.3). Cumulative hazard for the first day of appearance of cholestasis was significantly lower in the IIT group (p= 0.039 for the bili>3 mg/dL definition, p=0.05 for the AP >400 and GGT> 80 IU/L).

Conclusion:

IIT appears to reduce the incidence of cholestasis and biliary sludge in prolonged critical illness.

P158 STIMULATING EFFECTS OF SERA FROM SEVERE TRAUMA PATIENTS ON NF-κB ACTIVITY IN MACROPHAGE AND THEIR RELATIONSHIP WITH PATIENTS PROGNOSIS

Huaping Liang, Xiang Xu, Hao Xia, Jun Fei, Dan Wu, and Zhengguo Wang. State Key Laboratory of Trauma, Burns, and Combined Injury, Department 1, Research Institute of Surgery, Daping Hospital, Third Military Medical University, Chongqing 400042, China

Objective:

To investigate the stimulating effects of sera from severe multiple trauma patients within 24h post trauma on nuclear factor-κB (NF-κB) activity in macrophage and their relationship with patients prognosis.

Methods:

24h after transfection of the recombinant NF-κB plasmid containing luciferase reporter gene into the mouse macrophage line (RAW264.7), the cells were stimulated by different sera for 6h, then stimulating effects of sera on NF-κB were detected by luciferase action, The concentration of interleukin-1β, tumor necrosis factor α, interleukin-10, interleukin-1 receptor antagonist, and soluble tumor necrosis factor receptor I were detected with ELISA kits.

Results:

The stimulating activity of NF-κB was increased significantly in traumatic sera group, which is higher in MODS group, nonsurvivors group than that in non-MODS, survivors group respectively. These changes were correlated positively with APACHE II score, while had no relationship to the cytokine or endogenous antagonist levels. The area under the receiver operator characteristic (ROC) curve of NF-κB activity for predicting MODS and mortality was significantly higher than that of APACHE II score.

Conclusion:

Early measurement of NF-κB stimulating activity of sera from severe multiple trauma patients may show the predictive value for the late occurrence of MODS and mortality.

P159 TRANSSEROSAL LEAKAGE OF GUT DERIVED -INFLAMMATORY MEDIATORS DURING ABDOMINAL AORTIC ANEURYSM SURGERY

Daisuke Sagera, Naoki Unno, Hiroshi Mitsuoka, Naoto Yamamoto, Kazunori Inuzuka, and Hiroyuki Konno. Division of Vascular Surgery, Second Dept. of Surgery, Hamamatsu Univ. School of Medicine, Hamamatsu, Japan

Backgrounds:

Gut produces various inflammatory mediators which can enter systemic circulation via mesenteric lymphatic route or portal venous route. The aim of this study is to assess the third route of the mediator via the peritoneal cavity.

Methods:

During open surgery of abdominal aortic aneurysm (AAA), small intestine was placed in an intestinal bag in five patients. Ascites was sampled from the bag at the end of surgery and stored at -70°C. Three months after the surgery when the patients fully recovered, blood was sampled. By addition of the stored ascites, neutrophil activation was investigated to assess the pseudopod projection morphologically and CD11b expression by flow cytometry.

Results:

A significant increase in pseudopod formation was observed by adding the ascites. Moreover, ascites induced CD11b expression in neutrophil.

Figure
Figure

Conclusion:

Ascites derived from small intestine during AAA surgery included inflammatory mediators, which suggested the possibility of the third route of mediators from gut to the systemic circulation via peritoneal cavity.

Intensive Care Basic Research

P160 GLUTAMINE: A NOVEL LIGAND DEPENDENT ACTIVATOR OF PPARγ

K Ban, JM Sprunt, and RA Kozar. University of Texas-Houston, Houston, TX 77030

We have shown clinically and in the laboratory that glutamine possesses gut protective effects under conditions of hypoperfusion and that protection is mediated by an increase in the anti-inflammatory transcriptional regulator, peroxisome proliferator receptor gamma (PPARγ). Once activated, PPARγ binds to a PPARγ response element in the promoter of target genes. We hypothesized that glutamine activates PPARγ via a ligand-dependent mechanism.

Methods:

IEC-6 cells were co-transfected with PPRE-luciferase promoter/reporter constructs and Renilla expression vectors. Cells were treated with glutamine± GW9662 (specific inhibitor which binds to ligand binding site) then stimulated with H2O2 (0.125 μM) to induce oxidant stress. Lysates were analyzed for PPRE luciferase as an indicator of PPARγ activation and normalized to Renilla expression. PPARγ nuclear activity was measured by EMSA. Results are mean±SEM, n=3 experiments, ANOVA.

Results:

A concentration-dependent increase in luciferase activity was observed with increasing glutamine under normal and oxidant conditions. This effect was abrogated by the PPARγ inhibitor, GW9662, and paralled changes in PPARγ nuclear activity.

Conclusions:

Results suggest that transcriptional activation of PPARγ by glutamine occurs via a ligand-dependent mechanism, a novel finding which warrants further investigation.

P161 INFLUENCE OF INTRAVASCULAR VOLUME STATE ON RESPIRATION DEPENDENT PaO2 OSCILLATIONS

S Bierschock, M Bodenstein, A Vogt, H Wang, and K Markstaller. Dept. of Anesthesiology, Johannes Gutenberg-University Mainz 55131, Germany

Minor changes of intraarterial PO2 within respiratory cycles are known to exist in healthy, spontaneously breathing subjects. However, recent studies revealed much higher PaO2 oscillations in animal models of saline lavage ARDS, best explained by cyclic recruitment and derecruitment of atelectatic lung areas. This study was designed to investigate the influence of intravascular volume state on the amplitude of PaO2 oscillations. Therefore, eight pigs were anesthetized and ventilated via endotracheal tube in pressure controlled mode. Ultrafast PaO2 in-vivo measurements were performed by fluorescence quenching technique, using a fiberoptical, Ruthenium tipped catheter placed in the ascending aorta. After baseline measurements, different volume levels (hypo- and hypervolemia) were provoked by volume challenges of 10 ml / kg in randomized order. The oscillation amplitude of PaO2 decreased about 66% by inducing hypervolemia and increased about 34.2% during hypovolemia from baseline. A broad range of oscillation amplitudes was observed. Effect size analysis showed a medium effect between hyper- and hypovolemic state. Volume state dependent changes of the oscillation amplitude in PaO2 within respiratoy cycles can be explained by dynamic changes of physiological deadspace in overdistended areas during inspiration and consecutive redistribution of perfusion to shunt areas, specifically in volume depleted subjects.

Funding: DFG Ma 2398 / 3

P162 HEMORRHAGIC SHOCK-RESUSCITATION DOES NOT EXACERBATE VENTILATOR ASSOCIATED LUNG INJURY IN PIGS

H Wang, M Bodenstein, B Duenges, S Ghanaati, and K Markstaller. Dept. of Anesthesiology, Johannes Gutenberg-University, Mainz 55131, Germany

Mass bleeding and transfusion combined with non-protective ventilation may lead to ventilator associated lung injury (VALI). To explore whether this hemorrhagic shock-resuscitation exacerbates VALI, pigs were randomized to control (CTR), SHAM and hemorrhagic shock/resuscitation (HS/R) groups and ventilated by pressure controlled ventilation (FIO2 1.0, RR 8/min, VT 20 mL/kg). After 1.5hrs (CTR) or 5 hrs (SHAM and HS/R) of ventilation, pigs were executed by exsanguination. Wet-to-dry weight ratio (W/D, measured from the mid lobe of the right lung), total protein content in bronchoalveolar lavage fluid (BALF, sampled from the lower lobe of the right lung) and histopathology scoring for 9 injury categories (on eleven tissue sectors sampled from the left lower lobe) were used to evaluate lung injury. We found W/D in the SHAM group increased significantly versus CTR group. BALF total protein content showed no difference between the three groups. Only sporadic overdistension, atelectasis and congestion were found in the CTR group. The SHAM and HS/R groups showed specific patterns of lung injury, with prominent lesions of atelectasis, overdistension, inflammation, interstitial edema and congestion. Histopathology scores for atelectasis, inflammation and interstitial edema were significantly higher in SHAM and HS/R groups than in CTR. However, no difference was found either in the scores for the individual categories or in the total injury score between SHAM and HS/R. We conclude that a brief hemorrhagic shock-resuscitation does not exacerbate ventilator associated lung injury.

P163 COMPARISON OF RESPIRATION DEPENDENT ARTERIAL PO2 OSCILLATIONS WITH SpO2 OSCILLATIONS

S. Boehme, M. Bodenstein, J. Baumgardner, H. Wang, and K. Markstaller. Dept. of Anesthesiology, Johannes Gutenberg-University Mainz 55131, Germany

PaO2 oscillations during artificial ventilation in animal models of ARDS reflect cyclic recruitment of atelectasis. A non-invasive, bedside measurement technique like pulse oximetry, to detect oscillations in oxygenation in the intensive care setting could be of interest in order to depict cyclic recruitment of atelectasis. The aim of this study was to compare respiratory changes of SpO2 with changes of PaO2 within a respiratory cycle. With IRB approval, three pigs with differently injured lungs were mechanically ventilated. Using fluorescence quenching technique (Ocean Optics, Inc., USA), respiration dependent PaO2 oscillations were recorded when mean PaO2 reached values below 150 mmHg. Simultaneously SpO2 oscillations were measured by high resolution Fast Sat technology (Radical™, Masimo, USA). To compare both methods, a mathematical routine was adapted to calculate PaO2oscillations using SpO2 values by adjustment of the oxygen-hemoglobin dissociation curve in accordance with temperature and acid-base state. The relative deviation between measured and calculated PaO2 oscillations in the three pigs were: 10.6%, 12.3% and 32.1%. In this pilot-study PaO2 oscillations could be estimated with adequate accuracy using SpO2 measurement. Systematic validation studies may allow the use of pulse oximetry in future as a non-invasive method to detect cyclic recruitment in patients of risk to ventilator associated lung injury.

Funding: DFG Ma 2398 / 3

P164 H2S ATTENUATES OXIDATIVE DNA-DAMAGE DURING RENAL ISCHEMIA/REPERFUSION INJURY

M. Gröger1, F. Simon1,2, S. Öter1, G. Speit3, C. Szabó4, P. Radermacher1, and Calzia E1. 1Anästhesiologische Pathophysiologie und Verfahrensentwicklung, 2Thorax- und Gefäßchirurgie,3Humangenetik, Universitätsklinikum, 89070 Ulm, Germany; 4Ikaria, Seattle WA 98102, USA

In awake mice inhaled H2S caused hypothermia and reduced O2 demand and, thus, protected against lethal hypoxia [1]. Therefore we investigated whether intravenous H2S (sulfide) may attenuate oxidative DNA damage during ischemia/reperfusion (I/R) injury. After 2 hours of i.v. sulfide (2 mg/kg×h) anesthetized, ventilated and instrumented pigs underwent 2 hours of intra-aortic balloon-inflation-induced renal ischemia and subsequently 8 hours of reperfusion with additional sulfide infusion (1 mg/kg×h) during the first 4 hours of reperfusion. While sulfide did not affect the surgery and I/R-related oxidative DNA damage (single cell gelelectrophoresis; tail moment in the comet assay) in whole blood cells before and after ex vivo exposure to hyperbaric oxygen, it attenuated the significant I/R-induced increase of the tail moment in the kidney tissue samples (table: data are median(range); # p<0.05 vs. before clamping, §p<0.05 between groups)

Table 1
Table 1

Sulfide also reduced the norepinephrine requirements needed maintain target hemodynamic parameters during the early reperfusion period. Thus i.v. sulfide may be tissue-protective during visceral organ ischemia.

References

1. Szabó C: Hydrogen sulfide and its therapeutic potential. Nat Rev Drug Disc 2007;6:917-35.

Grant acknowledgement: Supported by Ikaria

P165 VIABILITY OF STAPHYLOCOCCUS AUREUS IN SUTURE-ASSOCIATED BIOFILM

M. Henry-Stanley, D. Hess, and C. Wells. University of Minnesota, Minneapolis, USA 55455

Surgical suture can facilitate biofilm formation by Staphylococcus aureus. Historically, viable colony-forming units (CFU's) have been used to quantify bacterial numbers within a biofilm. Assessment of suture-associated S. aureus viability during biofilm formation has largely been ignored. We studied bacterial viability within S. aureusbiofilms on suture. Two cm pieces of 3-0 vicryl and 3-0 silk suture were placed separately in tissue culture medium, inoculated with S.aureus, and incubated up to 3d at 37°C, with the medium changed daily. Sutures were removed 1, 2, and 3d later, rinsed, and sonicated. Bacterial viability in the sonicate was assayed using the fluorescent reagent BacLight™ and viable CFUs were quantifed. A large percentage of bacteria (27% to 38%, P=.2)) remained nonviable over the 3d time period, although the numbers of viable bacteria increased (P<.01, Table). Thus, a substantial and stable portion of S. aureus remained nonviable while the numbers of viable S. aureus continued to increase (reflecting an increase in biofilm mass).

Table 1
Table 1:
No caption provided.

P166 P38 MAP KINASE INHIBITION DECREASES GLUTAMINE MEIDATED HSP70 EXPRESSION AND CELLULAR PROTECTION

C.R. Hamiel, J. Henderson, A. Kallweit, D. Richter, and P.E. Wischmeyer. Anesthesiology, University of CO Health Sciences Center, Denver, CO, 80262

Objective:

Glutamine (GLN) protects via induction of Heat Shock Protein 70 (HSP70). The mechanism by which GLN enhances HSP70 expression is unknown. GLN is hypothesized to induce cell swelling and affect p38 MAP kinase (p38) activity in cellular injury models. Cell size increases and p38 may play a role in GLN-mediated HSP70 expression and protection.

Methods:

Fibroblast cells were treated with and without p38 inhibitors, SB203580 or SB202190. 0 or 8mM GLN was added and cells were subjected to lethal heat stress (HS). Cell survival was evaluated via MTS assay. Western blots were run for HSP70 expression. HSP72 transcriptional activation was assessed via luciferase activity. Cell size was measured via microscopy.

Results:

Cell survival increased 6 fold with GLN(p=0.009 vs. HS CT). P38 inhibition (p38 inh) decreased protection by 71% (p<0.03 vs HS GLN). HSP70 increased 250% with GLN (p<0.01 vs. HS CT). P38 inh decreased GLN's effects by 45-50% (p< 0.03 vs GLN). Luciferase activity increased 17 fold with GLN (p=0.0004) which decreased 50% with P38 inh (p<0.04). GLN increased cell size 46-52% (p<0.0008) in HS cells. p38 inh decreased GLN's effects by 50% (p=0.04).

Conclusions:

Cell swelling and P38 may be key mechanistic pathways in GLN-mediated HSP expression and cellular protection.

P167 ANTI-INFLAMMATORY ADJUVANT IN RESUSCITATION FLUIDS IMPROVES SURVIVAL IN HEMORRHAGE

B. Cai, F. Chen, W. Dong, E.A. Deitch, and L. Ulloa. Laboratory of Surgical Immunology. UMDNJ- New Jersey Medical School. Newark. NJ 07103. USA

Severe hemorrhage is a common cause of death despite the recent advances in resuscitation and critical care. Conventional resuscitation fluids are designed to reestablish tissue perfusion, but they fail to prevent inflammatory responses. Indeed, resuscitation can exacerbate the production of proinflammatory cytokines that can be more dangerous than the original hemorrhage. Our previous studies indicate that ethyl pyruvate is an anti-inflammatory compound that inhibits the production of TNF from macrophages. Here, we analyzed ethyl pyruvate as a potential supplement to provide a therapeutic anti-inflammatory value to resuscitation fluids. Resuscitation with Hextend supplemented with ethyl pyruvate rescued all the animals from both hemorrhage and hemorrhage with trauma induced by broken femur. Ethyl pyruvate improved survival in a concentration dependent manner. Unlike conventional fluids, ethyl pyruvate inhibited the production of proinflammatory and cardiodepressant factors such as TNF and HMGB1. From a pharmacological perspective, resuscitation with ethyl pyruvate was particularly effective at inhibiting TNF production in the spleen and theheart. However, unlike other anti-inflammatory compounds, ethyl pyruvate mitigated serum and cardiac TNF levels in both control and splenectomized animals. At molecular level, ethyl pyruvate triggers a composite anti-inflammatory mechanism by inhibiting both poly(ADP-ribose) polymerase, and p65RelA DNA-binding without affecting IκBα activation. Our studies suggest that ethyl pyruvate is a promising supplement to provide anti-inflammatory potential to resuscitation fluids and improve survival in severe hemorrhage. This study was funded by grants of the AHA and the USAMRMC to LU.

P168 MYOCARDIAL EFFECTS OF HYPOTHERMIA AND INHALED H2S IN VENTILATED MICE

K. Baumgart, V. Simkova, S. Weber, E. Barth, G. Albuszies, P. Radermacher, and E. Calzia. Sektion Anästhesiologische Pathophysiologie und Verfahrensentwicklung, Universitätsklinikum, 89070 Ulm;

Inhaling H2S induced a suspended animation"-like metabolic state with hypothermia and decreased heart, respiratory and metabolic rate, but preserved blood pressure (MAP) and stroke volume (SV) [1]. As suspended animation could be protective in critical illness, we studied the myocardial response to H2S inhalation and hypothermia. Mice were anesthetized, mechanically ventilated and instrumented with left ventricular pressure-volume conductance catheter and assigned to 5 hours of 100 ppm inhaled H2S or vehicle with core temperature maintained at 38°C or 27°C. Hypothermia, but not H2S inhalation alone, decreased MAP and heart rate (HR) while SV, ejection fraction (EF) and end-diastolic pressure (EDP) were not affected (data median (range), #p<.05 vs. Control 38°C).

Table 1
Table 1

In contrast to awake mice, anesthesia (ketamine, fentanyl) blunted the myocardial effect of inhaled H2S.

1. Szabó C: Hydrogen sulphide and its therapeutical potential. Nat Rev Drug Discov 2007; 6:917-35

GRANT ACKNOWLEDGEMENT: Supported by the Deutsche Sepsis-Gesellschaft.

P169 EXPERIMENTAL POLYMICROBIAL PERITONITIS-ASSOCIATED TRANSCRIPTIONAL REGULATION OF MURINE ENDOGENOUS RETROVIRUSES

Kiho Cho, Sophia Chiu, Young-Kwan Lee, David Greenhalgh, and Jean Nemzek1. Burn Research, Shriners Hospitals for Children Northern California and Department of Surgery, University of California, Davis, Sacramento, CA 95817, and 1Unit for Laboratory Animal Medicine and Department of Pathology, University of Michigan, Ann Arbor, MI 48109-0614

Despite advancements in understanding the pathophysiology of sepsis, the mortality rate for this illness remains high. The determinants underlying variations in clinical outcomes among sepsis patients are poorly characterized. Endogenous retroviruses (ERVs) constitute ~8 % of human genome and each individual has an unique ERV profile. In this study, we investigated whether ERVs are associated with the pathogenesis of sepsis. ICR mice were subjected to cecal ligation and puncture (CLP) and murine ERV (MuERV) expression were examined in the liver and lung. RT-PCR analysis ofthe 3' U3 regions revealed clear regulation (induced or repressed) of specific MuERVs in both tissues at 12 hours after CLP. Also, increased expression of several variant transcripts was observed, primarily in the liver, at 12 and 48 hours: nine splicing variants, including one with four junctions and one 5.06 kb non-spliced transcript. Four novel MuERV splicing signals (two donors and two acceptors) were identified from this study. Interestingly, six variants were presumed to be splicing products of the 5.06 kb transcript derived from a defective MuERV, while the other three were envelope variants (one intact and two defective) transcribed from at least five MuERV loci. Five different full-length envelope transcripts capable of encoding intact envelope polypeptides, were differentially regulated in the liver at 12 hours after CLP. Furthermore, tropism traits and transcription potentials of putative MuERVs associated with CLP were analyzed. The findings from this study suggest the involvement of certain MuERVs and their gene products in sepsis pathogenesis.

P170 IMPACT OF HYPOTENSION AT ICU ARRIVAL ON OUTCOME AFTER ROSC FROM CARDIAC ARREST

S. Trzeciak, A.E. Jones, J.H. Kilgannon, N.I. Shapiro, S.M. Hollenberg, R.P. Dellinger, and J.E. Parrillo, for the Emergency Medicine Shock Research Network (EMSHOCKNET)

Expert guidelines advocate hemodynamic optimization after return of spontaneous circulation (ROSC) from cardiac arrest. However, data on prevalence of post-ROSC hemodynamic abnormalities and effect on survival are few. We aimed to test the hypothesis that early post-ROSC hypotension predicts mortality. We analyzed the Project IMPACT database (ICU patients from 120 US hospitals). Inclusion criteria: 1) age >17; 2) non-trauma; and 3) CPR <24 hours prior to ICU admission. Subjects were divided into 2 groups: 1) Early exposure to hypotension - one or more systolic blood pressure (SBP) <90 mmHg within 1 hour of ICU arrival or 2) Non-exposure - all SBP >90. The primary outcome was in-hospital mortality; secondary outcome was re-arrest in ICU. Chi-2 and 95% CI for the difference in proportions were used to compare exposures to non-exposures. Multivariate logistic regression was used to determine the ability of early exposure to hypotension to independently predict death. 8736 subjects met inclusion criteria. The overall mortality rate was 50% and re-arrest rate was 5%. Early exposure to hypotension occurred in47% and was associated with significantly higher mortality (65% vs. 37%, difference 28% [CI 26%-30%]) and re-arrest (7% vs. 3%, difference 4% [3%-5%]) compared to non-exposures (p<0.001). On multivariate analysis, early exposure to hypotension was a strong independent predictor of death (OR 2.7 [2.5-3.0]). We conclude: Half of cardiac arrest patients that survive to ICU admission die in the hospital. Early hypotension after ROSC is common and is an independent predictor of in-hospital mortality. These data underscore the need for clinical trials of early hemodynamic optimization after ROSC.

P171 DOES SLEEP DEPRIVATION AFTER SEPTIC INSULT INFLUENCE SURVIVAL?

Brandon Bruns, Robert Barber, and Randall Friese. University of Texas Southwestern Medical Center, Dallas, TX 75390

Background:

Sleep deprivation is prevalent in intensive care unit (ICU) patients. Animal models have demonstrated that chronic sleep deprivation (SD) alone leads to immune suppression, septicemia, and death. However, the effects of SD associated with acute illness, such as a septic insult, have not been previously described. We hypothesized that SD after septic insult would result in decreased survival.

Methods:

C57/black mice underwent cecal ligation and puncture (CLP). Animals were then randomized to receive SD for 48 hours or standard recovery (no SD). Sham animals underwent laparotomy incision without CLP. SD was achieved by securing animal housing to an orbital shaker set to repeatedly cycle at 30 RPM over 120 seconds (30 seconds on/90 seconds off). This method of SD has been previously validated using electroencephalography. Primary outcome was survival at 5 days post CLP. Kaplan Meier survival analysis with log rank test was utilized.

Results:

Survival after CLP with SD (n=19) was 32% and survival after CLP without SD (n=23) was 65% (p=0.022). All sham animals survived. SD was associated with a two-thirds reduction in survival compared to controls [RR=0.33; 95%CI 0.130-0.856].

Figure 1
Figure 1

Conclusions:

Sleep deprivation after a septic insult decreases survival. These findings suggest that sleep deprivation may increase mortality in patients at high risk for septic insults cared for in the ICU.

P172 WHOLE BODY COOLING MAY IMPROVE SURVIVAL DURING EXPERIMENTAL HEAT STROKE BY REDUCING ACUTE LUNG INJURY

Hsi-Hsing Yang, Ching Ping Chang, Juei-Tang Cheng, and Mao-Tsun Lin. Department of Intensive Care Medicine, Chi-Mei Medical Center, Tainan, Taiwan 710; Institute of Basic Medical Sciences of National Cheng Kung University School of Medicine, Tainan, Taiwan 710; Department of Medical Research, Chi-Mei Medical Center, Tainan, Taiwan 710

Heatstroke is a life threatening syndrome characterized by hyperthermia, arterial hypotension, central nervous system disorders, respiratory distress, and even acute lung injury. When heatstroke complicated with acute lung injury leads to a high mortality. In the present study, we will test the hypothesis that whole body cooling may improve survival in heatstroke rats by reducing acute lung injury. The animals were divided into a control and a heatstroke group. Heatstroke was induced by exposing the animals to a high ambient temperature to 43°C.The heatstroke group was further divided into a group in which no cooling therapy was induced and another group that was treated by whole body cooling through setting the blanket to 16°C. The preliminary data showed that the whole body cooling group when comparing with the heatstroke untreated group, the values of arterial PaO2, HCO3, and SaO2% were higher and the survival time was longer (all P<0.005). In contrast the value of core temperature and PaCO2 were lower (all P<0.005). The bronchoalveolar lavage fluid tumor necrosis factor-alpha, interleukin-1 and interleukin-6 in whole body cooling heatstroke group were lower than untreated heatstroke group (all P<0.005). Whole body cooling may improve survival of heatstroke rat by attenuating acute lung injury. Use of whole body cooling could be exploited as a new approach for the management of acute lung injury.

P173 CYTOCHROME C STIMULATED RESPIRATION IN LIVER MITOCHONDRIA OF ANESTHETISED MICE: EFFECTS OF BODY TEMPERATURE AND H2S

V. Simkova, K. Baumgart, E. Barth, G. Albuszies, S. Weber, P. Radermacher, and E. Calzia. Sektion Anästhesiologische Pathophysiologie und Verfahrensentwicklung, Universitätsklinikum, 89070 Ulm

In a preliminary study we measured mitochondrial respiration by means of high-resolution respirometry (Oxygraph-2k, Oroboros, Austria) in mechanically permeabilised, small liver biopsies1 of anesthetised mice undergoing laparatomy. We studied 4 groups of animals (n=8); biopsies were sampled at the end of a 5h-observation period after laparatomy. During the experiment, body temperature was kept at 38°C or 27°C; at each temperature level, one group of animals additionally received inhaled H2S. Our protocol particularly focussed on the stimulation of oxygen flux induced by addition of cytochrome c during state 3 respiration with combined complex I and II substrates. Stimulation by cytochrome c should not occur in intact mitochondria and is an indicator of cytochrome c release owing to mitochondrial damage (2). Data are presented as mean ± SD as the ratio of oxygen flux after vs. before addition of cytochrome c.

Table 1
Table 1

Combining low body temperature and H2S-Inhalation reduced cytochrome c induced stimulation of mitochondrial respiration. Both interventions alone also tended to reduce cytochrome c induced stimulation, but these effects were much less pronounced and not statistically significant. The correlation between these effects and cell apoptosis / necrosis remains to be investigated.

Supported by the Deutsche Sepsisgesellschaft.

References

1. Kuznetsov et al, Anal Biochem (2002)305:186-194.

2. Kuznetsov et al, Am J Physiol (2004)286:H1633-H1641.

P174 EFFECT OF HYPERCAPNIA ON MICROVASCULAR GASTRIC MUCOSAL OXYGENATION UNDER PHYSIOLOGICAL AND COMPROMISED CIRCULATORY CONDITIONS IN DOGS

C. Beck, I. Bauer, I. Schwartges, T.W. Scheeren, A. Fournell, and O. Picker.

Aim of the study:

Permissive hypercapnia (PHC), a component of lung-protective ventilation (1), induces systemic and regional vasodilatation (1). It is yet unknown whether this vasodilation also affects the gastrointestinal mucosa and might thus improve microvascular oxygenation (μHbO2). This could be beneficial not only in hemorrhagic shock states, since an adequate μHbO2 appears to be crucial to maintain an intact mucosal barrier function (2). We therefore tested the effect of PHC on μHbO2 during hemorrhagic shock.

Methods:

The effect of PHC (etCO2=70mmHg) on gastric mucosal μHbO2 was measured using tissue spectrophotometry (3) and arterial lactate samples were taken. Hemorrhagic shock was induced by removal of 20% estimated blood volume. The ventilated dogs (1.5 MAC Sevoflurane,n=6) were randomized to each of the following three groups (n=6): 1.) PHC+shock, 2.) normocapnia (etCo2 35mmHg)+shock and 3.) PHC without shock. Statistics: Analysis of variance, Fisheŕs PLSD, p<0.05.

Results:

Under physiological conditions PHC significantly increased regional mucosal oxygenation (μHbO2), from 53 ± 3 to 60 ± 2%, with a decrease in arterial lactate levels. This increase in μHbO2 persisted for hours and returned to baseline after normalisation of etCO2. During normocapnia the induction of hemorrhagic shock significantly decreased μHbO2 (−14 ± 4%). However, this effect was significantly reduced during PHC (−3 ± 2%). In addition, the increase in lactate levels was significantly reduced during PHC compared to normocapnia (1.3 ± 0.1 v.s. 2.2 ± 0.1 mmol/l).

Conclusion:

PHC not only increases the gastrointestinal mucosal oxygenation during physiological conditions but also diminishes the decrease induced by hemorrhagic shock. Therefore, PHC might be a beneficial prophylactic intervention not only reducing lung injury, but also maintaining the mucosal gut barrier function in compromised haemodynamic situations.

References

1. Laffey JG, Kavanagh BP: Lancet 354:1283-6, 1999.

2. Sato N, Kamada T, Shichiri M, Kawano S, Abe H, Hagihara B: Gastroenterology 76:814-9, 1979.

3. Schwarte LA, Picker O, Schindler AW, Fournell A, Scheeren TWL: Crit Care Med 31:1999-2005, 2003.

P175 PRELIMINARY EVALUATION OF A NEW RAPID METHOD FOR MIGET

B. Duenges, A.P. Vogt, M. Bodenstein, H. Wang, A. Becher, B. Röhrig, J.E. Baumgardner, and K. Markstaller. Johannes Gutenberg-University, Mainz, Germany

Introduction:

MIGET by MMIMS is a new method to rapidly measure V/Q distributions in experimental models of lung injury. This novel technology has not yet been compared to traditional standards for assessing gas exchange. We compared shunt fraction, a single feature of the V/Q distributions measured by MIGET by MMIMS, to shunt fraction calculated from venous admixture.

Methods:

After IACUC approval, lung injury was produced in 8 anesthetized pigs, average weight 24 Kg, by saline lavage. Eight additional pigs served as controls. The animals were ventilated with 100% oxygen. Shunt fraction varied over a broad range (4% to 62%). Six inert gases were dissolved in saline and infused at 825 ml/hr. Arterial and pulmonary arterial blood samples were withdrawn for MIGET and for blood gas analysis. Venous admixture (VA) was calculated from the blood gas data by the Berggren method. Blood samples (5 ml each) were analyzed for inert gas partial pressures with the MIGET by MMIMS system in 12 minutes. Shunt (MM-S), and shunt plus low V/Q (MMLO) from MIGET were compared to VA by linear regression.

Results:

MM-S was highly correlated with VA (r2=0.83), but underestimated shunt (MM-S=−4.3+0.60*VA), whereas MMLO slightly overestimated shunt (MMLO=11.0+0.95 * VA, r2=0.70).

Conclusions:

MIGET by MMIMS rapidly measured V/Q distributions using small blood samples. This early version of this new technology underestimated true shunt fraction, suggesting inadequate discrimination between true shunt and low V/Q regions of the lung.

Support:

German Research Foundation DFG Ma 2398/3.

P176 EFFECTS OF MAJOR ABDOMINAL SURGERY AND ANESTHESIA ON MITOCHONDRIAL RESPIRATION

Tomas Regueira1, Siamak Djafarzadeh1, Sebastian Brandt2, Francesca Porta1, Jukka Takala1, Hendrik Bracht1, Philipp M. Lepper1, and Stephan M. Jakob1. Departments of Intensive Care Medicine1 and Anesthesiology2, Inselspital, Bern University Hospital, and University of Bern, CH-3010 Bern, Switzerland

Introduction:

Major abdominal surgery, anesthesia and prolonged sedation are frequently associated with postoperative complications. Mitochondrial dysfunction is a contributing factor for organ dysfunction, and surgical stress has been proposed to be associated with mitochondrial damage. We aimed to assess hepatosplanchnic hemodynamics and muscle mitochondrial respiratory efficiency after major abdominal surgery and prolonged anesthesia in pigs.

Methods:

65 pigs (weight: 37-42 kg) were anesthetized with pentobarbital (7 mgkg−1h−1), fentanyl (25 μgkg−1h−1) and pancuronium (1 mgkg−1h−1) for a protocolized 3-5 hours major abdominal surgery. Systemic (thermodilution) and splanchnic organ blood flows (ultrasound) were measured continuously during the 13-17 hours of post-operative stabilization period without any intervention but using continuous sedation based on pentobarbital (7 mgkg−1h−1), fentanyl (3 μgkg−1h−1) and pancuronium (1 mgkg−1h−1). Mitochondrial respiration was assessed polarographically from muscle biopsies taken from quadriceps before the operation and after the stabilization period.

Results:

Cardiac index (85 ± 20 to 81 ± 17 mlkg−1min−1), mean arterial pressure (71 ± 12 to 72 ± 13 mmHg) and heart rate (111 ± 16 to 108 ± 21 beats/min) did not change from postoperative values to the end of the stabilization period. Central venous pressure (3.6 ± 2 to 4.2 ± 2 mmHg), total liver blood flow (20 ± 5 to 23 ± 5 mlkg−1min−1) and total hepatosplanchnic oxygen delivery (2.7 ± 0.6 to 3 ± 0.8 mlkg−1min−1) and consumption (1.6 ± 0.4 to 1.72 ± 0.4 mlkg−1min−1) increased in the same period (p<0.02 for all). Arterial lactate concentration (1.1 ± 0.3 to 0.7 ± 0.3 mmol/l, p<0.001) and hepatic lactate influx decreased, while the hepatic lactate extraction ratio increased (42 ± 20 to 56 ± 14%, p<0.001). Muscle mitochondria complex I and II-dependent respiration efficiency (respiratory control ratio) remained stable between pre-operative and end of stabilization period [12 ± 4.4 to 11.1 ± 4.3 (complex I), 4.4 ± 1.5 to 4.5 ± 1.4 (complex II)].

Conclusion:

Major surgery and prolonged anesthesia did not affect the efficiency of muscle mitochondrial respiration. Whether the increase in liver oxygen consumption and lactate extraction are related to a selective improvement in liver mitochondrial function should be evaluated in further studies.

P177 HSD IS A BETTER RESUSCITATION FLUID FOR HEMORRHAGIC SHOCK WITH PULMONARY EDEMA AT HIGH ALTITUDE

Liang-Ming Liu, De-Yao Hu, XueWu Zhou, Jiang-Cang Liu, and Ping Li. State Key Laboratory of Trauma, Burns, and Combined Injury, The Second Department of Research Institute of Surgery, Daping Hospital, The Third Military Medical University, Chongqing 400042, P.R.China

Objective:

To investigate the fluid tolerance of hemorrhagic shock with pulmonary edema at high altitude in unacclimated rats and the beneficial effect of 7.5% hypertonic saline/6% dextran (HSD).

Methods:

One hundred and seventy six SD rats, transported to LaSa, Tibet, 3760 meters above the sea level, were anesthetized with sodium pentobarbital (30 mg/kg, ip) within one week. Hemorrhagic shock with pulmonary edema was induced by bloodletting (50 mmHg for 1 hour) plus intravenous injection of oleic acid(50μl/kg). Seventy seven rats were equally divided into eleven groups(n=7/group) including sham operated control group, hemorrhagic shock control group, hemorrhagic shock with pulmonary edema(HSPE) control group, HSPE plus 0.5-, 1.0-, 1.5-, 2.0-or 3.0-fold volume of LR groups and HSPE plus 4ml/kg, 6ml/kg and 8ml/kg of HSD groups. Hemodynamic parameters including mean arterial blood pressure (MAP), left intraventricular systolic pressure (LVSP) and the maximal change rate of intraventricular pressure rise or decline (±dp/dtmax) were observed at baseline, and at 15, 30, 60, and 120 min after infusion, blood gases were measured at 30 and 120 min after infusion and the water content of lung and brain was determined at 120 min after infusion. Additional ninety nine rats were used to observe the effect of above treatments on the survival time of HSPE rats.

Results:

0.5 volume of LR infusion slightly increased the MAP, LVSP and ±dp/dt max, and prolonged the survival time of HSPE animals as compared to the HSPE group (P<0.05-0.01), it did not increase the water content of lung and brain, and had no marked influences on blood gases. One volume of LR infusion had somewhat improvement on hemodynamic parameters for HSPE animals, but had no apparent effect on the survival time and the water content of lung and brain. 1.5-, 2-and 3vol LR infusion significantly deteriorated the hemodynamic parameters, increased the water content of lung and decreased the survival time of HSPE animals. HSD(4-8 ml/kg) significantly increased the hemodynamic parameters, improved the blood gases, decreased the water content of lung and brain, and prolong ed the survival time of HSPE rats. Among the three dosages of HSD, 6 ml/kg of HSD had the best effect.

Conclusion:

The tolerance of fluid infusion for hemorrhagic shock with pulmonary edema at high altitude is significantly decreased. Over 1 vol of LR infusion would aggravate the pulmonary edema and exacerbate the resuscitation effect, but only one vol of LR can not reach the effective volume resuscitation. Small volume of HSD could better resuscitate hemorrhagic shock with pulmonary edema at high altitude.

Keywords:

high altitude; hemorrhagic shock; pulmonary edema; fluid tolerance, HSD

P178 IDEAL PRESSURE OF FLUID RESUSCITATION FOR EARLY TREATMENT OF UNCONTROLLED HEMORRHAGIC SHOCK IN RATS

Liangming Liu, Youfang Diao, Ping Li, and Zifu Liao. State Key Laboratory of Trauma, Burns and Combined Injury, Department 2, Research Institute of Surgery, Daping Hospital, The Third Military Medical University, Chongqing 400042,China

Objective:

To explore the ideal fluid resuscitation pressure for early treatment of uncontrolled hemorrhagic shock.

Method:

Sixty four Wistar rats were anesthetized with sodium pentobarbital. Uncontrolled hemorrhagic shock was produced by spleen amputation and letting the mean arterial blood pressure (MAP) drop to 40 or 50 mmHg. The management was distributed into three phases. The first phase mimicked the prehospital emergency treatment phase, during which the shocked rats were resuscitated with lactated Ringer's solution and 6% dextran to a MAP at 40,50,60,70,80 and 100 mmHg for 1 hour respectively. The second phase mimicked the definitive treatment of hospital, during this phase, the spleen artery was ligated and the rats were resuscitated to a MAP at 100 mmHg for 2 hours with fluid and blood. The third phase was resuscitation effect observing period. The observed parameters included animal survival time, MAP, the hemodynamic parameters, the amount of infused fluid, the hematocrit and arterial blood gases.

Results:

The animals in the hypertensive (80,100 mmHg) resuscitation groups at the first phase had a short survival time, most of animals died during the second and the third phase. They had severe hemodilution and metabolic acidosis, their blood gases were inferior to the hypotensive resuscitation groups. In contrast, the animals in the hypotensive (50,60,70 mmHg) resuscitation groups at the first phase had a longer survival time, and better hemodynamic parameters. Their hemodilution was not severe and their blood gases were in normal or close to normal. The effect of 50 and 60 mmHg resuscitation group was best. Too low resuscitation pressure (40 mmHg) was not good to shock resuscitation, in this group, the mortality of animals was also high.

Conclusion:

Hypertensive resuscitation for uncontrolled hemorrhagic shock in prehospital phase will increase the blood loss and hemodilution and interfere with the resuscitation effect of shock.

Proper Hypotensive resuscitation in prehospital will benefit to shock resuscitation. The ideal infusion pressure was 50-60 mmHg. Too low resuscitation pressure was not good to shock resuscitation.

Keywords:

Uncontrolled hemorrhagic shock; prehospital emergency treatment; fluid resuscitation; resuscitation pressure.

email: [email protected]

P179 BENEFICIAL EFFECT OF AVP IN HEMORRHAGIC SHOCK AND ITS RELATIONSHIP TO VASCULAR REACTIVITY IMPROVEMENT

Tao Li, Guangming Yang, Jing Xu, Jiancang Liu, and Liangming Liu. State Key Laboratory of Trauma, Burns and Combined Injury, The 2nd Department of Research Institute of Surgery, Daping Hospital, The Third Military Medical University, Chongqing 400042, P.R.China

Backgroud:

The vascular reactivity and calcium sensitivity were decreased following hemorrhagic shock. Arginine vasopressin (AVP) was beneficial to endotoxic, infectious/septic and hemorrhagic shock. Our previous studies found that Rho kinase played an important role in the occurrence of calcium desensitization following shock. It was reported that AVP was with stimulation effect of Rho kinase. So we hypothesized that AVP might have beneficial effect on shock via activation of Rho kinase to regulate the calcium sensitivity and vascular reactivity.

Method:

Hemorrhagic shock (40 mmHg for 2 h) Wistar rats in vivo were adopted to observe the effects of small dose of AVP on hemodynamics, 24-h survival rate, the pressor effect of norepinephrine (NE) and the contractility of superior mesenteric artery (SMA). Isolated SMAs from hemorrhagic shock rats were adopted to observe the effects of AVP on vascular reactivity and calcium sensitivity and its relationship to Rho kinase with an isolated organ perfusion system.

Results:

The results showed that AVP at the concentration of 0.1U/kg and 0.4U/kg significantly improved the hemodynamic parameters and the 24-h survival rate of hemorrhagic shock rats, meanwhile, this dosage of AVP significantly increased the pressor effect of NE and the contractile response of SMA to NE. Y-27632 (30μg/kg), a Rho kinase specific inhibitor, abolished these beneficial effects of AVP. In vitro, the calcium sensitivity and vascular reactivity of SMA to calcium and NE were significantly decreased following hemorrhagic shock, AVP at the concentration of 0.5nmol/L and 5nmol/L significantly increased the calcium sensitivity and vascular reactivity. These effects of AVP were abolished by Y-27632 (10μmol/L).

Conclusion:

Taken together the results suggest that small doses of AVP are beneficial to hemorrhagic shock, which was closely related to its improving effect of vascular reactivity. AVP improving vascular reactivity and calcium desensitization needs the involvement of Rho kinase.

Keywords:

Hemorrhagic shock; Vascular reactivity; Calcium sensitivity; Rho kinase; AVP.

P180 EFFECT OF AVP ON VASCULAR REACTIVITY AND CALCIUM SENSITIVITY OF VASCULAR SMOOTH MUSCLE AND ITS RELATIONSHIP TO PKC FOLLOWING HEMORRHAGIC SHOCK IN RATS

Yang Guangming, Li Tao, Xu Jing, Ming Jia, Liu Liangming. State Key Laboratory of Trauma, Burns and Combined Injury, Department 2, Research Institute of Surgery, Daping Hospital, The Third Military Medical University, Chongqing 400042, China

Objective:

The present study was to investigate the effect of AVP on vascular reactivity and calcium sensitivity following hemorrhagic shock and its relations to PKC isoforms.

Methods:

The denuded endothelium superior mesenteric artery (SMA) rings from hemorrhagic shock rats were adopted to assay the vascular reactivity and calcium sensitivity via observing the contractile response of SMA to NE and to Ca2+under depolarizing conditions (120 mmol/L K+). The effects of AVP (5×10−11, 5×10−10, 5×10−9mol/L) on vascular reactivity and calcium sensitivity of SMA from hemorrhagic shock rats and its relationship to α and δ isoforms of PKC were observed.

Results:

AVP (5×10−11, 5×10−10, 5×10−9mol/L) pretreatment improved reactivity of SMA rings to NE and calcium following hemorrhagic shock with concentration-dependently. Gö 6976 (5×10−6 mol/L), the specific PKC-α isoform (Ca2+-dependent isoform) inhibitor, and Rottlerin (10−5 mol/L), the specific PKC-δ isoform (Ca2+-independent isoform) inhibitor, antagonized AVP-induced increase of vascular reactivity and calcium sensitivity of SMA following hemorrhagic shock.

Conclusions:

The AVP significantly restored the decreased vascular reactivity and calcium sensitivity of vascular smooth muscle following hemorrhagic shock, and its mechanisms may be related to both α and δ isoforms of PKC activation.

Keywords:

Hemorrhagic shock, Arginine vasopressin, PKC-α, PKC-δ, Vascular reactivity, Calcium sensitivity

email: [email protected]

P181 THE REGULATORY EFFECT OF PKCε ON VASCULAR HYPOREACTIVITY AND CALCIUM SENSITIVITY DURING HEMORRHAGIC SHOCK

Jing Xu, Guang-Ming Yang, Tao Li, Jia Ming, Wei Cheng, Yuan Zhang, and Liang-Ming Liu. State Key Laboratory of Trauma, Burns and Combined Injury, Department 2, Institute of Surgery Research,Daping Hospital,Third Military medical University, Chongqing, 400042, China

Objective:

To observe the regulatory effect of PKCε on vascular hyporeactivity and calcium sensitivity following hemorrhagic shock.

Method:

The superior mesenteric artery(SMA) from hemorrhagic shock rats of different time (immediately, 30min, 1h, 2h, 4h) was adopted to determine the mRNA expression of PKCε using RT-PCR technique, and the first class arborization of SMA was adopted to assay the vascular reactivity and calcium sensitivity via observing the contraction initiated by norepinephrine(NE) and Ca2+with isolated organ perfusion system. The agonist and antagonist of PKCε were used to observe the regulation of PKCε on vascular hyporeactivity following hemorrhagic shock.

Result:

(1) Vascular reactivity and calcium sensitivity of the first class arborization of SMA were increased at the early stage of hemorrhagic shock, and decreased at the late stage of hemorrhagic shock. PKCε mRNA exhibited a time-dependent increase following HS, peaked at 1h(p<0.01) and maintained at a high level at 4h(p<0.01) (2)The agonist of PKCε increased the vascular reactivity and calcium sensitivity of the first class arborization of SMA, and its antagonist decreased the vascular reactivity and calcium sensitivity of SMA at 2h after shock.

Conclusion:

PKCεmay be an important endogenous protective molecule, it plays an important role in the regulation of vascular reactivity and calcium sensitization following HS.

Keywords:

hemorrhagic shock; vascular reactivity; calcium sensitivity; PKCε

email: [email protected]

Intensive Care Clinical Research

P183 EXPOSURE TO CARDIOPULMONARY BYPASS IS ASSOCIATED WITH INCREASED MICROCIRCULATORY BLOOD FLOW

R.C. Arnold, S. Trzeciak, J. McCoy, V. Lotano, J.E. Parrillo, S.M. Hollengberg, and R.P. Dellinger. UMDNJ-RWJMS, Cooper University Hospital, NJ, USA 08103

Background:

Systemic inflammation can cause alterations in microcirculatory blood flow. Exposure to extracorporeal circulation can induce a profound systemic inflammatory response, but the impact on microcirculatory flow is unclear.

Objectives:

To test the hypotheses that, compared to pre-exposure and recovery measurements, cardiopulmonary bypass (CPB) would be associated with microcirculatory flow alterations.

Methods:

Prospective observational study of cardiac surgery patients exposed to CPB. We performed sublingual Sidestream Darkfield videomicroscopy at 5 sublingual sites at three time points: 1) pre-operative (PRE), 2) immediately after surgery (POST), and 3) after recovery (REC). Off-line and blinded to all clinical data, we determined the microcirculatory flow index (MFI) by assigning a semi-quantitative score (0=no flow to 3=brisk) to each quadrant of the image and averaging the 4 values. We compared mean MFI values for PRE, POST, and REC time points using ANOVA.

Results:

225 images were obtained in 15 patients at the 3 time points. Exposure to CPB was associated with a rise in MFI compared with pre-operative and recovery images, PRE: 2.18, POST: 2.49, REC: 2.24 (p<0.02). Mean arterial pressure (mmHg) was not significantly different between the three time points, PRE: 80, POST: 87, REC: 83 (p=0.27).

Conclusion:

In this sample of cardiac surgery patients, the post-CPB period was associated with an increase in microcirculatory blood flow compared to pre-operative and recovery measurements. Whether or not the uniform use of intravenous nitroglycerin (a nitric oxide donor agent) or other vasoactive agents in the post-CPB period had an impact on MFI is unknown. These data support the conclusion that further investigation is warranted to determine 1) the effect of CPB on microcirculatory flow, 2) the specific determinants of flow alterations, and 3) the association with clinical outcomes.

P184 INTRATHORACIC BLOOD VOLUME MEASUREMENT: A COMPARISON OF TRANSPULMONARY LITHIUM INDICATOR DILUTION WITH INDOCYANINE GREEN INDICATOR DILUTION

Ben Maddison, George Findlay, Peter Radermacher, Charles Hinds, and Rupert M. Pearse. Barts and The London School of Medicine and Dentistry, Queen Mary's University of London, UK

Introduction:

Intrathoracic blood volume (ITBV) is thought to be a superior measure of cardiac preload than intravascular pressure1. Transpulmonary indocyanine green (ICG) indicator dilution is regarded as the most reliable method of ITBV measurement but is no longer commercially available. Our previous work suggests lithium indicator dilution could be used to measure ITBV2.

Method:

Patients undergoing cardiac surgery with cardiopulmonary bypass who met inclusion criteria were enrolled to a single centre, observational study. Peri-operative care was standardised. Comparative ITBV measurements were performed 1, 2, 4 and 6 hours after surgery, using lithium indicator dilution via a radial artery catheter (LiDCOplus, LIDCO Ltd, UK) and ICG indicator dilution via a femoral artery catheter (COLD-Z, Pulsion, Germany). Data were compared by Bland-Altman analysis.

Results:

17 patients were recruited [age 69 yrs (54-87); Parsonnet score 10 (0-29)] providing a total of 68 paired measurements. 16 ICG measurements were excluded because of the poor quality of the indicator dilution curve, leaving a total of 52 paired comparisons with good quality data. Mean ITBV measured by lithium dilution was 2522 ml (±691) and by ICG dilution 1708ml (±432). The mean bias between paired measurements was 813 ml (LOA ±1248; p<0.001). However, for cardiac index, the bias between techniques was only 0.39 l/min/m2, (LOA ± 0.9 l/min/m2; p<0.0001). Thus the discrepancy between the techniques related to differences in the measurement of mean indicator transit time (MTT). There was a decreasing trend in the mean differences in ITBV and MTT (Li-ICG) from 1014ml and 16.1s at 1 hour 1 to 466ml and 10.6s at hour 6 (p=NS). This may relate to changes in temperature and cardiac index over the same time period.

Conclusion:

Poor agreement between ITBV measurements taken using ICG and lithium indicator dilution appears to be due to inaccurate measurement of mean indicator transit time. This may relate to the use of a radial as opposed to a femoral artery catheter in patients with poor peripheral perfusion at baseline.

References

1. Wiesenack C, et al: J Cardiothorac Vasc Anesth 15:584, 2001.

2. Maddison B, et al: Crit Care 11(Suppl 2):295, 2007.

P185 ACCURACY OF CYTOKINE-RELATED GENE POLYMOPRHISM ANALYSIS WITH A NEWLY DEVELOPED DNA CHIP ON CRITICALLY ILL PATIENTS

Shunsuke Otani, Shigeto Oda, Tomohito Sadahiro, Masataka Nakamura, Eizo Watanabe, Tomo Yamasaki, Takeshi Tokuhisa, and Hiroyuki Hirasawa. *Department of emergency and critical care medicine, graduate school of medicine, Chiba University; **Department of developmental genetics, graduate school of medicine, Chiba University

Objective:

To investigate the usefulness of a novel method foranalysis of single nucleotide polymorphism (SNP) as a rapid assay for polymorphisms in cytokine-related genes in critical care patients.

Patients:

Seventy-nine critically ill patients admitted to the ICU of a university teaching hospital.

Measurement and Main Results:

First, genomic DNA was extracted from blood samples collected from 59 unrelated patients after informed consent was obtained.

The DNA samples obtained were used for conditioning newly developed DNA chip assay, which consists of multiplex PCR reaction,single base extension(SBE) and chip hybridization. These DNA were previously examined for genetic polymorphisms in IL-6-596 G/A,-572 C/G,-174 G/C, TNF-ß-308G/A,-238G/A, IL-1α-511C/T, and-31T/C by TaqMan assay. The same genotyping result was obtained with the two assay methods. Then, 20 additional patients were subjected to genotype by both methods, and the validity of the DNA chip assay was confirmed.

Conclusions:

Genotyping with this new assay detected an allele of interest as accurately as a conventional assay, and with excellent reproducibility. Multiple loci of SNPs can be genotyped simultanously with this method, and it will be clinically applicable for future tailor-made medicine based on the genotyping of clinically relevant SNPs.

P186 TISSUE OXYGENATION AND MICROVASCULAR FLOW AFTER MAJOR ABDOMINAL SURGERY

S. Jhanji, C. Lee, D. Watson, C.J. Hinds, and R.M. Pearse. Royal London Hospital, UK

Background:

Therapies designed to increase global oxygen delivery (DO2I) perioperatively appear to improve outcome in high risk surgical patients, but the mechanism of benefit is unclear.

This study investigated the changes in tissue oxygenation and microvascular flow after major abdominal surgery.

Methods:

Patients received usual clinical care. Observational data were collected prior to, and for eight hours after surgery. Measurements included cutaneous tPO2 (Clark electrode), mean red cell flux (laser Doppler), microvascular flow index (MFI) from sublingual microcirculation images (sidestream darkfield imaging), DO2I (lithium indicator dilution) and mean arterial pressure (MAP). The study was approved by the local research ethics committee.

Results:

Data presented as median (IQR).25 patients (10 males) were recruited. 18 patients developed complications. There was one death.

Table
Table

Conclusions:

After major abdominal surgery increased heterogeneity of microvascular flow is associated with impaired tissue oxygenation.

P187 INVESTIGATION OF BLOOD LACTATE LEVEL AS AN INDICATOR FOR SEVERITY ASSESSMENT

Yoshihito Fujita, Tae Kato, and Hirotada Katsuya. Department of Anesthesiology and Medical Crisis Manegement, Nagoya City University, Japan 467-8601

Objectives:

Many parameters to be used as an indicator for severity assessment of a patient are often complicated in estimation of the values. To investigate that the blood lactate level is useful or not as an indicator for assessment of the clinical condition, we compared blood lactate levels with APACHE II scores by underlying disease.

Subjects and Methods:

Of the 802 patients who were hospitalized in our ICU through the period of 2 years from 2001 to 2002, 240 patients other than the postoperative patients, patients with cardiac anomaly, and infantile patients were included in the study. A correlation of APACHE II scores immediately after transfer in the ICU with blood lactate levels was investigated.

Results:

A scatter diagram was plotted with APACHE II scores on the horizontal axis and blood lactate levels on the longitudinal axis, and an approximate line was drawn. In all, the approximate line of y=2.50x−4.48 was obtained with R2=0.34, which indicates mild correlation. In circulatory disorders more potent correlation with y=1.56x-0.07 and R2=0.46 was observed, while in respiratory disorders the correlation was little observed with y=1.32x+2.45 and R2=0.18.

Discussion and Conclusion:

Blood lactate level was correlated with APACHE II score, and it was shown to be able to use as an indicator for the severity assessment of a patient. However, it was suggested that there was variation in the reliability as an indicator for assessment by disorders. Blood lactate level did not reflect the severity of respiratory disorders. In the circulatory disorders, it was suggested to be usable as an indicator of the severity.

P188 THE RELIABILITY OF IL-6 VALUES IN PRESERVED SAMPLES

Tomoyuki Nakamura#1, Osamu Nishida#1, Yoshitaka Hara#2, Naohide Kuriyama#1, Miho Yumoto#1, Hiroki Tubouchi#2, Tetsuya Tamura#2, Hiromi Kako#2, and Daisuke Niimi#2. #1Department of Anesthesiology and Critical Care Medicine, Fujita Health University, Japan #2Department of Intensive Care, Kainan Hospital, Japan

Background:

IL-6 is known to be an important indicator of early response after invasion.The value of measuring IL-6 lies in its property of elevating before CRP.Attaching a specialized cartridge for measuring human IL-6 to a preexisting measuring device (FUJIREBIO), our Intensive Care Unit is capable of measuring IL-6 within an hour; therefore making clinical application of IL-6 value possible. However on weekends and nights, if there is no need for urgency, the lab technician preserves the samples for later testing. In this report, we investigated the relationship between method of preservation and IL-6 values.

Method:

We randomly selected ten patients admitted to our ICU in whom IL-6 was measured. Blood samples taken immediately underwent plasmapheresis and IL-6 measurement. Samples were then preserved at room temperature; 4° and-30°. Each were measured 3 hours and 24 hours later.

Result:

For the convenience of comparing, immediate IL-6 values were computed to a value of 1.Samples preserved at room temperature for 3 hours were 0.961±0.059 (mean±SD), room temperature for 24 hours were 0.919±0.203, 4° for 3 hours were 0.964±0.066, 4° for 24 hours were 0.924±0.147,−30° for 3 hours were 1.021±0.115,−30° for 24 hours were 0.982±0.034.We did not find any significant difference (P=0.7047) after statistical examination using repeated measure ANOVA.

Conclusion:

In our institution, samples taken on weekdays are tested either immediately or within 3 hours preserved at 4°.Samples taken in night or on weekends are preserved at−30° until testing in the morning of the following weekday. We examined the reliability of IL-6 values in preserved samples. Our study did not show any significant difference in IL-6 values among preserved samples, indicating the adequacy of our method of preservation and measurement.

P189A INFLUENCE OF PRONE POSITIONING ON MEASUREMENT OF EXTRAVASCULAR LUNG WATER BY TRANSPULMONARY THERMODILUTION

Samir G. Sakka, Ulrike Brücken, Ulrike Glöckner, Dirk Knüttgen, and Frank Wappler. Department of Anesthesiology and Intensive Care Medicine, Medical Center Cologne-Merheim, University of Witten/Herdecke, Cologne

Introduction:

The transpulmonary thermodilution technique enables measurement of extravascular lung water index (EVLWI), which has been found in experimental and clinical studies to agree well with the reference method (transpulmonary double indicator technique)1,2. In this study, we analyzed the influence of prone positioning on the reliability of measurement of EVLWI by the transpulmonary thermodilution technique.

Patients and Methods:

10 mechanically ventilated patients (7♂, 3♀ age 20-64 years) with chest trauma or ARDS undergoing prone positioning and hemodynamic monitoring with the transpulmonary thermodilution technique (PiCCO®, Pulsion Medical Systems AG).All patients received a thermistor catheter (A. femoralis) which was connected to a monitor (PiCCO®plus, Version 7.0 nonUS). Measurement of cardiac output, intrathoracic blood volume (ITBV) and EVLWI were obtained by bolus injections of 15 ml NaCl (<8°C) before and 10 min after modified prone positioning (135°). No changes in ventilator settings or fluid management were made.

Results:

EVLWI was 5.0-16.0 and 5.0-17.0 ml/kg (r=0.92, mean difference 0.17 ± 1.2 ml/kg). Regression analysis for ITBV between both time points revealed r=0.97.

Conclusion:

Measurement of extravascular lung water by the transpulmonary thermodilution technique is not influenced by prone positioning.

References

1. Neumann P. Intensive Care Med 1999.

2. Sakka SG. Intensive Care Medicine 2000.

P189B MEASUREMENT OF CARDIAC OUTPUT BY PULSE CONTOUR ANALYSIS (VIGILEO®) IN CRITICALLY ILL PATIENTS WITH PRONE POSITIONING-A COMPARISON WITH TRANSPULMONARY THERMODILUTION

Samir G. Sakka, Ulrike Brücken, Ulrike Glöckner, Dirk Knüttgen, and Frank Wappler. Department of Anesthesiology and Intensive Care Medicine, Medical Center Cologne-Merheim, University of Witten/Herdecke, Cologne

Introduction:

Cardiac output (CO) and stroke volume variation (SVV) are clinically used for guiding hemodynamic treatment in critically ill patients. We analyzed the reliability of a pulse contour technique for continuous assessment of CO and SVV (Vigileo®).

Patients and Methods:

10 mechanically ventilated patients (7♀, 3♂ age 20-64 years) with chest trauma or ARDS who underwent modified prone positioning (135°) and were monitored by the transpulmonary thermodilution technique (PiCCO®, Pulsion Medical Systems AG) using a thermistor catheter (A. femoralis) and PiCCO®plus monitor. In addition, a FloTrac® sensor (A. radialis) with a Vigileo® monitor (Version 1.10, Edwards Lifesciences) was used. Measurements were obtained by bolus injections of 15 ml NaCl (<8°C) before and 10 min after proning.

Results:

At begin, CO was 4.77-8.97 l/min (Vigileo®) and 3.00-9.32 l/min (PiCCO®): r=0.68 (p=0.03), mean difference 0.45±1.48 l/min. SVV was 6-24% (Vigileo®) and 7-24% (PiCCO®): r=0.85. After proning, CO was 4.80-9.23 l/min (Vigileo®) and 4.77-9.98 l/min (PiCCO®): r=0.16 (p=0.66), mean difference 0.18±2.02 l/min. SVV was 5-10% (Vigileo®) and 4-14% (PiCCO®): r=0.91. Overall, r=0.45 (p=0.04) for CO and r=0.87 for SVV.

Conclusion:

Assessment of SVV by the Vigileo® system seems reliable, however, measurement of CO with this technique cannot be recommended in critically ill patients undergoing prone positioning.

P190 PLASMA DISAPPEARANCE RATE OF INDOCYANINE GREEN (PDR-ICG) AND THE CYTOKINE ACTIVATION PATTERN AND COMPLICATIONS AFTER PERITONECTOMY

Michael O'Leary, Mun Lin Tiong, Theresa Jacques, and David Morris*. Departments of Intensive Care and *Surgery, The St George Hospital, Kogarah, NSW, Australia

Severe morbidity occurs in 20% patients, and mortality in 4%, after cytoreductive surgery and heated intraperitoneal chemotherapy (peritonectomy) for peritoneal surface malignancy1. SIRS and gastrointestinal dysfunction are common2. As PDR-ICG is a marker of hepatosplanchnic perfusion3, and is prognostic in critically ill patients4, we postulated that it might identify peritonectomy patients at risk of systemic and abdominal complications. 12 patients scheduled for peritonectomy had PDR-ICG and plasma IL-6 and IL-10 levels measured pre-operatively, immediately post-operatively and on the1st, 3rd and 6th post-operative mornings. SOFA scores, complications and outcomes to 21 days were collected. Initial mean (±SEM) PDR-ICG was 25.0±3.0% and did not change post-operatively. The only 2 patients with PDR-ICG<16% were inoperable. IL-6 and IL-10 were elevated post-operatively. Post-operative mean SOFA score was 2.3, falling to 0.6 on day 6. 2 patients developed serious abdominal complications but there was no mortality. We found no relationship between PDR-ICG and IL-6 and IL-10 levels, SOFA scores or the development of complications. PDR-ICG may identify patients with inoperable disease but does not predict the development of post-operative complications.

References

1. Yan TD, et al: Ann Surg Oncol 14:2270-80, 2007.

2. Yan TD, et al: J Surg Oncol 92:93-4, 2007.

3. Sander M, et al: Cardiovasc J Afr 18:375-9, 2007.

4. Sakka SG, et al: Chest 122:1715-20, 2002.

P191 A SURVEY OF TRANSFUSION PRACTICES IN OBSTETRICS PATIENTS IN KASHAN SHABIH-KHANI HOSPITAL, IRAN, 2005-2006

Mehrdad Mahdian* and Zohreh Tabasi. Kashan University of Medical Sciences, Kashan, Iran

Introduction:

Hemorrhage is the leading cause of shock and even dead in obstetric patients. The aim of this study was to assess the obstetric risk factors for hemorrhage that needed blood component transfusion in our hospital.

Methods:

This hospital based descriptive study was done at the Kashan University of Medical Sciences. The data were collected from medical records of pregnant women who were admitted during the study period (year 2005-2006). Descriptive statistics were used to analyze the results.

Results:

Out of 5276 cases who were admitted during the study period 90 women (1.57%) received transfusion. Transfusion rate in women who were delivered by cesarean section (1.69%) were more than in women who had normal vaginal delivery (1.01%). Nulliparas and primigravidas and women with history of low hemoglobin (=10mg/dl) before pregnancy, had more transfusion rates than the other cases. The most causes of hemorrhage that leaded to transfusion were as follows: abruptio placentae (16.6%), severe pre-eclampsia (16.6%), abortion (13.3%) and uterine atonia (10%).

Conclusion:

Because of transfusion risks, effort should be made to reduce blood transfusion without increasing maternal morbidity and mortality. Therefore, recognition of risk factors for hemorrhage and transfusion during pregnancy and rapid management of such problems is essential.

*Mehrdad Mahdian

Paramedical School

Kashan University of Medical Sciences

Postal code: 8715985131

Kashan, Iran

P192 SESAME SEEDS - JUST ONE OF MANY FOOD ALLERGENS OR A HIDDEN KILLER?

B. Rymarczyk, B. Rogala, and J. Gluck. Chair and Clinical Departament of Internal Diseases, Allergology and Clinical Immunology, Medical University of Silesia, Poland

The prevalence of sesame allergy increases due to the fact that its consumption is continuously increasing thorughout the world. Ingestion of seseme seeds may elicite severe, life threatening reactions but also may cause delayed hypersensitivity reactions expressed as contact dermatitis. Diagnostic procedures of that kind of food allergy are very difficult as the medical history is often unclear, especially in cases of unadvertent ingestion of food containing sesame or unexpected contamination with trace amounts of it. Physical examination, skin prick tests, laboratory measurements - specific allergen IgE serum level sometimes turn to be ineffective in establishing a proper diagnosis. Open and in selected cases single or double blind placebo - controlled food challenge remain the only reliable method in searching of the offending food. Biphasic and late reactions may also make the diagnosis more difficult. Appropriate use of the combination of these methods contributes to the quality of the diagnosis and treatment of food allergy.

A 40-years old man, in good health so far, exhibited generalized urticaria, dyspnea caused by bronchospasm and hypotension with loss of consciousness shortly after eating industrially prepared waffle containing eggs, sugar, flour and a cocoa filling. Therefore we purchased cocoa, sesame and several kinds of nuts and used them for skin testing (prick-by-prick). As they yielded negative for all kind of nuts (peanuts, walnuts, hazelnuts, cashew, brazilian nuts, almonds) and sesame seeds an open challenge test with all those allergens was performed. Shortly after ingestion of a small amount of a cake containing sesame the patient manifested distress, generalized urticaria, dyspnoea and a significant hypotension. After 4 hours a late response was observed (generalized urticaria with burning and itching).

Conclusion:

The case we reported highlight that in some cases of sesame immediate hypersensitivity the skin prick tests and specific allergen IgE serum level may remain negative and the proper diagnosis might be confirmed only by challenge test.

P194 EFFECTS OF COLLOID RESUSCITATION ON THE SUBLINGUAL MICROCIRCULATION DURING EXPERIMENTAL HEMORRHAGIC SHOCK

József Kaszaki1, Domokos Boda2, Balázs Sütõ3, Andrea Rostás1, András Nagy1, Szilvia Rokolya1, and Mihály Boros1. Institute of Surgical Research1, Department of Anaesthesia and Intensive Therapy3, University of Szeged and Department of Paediatrics2, University of Pécs, Hungary

Background:

Detection of sublingual (SL) pCO2 gap changes might be of diagnostic value to evaluate the effectiveness of resuscitation strategies targeting the gastrointestinal (GI) microcirculation.

Methods:

Red blood cell velocity (RBCV) and capillary perfusion rate (CPR) were monitored in the oral cavity by means of orthogonal spectral polarization imaging, while SL and small intestinal pCO2 gap changes were determined by indirect tonometry in pentobarbital-anesthetized minipigs during hemorrhagic shock (HS) and volume resuscitation (25 ml/kg iv) with 5% albumin (AB), 6% hydroxyethyl-starch 130/0.4 (HES) and 6% dextran-60 (DX) solutions.

Results:

Both intestinal and SL pCO2 gap values were increased significantly (2.6 and 3.6 fold, respectively) during HS, while RBCV and CPR of the oral mucosa were decreased by 51% and 35% respectively. Administration of colloids significantly improved macrohemodynamics, however RBCV in the SL area was restored only by HES and AB (944±124 and 980±57 μm s−1, respectively), while DX was ineffective (620±65 vs 1080±134 μm s−1 baseline). As compared to HS-induced changes, the pCO2 gap values were decreased by 51% (DX), 68% (HES) and 72% (AB) in the SL region.

In conclusion: pCO2 gap changes are associated with characteristic microcirculatory alterations in the SL region. As compared to DX, HES and AB significantly ameliorated the HS-caused microcirculatory deterioration in the oral cavity during the early phase of resuscitation.

Supported by ETT 594/2006 and NKTH 08/2004 grants.

Inflammation / Immunodysfunction Basic Research

P195 ROLE OF TLR2 SIGNALING IN HEMORRHAGIC SHOCK-PRIMED IL-1β PROCESSING

Yuehua Li, Yujian Liu, Jian Zhang3, Guozhi Xiao3, Yoram Vodovotz2, Timothy Billiar2, Mark Wilson1,2, and Jie Fan1,2. 1Surgical Research, VA Pittsburgh Healthcare System; 2Dept. Surgery, and 3 Dept. Medicine, University of Pittsburgh, PA 15213

Background:

IL-1β is a key inflammatory mediator contributing to post-trauma sepsis. The release of active IL-1β from macrophages requires proteolytic maturation by caspase-1, which is activated in inflammasomes. Hemorrhagic shock (HS) primes for IL-1β release in response to LPS via currently unknown mechanisms. We have reported that LPS/TLR4 signaling up-regulated TLR2 expression in alveolar macrophages (AM), thereby augmenting the expression of cytokines by AM in response to the bacterial products LPS (a TLR4 agonist) and PGN (a TLR2 agonist) causing increased lung inflammation. In this study, we addressed the role of TLR4-induced TLR2 upregulation in shock-primed caspase-1 and IL-1β maturation.

Methods:

In vivo experiments: Wild-type (WT, C3H/HeOUJ), C3H/HeJ and TLR2 knockout (TLR2−/−) mice were bled to a MAP of 40 mmHg, maintained for 1h, then resuscitated with shed blood and Ringer's lactate. HS was followed by LPS (30 μg/kg, intratracheal [i.t.]) at 2h after resuscitation, and sequential treatment of PGN (30 μg/kg; i.t.) 2h after LPS. At 2h after PGN i.t., bronchoalveolar lavage fluids (BALF) were collected for detection of IL-1β in the AM and the BALF by ELISA. In vitro AM-PMN co-culture experiments: AM from WT mice were co-cultured with PMN that were isolated from either sham-operated or HS mice. LPS was added at 0h, followed by PMN removal and addition of PGN to the AM at 2h. TLR2 expression in the AM was confirmed by immunoblot. IL-1β in the AM lysates and medium, as well as caspase-1 and IL-1β processing in the AM, were then assessed.

Results:

Sequential treatments of LPS+PGN caused a significant increase in IL-1β in BALF in the WT HS mice as compared to that in WT sham animals, HS C3H/HeJ or HS TLR2−/−mice. AM-PMN co-culture experiments showed that PMN derived from shocked animals increased TLR2 expression in AM in response to LPS stimulation, and subsequently caused a significant increase in caspase-1 and IL-1β maturation in the AM as well as IL-1β release from the AM in response to TLR2 agonist PGN. Furthermore, increasing extracellular K+concentration failed to prevent the TLR2 signal-induced IL-1β maturation.

Conclusion:

amplified TLR2 signaling in AM, which is induced by LPS/TLR4 and enhanced by HS, acts as a second signal that stimulates caspase-1 activation and IL-1β maturation via mechanisms independent of K+efflux.

P196 HYPERTONIC SALINE INCREASES THE CLEARANCE OF INFLAMED NEUTROPHILS BY T CELLS

Mark I. Hirsh, Yoshiaki Inoue, Yu Chen, Reinhard Pauzenberger, and Wolfgang G. Junger. Department of Surgery, Trauma and Surgical Critical Care, BIDMC, Harvard Medical School, Boston, MA 02215

Hypertonic saline (HS) fluids used to resuscitate trauma patients can prevent neutrophil (PMN)-mediated lung tissue damage, making them attractive alternatives to conventional resuscitation fluids. HS blocks PMN activation. In addition, γδT cells-a small T lymphocyte subset-reduce tissue damage by eliminating inflamed PMN that express heat shock protein (Hsp) 72 on their cell surface. Here we studied how HS influences the ability of γδT cells to kill PMN.

Human γδT cells and PMN were isolated from the peripheral blood of healthy humans and treated with clinically relevant HS concentrations (20 mM) in the presence or absence of endotoxin (LPS). Hsp72 expression and PMN killing in co-cultures with γδT cells were measured.

Stimulation of PMN with 1-1,000 ng/ml LPS increased Hsp72 expression on the cell surface more than 3-fold. HS significantly augmented this response. Under baseline conditions, γδT cells killed 15.6±3.4% of all PMN within 120 min. In the presence of LPS (1 μg/ml), this percentage increased to 23.7±2.1%, and when HS was present 31.8±3.1% PMN were killed.

Our findings indicate that HS enhances killing of inflamed PMN by γδT cells through upregulation of Hsp72 expression by PMN. HS resuscitation therefore protects lung tissues during sepsis at least in part by enhancing the clearance of PMN from inflamed host tissues. (Funding: Novo Nordisk, NIH, and DoD).

P197 GENERATION OF ADENOSINE FROM RELEASED ATP, BY THE ECTOENZYMES CD39 AND ALKALINE PHOSPHATASE, REGULATES CHEMOTAXIS

Ross Corriden, Yu Chen, Yoshiaki Inoue, Guido Beldi, Simon Robson, and Wolfgang G. Junger. Depts. of Surgery and Medicine, BIDMC/Harvard Medical School, Boston, MA 02215; and Depts. of Surgery, Pharmacology, and Medicine, UCSD, San Diego, CA 92103

Our previous work has shown that polymorphonuclear neutrophils (PMN) release ATP in response to chemoattractants and that chemotaxis requires adenosine formation and feedback through A3 adenosine receptors. Here we studied the ecto-enzymes of PMN that are responsible for generating extracellular adenosine.

Human PMN efficiently hydrolyze ATP and ADP to AMP, while the conversion of AMP to adenosine is significantly slower. We note that PMN express CD39 (ecto-NTPDase1) and alkaline phosphatase (ALP). Inhibitors of CD39 (NaN3, ARL67156) suppress phosphohydrolysis of ATP, while inhibitors of ALP (Na3VO4, levamisole) block the conversion of AMP to adenosine. ALP is uniformly distributed across the cell membrane of human PMN, while CD39 is localized at the leading edge of PMN placed into a chemotactic gradient field. Inhibitors of CD39 significantly slow the migration velocity but not gradient sensing of human PMN. Migration speeds of PMN isolated from the bone marrow of CD39 knockout (CD39 KO) mice are significantly slower, compared to wild-type (WT) cells. Similarly, in vivo migration of cells towards the chemotactic peptide injected into the peritoneal cavities of CD39 KO mice are significantly diminished, when compared to WT mice.

Our data suggest that CD39 and ALP modulate ATP phosphohydrolysis to control PMN chemotaxis by generating extracellular adenosine from endogenously released ATP (Funding: Novo Nordisk, NIH, and DoD).

P198 VASCULAR RESISTANCE TO VENOUS RETURN INCREASES DURING RAT ANAPHYLACTIC SHOCK

Toshishige Shibamoto, Sen Cui, Wei Zhang, and Yasutaka Kurata. Department of Physiology II, Kanazawa Medical University, Uchinada Ishikawa 920-0293, Japan

Anaphylactic shock is a sudden, life-threatening allergic reaction associated with severe hypotension. The increased vascular resistance to venous return accounts for the anaphylactic hypotension in anesthetized dogs. However, the change in peripheral vascular resistances during anaphylactic hypotension in other animals, such as rats, is not known. We measured the mean circulatory filling pressure (Pmcf) using the mechanical occlusion method of inflation of the right atrial balloon, along with systemic arterial pressure (Psa), central venous pressure (Pcv) and portal venous pressure (Ppv). Cardiac output (CO) was also measured with the thermodilution method. From these hemodynamic variables we calculated the total peripheral (Rt) and venous (Rv) resistances during anaphylactic hypotension in anesthetized rats. These hemodynamic variables were compared with those in the hemorrhagic shock. After an intravenous injection of antigen ovalbumin 0.6mg in sensitized rats, Psa decreased from 119 ± 4 to 43 ± 2 mmHg, CO decreased from 84.5 ± 5.7 to 37.8 ± 2.1 ml·min−1, Pcv decreased from 0.9 ± 0.1 to 0.1 ± 0.1 mmHg, and Pmcf also decreased from 6.0 ± 0.2 to 5.2 ± 0.3 mmHg. Thus, the venous resistance (Rv) increased from 0.06 ± 0.05 to 0.15 ± 0.02 mmHg·ml−1·min, but Rt did not significantly change. Ppv also increased from 5.6 ± 0.5 to 21.5 ± 0.9 mmHg. During hemorrhagic shock Psa decreased in the manner similar to anaphylactic shock; however, Rv did not significantly change and Ppv decreased. In conclusion, in rat anaphylactic shock, a substantial increase in vascular resistance to venous return presumably due to hepatic venoconstriction may decrease venous return, resulting in systemic hypotension.

Keywords:

Anaphylactic shock, hemorrhagic shock, mean circulatory filling pressure, circulatory vascular resistance, portal venous pressure

P199 GREEN TEA POLYPHENOL AMELIORATES PANCREATIC INJURY IN CAERULEIN-INDUCED MURINE ACUTE PANCREATITIS

B.I. Babu1, T. Genovese2, E. Mazzon2, R.D. Paola2, C. Crisafulli2, G. Malleo2, R. Caminiti2, A.K. Siriwardena1, and S. Cuzzocrea2. 1Dept of Surgery, Manchester Royal Infirmary, UK; 2Dept of Clinical and Experimental Medicine and Pharmacology, Torre Biologica, Policlinico Universitario, 98123, Messina, Italy

Green tea is rich in polyphenols - naturally occurring anti-oxidants which have powerful anti-inflammatory properties mediated by action at a variety of pathways including superoxide radical generation (ROS), nitric oxide and nuclear factor κB (Nf-κB). Oxidative stress mediated by ROS and NF-kB mediated pathways are known to be mediators of acinar injury in acute pancreatitis. This study investigates the effect of GTE in a caerulean-induced murine model of acute pancreatitis (AP). Studies were carried out in accordance with EU animal experimentation regulations. Male CD mice (Median weight 37.7g(Range 33-41g))were divided in to four groups. Caerulein and vehicle {C and V}, Caerulein and Green tea {C and G}, Vehicle and Saline {V and S}, Vehicle and Green tea {V and G}). AP was induced by serial Intraperitoneal (IP) administration of Caerulein (50μg/kg (x6)). 25μg/kg of GTE was administered intraperitoneally on the 1st, 3rd and 6th hour after induction of pancreatitis in the C and G and V and G groups. Animals were sacrificed at 24 hours after the first administration of Caerulein and the following parameters were measured: 1) expression of the adhesion molecule ICAM-1, 2)nitrotyrosine, 3)inducible nitric oxide synthase (iNOS),4) Poly (ADP-ribose) synthetase (PARS), 5)Myeloperoxidase, 7)Malondialdehyde, 8)Amylase, 9)Lipase and 10) histologic evidence of pancreatic injury. Intraperitoneal injection of Caerulein in mice resulted in severe acute pancreatitis characterised by oedema, neutrophil infiltration, tissue haemorrhage and necrosis and elevated serum levels of amylase and lipase. Infiltration of the pancreas with neutrophils (measured as increase in myeloperoxidase activity) was associated with enhanced lipid peroxidation (increased tissue levels of malondialdehyde). Immunohistochemical examination demonstrated a marked increase in immunoreactivity for nitrotyrosine, iNOS and poly (ADP-ribose) synthetase (PARS) in the pancreas and lung of caerulein-treated mice. In contrast, the above mentioned parameters were markedly reduced by treatment with GTE. Our results have shown that GTE has significantly ameliorated the effects of Caerulein induced pancreatitis. This study demonstrates the protective effect of GTE and raises the prospect of a novel therapeutic mode in the management of acute pancreatitis.

P200 THE NON-GENOMIC PATHWAY MEDIATES SOME OF THE EFFECTS OF ESTROGEN ON CYTOKINE PRODUCTION BY T CELLS FOLLOWING TRAUMA-HEMORRHAGE

Takao Suzuki, Huang-Ping Yu, Ya-Ching Hsieh, Mashkoor A. Choudhry, Kirby I. Bland, and Irshad H. Chaudry. Center for Surgical Research and Department of Surgery, University of Alabama at Birmingham, AL 35294

Studies have shown that administration of 17β-estradiol (E2) following trauma-hemorrhage (T-H) attenuates the T-H-induced suppression in T cell cytokine production. However, it remains unknown whether E2 mediates its effects via genomic or non-genomic pathway. To determine the role of non-genomic pathway and the role of MAPK in mediating the non-genomic effects of E2, E2 conjugated to BSA (E2-BSA) which acts on only cell surface membranes was used. Male rats underwent T-H (mean BP 40mmHg for 90min, then resuscitation). E2, E2-BSA with or without an estrogen receptor antagonist (ICI 182,780), or vehicle was injected intravenously during resuscitation. IL-2 and IFN-γ productive capacity and the activation of MAPK (p38, ERK1/2, JNK) in response to anti-CD3 in T cells decreased at 2 h after T-H (Fig.). However, E2 administration restored the values to normal. Although E2-BSA administration also significantly attenuated the suppression in T cell cytokine production, the values were still lower than sham. In contrast, administration of E2-BSA prevented T-H-mediated changes in MAPK activation in T cells to the same extent as E2 treatment. Co-administration of ICI 182,780 abolished the effects of E2-BSA. Moreover, IL-2 and IFN-γ production was further suppressed in vitro by selective inhibitors of ERK1/2 (PD98059) or JNK (SP600125) pathway in T cells, while p38 inhibitor (SB203580) had no significant effects. Thus the salutary effects of E2 on T cell cytokine production are mediated at least in part via the non-genomic pathway and these non-genomic effects are likely mediated via MAPK.

Figure 1
Figure 1

P201 THE PEROXISOME PROLIFERATOR ACTIVATED RECEPTOR-γ (PPARγ) LIGAND 15d-PGJ2 REDUCES THE PRO-INFLAMMATORY RESPONSE IN MYOCYTES: A MICROARRAY ANALYSIS

A. Denenberg*, P.W. Hake*, M. O'Connor*, and B. Zingarelli. Critical Care Med., Cincinnati Children's Hosp. Med. Ctr., University of Cincinnati, Cincinnati, OH 45229, USA

The nuclear factor PPARγ has been implicated in the regulation of the inflammatory response in sepsis and reperfusion injury. Here, we investigated the role of PPARγ on gene expression in rat myoblasts, which were treated with TNFα in the presence or absence of the PPARγ ligand 15d-PGJ2. Gene expression was evaluated by microarray analysis using Affymetrix 230 2.0 Array chips. Analysis was restricted to genes exhibiting a statistical difference (P<0.05 by Welch t test) within vehicle and 15d-PGJ2-treated groups. Treatment with 15d-PGJ2 significantly reduced several TNFα-induced pro-inflammatory genes, such as adhesion molecule genes (Ccl2 chemokine ligand 2, NM_03153; Icam1 intercellular adhesion molecule-1, NM_012967; Cinc2 cytokine-induced neutrophil chemo-attractant-2, NM_138522), genes for cytokines and related signaling pathway (Il6st interleukin signal transducer, AY310138; Il6 interleukin 6, NM_012589) and genes for transcription factors and inhibitors (Nfkb1a nuclear factor-κB inhibitor-a, XM_343065; EST similar to NF-κB subunit p100, XM_219954). Notably, treatment with 15d-PGJ2 significantly increased anti-inflammatory genes, such as heat shock protein (HSP) genes (Hspa1a HSP 70kD 1A, NM_031971; EST similar to HSP 105kD a, XM_213699; EST similar to HSP40, XM_341663; Hspb1 HSP 27kD, NM_031970; Hspca heat shock protein 1 a, NM_175761). The data suggest that 15d-PGJ2 regulates inflammation by selective down-regulation of pro-inflammatory genes and up-regulation of the cytoprotective heat shock response. Supported by NIH (R01 GM-067202; R01 AG-27990).

P202 EX-VIVO CYTOKINE RESPONSE TO STIMULATION IN HEALTH AND DISEASE

Axel Nierhaus, Daniel Frings, Barbara Montag, Jo Linssen, and Georg Kreymann. Dept. of Critical Care, University Medical Center Hamburg-Eppendorf, Martinistr. 52, D - 20246 Hamburg, Germany

Introduction:

Trauma and sepsis lead to SIRS and to a Compensatory Anti-inflammatory Response Syndrome (CARS) with impaired host defense. GM-CSF in patients with major surgery or sepsis can improve host defense. In this study we investigated the effects of GM-CSF and LPS on the ex-vivo TNF-α production.

Methods:

Whole blood of 40 healthy donors (mean age 54 y) was used to determine optimal concentrations and incubation time for LPS. The immunomodulating properties of GM-CSF (Leukine® (sargramostim)) were investigated in whole blood of 28 healthy donors (mean age 51 y) and 12 ICU patients suffering from sepsis. Six patients had immunoparalysis as defined by a monocytic HLA-DR expression of < 150 MFI and an ex-vivo stimulation test of < 175 pg/ml after LPS incubation, whereas the other 6 had an HLA-DR expression of > 150 MFI and an ex-vivo stimulation test of > 175 pg/ml. Samples were primed either with GM-CSF or LPS prior to incubation. TNF-α and IL-8 concentrations were determined with the IMMULITE immunoassay. Leukocyte phenotyping was performed by flow cytometry.

Results:

In healthy donors, stimulation with LPS leads to an increase of TNF-α production. However, if whole blood is incubated with GM-CSF 3 hours prior to the LPS challenge, the TNF-α production is increased. The simultaneous incubation with LPS and GM-CSF leads to a decrease in TNF-α levels in the same patient population. GM-CSF stimulation of whole blood 3 hours after LPS causes no change in TNF-α production. In patients with sepsis and TNF-α levels of < 30pg/ml, GM-CSF pre-incubation leads to an increase in ex-vivo TNF-α production, whereas patients with higher levels of TNF-α had a blunted reaction to LPS.

Conclusion:

Both the sequence of stimulation with either GM-CSF or LPS and the presence of systemic TNF-α determine the ex-vivo cytokine response of whole blood. Hence, it may be speculated that (1) the administration of GM-CSF prior to the inflammatory stimulus would be most efficient, and that (2) the lack of stimulation effect in patients with high endogenous TNF-α may mirror endotoxin tolerance.

P203 X-CHROMOSOME MOSAICISM AND THE FEMALE ADVANTAGE DURING INFECTIONS

Z. Spolarics, E.A. Deitch, G. Hasko, and R. Chandra. Dep. Surgery, UMDNJ-New Jersey Medical School, Newark USA

Cells from females carry both parental X chromosomes whereas males carry the maternal X chromosome only. As a result of dosage compensation and random X chromosome inactivation, females are cellular mosaics for X-linked polymorphic proteins. We hypothesized that X-mosaicism in females represents a functionally adaptive condition. This was tested using an X-linked gp91phox-deficient mouse model in which neutrophil activation and oxidative burst are compromised. LPS-induced changes (E. coli, 20mg/kg ip) in bone marrow (BM), blood and spleen CD11b+ neutrophil content (PMN) were compared between female gp91phox mosaic (−/+) and male hemizygous deficient (j/y) or WT (+/y) animals. Changes in WT/deficient PMNratios in mosaic mice were tested using an anti-gp91phox antibody and flow cytometry. In controls, BM, blood and spleen PMN was increased in hemizygous deficient animals as compared to WT or mosaic mice. LPS depleted BM PMN whereas increased blood and spleen PMN in WTandmosaic animals similarly. In contrast, LPS caused no changes in BM, blood or spleen PMN in hemizygous deficient mice indicating compromised PMN mobilization and recruitment. WT/deficient PMNratio was ~0.9 in BM, blood and spleen from control mosaic mice. LPS treatment increased WT/deficient PMN-ratios in blood and spleen indicating that cell recruitment was skewed toward the WT mosaic subpopulation in females. The observations suggest that female Xmosaicism represents a broadened cellular repertoire and a functionally adaptive cellular system in this model. The study also supports the hypothesis that human mosaicism associated with X-linked genetic polymorphisms may contribute to the female gender benefit observed during the clinical course of infection.

P204 NEAR INFRARED DYES VERSUS CHROMOGENIC SUBSTRATES IN CTOKINE ELISAS

Elizabeth Schuller, and Daniel Remick. Boston University School of Medicine Dept. of Pathology Boston, MA 02118

Standard enzyme-linked immunosorbent assays (ELISA) have been shown to be a sensitive and accurate way of measuring protein levels in serum. In most standard ELISAs a chromogenic substrate is used to quantify the amount of protein in the sample. The purpose of this study was to determine if using near-infrared dyes in a standard ELISA would be as sensitive and accurate as chromogenic substrates, while also reducing the effect of photo-bleaching, which can occur with chromogenic substrates.

Methods:

Blood, taken from healthy donors, was stimulated with 50ng/ml lipopolysaccharide (LPS). Samples were incubated for 3, 6, 12 and 24 hours, then the plasma was taken and stored at 20°C until analysis. The new protocol uses strepavidin-conjugated IRDye in the place of horseradish peroxidase (HRP) and TMB and uses a scanner that can read near-infrared. The two protocols were run side by side using the same matched-antibody pairs. The IRDye plate was also saved and rescanned two months later. The strength of correlation was determined by linear regression.

Results:

IL-8 and MCP-1 showed strong correlation between IRDye and the colorimetric substrate (R2 0.92, slope 1.03 and R2 0.96, slope 0.92 respectively.)

Figure
Figure

Discussion:

Near-infrared dyes have been shown to be as sensitive and accurate as chromogenic substrates.

P205 IMPACT OF VARYING HYPERTONICITY ON THE IMMUNE-MODULATING EFFECTS OF LIPOPOLYSACCHARIDE (LPS) AND PEPTIDOGLYCAN (PEPG)

Tom Erik Ruud, Yngvar Gundersen, and Ansgar Aasen. Institute for Surgical Research, Rikshospitalet University Hospital, Oslo, Norway

The immune-modulating qualities and possible mechanisms of action of hypertonic saline have been debated. The aim of the present study was to investigate how varying NaCl concentrations would influence the immune effects of two different stimulators (LPS and PepG) with distinct site of action.

Methods:

Freshly tapped blood from ten healthy donors was stimulated by the bacterial wall constituents LPS 10 ng/ml or PepG 1 μg/ml at 37 °C in an ex vivo whole blood model. The incubation lasted for 6 h and concentrations of NaCl from 0.9 to 6.0 % were employed. The leukocyte response was evaluated according to the concentration of TNF-α in the supernatant.

Results:

After activation with PepG, increasing hypertonicity led to a steady drop of TNF-α values relative to normal saline. At NaCl 3%, statistical significance was reached (See figure). The stimulating effects of LPS, in contrast, rather tended to be boosted by hypertonicity (NS). At 6% an abrupt reduction of TNF-á levels was found for both activators, coinciding with reduced cellular viability.

Figure
Figure

Conclusions:

Hypertonic saline has immune-modulating properties. Concentrations normally found invivo (corresponding to 1.1 to 1.5 %) had only weak and insignificant effects on the immune response. Cells stimulated with PepG were more affected than those stimulated with LPS, possibly related to their distinct sites of action (intracellular or membrane-bound).

P206 DELAYED ADDITION OF DEXAMETHASONE FAILS TO REDUCE PRO-INFLAMMATORY CYTOKINES

Horton Devin1,2, and Remick Daniel1. Dept. of Pathology, Boston University Medical School, Boston, MA, Program in Cellular & Molecular Biology, University of Michigan Medical School, Ann Arbor, MI

Numerous studies have investigated the mechanisms of inflammation using the potent glucocorticoid, Dexamethasone (Dex), as an anti-inflammatory therapy either prior to or simultaneously with an inflammatory stimulus. Patients generally present in a clinical setting following the onset of inflammation, thus a more clinically relevant model is needed. To determine the underlying mechanisms of persistent or, ongoing inflammation, we used Dex to modulate the inflammatory response after its onset. Heparinised whole blood from normal volunteers was stimulated with 50 ng/ml LPS. Dex was added (10−6M) either simultaneously or 6 h after the LPS stimulus. Preliminary data show that, adding Dex simultaneously with LPS suppressed pro-inflammatory cytokine levels (measured at 24 hours), while delaying addition of Dex by just 6 h dramatically diminished its capacity to suppress both IL-6 and IL-1β. These data suggest that different mechanisms regulate the onset of inflammation compared to maintaining ongoing inflammation. Further investigation of the mechanisms of the clinically relevant model of ongoing inflammation is imperative to develop better anti-inflammatory therapies.

Table
Table

P207 PRESENCE OF POTENTIAL TARGET ALTERS SENTRY BEHAVIOR OF NATURAL KILLER T (NKT) CELLS IN THE LIVER IN VIVO.

Eric Norris1, Jerrod Kraftchick2, Cathy Culberson1, Sang-Ho Lee1, Min Shin2, and Mark G. Clemens1. Dept of 1Biology and 2Computer Science, University of North Carolina at Charlotte, Charlotte NC, 28223 USA

There is growing recognition of the importance of NKT cells in shock. NKT cells sequester in the liver and migrate through the sinusoids exhibiting an apparently random sentry pattern. We tested the hypothesis that the sentry behavior of liver NKT cells is influenced by the presence of a potential target. CXCR6gfp+/+ transgenic mice in which NKT cells specifically express green fluorescent protein were used in the presence or absence of fluorescently labeled M38 colon cancer cells as potential target to study sentry behavior by time-lapsed in vivo microscopy with automated image acquisition, processing and analysis. M38 liver metastases were produced by intrasplenic injection. Sentry behavior existed in two distinct patterns: roaming", in which NKT cells migrated over long distances in a relatively straight pattern; and dancing", in which NKT moved within a constrained dance floor". Total NKT cells per field were similar with or without target (19.9vs 21.6, respectively) but mice with M38 cells showed a higher proportion of roaming cells compared to controls (82% vs 71%). To assess the behavior of NKT cells as they approached a potential target, acceleration of NKT cells was assessed in livers with M38 cells or latex beads. There was no change in velocity (acceleration = 0) as NKT approached beads but a significant slowing as they approached M38 (p< 0.001). These results indicate that the sentry behavior of NKT cells is sensitive to the presence of target in the liver microcirculation. Moreover, these imaging techniques can provide valuable tools for the study of NKT cell behavior in shock. Supported by GM 77501.

P208 LUNG INFLAMMATION FOLLOWING CARDIOGENIC SHOCK

X. Lin, K. Koga, Y. Zhang, K. Takahashi, H. Linge, C.N. Metz, K. Ojamaa, and E.J. Miller. The Feinstein Institute for Medical Research, Manhasset, NY, 11030

Acute myocardial infarction (AMI) is the most common cause of cardiogenic shock, and activation of the inflammatory response plays an important role in its pathogenesis and outcome.

Our objective:

was to study the inflammatory changes in the lung after AMI.

We used ligation of the left coronary artery in male Sprague Dawley. After 48hrs, animals were euthanized and bronchoalveolar lavage (BAL) samples collected. Lung and cardiac tissue were harvested for histology and infarct size evaluation. Animals were then grouped with respect to infarct size as a percent of myocardial volume (I 50: 50%; I<30: <30%) and compared to sham operated animals.

Results:

There were increased numbers of neutrophils and foamy macrophages within the I50 group as well as a seven fold increase in Gro/KC (CXCL1; Table 1).

Table
Table

Conclusion:

While not recapitulating all of the published in vivo data, these results demonstrate this in vitro assay shares a key feature with in vivo models of sepsis, namely splenocytes over-expressing the anti-apoptotic protein Bcl-2 are protected from apoptosis.

P209 SPLENOCYTES FROM BCL-2 OVER-EXPRESSORS ARE RESISTANT TO E. COLI-INDUCED APOPTOSIS

A.H. Walton*, C.G. Davis, K. Chang*, R.S. Hotchkiss, and J.E. Mcdunn. Washington University School of Medicine, 660 S.Euclid Ave., Campus Box 8109, Saint Louis, MO 63110

Introduction:

Extensive lymphocyte apoptosis is one of the hallmarks of sepsis. Furthermore, transgenic mice with apoptosis-resistant lymphocytes display a significant survival advantage compared to wild type littermates in sepsis models. Recently, we have developed an in vitro model of E. coli-induced lymphocyte apoptosis to enable screening of over 32,000 small-molecule therapeutic candidates.

Hypothesis:

This in vitro assay captures essential features of the disease process it was designed to model. To test this hypothesis, we evaluated whether splenocytes from transgenic animals displaying attenuated sepsis-induced lymphocyte apoptosis were resistant to E. coli-induced apoptosis.

Methods:

Splenocytes were harvested from transgenic mice and wild type littermates, cultured overnight in the presence of lyophilized E. coli, stained for CD3 (T-cells) and TUNEL (apoptosis), then evaluated by flow cytometry. Dose-response curves were generated and LD50s calculated.

Results:

For Bcl-2 over-expressers (M22, and TBJ) there was a marked change in the dose-response and a 3-4 fold increase in the LD50s (Figure). For other transgenic mice (Bim −/−, Bid −/−, MyD88 −/−, FADD-DN, and myr-AKT, E. coli sensitivity was identical to wild-type splenocytes.

Figure
Figure

Conclusion:

While not recapitulating all of the published in vivo data, these results demonstrate this in vitro assay shares a key feature with in vivo models of sepsis, namely splenocytes over-expressing the anti-apoptotic protein Bcl-2 are protected from apoptosis.

P210 LIPOPOLYSACCHARIDE REGULATES ADENYLYL CYCLASE EXPRESSION IN HUMAN MONOCYTES

Petter K. Risøe, Una Ryg, Ansgar O. Aasen, Jacob E. Wang, and Maria K. Dahle. Institute for Surgical Research, University of Oslo and Rikshospitalet, 0027 Oslo, Norway

Phosphodiesterase (PDE) inhibition by pentoxifylline has shown promising signs as a treatment modality in animal models of septic shock, and exerts its effect through elevating levels of the intracellular signaling mediator cyclic adenosine monophosphate (cAMP). By utilizing several isoforms of adenylyl cyclases (ACs) and phosphodiesterases (PDEs), this ubiquitous system provides the cell with fine tuned cAMP signaling, which is crucially involved in maintaining physiological functions commonly impaired in sepsis. We have demonstrated that lipopolysaccharide (LPS) attenuates AC mRNA in an isoform-specific manner in a rat model of endotoxemia and in cultured rat Kupffer cells. Here, we have studied the effects of LPS on AC expression in human monocytes.

Method:

We have exposed monocytes to LPS and cAMP-elevating agents in a whole blood from healthy volunteers of both genders, followed by plasma collection or monocyte isolation by magnetic beads, RNA isolation and RT-PCR.

Results:

In whole blood, cAMP elevating drugs clearly limit the release of proinflammatory tumor necrosis factor á (P>0.01) and interleukin 6 (P>0.05) levels. Human monocytes express AC4, 6, 7, 9 and soluble AC, of which AC4 and AC7 follow a pattern of attenuation by LPS at 1 and 3 hours compared to untreated cells (AC4 p<0.05; AC7 p=0.08), followed by upregulation maximized at 24 hours (p<0.05). The results suggest stronger regulation in male compared to female donors.

Conclusion:

LPS leads to alterations in AC expression in human monocytes, primarily from male donors. Such regulation may affect cAMP production and thereby cAMP-mediated attenuation of LPS-mediated production of proinflammatory cytokines.

P211 CLP SEPSIS IS ASSOCIATED WITH REDUCED ADENYLYL CYCLASE EXPRESSION

Una Ryg, Petter K. Risøe, Yun Yong Wang, Jacob E. Wang, Ansgar O. Aasen, and Maria K. Dahle. 1Institute for Surgical Research, University of Oslo and Rikshospitalet, 0027 Oslo, Norway

Introduction:

One reason for the fatal inflammatory response causing organ injury and death in sepsis is the loss of immune modulatory mechanisms. The intracellular signaling mediator cyclic adenosine monophosphate (cAMP) is a potent modulator of innate immune cell activation, and elevation of cAMP by phosphodiesterase inhibitor treatment can increase survival in endotoxemia. We have previously demonstrated that expression of cAMP-producing adenylyl cyclases (AC) are attenuated by bacterial lipopolysaccharide (LPS) in vivo and in Kupffer cell cultures. Here we explore AC gene expression and regulation in a rat model of cecal ligation and puncture (CLP) sepsis and in liver cells isolated from this model.

Methods:

Wistar rats were subjected to CLP procedure or sham operated, and after 10 or 18 hours organs were removed. Hepatocytes, liver sinusoidal endothelial cells (LSEC) and Kupffer cells were isolated 18 hours after CLP/sham-operation. RNA was isolated and analysed for AC mRNA expression.

Results:

We found significantly attenuated liver expression of AC6 (P ≤ 0.0336) and AC9 (P ≤ 0.0144) mRNAs in CLP animals compared to sham, an attenuation also found in Kupffer cells (AC6 P≤0.0134; AC9 P≤0.0623) isolated from the model. AC9 was also attenuated in spleen (P≤ 0.0154) and kidney (P≤ 0.0091) after 18 hours. AC7 mRNA was only attenuated in spleen (P≤ 0.0183, 18h), and appeared to increase in kidney, liver and LSEC.

Conclusion:

CLP sepsis is associated with reduced AC mRNA expression in spleen, kidney, liver and Kupffer cells, which may reduce cAMP production and impair cAMP-mediated immune modulation.

P212 MONONUCLEAR CELLS ACCUMULATE IN ALVEOLI AFTER LUNG CONTUSION - ROLE OF MEDIATORS RELEASED BY MACROPHAGES

D.H. Seitz, A. Palmer, U. Niesler, M. Sulger, M. Perl, M.S. Huber-Lang, F. Gebhard, and M.W. Knöferl. Dept. of Trauma Surgery, University of Ulm, 89075 Ulm, Germany

Lung contusion induces local and systemic inflammation. Previous studies demonstrated an increase of alveolar macrophages (AM) in traumatized lungs. Supernatants of AM isolated after blunt chest trauma have been shown to be rich in chemotactic mediators. Our hypothesis was that mononuclear cells migrate to lungs after chest trauma and that mediators of AM are involved. To study this, male Sprague Dawley rats were subjected to sham procedure or blunt chest trauma induced by a single blast wave. At 6, 24 or 48h after the insult, AM were isolated and cultured for 24h without stimulation. Mononuclear cells (PBMC) were isolated from untreated rats. Migratory activity of PBMC to AM supernatants was determined using a Boyden-Chamber. PBMC showed a significantly increased migratory activity after addition of AM supernatants isolated at 6 or 48h after chest trauma, compared to sham. In another set of animals, resident AM, but not circulating PBMC were selectively stained by intravenous application of PKH26. 48h later, animals were subjected to blunt chest trauma. At 4, 24 or 48h after the insult, alveolar macrophages were isolated. Staining for PKH26 was evaluated by FACS. The content of PKH26 negative cells in the cellular fraction of the BAL was markedly increased at 48h after lung contusion, when compared to sham. These results indicate that mononuclear cells accumulate in the alveoli after chest trauma. Furthermore, our results point out that mediators released by AM after lung contusion exhibit a marked chemotactic potential on PBMC. (DFG KN 475/3-2)

P213 RAPID SCREENING ASSAYS FOR 5_LIPOXYGENASE GENETIC POLYMORPHISMS

Emanuel V. Geiger, MD1,2, Alexandra Doehring, PhD1, and Jörn Lötsch, MD. 1pharmazentrum frankfurt/ZAFES, Institute of Clinical Pharmacology, Johann Wolfgang Goethe-University, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany 2Department of Trauma, Hand, and Reconstructive Surgery, Johann Wolfgang Goethe-University, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany

Objective:

Leukocyte chemotaxis, cytokine release, tissue edema, bronchoconstriction, and vasodilation are believed to contribute to the acute respiratory distress syndrome (ARDS). Leukotrienes (LT), an important family of eicosanoids formed from arachidonic acid via the key enzyme 5-lipoxygenase importantly contribute to pathogenesis of ARDS. Therefore, polymorphisms of the ALOX5 gene are candidate modulators of the individual risk to develop ARDS or may modulate the severity of the clinical symptoms. Hence, we developed rapid and reliable screening methods for ALOX5 variants spanning the whole range of the gene, with inclusion of previously reported clinical functional polymorphisms.

Material and Methods:

Single nucleotide polymorphisms (SNP) with a minimum minor allelic frequency of 10% or reported functional relevance were included, which led to ten SNPs providing a comprehensive coverage of the whole ALOX5 gene from the promoter to the 3'UTR region. Pyrosequencing™ screening assays were established and validated in DNA samples from 180 healthy unrelated Caucasians.

Results:

ALOX5 polymorphisms were identified correctly as proven by conventionally sequenced control samples. Frequencies of homozygous, heterozygous and non-carriers of the minor alleles agreed with the Hardy-Weinberg equilibrium. Minor allelic frequencies were: rs4986832G>A = 0.15, rs4987105C>T = 0.06, rs2115819T>C = 0.45, rs3740107G>A = 0.25, rs156096A>G = 0.22, rs2291427G>A = 0.31, rs10751382A>G = 0.31, rs2242334G>T = 0.34, rs2229136A>G = 0.07 and rs3802548A>T = 0.26.

Conclusions:

The here developed Pyrosequencing™ assays allow for quick and reliable detection of ALOX5 genotypes and may facilitate further investigations of ALOX5 genetic functional associations in ARDS and other clinical conditions that involve regulation of 5-lipoxygenase.

P214 INHALED H2S AND HYPOTHERMIA COMPARABLY ATTENUATE SURGERY-RELATED INFLAMMATION

K. Baumgart, V. Simkova, M. Perl, D.H. Seitz, M. Huber-Lang, S. Weber, P. Radermacher, and M.W. Knöferl. Sektion Anästhesiologische Pathophysiologie and Klinik für Unfallchirurgie, Universitätsklinikum, 89070 Ulm, Germany

Inhaling H2S induced hypothermia and suspended animation and thus protected mice against lethal hypoxia [1]. Since H2S was referred to exert both pro- and anti-inflammatory properties [1], we studied the effect of inhaled H2S on the surgery-induced lung inflammatory response. 24 hours after laparotomy, lung tissue TNF-α, IL-6, MCP-1, MIP-2 and GRO/KC were measured in anesthetized and mechanically ventilated mice assigned to 6 hours of 100 ppm inhaled H2S or vehicle with core temperature maintained at 38°C or 27°C, respectively. Inhaled H2S and hypothermia comparably attenuated MCP-1, MIP-2 and GRO/KC expression, while TNF-α and IL-6 were not affected (data median (range), # p<0.05 vs. Control 38°C).

Table
Table

Inhaled H2S has similar anti-inflammatory properties as profound hypothermia without major synergistic effects.

1. Szabó C: Hydrogen sulphide and its therapeutical potential. Nat Rev Drug Discov 2007; 6:917-35

GRANT ACKNOWLEDGEMENT:

Supported by the Deutsche Sepsis-Gesellschaft.

P215 INHALATION OF 100% OXYGEN IMPROVES PLASMA OXIDATIVE STATUS AFTER SPLANCHNIC ISCHEMIA/REPERFUSION IN RATS

Vera Brod, Sergei Shnizer, Igor Sukhotnik, and Haim Bitterman. Carmel Hospital. Faculty of Medicine. Technion. Haifa 34362, Israel

Objective:

Inhalation of oxygen improves hemodynamics, attenuates the inflammatory response, and improves intestinal rehabilitation after splanchnic ischemia/reperfusion (IR). Yet, the use of hyperoxia in IR is hindered by concerns that it could exacerbate reperfusion injury by increasing free radicals formation. We used the thermochemiluminescence oxidizability assay to evaluate the effect of hyperoxia on plasma oxidative status after splanchnic IR in rats.

Methods:

Animals were assigned to four groups: 1) Sham-operated controls breathing air, 2) Sham-operated controls breathing 100% oxygen 3) IR-air rats with occlusion of the superior mesenteric artery and portal vein for 30 minutes breathing air, and 4) IR-O2 breathing 100% oxygen starting 10 minutes before and continuing for 6 hours after reperfusion. The thermochemiluminescence (TCL) oxidizability of plasma was determined at baseline, and 6 and 24 hours after IR. TCL ratio values represent the oxidative potential of the sample. Hence, higher values reflect lower oxidative status.

Results:

Untreated IR rats (IR-air) exhibited a significant 25% decrease in TCL ratio after six hours (P<0.05 from the baseline and sham groups). In contrast, after six hours, IR-O2 rats maintained TCL ratios at significantly higher values that were not significantly different from those of the sham rats. TCL ratio values were not statistically different among the three experimental groups at the end of the 24 hours protocol.

Conclusions:

Improved plasma oxidative status may be an important mechanism of the beneficial effects of hyperoxia in IR.

P216 GENDER-RELATED DIFFERENCES IN IMMUNE RESPONSES FOLLOWING MAJOR SURGICAL TRAUMA ARE DIMINISHED BY PREOPERATIVE STEROID ADMINISTRATION

T. Matsutani, A. Matsuda, K. Sasajima, M. Miyashita, T. Tajiri, K. Tamura, H. Kogo, and I.H. Chaudry. Dept. Surgery, 1Nippon Medical School Tama-Nagayama Hospital, Tokyo 206-8512, Japan; 2Nippon Medical School, Tokyo; 3Dept. Endocrine Pharmacology, Tokyo Univ. Pharmacy and Life Sciences, Tokyo; 4Center for Surgical Research, Univ. Alabama at Birmingham, AL 3594, USA

Object:

To determine whether gender differences are observed in circulating inflammatory mediator levels by the preoperative methylprednisolone (MP) administration following major surgery.

Methods:

In 75 patients who underwent thoracic-abdominal surgery, 25 patients (male; n=20, female; n=5) were given 10 mg/kg of MP intravenously and 50 (male; n=39, female; n=11) were served as controls. IL-6 and CRP were measured following surgery.

Results:

IL-6 and CRP levels in control male group increased significantly compared to the female control group. IL-6 and CRP levels in MP group were significantly lower than those in conrol group. However, no difference in those levels was found between the male and female MP groups.

Figure
Figure

Conclusion:

These results collectively indicate that proinflammatory mediator release is significantly higher in males than females following major surgery. However, preoperative MP administration eliminates the gender-related differences in inflammatory response (supported by Grant for Japanese Scientific Research (C) 17591356).

P218 PGC1 MODULATES HNF4 BINDING ABILITY IN THE CYTOKINE INDUCED ACUTE PHASE RESPONSE

Z. Wang, and P.A. Burke. Department of Surgery, Boston University School of Medicine, Boston, Massachusetts 02118, USA

Objective:

HNF4 is a crucial regulator of liver-specific gene expression and its binding ability is quickly reduced after injury. However, the molecular mechanism of this response remains to be defined. In this study, the effects of the transcription coactivator PGC1 on HNF4 binding ability during the acute phase response (APR) are investigated.

Methods:

Utilizing a cytokine induced HepG2 cell injury model, the impact of PGC1 on the binding ability of HNF4 was elucidated by EMSA and ChIP assays. HepG2 cells were transfected with coactivator expression plasmid and treated with cytokines overnight. P32 labeled oligonucleotides containing HNF4 binding sites derived from the APR genes, apolipoprotein B (ApoB), transthyretin (TTR) and α1-antitrypsin (α1-AT), were used as probes in EMSA. To test how PGC1 affects HNF4 binding in vivo, ChIP assays were performed using PGC1 antibody to precipitate formaldehyde cross-linked chromatin. The interaction of PGC1 with HNF4 and the ability of PGC1/HNF4 complex to bind HNF4 binding sites in APR genes were determined.

Results:

Cytokine treatment caused a reduction in HNF4 binding ability in a binding site specific manner. The greatest suppression was observed to the TTR, followed by α1-AT, and ApoB. The provision of exogenous PGC1 reversed the binding inhibition induced by cytokines in all three genes as did the provision of excess HNF4. ChIP assays showed similar effects of PGC1 on HNF4 binding ability.

Conclusion:

Cytokine treatment results in reduction of HNF4 binding ability. The mechanism of this response is due to an alteration in the formation or affinity of the PGC1/HNF4 complex. This study establishes the importance of PGC1 for HNF4 function and describes a new HNF4-dependent regulatory mechanism that is involved in the transcriptional regulatory response to injury.

P219 PEPTIDOGLYCAN-INDUCED TOLERANCE IMPROVES BACTERIAL CLEARANCE AND SURVIVAL IN MICE AFTER GRAM-AND GRAM+BACTERIAL CHALLENGES

E.D. Murphey, and Edward Sherwood. Dept of Anesthesiology and Shriners Burns Institute, Univ. of Texas Medical Branch, Galveston, TX USA 77555

This study was conducted to determine if peptidoglycan (PGN), a component of the cell wall of Gram+bacteria, could induce tolerance similar to that induced by endotoxin in which bacterial clearance and survival are improved after challenge with either Gram-or Gram+bacteria; and to determine if TLR2 or TLR4 signalling are necessary for those results. C57BL6/J; TLR2−/; and C3H/HeJ mice (male, 8-10 weeks) were pretreated with PGN (1mg, I.P.) on 2 consecutive days. 48 hours later, the mice were challenged with live S. aureus or Ps. aeruginosa bacteria (1 x 108 cfu i.v.). In each group, pre-treatment with PGN was associated with decreased bacterial colonization, decreased inflammatory cytokine burden, and decreased mortality after challenge with S. aureus when compared to their respective saline pre-treated controls:

Table
Table

Similar results were obtained in PGN pretreated C57BL6 mice challenged with Ps. aeruginosa. Peptidoglycan can induce tolerance and non-specific enhancement of innate immune function independent of TLR2 and TLR4.

Funded by NIH-NIGMS and Shriners of North America.

P220 ROBUSTNESS OF EARLY INFLAMMATION MAY PREDISPOSE TO SURVIVAL: STUDIES IN A PORCINE MODEL OF HEMORRHAGIC SHOCK

Rajaie Namas, Andres Torres, Hernando Gomez, Derek Barclay, Sven Zenker, Gilles Clermont, Juan Carlos Puyana, Michael R. Pinsky, and Yoram Vodovotz. University of Pittsburgh, PA, 15213

Introduction:

Hemorrhagic shock (HS) results in acute inflammation that is generally considered detrimental. Yet, inflammation is necessary for healing. We wished to determine the appropriate inflammatory mediator response to severe decompensated HS in swine.

Methods:

12 anesthetized female Yorkshire/Durock pigs were rapidly bled to a MAP of 30 mmHg and kept at this pressure by re-bleeding via peristaltic pump. Swine remained hypotensive for 45-90 min. Mixed venous blood samples were taken at pre-surgery, baseline post-surgery, beginning of HS, and every 15 min thereafter until 75 min, the longest time point common to both groups. Plasma samples were assayed for TNF, IL-6, IL-10, and NO2/NO3The data were analyzed by Kruskall-Wallis ANOVA on ranks followed by Dunn's post-hoc test.

Results:

Mean post-surgery ± HS TNF values were higher in the survivors vs. non-survivors. Survivor mean post-surgery ± HS TNF values were elevated vs. pre-surgery baseline (p=0.009). Non-survivors had no measurable change in TNF levels over the same interval (P=0.246). No differences in IL-10, IL-6, or NO2/NO3were found between survivors or non-survivors or within groups.

Conclusions:

While additional studies are necessary, survivors of severe, acute HS appear to exhibit an immediate increase in serum TNF levels not seen in non-survivors. It is currently unclear if the observed robust HS TNF response was the cause of this protection, a marker of survival, or a feature of the experimental protocol used.

P221 RADIATION WORSENS SURVIVAL IN PNEUMONIA: DEVELOPING AN ANIMAL MODEL OF POST-RADIATION SEPSIS

P. Brahmamdam, C.G. Davis, E.E. Perrone, J.S. McDonough, D.F. Osborne, J.T. Muenzer, C.M. Coopersmith, and R.S. Hotchkiss. Washington University School of Medicine. St. Louis, MO

Introduction:

The threat of terrorist attacks on nuclear facilities or the use of "dirty bombs" place large civilian populations at risk of radiation injury. Initial survivors of radiation may later succumb to infections due to bone marrow failure and lymphocyte apoptosis. The purpose of this study was to develop a murine model of post-radiation sepsis that can be used for the development of potential therapies.

Methods:

Male, 6-8wk old C57/Bl6 mice underwent 7.0Gy Cs-gamma irradiation. 96 hrs later, irradiated and non-irradiated controls were given 6x106 CFUs of Streptococcus pneumonia (S.p) intranasally and followed for 7 days (n=27). Complete blood counts (CBC) were done prior to infection. Cytokine levels from serum and bronchio-alveolar lavage (BAL) prior to and 48 hrs after infection were obtained.

Results:

CBCs 96 hrs after radiation show lymphopenia compared to controls (mean WBC 570 vs. 5333/mm3, p<.001). Histology revealed profound cell loss in spleens and thymi of irradiated mice. Irradiated mice have significantly worse survival from pneumonia than controls (3.7% vs. 37%, p=.008). Irradiated mice exhibited a blunted inflammatory response compared to controls: TNF-α (24.4 vs. 6061pg/ml in BAL, p=.037) and IFN-γ (5.53 vs. 14.84pg/ml in serum, p=.047; 2.75 vs. 695.3pg/ml in BAL, p=.007).

Conclusion:

Radiation severely compromised the host immune system and worsened survival in S.p. pneumonia. Irradiated mice were unable to produce a robust inflammatory response locally or systemically. This model can be used to further elucidate the pathophysiology of post-radiation sepsis and to test potential treatments that boost the host response.

P222 DIVERGENT FUNCTIONS OF BONE MARROW DERIVED FIBROCYTES IN SEPSIS: ANTIGEN PRESENTATION AND COLLAGEN PRODUCTION

J.A. Nemzek, C. Fry, and O. Abatan. University of Michigan, Ann Arbor, MI 48109

First described in 1994, bone marrow-derived fibrocytes are unique cells expressing both hematopoeitic and mesenchymal characteristics. Recent studies indicate that circulating fibrocytes have an intriguing role in a diverse array of inflammatory processes. However, fibrocytes have not been examined in the sepsis syndrome. This study characterized the potential for antigen presentation as well as extracellular matrix production by fibrocytes in septic peritonitis. Non-lethal cecal ligation and puncture (CLP) or Sham surgeries were performed on mice which were euthanized at acute (<5 days) or chronic time points. Cells from peritoneal fluid, mesenteric lymph nodes and abscess wall were processed to identify fibrocytes (surface CD45+and intracellular Collagen I+) and surface proteins associated with antigen presentation. Flow cytometry revealed that fibrocytes are constitutive in the peritoneal cell population and subpopulations express molecules associated with antigen presentation, CD80 (11%), CD86 (20%), and MHC Class II (52%). Peritoneal fibrocytes increased significantly within 24 hours of CLP and the total number expressing co-stimulatory molecules also increased. Mesenteric lymph nodes contained fibrocytes expressing co-stimulatory molecules at acute and chronic time points. Abscess wall tissue contained more fibrocytes than fibroblasts one week after CLP but relatively few expressed markers associated with antigen presentation. These studies demonstrate that bone marrow derived fibrocytes are recruited in response to polymicrobial peritonitis. The cells appear to develop different phenotypes which could significantly impact long term consequences of sepsis through divergent functions.

P223 BACTERIAL CLEARANCE AND MORTALITY ARE IMPROVED BY PLATELET-ACTIVATING FACTOR ACETYLHYDROLASE (PAF-AH) ADMINISTRATION: INVOLVEMENT OF MCP-1/CCL2

Rachel N. Gomes1, Michelle Gomes1, Mariana Cunha1, Daianne Torres1, Fernando A. Bozza1, Guy A. Zimmerman2, Stephen M. Prescott3, Patrícia T. Bozza1, and Hugo C. Castro-Faria-Neto1. 1Laboratório de Imunofarmacologia-IOC-FIOCRUZ, RJ; 2Program in Human Molecular Biology and Genetics; 3Oklahoma Medical Research Foundation, OMRF,USA

Current evidence indicates that dysregulation of the host inflammatory response to infectious agents is central to the mortality of patients with sepsis. Strategies to block inflammatory mediators, such as PAF are currently being investigated as new adjuvant therapies for sepsis. Recently, PAF-AH, the enzyme responsible for PAF degradation, was cloned and its anti-inflammatory effects were demonstrated. In this study we aimed to investigate the protective mechanism of PAF-AH in sepsis, using the murine model cecal ligation and puncture (CLP). Mice were subjected to CLP and treated with rPAF-AH. The mortality rate, levels of cytokines and clearance of bacteria were evaluated 6 hours after CLP. Our results demonstrated that rPAF-AH protects animals from CLP. Treatment with rPAF-AH increased peritoneal fluid levels of the anti-inflammatory mediators MCP-1/IL-10, decreased pro-inflammatory mediators MIF/IL-6/TNF levels after CLP. The numbers of bacteria (CFU) into peritoneal cavity was inhibited in rPAF-AH treated group after CLP, suggesting the more clearance of bacteria after rPAF-AH treatment. When we submitted the CCR2 (receptor for MCP-1/CCL2) deficient mice to CLP, we did not observe the protective effect after rPAF-AH treatment and the effect in clearance of bacteria was abolished. In this study we observed the protective effect of rPAF-AH treatment in a murine model of sepsis. This effect was correlated with the effective clearance of bacteria in peritoneal cavity after rPAF-AH administration and this phenomenon involved the increase in MCP-1/CCL2 levels. We conclude that administration of exogenous rPAF-AH reduces inflammatory injury, altered cytokine levels and mortality in models relevant to the clinical syndrome.

NIH-FIRCA/CNPq/FAPERJ/FIOCRUZ.

P224 RED BLOOD CELL SUPERNATANT GENERATES TRANFUSION-RELATED IMMUNOSUPPRESSION VIA INDUCTION OF REGUALTORY T CELLS

J. Baumgartner, E. Moore, C. Silliman, A. Banerjee, and M. McCarter. University of Colorado Denver, CO, 80262

Allogeneic blood transfusion often provokes an immuno-suppressive response implicated in trauma-related organ injury and infections. Regulatory T cells (Tregs) are potent immunosuppressive T cells involved in the promotion of tolerance and the prevention of autoimmunity. We hypothesized that transfusion-related immunosuppression is in part mediated through induction of Tregs.

Methods:

Normal human peripheral blood mononuclear cells (PBMCs) were exposed to packed red blood cell (PRBC) supernatant from stored units with (LR) or without (NLR) pre-storage leukoreduction. After five days ± anti-CD3 antibody, the PBMCs were analyzed by flow cytometry for the percentage of CD4+Foxp3+Tregs or activated CD25+Foxp3T cells.

Results:

Exposure to PRBC supernatants alone did not significantly increase the induction of Tregs. With anti-CD3 stimulation, both PRBC supernatant groups resulted in a significantly greater proportion of Tregs than control media. The percentage of activated non-Treg CD25+T cells was not different between stimulated groups.

Figure
Figure

Conclusion:

PRBC supernatant induces Tregs from stimulated PBMCs, which is not altered by pre-storage leukoreduction. These findings suggest that soluble factors in allogeneic blood transfusions have the capacity to induce immunosuppression.

P225 AGE-RELATED AUGMENTATION OF THE MYOCARDIAL INFLAMMATORY RESPONSE TO ISCHEMIA: A CRITICAL ROLE OF TOLL-LIKE RECEPTOR 4

Lihua Ao, Joseph C. Cleveland Jr, Ning Zou, Xiaoping Yang, Xin Su, David A. Fullerton, and Xianzhong Meng. Department of Surgery, University of Colorado Denver, Denver, Colorado, USA

Background:

Aging is associated with exaggerated cardiac functional deficits following myocardial ischemia and reperfusion (I/R). Although cytokines are known to depress cardiac contractility, the influence of aging on myocardial cytokine production following I/R remains to be characterized. We earlier found that Toll-like receptor 4 (TLR4) plays an important role in the myocardial inflammatory response to I/R in adult mouse hearts. However, it is unknown whether this innate immunoreceptor is involved in the inflammatory response to I/R in aged hearts and whether its expression and signaling change with age. This study tested the hypothesis that aging augments the myocardial inflammatory response to I/R through TLR4.

Methods and Results:

We examined the myocardial inflammatory response to I/R in patients who had heart surgery with obligatory global ischemia and in mice subjected to global I/R (20 min/60 min) via the Lagendorff method. In humans, we found a greater elevation in levels of IL-6 and IL-8 in coronary venous blood during reperfusion in elderly patients (65 years and older) than in younger patients (less than 65 years old). In mice, we found that myocardial production and release of IL-6 and KC following I/R were significantly increased in aged mice (18 to 24 months old) compared to young adult mice (4 months old). In mice, TLR4 deficiency reduced myocardial production and release of these factors following I/R in both young adult and older hearts, and diminished the age-related difference. Murine myocardial TLR4 levels were not significantly increased, but TLR4 signaling, as measured by NF-κB activation, was enhanced in aged hearts following I/R.

Conclusions:

These results demonstrate that aging augments the myocardial inflammatory response to I/R in both humans and mice, that TLR4 plays a critical role in the myocardial inflammatory response to I/R in both young adult and aged mouse hearts, and that enhanced TLR4 signaling appears to contribute to the mechanisms underlying the age-related augmentation of the myocardial inflammatory response following I/R.

P226 AGMATINE CAUSES AMELIORATION OF GLIOSIS AND INDUCIBLE NITRIC OXIDE SYNTHASE EXPRESSION AFTER TRANSIENT FOCAL CEREBRAL ISCHEMIA IN RATS

Che-Chuan Wang1,2, Jinn-Rung Kuo1,2,3, Bor-Chih Cheng2,4, Mao-Tsun Lin2,5, Yun-Chin Yao5, and Ching-Ping Chang2,5. 1Department of Surgery, Chi-Mei Medical Center Tainan 710, Taiwan; 2Department of Biotechnology, Southern Taiwan University, Tainan 710, Taiwan; 3Institute of Clinical Medicine, School of Medicine, National Cheng-Kung University, Tainan 710, Taiwan; 4Department of Cardiovascular Surgery, Chi-Mei Medical Center, Tainan, Taiwan; 5Department of Medical Research, Chimei Medical Center, Tainan 710, Taiwan

The aim of present study was to evaluate the temporal profiles of gliosis and inducible nitric oxide synthase (iNOS) expression in brain after transient focal cerebral ischemia in rats treated with or without agmatine. Here we demonstrate by quantitative immunohistochemistry that ischemia cortex and striatum show similar patterns of both gliosis (evidenced by increased expression of glial fibrillary acidic protein, GFAP) and iNOS over-expression in the reperfusion phase after 90 minutes of middle cerebral artery occlusion in the rat. The cortical and striatal cores display gliosis, iNOS over-expression, infraction, and apoptosis at 1 hour of reperfusion. The cortical and striatal over-expression of both GFAP and iNOS is associated with cerebral infarction and apoptosis. This abnormalities show no recovery toward 72 hours of reperfusion. Agmatine (100mg/kg body weight, i.p.) administered immediately after middle cerebral artery occlusion attenuates the transient focal cerebral ischemia-induced gliosis, iNOS over-expression, and apoptosis evaluated 72hours after reperfusion. These results suggest that agmatine may attenuate transient focal cerebral ischemia by reducing gliosis and iNOS over-expression.

Keywords:

agmatine, stroke, ischemia, apoptosis, nitric oxide synthase, gliosis

P227 DEPRESSED ADIPOCYTE FUNCTION IS A RISK FACTOR FOR POSTOPERATIVE INFECTION FOLLOWING COLORECTAL CANCER SURGERY

A. Matsuda1, T. Matsutani1, K. Sasajima1, K. Furukawa2, M. Miyashita2, T. Tajiri2, K. Tamura3, and H. Kogo3. Dept. Surgery, 1Nippon Medical School Tama-Nagayama Hospital, Tokyo 206-8512, Japan; 2Nippon Medical School, Tokyo; 3Dept. Endocrine Pharmacology, Tokyo Univ. Pharmacy and Life Sciences, Tokyo

Object:

To clarify the association between the perioperative endocrine functions of adipocytes and the development of postoperative infection following colorectal cancer surgery.

Methods:

In 41 patients who underwent colorectal cancer surgery, peripheral blood samples were collected perioperatively. Plasma adiponectin and leptin levels were measured by ELISA. The patients were divided into a group with postoperative infections (n=12) and an uninfected group (n=29).

Results:

The postoperative plasma adiponectin levels decreased transiently and then gradually recovered. The infected group had significantly lower adiponectin levels throughout the perioperative period than theuninfected group. Logistic regression analysis revealed thatlowpreoperative adiponectin level was an independent riskfactorfor postoperative infection.

Conclusion:

Preoperative adiponectin levels may be useful for anticipating the developmentofpostoperative infection following colorectal cancer surgery. (supported by Grant for Japanese Scientific Research (C) 19591518).

Table
Table

P228 EFFECTS OF PHOSPHATIDYLCHOLINE IN CARRAGEENAN-INDUCED SUBACUTE ARTHRITIS

Petra Hartmann, Andrea Szabó, Dóra Gurabi, Renáta Varga, Gábor Erös, and Mihály Boros. Institute of Surgical Research, University of Szeged, Hungary

Phosphatidylcholine (PC) is a major membrane phospholipid and an important supplier of biologically active molecules. Our aim was to compare the anti-inflammatory potential of oral PC treatment (150 mg/kg gavage twice daily) and a non-steroidal antiinflammatory drug (0.5 mg/kg diclofenac twice daily) in a rat model of subacute arthritis.

Methods:

A solution of 2% λ-carrageenan and 4% kaolin was injected into the right knee joint, whereas saline was administered into the contralateral knee. Synovial leukocyte-endothelial cell interactions were visualized by means of fluorescent intravital videomicroscopy 6 hr after the challenge. Functional changes were assessed 24 hr later by nociceptive tests (thermal and mechanical hyperalgesia), followed by knee volume measurements at 48 hr.

Results:

Joint inflammation was accompanied by a significant (approx. 6-fold) increase in the number of adherent cells in the synovial postcapillary venules. This reaction was significantly (by 40%) reduced by PC, and less effectively (22%) by diclofenac treatment. The significantly decreased (45%) thermal nociceptive latency and the approx. 3-fold increase in mechanical touch sensitivity (von Frey test) were significantly ameliorated by both treatments. The knee cross-section area was increased by 35%, and this was also reduced by both approaches.

Conclusions:

This study confirms potent anti-inflammatory effects for PC, as evidenced by the reduction of leukocyte-endothelial interactions and functional improvements. Since the knee joint is regarded as body compartment, oral PC could possibly offer a novel therapeutic approach to treat localized inflammation (Supported by OTKA K 60752).

P229 PHOSPHATIDYLCHOLINE PRETREATMENT DECREASES ISCHEMIA-REPERFUSION-INDUCED METHANE GENERATION AND THE INFLAMMATORY RESPONSE IN THE SMALL INTESTINE

Mihály Boros, Miklós Ghyczy, Csilla Torday, Andrea Szabó, Miklós Czóbel, and József Kaszaki. Institute of Surgical Research, University of Szeged, Hungary

The aims were to establish whether the dietary administration of phosphatidylcholine (PC) can protect the reperfused small bowel mucosa by its acting as an anti-inflammatory agent, and to investigate this possibility in association with in vivo methane generation.

Methods:

Group 1 of anesthetized dogs served as sham-operated controls, while in groups 2 and 3, complete small intestinal ischemia was induced by occluding the superior mesenteric artery for 60 min. Groups 1 and 2 were fed with normal laboratory chow for 1 week, while group 3 received a special diet containing 1% PC. Intestinal superoxide production and myeloperoxidase (MPO) activity (a marker of tissue PMN leukocyte infiltration) were ascertained on ileal biopsy samples 180 min after reperfusion. The content of methane in the exhaled air was determined by gas chromatography.

Results:

I-R was characterized by ROS generation and elevated MPO activity. These changes were accompanied by increased methane production in the exhaled air during reoxygenation. The PC-enriched diet diminished the intestinal superoxide generation and the MPO activity, and significantly decreased the exhaled methane concentration.

Conclusions:

The increased dietary uptake of PC exerts an anti-inflammatory influence in the gastrointestinal tract. Exhaled methane is linked to abnormal ROS generation; a decreased methane production is associated with significantly reduced inflammatory activation during I-R (supported by NKTH 08/2004).

P230 EFFECTS OF COLLOID RESUSCITATION ON THE SUBLINGUAL MICROCIRCULATION DURING EXPERIMENTAL HEMORRHAGIC SHOCK

József Kaszaki1, Domokos Boda2, Balázs Sütö3, Andrea Rostás1, András Nagy1, Szilvia Rokolya1, and Mihály Boros1. Institute of Surgical Research1, Department of Anaesthesia and Intensive Therapy3, University of Szeged and Department of Paediatrics2, University of Pécs, Hungary

Background:

Detection of sublingual (SL) pCO2 gap changes might be of diagnostic value to evaluate the effectiveness of resuscitation strategies targeting the gastrointestinal (GI) microcirculation.

Methods:

Red blood cell velocity (RBCV) and capillary perfusion rates (CPR) were monitored in the oral cavity by means of orthogonal polarization spectral imaging, while SL and small intestinal pCO2 gap changes were determined by indirect tonometry in pentobarbital-anesthetized minipigs during hemorrhagic shock (HS) and volume resuscitation (25 ml/kg iv) with 5% albumin (AB), 6% hydroxyethyl-starch 130/0.4 (HES) and 6% dextran-60 (DX) solutions.

Results:

Both intestinal and SL pCO2 gap values were increased significantly (2.6 and 3.6 fold, respectively) during HS, while RBCV and CPR of the oral mucosa were decreased by 51% and 35% respectively. Administration of colloids significantly improved macrohemodynamics, however RBCV in the SL area was restored only by HES and AB (944±124 and 980±57 μm s−1, respectively), while DX was ineffective (620±65 vs 1080±134 μms−1 baseline). As compared to HS-induced changes, the pCO2 gap values were decreased by 51% (DX), 68% (HES) and 72% (AB) in the SL region.

In conclusion:

pCO2 gap changes are associated with characteristic microcirculatory alterations in the SL region during experimental HS; HES and AB significantly ameliorate the HS-caused microcirculatory deterioration in the oral cavity during the early phase of resuscitation. (Supported by ETT 594/2006 and NKTH 08/2004 grants)

P231 KINETICS OF NO DEGRADATION IN INTACT MACROPHAGES USING MICROFLUIDICS

M.A. Robinson, J.E. Baumgardner, V. Good, and C.M. Otto. University of Pennsylvania, Philadelphia, 19104

Introduction:

The amount of NO available to effect its many biologic actions is a balance between NO production and NO degradation, but very little is known about the kinetics or pathways of NO degradation in intact cells. We previously reported the use of forced convection cell culture to study the oxygen dependence of NO production in RAW 264.7 cells. In the current study, we used this system to deliver varying amounts of NO to live, intact cells, and quantitatively studied the overall reaction kinetics of NO degradation.

Methods:

Forced convection cell culture was carried out as previously described. NO production in macrophages growing on the inside of fused silica capillary tubing was suppressed with administration of 1400W. The cells were then exposed to a series of NO and O2 partial pressures, and the amount of NO leaving the column of cells was measured with an NO electrode. The difference between NO entering the column of cells and NO exiting the column of cells was related to consumption kinetics by a mass balance on the column. Dependence of NO consumption on NO and O2 partial pressures was tested by linear regression.

Results:

NO consumption was first order in NO partial pressure (p<0.05) and first order in O2 partial pressure, with a rate constant of 0.038 pmol NO/(sec-106 cells-Torr NO-Torr O2).

Conclusions:

NO consumption in intact macrophages was most closely described by first order dependence in both NO and O2, with an overall rate constant approximately 40 fold smaller than the rate constant reported for cultured rat hepatocytes. Forced convection cell culture is a valuable tool for investigating the largely unexplored pathways and kinetics of NO degradation.

Supported by NIH GM64486.

P232 THE ROLE OF IL-18 IN APOPTOSIS INDUCTION OF BONE MARROW GRANEULOCYTE DURING SYSTEMIC INFLAMMATION IN MICE

Michiko Aoyama1, Joji Kotani2, Makoto Miyoshi1, Hiroe Sakaki1, Katsuhito Shuno1, Mari Watanabe1, Makoto Usami1, and Seisiro Marukawa2. 1Faculity of Health Science, Kobe University School of Medicine, Kobe, Japan. 2Department of Emergency and Critical Care Medicine, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan

Introduction:

Decreased apoptosis (Ap) in neutrophils, which are produced in the bone marrow (BM), is implicated in presistant inflammatory states, leading to multiple organ dysfunction syndromes (MODS). Serum IL-18 levels correspond to severity of systemic inflammation.

Purpose:

To elucidate the roles of IL-18 in the BM neutrophils apoptosis during endotoxin-induced systemic inflammation.

Methods:

Wild-type (WT) or IL-18 knock-out (KO) mice were intraperitoneally (i.p.) injected with PBS or LPS (40mg/kg body weight), followed by sacrifice and collection of the BM cells from their femur bones at 24 hrs later. Ap was assessed by Annexine V staining using two-color flow cytometry with mice graneulocyte-spesific Gr-1.

Results:

Although only 30% of the WT mice injected with LPS could survive, 60% of the IL-18 KO mice survived for 24hrs. LPS i.p. decreased the cell numbers and induced Ap in the BM Gr-1 positive cells both mice groups, however, theGr-1 positive cell numbers were significantly lower despite of lowerAp the KOmice group.

Table
Table

Conclusion:

IL-18 may play a role in granulopoiesis or alternatively, retaining cells with increased Ap potential hence releasing a population of cellswith decreased Ap potential, leading to MODS during systemic inflammation.

P233 TUMOR NECROSIS FACTOR-α GENE-238 SINGLE NUCLEOTIDE POLYMORPHISMS (SNPS) ARE ASSOCIATED WITH LOW TNF-α PRODUCTION FROM MONONUCLEAR CELLS IN JAPANESE

Katsuhito Shuno1*, Michiko Aoyama1*, Shoko Fudo1*, Hiroe Sakaki1*, Makoto Miyoshi1*, Makoto Usami1*, Seishiro Marukawa2*, and Joji Kotani2*. 1Facility of Health Science, Kobe University School of Medicine, Kobe, Japan; 2Department of Emergency and CCM, Hyogo College of Medicine, Nishinomiya, Japan

Introduction:

TNF-α and IL-18 levels in the sera correspond to severity of the diseases during systemic inflammation. SNPs affect protein expression and show ethnic difference.

Purpose:

To determine the relations between expression and SNPs of these cytokines in Japanese population.

Methods:

Blood was obtained from 20 and 15 healthy volunteers for TNF-α and IL-18, respectively. DNA was obtained from whole blood using DNA extractor kit. The promoter SNPs (TNFα-238,-308, and -1031, and IL18-137 and -607) were detected by sequence specific polymerase chain reaction. Isolated mononuclear cells were cultured for 24 hrs with or without LPS (1 μg/mL) and cytokines levels in the medium were measured by ELISA.

Results:

TNFα-238 GG,-308 GG, and-1031 TT were most common genotypes (n=11). The TNF-α level in TNFα-238 GA genotype (n=2) were significantly lower than in other common genotypes (667 ± 343 pg/mL vs 1610 ± 638 pg/mL, respectively). IL18-137 GG,-607 AC genotypes were most common genotypes (n=11). For position-607, the frequencies were AC: 0.73, AA: 0.13, CC: 0.13. However, there was no difference in IL-18 production between genotypes.

Conclusion:

SNPs of TNF-α and IL-18 in Japanese population are different from the previous reports in Caucasians. TNFα-238 GA genotype has low TNF-α production and IL18-607 polymorphisms does not affect production of IL-18.

P234 EFFECT OF OXYGEN RADICAL SCAVENGING ON TRAUMA, HEMORRHAGE, AND RESUSCITATION (THR) RELATED INFLAMMATORY RESPONSE IN RATS

M. Jafarmadar, C. Zifko, A. Postl, B. Reipert*, N. Rammal, A. Kozlov, H. Redl, and S. Bahrami. Ludwig Boltzmann Institute f.Traumatology and the Research Center of AUVA, Vienna, Austria, *Baxter BioScience, Vienna, Austria

THR is often associated with systemic inflammatory response syndrome (SIRS). SIRS is characterized by activation of humoral/cellular systems and release of inflammatory mediators, including reactive oxygen species (ROS) and cytokines acting in a complex network. The aim of the present study was to investigate the role of ROS formation on the inflammatory response following THR. Anesthetized rats (n=5) were subjected to a THR-model, including laparotomy, bleeding and resuscitation (adequate/ inadequate phase) followed by a 2h observation. CPH, a non-toxic ROS scavenger, was infused starting at the onset of resuscitation up to the end of the experiment. CD11b (cell activation) and cytokines (inflammation) were assessed by flow cytometry (whole blood) and ELISA. Data as median with Q1/Q3. THR alone increased CD11b by 132% (25/51) vs. baseline (100%). THR related inflammatory response was reflected in plasma IL-1ß, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ, and TNF-α levels. Scavenging of ROS increased CD11b to 171% (38/107) vs. baseline and enhanced release of IL-1ß 81 (62/124) vs. 52 (38/62)-controls], GM-CSF [132 (76/142) vs. 2 (2/2)] and TNF-α [511 (204/586) vs. 94 (76/94) pg/mL]. Our data suggest that the activation level of THR-induced immune response is partially under the control of ROS.

P235 ANDROSTENEDIOL MODULATES LPS-INDUCED CHANGES IN NEUTROPHIL CHEMOKINE RECEPTOR EXPRESSION IN VITRO

Tanja Barkhausen, Frank Hildebrand, Christian Krettek, and Martijn van Griensven. Experimental Trauma Surgery, Hannover Medical School, D-30625 Hannover

Objective:

Neutrophils are an important cell type in the pathogenesis of posttraumatic diseases as their hyper-activation often induces tissue damage. Androstenediol is a steroid hormone exhibiting protective properties in experimental trauma and sepsis. However, mechanisms of androstenediol action, especially regarding neutrophils, are not well understood. Thus, we investigated the influence of androstenediol on one of the first steps in neutrophil activation, the neutrophil chemokine receptor expression.

Methods:

Neutrophils freshly isolated from human blood were stimulated withLPS and/or androstenediol in three concentrations (1 ng/ml; 10 ng/ml; 100 ng/ml). Expression of CXCR-1 and CXCR-2 was determined after 2 h and 4 h by flow cytometry. Unstimulated cells served as controls.

Results:

LPS alone reduced the expression of CXCR-1 and CXCR-2 at both time points. Co-stimulation of LPS/androstenediol (10 ng/ml) resulted in an elevated expression of CXCR-1 after 2 h and CXCR-2 after 4 h compared to single LPS stimulation. Androstenediol alone as well as co-stimulation with the concentrations 1 ng/ml and 100 ng/ml had no influence on expression levels.

Conclusions:

Androstenediol counter-regulates LPS-induced decreases in chemokine receptor expression time-and concentration-dependent. We conclude an indirect action of androstenediol on chemokine receptors, possibly via modulation of LPS-induced signalling pathways, as androstenediol alone has no influence on expression patterns. Furthermore, this effect on neutrophil activation might contribute to protection in trauma and sepsis.

P236 TOLERANCE IN CHRONIC MURINE PERITONITIS

A. Lenz, and W. Cheadle. VAMC and Dept of Surgery, University of Louisville, Louisville, KY

Background:

We have shown that induction of tolerance to Klebsiella at 105 CFU, but not 103, is likely the reason for bacterial persistence in chronic murine peritonitis. Bacterial products BLP and LPS are recognized through TLR-2 and-4 respectively, and we aimed to discern whether these pathways are responsible.

Methods:

Resident peritoneal cells were harvested from C57BL/6 mice. Macrophages were enriched by plastic adherence and stimulated with different concentrations of BLP (ng), LPS (ng) or Klebsiella 105. A 1st stimulus was applied for 24h, then removed and followed by a 2nd stimulus for another 24h. Supernatants were collected at 24h and 48h. TNF-α was measured by ELISA. Cells without stimulus served as controls.

Results:

1st stimulus with BLP 10, 100, 500 or LPS 10, 100, 500 or Klebsiella induced tolerance against the second stimulus, if the same. Cells treated with BLP 10 first, showed increased TNF-α secretion when BLP 100 was applied second [BLP 10 / BLP 100]. Conversely TNF-α secretion by LPS 10 treated cells was decreased after LPS 100 was given as 2nd [LPS 10 / LPS 100]. Cells [LPS 10 / Klebsiella 105] showed less response after the 2nd stimulus. Interestingly, cells [BLP 10 / Klebsiella 105] showed similar response to cells [-/ Klebsiella 105] after the second stimulus.

Conclusions:

LPS and Klebsiella 105, but not BLP or Klebsiella 103, induced tolerance to Klebsiella 105. Tolerance against Klebsiella is most likely mediated by the TLR-4 pathway and not TLR-2. Bacterial concentrations may influence the balance between TLR-4 and other pattern recognition receptor activation by Klebsiella, which ultimately determines induction of tolerance.

P237 POST-SHOCK HIGH-LIPID NUTRITION REDUCES INFLAMMATION AND INTESTINAL DAMAGE

J. de Haan, T. Lubbers, M. Hadfoune, M.D. Luyer, C.H. Dejong, W.A. Buurman, and J.W. Greve. Maastricht University Medical Center, Maastricht, Netherlands

Control of the inflammatory status of trauma patients forms a major clinical problem since the inflammatory response is already ongoing upon presentation. Previously, we showed using a shock model that pretreatment with high-lipid feeding abrogated the negative consequences of shock in a cholecystokinin (CCK)-receptor dependent manner. This study investigates a high-lipid nutritional intervention in a setting of ongoing inflammation and tissue damage. After induction of hemorrhagic shock, rats were fasted or submitted to an intervention with lipid-enriched enteral nutrition containing 50.4 en% lipids (of which 30% phospholipids) or control low-lipid feeding at 30 and 180 min after shock. CCK-receptor antagonists were administered 10 minutes before feeding. Inflammation and intestinal integrity were assessed at 4 hours after shock. High-lipid nutrition after shock reduced plasma IFN-γ compared to fasted and low-lipid treated animals (p<0.001 vs p<0.01) (Mann-Whitney U). Enterocyte damage, measured as circulating ileal lipid binding protein (ILBP) was prevented by high-lipid intervention (p<0.001). Moreover, high-lipid nutrition preserved intestinal integrity, as assessed by bacterial translocation to distant organs (p<0.001) and leakage of horse radish peroxidase in ileal segments (p<0.001). The effects of high-lipid feeding were abrogated by CCK-receptor antagonists (all parameters: p<0.05). In conclusion, post-shock lipid-enriched nutrition reduces ongoing inflammation and tissue damage via a CCK-receptor dependent mechanism. This study implicates lipid-rich nutrition as a potential therapeutic option in settings in which inflammation and tissue damage are already present, such as in trauma patients.

P238 STROMAL CELL DERIVED FACTOR-1 MEDIATES MYELOID DERIVED SUPPRESSOR CELL EXPANSION DURING POLYMICROBIAL SEPSIS

Matthew J. Delano, Robert D. Winfield, Philip O. Scumpia, James L. Wynn, Kindra M. Kelly-Scumpia, Robert M. Strieter, and Lyle L. Moldawer. Department of Surgery, University of Florida College of Medicine, Gainesville, FL. U.S.A.

Myeloid derived suppressor cells (MDSCs) expand over 10 fold, suppress antigen specific CD8+T cells, augment the TH1 to TH2 immune polarization, and contribute to post-sepsis immune suppression. The signaling pathways that mediate MDSC expansion are yet unknown. In this study, we investigated the role of stromal cell derived factor-1 (SDF-1) in MDSC expansion during sepsis. Female, 8 wk old C57BL/6 mice underwent cecal ligation and puncture (CLP) (LD20) or sham procedure (n=5 mice/group) with and without anti-SDF-1 (1mg, i.p.) treatment daily. At 12 hours and 7 days post-procedure, total splenocytes, bone marrow cells, and whole blood were harvested and analyzed via flow cytometry. At 12 hours after sepsis, SDF-1 inhibition prohibited the bone marrow egression (p<0.01) and blood accumulation (p<0.01) of myeloid lineage (GR-1+) and progenitor cells compared with CLP alone, and had no effect on splenic myeloid cell numbers. After 7 days of SDF-1 blockade, both the percentage and absolute number of splenic MDSCs were reduced over 60% compared to controls (p<0.01). In addition MDSC precursors, splenic common myeloid progenitor (CMP) (Lin-ckit+Sca-1CD34+FcγRlow) cells were reduced to baseline compared to controls (p<0.01). There was no effect on the bone marrow hematopoetic stem cell (Lin-ckit+Sca-1+), CMP, or myeloid cell population. We conclude that SDF-1 plays an integral role in early bone marrow egression and blood accumulation of myeloid and progenitor cells that results in splenic MDSC expansion during prolonged sepsis.

P239 HEME-OXYGENASE-1 ATTENUATES MACROPHAGE CYTOKINE RELEASE BY INHIBITING NF-κB NUCLEAR TRANSLOCATION

J.P. Roach, E.E. Moore, D.A. Partrick, S.S. Damle, C.C. Silliman, F. Gamboni-Robertson, and A. Banerjee. University of Colorado Denver, Denver, CO, 80262

Heme-oxygenase-1 (HO-1) attenuates post-shock organ dysfunction. Because the cellular mechanisms are not clearly defined, we hypothesized that HO-1 induction in macrophages would decrease LPS-stimulated cytokine secretion by decreasing NF-κB nuclear translocation.

Methods:

HO-1 was induced in RAW 264.7 cells with a hemoglobin based oxygen carrier [20%] (HBOC). HO-1 induction was visualized by Western blot. LPS was used to stimulate cells, and zinc protoporphyrin (ZnPP) to inhibit HO-1. IL-6, IL-10, TNF-α, and MCP-1 release was determined by ELISA. NF-κB nuclear translocation was determined by immuno-flourescent microscopy.

Results:

HBOC induced HO-1 in macrophages which significantly decreased (p<0.05) LPS-stimulated secretion of MCP-1 to 22±2%, IL-6 to 57±6%, IL-10 to 80±3%, and TNF-α to 84±4% of control. ZnPP partially restored release of MCP-1 (54±4% vs. 17±2% of control) and IL-6 (80±5% vs. 61±7% of control) (p<0.05). HBOC treated macrophages had decreased LPS-stimulated NF-κB nuclear translocation (figure).

Figure
Figure

Conclusions:

HO-1 induction inhibits macrophage cytokine secretion through decreased nuclear translocation of NF-κB. These data demonstrate an additional mechanism by which HO-1 confers a protective effect.

P240 THE RESORPTION CAPACITY OF PERITONEUM FOR INFLAMMATORY MEDIATORS IN ACUTE GRAM POSITIVE PERITONEAL MODEL

Fawaz Torab, Noura Al Ali, Fatma Srour, Rowaya Obaid, Noura Ali, Kirshna Padmanabahn, and Frank Branicki. Faculty of Medicine and Health Sciences, UAE University, Al-Ain, United Arab Emirates

Aim:

To evaluate the resorption capacity of peritoneum for etox and Il-6 in a model of bacterial (gram-positive) peritonitis.

Animals and methods:

In 200 male Wistar rats, intraperitoneal injection of 108 CFU Mucin-pretreated staphylococci in Phosphate Buffered Saline (PBS) or PBS alone was performed. Studies of resorption were done at time points of 0 (no inflammatory changes), 4h, 8h, 12h and 24 hours. In 100 rats (50 peritonitis and 50 controls) etox and in another 100 rats (50 peritonitis and 50 controls) IL-6 were intraperitoneally injected. After 0, 5, 10, 15, 30 and 60 minutes, 0.5 ml blood was sampled. Etox and IL-6 were measured using the Limulus-Lysate-Test and ELISA technique respectively.

Results:

No etox or IL-6 was measured in the blood of controls in all 5 groups and at 0 h in the peritonitis group. The median etox and Il-6 in the peritonitis groups before the intraperitoneal injection of these mediators were after 0 h: etox: 0 EU/ml, IL-6: 0 pg/ml; 4 h: etox: 102.5 EU/ml, IL-6: 126 pg/ml; 8 h: etox 159 EU/ml, IL-6: 0 pg/ml; 12 h: etox: 65 EU/ml, IL-6: 0 pg/ml; and 24h: etox: 21 EU/ml, IL-6: 0 pg/ml. After injection of etox or IL-6, no significant change of their concentration in the blood was noticed during the time of sampling (60 min) in all groups.

Conclusion:

There was a clear reduction of resorption capacity of inflamed peritoneum for inflammatory mediators with an increase in severity and duration of peritonitis.

P241 ALPHA7 NICOTINIC ACETYLCHOLINE RECEPTOR AGONIST IMPROVES SURVIVAL DURING ENDOTOXEMIA

F. Chen, B. Cai, W. Dong, E.A. Deitch, and L. Ulloa. Laboratory of Surgical Immunology. UMDNJ-New Jersey Medical School. Newark. NJ 07103. USA

Sepsis is systemic inflammatory response syndrome that leads to acute organ dysfunction. Severe sepsis is a common cause of death despite the recent advances in critical care. Our previous studies indicated that the nervous system can modulate systemic inflammation via acetylcholine and the alpha7 nicotinic acetylcholine receptor. Here, we report that choline, a specific alpha7-nAChR agonist, can improve animal survival during lipopolysaccharide (LPS)-induced and cecum ligation puncture (CLP)-stimulated inflammatory responses in mice in vivo. Choline inhibits LPS-induced increases of systemic TNF and HMGB1, cytokines of inflammatory response. Further more, in animal spleen, choline inhibits LPS-induced activation of caspase 3, a downstream of apoptosis signaling. In RAW264.7 cells, LPS-induced release of TNF and HMGB1 is blocked by pre-treatments of these cells with choline. Further more, choline inhibits LPS-induced increase of NFkB-DNA binding. These data indicate that addition of alpha7-nAChR agonist can provide a therapeutic anti-inflammatory potential to improve survival during endotoxemia.

This study was funded by grants of the AHA and the USAMRMC to LU.

P242 SECRETORY IGA LEVELS ARE INCREASED FOLLOWING STRESS CONDITIONS: DUE TO INTRINSIC PROTEASE ACTIVITY

Lawrence N. Diebel, and David M. Liberati. Wayne State University, Detroit, MI 48201

Introduction:

Secretory immunoglobulin A (sIgA), the principle humoral defense at mucosal surfaces is produced locally as a dimeric form (dIgA) and then transported across mucosal epithelial cells to the apical side where it is proteolytically cleaved and released as sIgA. A recent study demonstrated that injury stimulates an innate respiratory sIgA response in humans. Other studies have shown that shock conditions activate luminal proteases. We postulated that ischemia/reperfusion (I/R), a common event in shock resuscitation, would enhance protease activity and increase luminal IgA levels.

Methods:

Dimeric IgA was added to either the basal or apical chambers of monolayers of MDCK cells transfected with cDNA for pIgR at 4oC to allow maximal binding of dIgA to pIgR. Monolayers were then subjected to control (21%O2) or 5%O2 for 90 minutes followed by 21%O2 (H/R). The protease inhibitor leupeptin (Leu) was added in subsets. Apical or basal media was sampled at 1, 3, and 12 hours and sIgA levels determined by ELISA. Monolayer integrity was monitored by measurement of transepithelial electrical resistance (TEER).

Results:

Apical chamber sIgA (mean±S.D., ng/ml, N=4 in each group).

Table
Table

Basal recovery of sIgA when added to the apical chamber wasnegligible in all treatment groups, confirming pIgR mediated IgA transport only. TEER remained stable throughout the study.

Conclusions:

IgA protease activity is a non-specific protective response of mucosal epithelial cells under shock and other stress conditions.

P243 THE ROLE OF αDβ2 INTEGRIN IN SALMONELLA TYPHIMURIUM AND STAPHYLOCOCCUS AUREUS INFECTION

D.O. Nascimento1, A. Vieira-de-Abreu1, P.S. Pacheco1, P.T. Bozza1, G. Zimmerman2, and H.C. Castro-Faria-Neto1. 1Lab. Imunofarmacologia, DFF-IOC/ FIOCRUZ;2 Huntsman Cancer Institute, UTAH

Different members of leukocyte integrin family, also known as β2 integrin, are involved in inflammatory response to infection. The αDβ2 Integrin, the most recently identified member of this integrin family, is preferentially expressed in macrophages in normal and inflamed tissues, although the role of αDβ2 remain largely undefined. In this study, we analyzed the role of αDβ2 in the intense inflammatory response induced by Salmonella Typhimurium or Staphylococcus aureus. Our results demonstrated that αDβ2−/− mice were more susceptible to S. Typhimurium infection, characterized by decreased infiltration of mononuclear cells and neutrophils, inhibition in lipid body formation and PGE2 synthesis and increased levels of TNF-α/MIP1-α/IL1β in the peritoneal lavage 24 and 72 hours after infection when compared to the Wild Type. The numbers of CFU were also augmented in the peritoneal fluid from αDβ2−/− mice after 24 hours after infection. The numbers of CFU and the levels of TNF-α/MIP1-α/IL1β were increased in the supernatant of αDβ2−/− macrophage culture after Salmonella challenge. However, mice infected with S.aureus did not show differences in the cell migration to peritoneal cavity 72 hours after infection and the numbers of CFU were not altered in αDβ2−/−. The intensity of fluorescence of infected cells with Salmonella labeled or S.aureus labeled in macrophage culture did not show differences, suggesting that phagocytosis is equal in WT and αDβ2−/−mice. We suggest a different role of αDβ2 integrin modulating the inflammatory response in Gram negative and Gram positive bacterial infections.

Support:

CNPq/FIOCRUZ

P244 HEMORRHAGE/RESUSCITATION INDUCED MORTALITY IN RATS IS DECREASED BOTH AFTER BINGE ETHANOL CONSUMPTION AND SIMVASTATIN PRETREATMENT WHEREAS EARLY PROINFLAMMATORY CHANGES BECOME DIFFERENTIALLY ACTIVATED

C. Höhn, B. Relja, F. Bormann, D. Henrich, C. Czerny, M. Lehnert, and I. Marzi. Trauma Surgery, JW Goethe University, Frankfurt

Background:

Hemorrhage/resuscitation (H/R) may result in multiple organ failure and trauma patients with an alcohol related comorbidity are more susceptible for a bad outcome. Therefore, we induced an acute fatty liver in a rat model of acute ethanol exposure before H/R. We studied effects of binge ethanol on mortality and inflammation after H/R and the effects of Simvastatin pretreatment.

Methods:

Before H/R, female Lewis rats were pretreated for 6 days with Simvastatin (5mg/kg per day i.p.) or vehicle and 12h before H/R, rats received an ethanol gavage (5g/kg, 30%). Rats were hemorrhaged to an arterial blood pressure of 30±2 mmHg for 60min and resuscitated. 2h later hepatic neutrophil infiltration and TNFα production of LPS-stimulated whole blood were measured. Mortality was assessed after 72h; p<0.05 was considered significant (ANOVA, Fisher's exact test, mean±S.E.M).

Results:

H/R induced mortality (80%) was only 20% both after ethanol gavage or Simvastatin pretreatment (p<0.05). Ethanol blunted hepatic neutrophil infiltration and whole blood TNFá production by 73% and 54%, respectively (p<0.05), effects reversed by Simvastatin pretreatment.

Conclusion:

Both ethanol gavage and Simvastatin reduce H/R induced mortality at 72h. Since 2h after H/R ethanol and Simvastatin affect inflammatory parameters differentially, the comparable effects on mortality may be due to an intervention-and time specific regulation of inflammation. (Supported by DFG MA 119/3-3)

P245 INCREASED SUSCEPTIBILITY TO LIVER INJURY AFTER HEMORRHAGE/RESUSCITATION IN RATS ACUTELY FED ETHANOL-ROLE OF PRE-TREATMENT WITH PLANT POLYPHENOLS

E. Toettel, L. Breig, B. Relja, D. Henrich, C. Czerny, H. Schneider, M. Lehnert, and I. Marzi. Trauma Surgery, JW Goethe University, Frankfurt

Background:

Ethanol modulates immune function partly due to generation of oxidative stress, and pre-existing hepatic dysfunction is a known predictor for worse outcome after trauma/hemorrhage. Accordingly, we induced an acute fatty liver in a rat model of acute ethanol exposure before hemorrhage/resuscitation (H/R) to study effects of acute ethanol intoxication after pre-treatment with antioxidative plant polyphenols (green tea extract (GTE)) on liver damage and inflammation after H/R.

Methods:

Female Lewis rats received daily chow containing 0.1% GTE or regular chow 5 days before H/R. 12h before H/R, rats underwent an ethanol gavage (5g/kg, 30%). Then, rats were hemorrhaged to an arterial blood pressure of 30±2 mmHg for 60min and resuscitated. 2h later, serum ALT and serum IL-6 were measured. (ANOVA, mean±S.E.M, p<0.05 was significant).

Results:

ALT strongly increased after H/R as compared to sham operation (1385±191 vs. 97±13 [IU/ml], p<0.05) and ethanol gavage further aggravated H/R induced ALT release (3192±560, p<0.05). GTE blunted ALT levels after H/R by 62% and after ethanol gavage by 86% (p<0.05). After H/R, serum IL-6 levels increased irrespective of ethanol gavage, an effect largelyblocked by GTE (1272±220 vs. 565±193 [pg/ml], p<0.05 and 1192±256 vs. 154±6 [pg/ml], p<0.05)

Conclusion:

Acute ethanolintoxication aggravates hepatic injury and modulates systemic pro-inflammatory changes after H/R. Plant polyphenols decrease hepatic injury after H/R also in the setting of acute ethanol intake, probably due to its high antioxidative capacity. (Supported by DFG MA 119/3-3)

P246 LYMPHOPROTECTIVE MOLECULES POTENTIATE IL-6 PRODUCTION BY STIMULATED CD4+T-CELLS

J.E. McDunn, A.H. Walton*, and R.S. Hotchkiss. Washington University School of Medicine, 660 S. Euclid Ave., Campus Box 8054, Saint Louis, MO 63110

Introduction:

Prevention of lymphocyte apoptosis confers a significant survival advantage in murine models of sepsis. Previously we identified 15 lymphoprotective compounds from a 32,087 member small molecule library. Here we demonstrate that a subset of these compounds similarly alter the CD4+T-cell response to canonical stimulation.

Methods:

Human peripheral blood mononuclear cells were cultured (5x105/ml; 37°C; 5% CO2), treated with an anti-apoptotic agent (10 μM) or vehicle (DMSO) control and stimulated with one or a combination of anti-CD3 and anti-CD28 monoclonal antibodies. Culture supernatants were harvested after 18 hours and cytokine concentrations were measured using a cytometric bead array.

Results:

Vehicle-treated, anti-CD3/anti-CD28 stimulated T-cells produced 66 pg/ml of IL-6. Five anti-apoptotic compounds increased this IL-6 release 3-8 fold while causing less than ±2-fold changes in the release of TNF-α, IFN-γ, IL-10, IL-4 and IL-2. Interestingly, while no cytokine release was observed in compound-treated, anti-CD28 stimulated cells, treatment with all compounds caused modest increases in cytokine release in anti-CD3 stimulated cells.

Conclusion:

We have identified five lymphoprotective compounds that in part replace the T-cell co-stimulatory signal and increased IL-6 release from CD4+T-cells in response to CD3/CD28 co-stimulation. These results suggest that providing a modest immunologic stimulus may preserve lymphocyte populations during sepsis and other acute disease processes.

P247 P38MAPK MODULATES THE PULMONARY ANTI-INFLAMMATORY EFFECTS OF HYPEROSMOLARITY AGAINST IL-1 STIMULATION

F. Wright, E.E. Moore, F. Gamboni, and A. Banerjee. University of Colorado Denver and Health Sciences Center, Denver CO, 80262

Recent evidence suggests that hyperosmolarity (HOsm) attenuates lung inflammation in epithelial cells, but the mechanism remains unclear. In other cell lines, HOsm modulates inflammation via p38 mitogen-activated protein kinase (MAPK). Therefore we hypothesizethat the p38MAPK pathway represents a vital component ofHOsmmodulation of lung inflammation.

Methods:

Human pulmonary epithelial cells were incubated with hypertonic saline (HTS) or sorbitol (SOR) at 400 mOsm 30 minutes prior to IL-1 (10ng/ml). Cytokines and p38MAPK phosphorylation were measured using ELISA. p38MAPK specific inhibitor SB 203580 was used.

Figure
Figure

Results:

Both HTS and SOR inhibit IL-1 stimulated release of RANTES in pulmonary epithelial cells (p<0.001). Inhibiting p38MAPK attenuates the effect of HOsm on RANTES (3-fold increase, p<0.05). HOsm also provokes p38MAPK phosphorylation (p<0.05).

Conclusion:

Hyperosmolar regulation of human pulmonary epithelial cell inflammatory response is dependent on p38 MAPK.

P248 ANTIOXIDANT DEFENSE SYSTEM IN ENDOTOXEMIC RATS WITH OBSTRUCTIVE JAUNDICE

Sz. Ábrahám1, E. Hermesz2, Á. Ferencz2, A. Szabó3, Gy. Lázár4, M. Boros3, Gy. Lázár Jr1. Dept. of Surgery1, Dept. of Biochemistry and Molecular Biology2, Inst. of Surgical Research3, Inst. of Pathophysiology4, University of Szeged, Hungary, H-6720, Pécsi u. 4., Szeged

Liver expression of the heme oxygenize (HO) and metallothionein (MT) genes is rapidly upregulated by proinflammatory cytokines and/or endotoxin (ETX), suggesting a protective role for them against cellular stress and inflammation. Gadolinium-chloride (GdCl3)-induced Kupffer cell (KC) blockade has beneficial consequences in endotoxemia following experimental bile duct ligation. The aim of this study was to characterize a possible link between KC blockade and the expression of HO and MT genes in endotoxemic rats with obstructive jaundice. Isoform specific expression of MT and HO genes was followed on RNAs, isolated from the liver of bile duct ligated (BDL), ETX and BDL+ETX treated animals, with/without GdCl3pretreatment. The expression of MT genes was altered in a stress-and isoform-specific manner: ETX and GdCl3 treatment by themselves led to a significant increase in the MT-2 expression, while BDL alone resulted in a drastic decrease in both MT-1 and MT-2 mRNA levels. Activation of HO genes by BDL and ETX was also isoform specific inducing HO-1 expression by 3.5-and 10-fold, respectively. GdCl3 decreased the ETX-and BDL-mediated HO-1 mRNA accumulation by approx. 50%. Our data suggest that experimental obstructive jaundice aggravated by endotoxemia significantly upregulates the expression of MT-2 and HO-1 isoforms, and Kupffer cell blockade influences this antioxidant defense system, already activated.

P249 DIFFERENTIAL IMMUNORESPONSES FOLLOWING EXPERIMENTAL TRAUMATIC BRAIN INJURY, BONE FRACTURE AND "TWO-HIT"-COMBINED NEUROTRAUMA

Marc Maegele, Stefan Sauerland, Bertil Bouillon, Ute Schäfer, and Edmund Neugebauer. Institute for Research in Operative Medicine (IFOM), Department of Trauma and Orthopedic Surgery, University of Witten-Herdecke, Cologne (Germany)

Cytokine-mediated immunoresponses are consequences of isolated traumatic brain injury (TBI) and muskuloskeletal trauma but little is known when both impacts occur simulanteously in combined neurotrauma (CNT), i.e. TBI+muskuloskeletal trauma (bone fracture). A "two-hit"-experimental model of CNT (TBI+tibia fracture) was used to investigate circulating cytokine interleukin-1-beta,-6,-10 and sTNF-R1 concentrations following peripheral bone fracture only, TBI only and CNT. Blood samples were drawn at 30 min, 6 h, 24 h, 48 h, and 7 days following trauma and circulating cytokine concentrations were determined via immunoassay. Circulating cytokines were increased after trauma (p<0.001 vs. controls), but peaked at different time points. sTNF R1 peaked first at 30 min, followed by IL-6 at 6 h after trauma. IL-10 levels were highest at 24 h, and those for IL-1beta at 48 h after trauma. Circulating IL-6 and IL-10 levels were highest in CNT at 8/10 time points studied (p<0.001). Circulating cytokine IL-1-beta,-6,-10 and sTNF-R1 concentrations are increased after trauma (TBI, fracture and CNT) but peak at different time points. Pronounced IL-6 and IL-10 responses after CNT may contribute to the increased susceptibility for complications in CNT versus monotrauma.

P250 HYPEROSMOLARITY INHIBITS TNF AND IL-1 SIGNALING BY DISTINCT MECHANISMS

F. Wright, E.E. Moore, F. Gamboni, and A. Banerjee. University of Colorado Denver and Health Sciences Center, Denver CO, 80262

The mechanism of hyperosmolar (HOsm) anti-inflammatory benefits on pulmonary epithelium remains obscure. Both TNF and IL-1 receptors activate transcription by NF-κB, which is known to be necessary for acute lung injury. We examined the role of HOsm on NF-κB signaling in human pulmonary epithelial cells stimulated with TNF and IL-1.

Methods:

Human pulmonary epithelial cells were incubated with hypertonic saline (HTS) or sorbitol (SOR) at 400 mOsm for 30 minutes prior to TNF or IL-1 (10ng/ml) stimulation. I-κB phosphorylation was measured using ELISA. Immunofluorescent microscopy quantified NF-κB p65 subunit mean fluorescence intensity within the nuclei. Cytokines were measured using ELISA and immunofluorescent bead arrays.

Results:

The inhibitory effects of HTS and SOR were similar on TNF and IL-1 stimulation of RANTES (90-95% inhibition vs normal media, p<0.001), IP-10 (40-65%, p<0.05), and MIP-1b (50-60%, p<0.05) production. MCP-1 triggered by TNF was inhibited by HOsm (40-50%, p<0.05), but MCP-1 production by IL-1 was unaffected. Furthermore HOsm decreased I-κB phosphorylation with both stimuli and inhibited p65 translocation following TNF (p<0.01) but not IL-1.

Conclusion:

In lung epithelium, HOsm inhibits TNF I-κB phosphorylation, p65 translocation and the subsequent production of multiple cytokines; whereas HOsm does not effect IL-1 p65 translocation and has a less complete inhibitory effect on cytokine generation. Thus HOsm influence on the NF-κB pathway in lung epithelium is a selective process which can effect signaling cascades at different steps.

P251 HYPERTONIC SALINE ALTERS IKK COMPLEXES: A MULTI-FRET STUDY

A. Banerjee, T. Nydam, E.E. Moore, F. Wright, and F. Gamboni. University of Colorado, Denver, CO 80262

Hypertonic saline inhibits pro-inflammatory stimuli including cytokine or ICAM production, by yet unknown mechanisms. We have shown biochemically that HTS retards signaling through the IKK complex required for NF-κB translocation, and used FRET to statistically depict binary and ternary complexes with IKKγ/NEMO, Hsp90 and Clathrin (CHC) in intact, resting cells. We hypothesized that HTS alters the binding efficiencies of IKKα and IKKβ with proteins of the active signalosome.

Methods:

FRET measurements in intact pulmonary epithelial A549 cells (n>49) were made with or without exposure to HTS (30 mins, 180mM NaCl, 400mOsm). Detection was by anti-species antibodies each carrying a different fluorescent dye (Cy5, Cy3, or ALEXA 488).

Results:

We examined 4 complexes of IKKα or IKKβ with either IKKγ/NEMO or CHC. 30 mins of HTS alone changed nFRET efficiencies (mean±sem) for IKKβ-IKKγ from 0.35±0.02 to 0.57±0.04 (p<0.01) and IKKβ-CHC (both in perinuclear arcs), increased from 0.56±0.01 to 0.75±0.02 (p<0.001). In contrast, nFRET for IKKα with either IKKγ or CHC, were not significantly affected.

Conclusion:

HTS induces dramatic (50%) but selective change of FRET efficiency of IKKβ (but not IKKα) with both IKKγ and CHC. Altered assembly of complexes after hyperosmolarity may correlate with disrupted TNF signaling but suggests testable implications forthe IL-1, LPS, GPCR and other pathways that also utilize this common step.

P252 NATURAL KILLER, BUT NOT CD1-RESTRICTED NATURAL KILLER T, CELLS FACILITATE SYSTEMIC INFLAMMATION DURING SEPTIC SHOCK CAUSED BY CLP

Edward R. Sherwood, Anthony Etogo, Jesus Nunez, Cheng Lin, and Tracy Toliver-Kinsky. Department of Anesthesiology, The University of Texas Medical Branch, Galveston, Texas, USA, 77555-0591

Introduction:

The goal of the present study was to evaluate the contribution of NK and NKT cells to inflammation and injury during septic shock caused by cecal ligation and puncture (CLP).

Methods:

Mortality, cytokine production and bacterial dissemination were evaluated in NK and NKT cell-deficient mice after CLP. Further studies were undertaken to evaluate NK cell trafficking and interaction with myeloid cell populations.

Results:

Mice treated with anti-asialoGM1 (NK cell deficient, 30% longterm survival) or anti-NK1.1 (NK/NKT cell-deficient, 20% longterm survival) showed improved survival after CLP compared to IgG-treated controls (0% survival). CD1 KO mice (NKT cell-deficient, 0% survival) did not show a survival benefit compared to wild type controls (0% survival). NK, but not NKT, cell-deficient mice showed lower levels of CLP-induced IL-6 and MIP-2 protein in blood and peritoneum as well as decreased expression of IL-6 and MIP-2 mRNAs in lung, spleen and heart compared to controls. Systemic bacterial counts were not significantly different between control and NK/NKT cell-depleted mice. A significant increase in the number of NK, but not NKT, cells was seen in the peritoneal cavities of mice after CLP. Evidence of intraperitoneal NK cell proliferation was not observed as determined by BrDU incorporation. Conversely, NK cell numbers in the blood and spleen decreased. The overall decline of splenic NK cells was not due to apoptosis or cell death as determined by annexin V and propidium iodide staining. NK cells in the peritoneum of CLP mice showed a high level of activation compared to NK cells from sham mice as shown by increased expression of CD69, FasL, CD11b and IFNγ. In addition, depletion of NK cells decreased the production of IL-6 by peritoneal macrophages (F4-80+Gr-1) and a population of F4-80+Gr-1+myeloid cells.

Conclusions:

These findings indicate that NK, but not CD1-restricted NKT, cells facilitate acute inflammation during CLP-induced septic shock. NK cells mediate their pro-inflammatory functions, in part, by migration into the peritoneal cavity from spleen and blood where they facilitate myeloid cell activation. Supported by NIH R01 GM66885 and Shriners Grant 8780.

P253 ANTITHROMBIN (AT) AND ACTIVATED PROTEIN C (APC): IMMUNOMODULATION AFTER TRAUMA?

M. Burggraf, H. Trentzsch, S. Zedler, J. Hoffmann, and E. Faist. Dept. of Surgery, Grosshadern, University of Munich

The anticoagulants AT and APC have shown anti-inflammatory effects in various studies on experimental sepsis. We have asked for the immunomodulatory capabilities of these substances on human mononuclear cells following tissue trauma. Venous blood was drawn from volunteers (CO; n=12) and patients undergoing abdominal surgery (OP; n=12) or after severe trauma (PT; n=9; mean ISS=32) on days pre-(d-1; OP only), 1 (d1), 3 (d3) and 7 (d7) after trauma. Peripheral blood mononuclear cells (PBMC) were obtained by Ficoll separation. PBMC were pre-treated 1h with AT (1 or 20IU/ml), APC (4 or 100μg/ml) or in combination and were challenged for 20 hours in serum-free media with bacterial LPS (1μg/ml). TNF-α and IL-10 were measured in culture supernatants using a Luminex 100 system. In LPS-stimulated cultures of CO incubated with AT20 (including combinations) TNF-α and IL-10 levels were highly significantly reduced (p<0,01). In terms of TNF-α this effect is sustained in both, surgical (p<0,01) and trauma (p<0,05) subgroups except PT d7. IL-10, though showing a trend towards reduced levels, didn't reach statistical significance. Sole APC showed no significant effect on cytokine liberation in neither concentration. The inflammatory response of human PBMC is modulated by AT20. Effects are stable irrespective of the underlying type of tissue trauma. The missing capability of APC in reducing the inflammatory response might be due to missing co-factors or endothelium interactions in a serum-free ex-vivo model. Combining both substances yielded no additional influence. These findings support the hypothesis of AT having immunomodulatory potential in settings of posttraumatic immunalteration. Modulatory actions of APC have to be further elucidated.

P254 HPS70 IS CRITICAL TO REGULATED IL-8 PRODUCTION BY LPS: THE ROLE OF MRNA STABILIZATION

S. Sakr*, S. Knutson*, V. Schaeffer, M. Knoll, and J. Cuschieri. University of Washington, Seattle, WA 98104

Background:

Macrophage activation by bacterial products, such as LPS, leads to the liberation of various inflammatory mediators that orchestrate subsequent innate immune responses. Recently, we have demonstrated that macrophage activation by LPS results in the clustering of a number of receptors in addition to CD14 and TLR4, in particular HSP70. The role that HSP70 plays in LPS signaling, however, remains unknown. The purpose of this study is to determine the role that HSP70 plays in LPS signaling.

Methods:

Differentiated THP-1 cells were stimulated with 100 ng/ml of LPS. Selected cells were pretreated with 1 mM 4-PhenylButyrate (4PB) for 24 hours to reduce expression of HSP70 or 1 μg HSP70 prior to LPS with/without 4BP treatment. Following stimulation total cellular and nuclear protein was extracted and analyzed by immunoblot for the activation of ERK 1/2, p38, JNK/SAPK and NF-κB. Harvested supernatants were analyzed by ELISA for the production of TNF-α, IL-6, IL-8 and IL-10. Determination of mRNA binding to HSC70 and decay was performed by subjecting cells to HSC70 immunoprecipita-tion followed by RtPCR.

Results:

LPS exposure led to the activa-tion of the MAPKs and NF-κB. This was followed by strong binding of HSC70 to IL-8 mRNA and weak binding to TNF-α mRNA, and the production of TNF-α, IL-6, IL-8 and IL-10. 4BP treatment was associated with a reduction in HSP70 expression. 4BP followed by LPS was associated with increased p38 and NF-κB activation, IL-8 mRNA stabilization, and increased IL-8 and TNF-α production. These changes were reversed in the presence of HSP70.

Conclusion:

LPS results in the activation of a number of receptor associated proteins, including HSP70. It appears that the HSP70 activation is critical to selective regulation of IL-8 production by LPS. The mechanism in this regulation appears to occur through selective down-regulation of p38 and destabilization of IL-8 mRNA. This data is consistent with previous observations seen in patients with acute lung injury implicating a role of both p38 and IL-8, and protection by HSP70.

P255 THE ROLE OF TRIF IN TLR-MEDIATED COMPLEMENT FACTOR B UPREGULATION

David J. Kaczorowski, Amin Afrazi, Melanie J. Scott, Roop Gill, Rebecca D. Edmonds, and Timothy R. Billiar. Dept. of Surgery, Univ. of Pittsburgh, Pittsburgh, PA 15213

TLR-signalling and complement activation are critical to the host responses in sepsis and trauma. We have shown that stimulation of macrophages (MΦ) with lipopolysaccharide (LPS:TLR4-ligand) or polyI:C (TLR3-ligand) causes robust upregulation and release of complement Factor B, a key component of the alternative pathway. Of TLRs, only TLR4 and TLR3 use TRIF-mediated signalling. We hypothesized that Factor B upregulation is TRIF-dependent. We usedRAW264.7 MΦ and thioglycollate-elicited peritoneal MΦ fromWT (C57BL/6), TRIF KO and MyD88 KO mice. Cells were stimulated for 6h with LPS (10ng/mL), polyI:C (10μg/mL) or MPLA (10ng/mL), a lipid A analogue that signals preferentially through TRIF. Quantitative RT-PCR determined mRNA levels of Factor B, and protein levels were detected by Western blot. Both LPS and polyI:C significantly upregulated Factor B mRNA and protein in RAW MΦ. Similar upregulation occurred in WT MΦ after LPS. This was partially abrogated in MyD88ko MΦ and significantly abrogated in TRIFko MΦ (p<0.05). Similar upregulation of Factor B was observed in RAW MΦ stimulated with MPLA. This suggests TLR4 upregulates Factor B largely through TRIF. However, there were no differences in Factor B levels after polyI:C in WT, TRIFko and MyD88ko MΦ, suggesting a role for receptors other than TLR3 forpolyI:C. These data indicate Factor B upregulation is regulated through TRIF-dependent (LPS) and TRIF-independent (poly I:C) pathways. Identifying mechanisms of TLR-mediated complement production may improve treatment strategies in sepsis and trauma.

P256 TREGS CAN DIRECTLY SUPPRESS MACROPHAGE TLR RESPONSES

Fionnuala O'Leary, Goro Tajima, Ann McKenna, Malcolm P. MacConmara, John A. Mannick, and James A. Lederer.

Mice lacking CD4 T cells develop a more pro-inflammatory phenotype when injured. We subsequently showed that CD4+CD25+regulatory T cells (Tregs) were required to control this injury-induced pro-inflammatory response. We now demonstrate that injured Treg-deficient mice are more susceptible to LPS-induced shock suggesting that Tregs play an active role in controlling Toll-like receptor 4 (TLR4) reactivity. Since macrophages are the primary cells responsible for enhanced LPS reactivity after injury, we wished to test whether Tregs might directly control macrophage responses to innate stimuli. To accomplish this, we first developed a macrophage/Treg suppression assay. Macrophages were purified from the spleens and CD4+CD25+T cells (Tregs) were purified from the lymph nodes of sham or burn C57BL/6J mice. Cells were then mixed at various Treg:macrophage ratios and stimulated with pure LPS (a TLR4 agonist) or Pam3Cys-Ser-(Lys)4 (a TLR2 agonist) and anti-CD3 antibody. Supernatants were tested for TNFα, IL-6, and IL-10 levels. Supernatants were also tested for their ability to transfer suppressive activity. Our results indicate that Tregs can directly suppress TLR4-and TLR2-induced inflammatory cytokine production by macrophages. We also discovered that supernatants were able to transfer Treg-dependent suppressive activity. Finally, we demonstrate that Tregs prepared from burn-injured mice are more potent than sham Tregs at suppressing macrophage TLR2 and TLR4 responses. Taken together, our findings show that Tregs can actively suppress macrophage TLR responses and provide new insight into how Tregs control innate immune responses to injury and inflammation.

P257 BINGE LIKE ETHANOL EXPOSURE AUGMENTS THE HEMORRHAGE/RESUSCITATION INDUCED LIVER INJURY AND THE PROINFLAMMATORY RESPONSE IN RATS - EFFECTS OF SIMVASTATIN PRE-TREATMENT

F. Bormann, B. Relja, C. Höhn, D. Henrich, C. Czerny, M. Lehnert, and I. Marzi. Trauma Surgery, JW Goethe University, Frankfurt

Background:

Since pre-existing hepatic dysfunction is a known predictor for worse outcome after trauma / hemorrhage, we induced an acute fatty liver in a rat model of acute ethanol exposure before hemorrhage/resuscitation (H/R). The aim was to study effects of binge ethanol on liver damage and inflammation after H/R and to test effects of Simvastatin pretreatment.

Methods:

Before H/R, female Lewis rats were pretreated for 6 days with Simvastatin (5 mg/kg per day i.p.) or vehicle and 12h before H/R, rats received an ethanol gavage (5g/kg, 30%). Then, rats were hemorrhaged to an arterial blood pressure of 30±2 mmHg for 60min and resuscitated. 2h later, serum ALT, serum IL-6 and IL-1β levels were measured; p<0.05 was considered significant (ANOVA, mean±S.E.M).

Results:

ALT was largely increased after H/R compared to sham operation (1618±256 vs. 118±17 [IU/ml], p<0.05). Ethanol further amplified H/R-induced ALT release (2863±554, p<0.05). Simvastatin decreased ALT release by 53% without ethanol gavage and by 83% with ethanol gavage (p<0.05). Simvastatin reduced serum IL-6 levels both after H/R with and without ethanol gavage (1596±816 vs. 217±73 [pg/ml] and 1127±410 vs. 57±57, p<0.05). IL-1β was only present after H/R in ethanol gavaged rats, an effect reduced by Simvastatin (560±204 vs. 29±29 [pg/ml], p<0.05).

Conclusion:

Binge like ethanol exposure in conjunction with H/R augments hepatic damage and the inflammatory response after H/R-effects that areblunted by Simvastatin pre-treatment. (Supported by DFG MA 119/3-3)

P258 A MATHEMATICAL MODEL OF ACUTE INFLAMMATION WHICH ACCOUNTS FOR BLOOD AND TISSUE INTERACTIONS

Angela Reynolds, Gilles Clermont, Jonathan Rubin, and Bard Ermentrout. University of Pittsburgh, Departments of Mathematics and Critical Care, Pittsburgh, PA 15260

Objective:

During an inflammatory response to infection associated with severe inflammation, the systemic nature of the response is responsible both for eliminating the infection and jeopardizing organ function. Therefore, we expanded our previous acute inflammation model to include a distinction between immune components in the blood and those in the tissue. Additionally, we explore the spread of infection between organs.

Methods:

We derived a differential equations model for acute inflammatory response containing components in both the tissue and blood. This model includes both pro and anti-inflammatories involved in the recruitment of neutrophils to tissue. The residual damage caused by the influx of neutrophils is modeled and multiple tissues were linked to the same blood compartment.

Results:

The model has three stable fixed points which represent the biological outcomes of acute inflammation: health, a return to homeostasis; aseptic death, elimination of pathogen with elevated immune mediators; and septic death, elevated pathogen and immune mediators. At low pathogen growth rate, the model allows a return to homeostasis and pathogen free runaway inflammation, while at high pathogen virulence, septic death becomes a stable outcome resulting in tristability. Linking two tissues to the same blood supply provides a survival advantage compared to a single tissue. The simulations are sensitive to the relative resistance to invasion of the different tissues.

Conclusion:

This multiple tissue compartment model is a first step in examining the spread of inflammation between organs and the impact of spread on the intensity of acute inflammation.

P259 ERYTHROPOIETIN PROTECTS IN HEPATIC ISCHEMIA-REPERFUSION (IR) INJURY THROUGH JAK-STAT CYTOKINE REGULATION

L.A. Langdale, V. Hoagland*, W. Benz*, D. Liggitt*, and J.S. Campbell*. VA-PSHC and University of Washington, Seattle WA 98145

The duration and intensity of cytokine-mediated signaling in hepatic IR injury are regulated, at least in part, through negative feedback by the suppressors of cytokine signaling, SOCS1 and SOCS3. We have shown that both Socs3 and Socs1 are induced during IR injury and appear to act as a complimentary regulatory mechanisms to limit cytokine-mediated inflammation as the severity of hepatic IR injury increases. Erythropoietin (rhEPO), a hormone known to induce SOCS1 and SOCS3, has been shown to protect a variety of organs subjected to IR. We hypothesized that rhEPO mediates its protective effects, in part, by inducing these regulators of cytokines signaling.

Methods:

C57Bl6 mice underwent severe (90min) partial liver IR. Mice treated with rhEPO 4U/g SQ at the onset of ischemia were compared to untreated controls for evidence of injury (ALT/AST and histology) 1, 2, 4, and 24h after reperfusion. Expression of inflammatory cytokines important to IR (Tnfα, IL-1β, and IL-6), and Socs1, Socs3, and Cis expression was analyzed in mRNA from ischemic and perfused lobes by RT-PCR. Activated STAT1, 3, and 5 were determined by phospho-immunoblotting, and apoptosis was assessed by caspase3. Data were analyzed by two-way ANOVA.

Results:

rhEPO provided significant protection after severe IR as evidenced by reduced ALT/AST levels and hepatic necrosis. TNFα, IL-1β, and IL-6were significantly reduced compared with untreated controls. rhEPO treatment correlated with down-regulation of STAT3 and STAT5 signaling, decreased caspase3 and cytokine expression, and induction of SOCS/CIS mechanisms.

Conclusions:

EPO protects liver after severe IR both through caspase3 inhibition and negative regulation of cytokines signaling via the JAK-STAT pathway.

Acknowledgement:

Dept. of Veterans Affairs Merit Review.

P260 ROLE OF STORE-OPERATED CALCIUM ENTRY IN DEGRANULATION OF HUMAN PMN

Qin Zhang, Mustafa Raoof, Bozena Antoniu, Kiyoshi Itagaki, and Carl J. Hauser. Department of Surgery, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston MA 02215 USA

Background:

Neutrophil (PMN) degranulation of matrix metalloproteinases (MMPs) in the lung after injury may play an important role in acute lung injury (ALI). MMP8 is stored in PMN granules and released by activation of G-protein coupled receptors (GPCR). Some GPCR cause only Ca2+release where others activate store-operated Ca2+entry (SOC). But full PMN activation often requires SOC, so we isolated and studied the role of SOC in MMP8 release.

Methods:

PMN isolated from volunteers were activated by N-met-leu-phe (fMLP, which causes Ca2+release) and/or thapsigargin (TG, which causes SOC) in the presence or absence of extracellular Ca2+and Ca2+channel blockers. MMP-8 secretion was measured by immunoassay. Ca2+flux was assessed as the area under the curve (AUC) of fura fluorescence over the same time in identical PMN isolates.

Results:

fMLP and TG induce synergistic degranulation of MMP-8. In Ca2+-free media PMN do not release MMP8. Under all conditions MMP8 release corresponds to the magnitude of SOC. Ca2+entry is inhibited in a dose responsive fashion by SOC blockade using Gd3+and La3+. Once again, quantitative MMP8 release parallels SOC in the presence of influx inhibition by Gd3+and La3+.

Conclusion:

These results indicate that Ca2+influx via SOC pathways plays a critical role in the control of PMN degranulation of MMP. Control of MMP mediated lung damage through modulation of PMN SOC may therefore represent a new approach to preventing or treating ALI.

P261 MACROPHAGES REMEMBER NEURAL SIGNALS THAT TEACH ATTENUATION OF RESPONSES TO BACTERIAL ENDOTOXIN

W.R. Parrish, M. Ochani, M. Rosas-Ballina, H. Khalili, A. Damle, M. Gallowitsch-Puerta, Y. Harris, K. Ochani, X. Lin, E.J. Miller, J. Huston, C.J. Czura, W. Li, F.M. Batliwalla, P.K. Gregersen, and K.J. Tracey. 1Laboratory of Biomedical Science, 2Dept of Surgery, 3Robert S. Boas Center for Genomics and Human Genetics, Feinstein Institute, Manhasset, NY 11030

Macrophage cytokine production is inhibited by vagus nerve signals that are transduced through the α7 nicotinic acetylcholine receptor subunit (α7nAChR) dependent pathway termed the "cholinergic anti-inflammatory pathway" (Nature 420:853-9, 2002). Here we address the hypothesis that macrophages can "remember" the anti-inflammatory stimulus provided by vagus nerve signals. Vagus nerve stimulation (VNS) (5V, 2 ms, 1Hz, 2.5 min) in rats 24 h prior to endotoxin (LPS) (1 mg/Kg I.P.) significantly reduced serum TNF (sham=767 pg/ml; VNS=358 pg/ml, p<0.01). This TNF-suppressing effect persists even when LPS exposure is delayed for 48 h after VNS (sham TNF=1327; VNS TNF=824, p<0.05). The molecular mechanism is attributable to α7nAChR signaling, because VNS fails to reduce TNF levels in α7nAChR KO mice. This cellular memory can be recapitulated in human macrophage cultures pulsed with acetylcholine (100 μM Ach; 60 min pulse; 24 h prior to LPS). LPS-induced TNF release was reduced 54% as compared with vehicle controls (p<0.05); pulsing also significantly suppressed activation of NF-κB (74%). ACh-pulsing significantly altered the macrophage transcriptome response to LPS (498 differentially expressed genes, p<0.01), indicating that suppressed macrophage TNF responses following cholinergic stimulation persist as a memory-like response. Evidence that discrete neural signals can mediate memory responses in immune cells via α7nAChR has surprising implications for understanding innate immunity. Supported in part by NIGMS.

P262 HETEROGENEITY OF ENDOTHELIAL CELL CALCEMIC RESPONSES TO COAGULATION AND INFLAMMATION

Kiyoshi Itagaki, Mustafa Raoof, and Carl J. Hauser. Department of Surgery, Beth Israel Deaconess Medical Center, Boston, MA 02215 USA

Background:

We have shown the dual functional roles of sphingosine 1-phosphate on EC calcium mobilization and permeability changes using human umbilical endothelial cells (HUVEC). We here examine the characteristics of another EC, human pulmonary artery endothelial cells (HPAEC) to evaluate the heterogeneity of ECs.

Objective:

To evaluate the heterogeneity of EC using HUVEC and HPAEC.

Methods:

Thrombin and histamine were applied to determine store-operated calcium entry mechanism in the cells by using fura-2 as a calcium indicator. TNF-α-induced sphingosine kinase-1 expression both as a protein and as a gene were evaluated by Western Blot and Real Time PCR, respectively.

Results:

Histamine induced calcium mobilization similarly to both cell types, however there was no response in HPAEC to thrombin administration. HUVEC responded thrombin similar to histamine. TNF-α increased level of SK-1 in protein level in a dose-dependent manner, however there was no difference in a message level in HPAEC. TNF-α treated HPAEC were subjected to thrombin stimulation, however no calcium depletion was detected.

Conclusions:

Results suggest that pulmonary vasculature may be less of a thrombotic surface than the systemic vasculature. Although TNF-α induced SK-1 in HPAEC, there was no change to thrombin response or no change in the magnitude of calcium influx by histamine or thapsigargin. Increased SK-1 protein has least effect on calcium influx suggesting that number of SK-1 being translocated to plasma membrane as an active enzyme is limited and does not require additional SK-1 to trigger calcium influx.

P263 MAMMALIAN TARGET OF RAPAMYCIN: AN ENDOGENOUS MODULATOR OF ENDOTHELIAL ICAM-1 AND LUNG IFLAMMATION?

M. Minhajuddin, F. Fazal, K.M. Bijli, and A. Rahman

We have shown that mammalian target of rapamycin (mTOR) down-regulates thrombin-induced ICAM-1 expression in endothelial cells by controlling a delayed and transient activation of NF-κB. We have also shown that PKC-δ, PI 3-kinase, and the downstream target Akt are important signaling intermediates in mediating NF-κB activation and ICAM-1 expression in these cells. We now provide the evidence that these pathways also activate mTOR to ensure a tight regulation of NF-κB activation and ICAM-1 expression. Stimulation of human vascular endothelial cells with thrombin resulted in phosphorylation of mTOR and its downstream target p70S6 kinase in a PKC-δ- and PI 3-kinase/Akt-dependent manner. Consistent with this, we observed that expression of constitutively active PKC-δ (PKC-δ-CAT) or Akt (Akt-CAT) mutant was sufficient to induce NF-κB activation and ICAM-1 expression, and that co-expression of mTOR suppressed whereas inhibition or depletion of mTOR augmented these responses. Increasing or impairing mTOR signaling by the above approaches produced similar effects on NF-κB activation and ICAM-1 expression induced by thrombin. These data reveal an important role of mTOR as an endogenous modulator that may be engaged in PKC-δ- and PI3K/Akt-dependent manner to dampen thrombin-induced NF-κB signaling of ICAM-1 expression in endothelial cells. We also determined the in vivoconsequence of mTOR overexpression in a mouse model of sepsis. Introduction of the construct encoding mTOR via cationic liposomes (which results in specific expression of the transduced gene in lung endothelium) caused a marked reduction in lung PMN infiltration in mice challenged i.p. with LPS. Together, these results indicate that mTOR may be a general modifier of lung inflammatory responses. (Supported by: ALA, NIH HL067424).

Figure
Figure

P264 ASSESSMENT OF DISTINCT ISOFORMS OF THE RECEPTOR FOR ADVANCED GLYCATION END-PRODUCTS EXPRESSED IN TISSUES AND CELL LINES

Julia V. Gefter, Mitchell P. Fink, and Russell L. Delude. Department of Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15261

Recent developments in inflammation research have focused attention on the receptor for advanced glycation end-products (RAGE). RAGE is reported to bind numerous ligands and is expressed as numerous protein isoforms. Northern blotting, immunoblotting and posttranslational analysis were used to survey RAGE isoform expression in various cell lines and tissues to provide a better understanding of the complexity of the RAGE expressome. "Full-length" transmembrane and soluble RAGE isoforms were expressed in lung which also expressed a novel LPS-inducible isoform of RAGE we refer to as X-RAGE. Non-pulmonary tissues expressed a unique "tissue" RAGE isoform. Cell lines expressed their own RAGE variant. Pulmonary RAGE mRNA is smaller than cell line and tissue RAGE mRNA. Human lung RAGE contained 3 N-linked glycans, human tissue RAGE contained 1, mouse lung RAGE contained 2, whereas mouse tissue RAGE and cell line RAGE were not N-glycosylated. Transfecting cell lines with lung RAGE cDNA led to co-expression of pulmonary and cell line RAGE isoforms. S100B up-regulated and HMGB1 down-regulated MCP-1 expression in HMEC-1 cells in a dose-dependent manner, but this effect was independent of transfection with RAGE cDNA and was inhibited by HMGB1. In conclusion, several RAGE protein isoforms are detected in tissues and cell lines. Despite the apparent complexity, isoform expression appears to be predicated on the pulmonary versus non-pulmonary source of a tissue or cell line. This research was supported by NIH R01 GM-37631 and GM68481.

P265 HEAT SHOCK COGNATE PROTEIN 70 INDUCES MYOCARDIAL INFLAMMATORY RESPONSE THROUGH MECHANISMS DEPENDENT AND INDEPENDENT OF TOLL-LIKE RECEPTOR 4

N. Zou, L. Ao, J.C. Cleveland Jr, D.A. Fullerton, and X. Meng. Department of Surgery, University of Colorado Denver, Denver, CO 80262, USA

Inducible heat shock protein 70 activates Toll-like receptor 4 (TLR4) and is capable of inducing cytokine production in monocytes. Several studies suggest that cell injury results in the release of constitutive heat shock proteins, including the 70 kDa heat shock cognate protein 70 (HSC70). However, it is unknown whether extracellular HSC70 exerts an influence on myocardial TLR4 signaling and whether it induces the myocardial inflammatory response. We tested the Hypothesis: that extracellular HSC70 induces the myocardial inflammatory response through TLR4.

Methods and Results:

We applied recombinant bovine HSC70 (0.5 μg/ml) to treat isolated/perfused mouse hearts. In wild-type (WT) hearts with competent TLR4, HSC70 induced NF-κB activation, p38 MAKP phosphorylation and the production of the pro-inflammatory mediators IL-1, IL-6, KC and MCP-1. In hearts with defective TLR4 (TLR4d), the effect of HSC70 on IL-1 and IL-6 was abrogated while HSC70 effect on KC and MCP-1 was attenuated.

Conclusions:

Extracellular HSC70 induces the myocardial inflammatory response. TLR4 mediates myocardial expression of IL-1 and IL-6 in response to extracellular HSC70, and it also contributes to the mechanisms of HSC70-induced myocardial expression of chemokines KC and MCP-1. Thus, extracellular HSC70 induces the myocardial inflammatory response though TLR4-dependent and -independent mechanisms.

Myocardial cytokine and chemokine levels (pg/mg protein)

P267 TLR4 AND TLR2 DIFFERENTIALLY REGULATE MIGRATION OF IMMUNE CELLS TO THE PERITONEUM

Daniela Plitzko, Katja Butterbach, Gesine Hoffmann, and F. Ulrich Schade. Surgical Research-Trauma Surgery, University Hospital Essen, Virchowstr. 171, 45147 Essen, Germany

Lipopolysaccharide (LPS, endotoxin) and lipopeptides are highly conserved bacterial structures of Gram-negative or Gram-positive bacteria capable of stimulating immune responses. While TLR4 has been identified as the transmembrane signal transducer for LPS, TLR2 plays a major role in recognizing the synthetic bacterial lipopeptide Pam3CSK4. To get insight into the cellular basis of TLR-dependent immune regulation, we determined the population of peritoneal immune cells 4 days after LPS-or Pam3CSK4-stimulation by flow cytometry. This specific time point was chosen since a single bolus of LPS on day 0 induces a state of low responsiveness to its own action on day 4. The results therefore are expected to provide further information on the involvement of TLR4 and TLR2 in LPS-tolerance.

While the percentage of CD19+B220+B lymphocytes is significantly upregulated in mice stimulated with Gram-negative bacteria (LPS), the percentage of CD11b+F4/80+CD14+macrophages is downregulated with regard to normal controls. In contrast to these findings, stimulation with Gram-positive bacteria (Pam3CSK4) involves no significant alterations in the percentage of B cells or macrophages. We investigated the expression pattern of co-stimulatory molecules as well as maturation markers on B lymphocytes and macrophages of either TLR4-or TLR2-activated mice in comparison to normal controls. Corresponding results will be presented.

P268 HYPEROXIA INDUCES CYTOKINE PRODUCTION AND RELEASE OF REACTIVE OXYGEN SPECIES BY HUMAN BLOOD LEUKOCYTES IN THE CONTEXT OF VACUUM-ASSISTED CLOSURE

Stephan Brauckmann, Daniela Plitzko, Sven Lendemans, F. Ulrich Schade, and Sascha Flohé. Surgical Research-Trauma Surgery, University Hospital Essen, Virchowstr. 171, 45147 Essen, Germany

Vacuum-assisted closure (VAC) is a non-invasive therapy using subatmospheric pressure to accelerate wound healing. Previous clinical studies have demonstrated positive effects on proliferation, tissue formation and modulation of inflammation in chronic wounds; the physiological and molecular mechanisms by which VAC therapy influences the immune response in addition to removal of infections agents are largely unknown. Application of subatmospheric pressure in vivo leads to hyperoxia, characterized by an increased oxygen partial pressure altering cytokine production of immune cells. To get insight into the in vitro situation of interactions of hyperoxia on immune cells, we determined cytokine production and generation of reactive oxygen species by human blood leukocytes and isolated polymorphonuclear neutrophils (PMN). To investigate the influences on the signalling pathway of Gram-negative as well as Gram-positive stimuli, we employed both lipopolysaccharide (LPS) and peptidoglycan (PGN) stimulated whole blood cultures compared to unstimulated controls. The application of hyperoxia on human blood leukocytes yields to a significant increase in the release of the pro-inflammatory cytokines tumor necrosis factor (TNF) alpha, Interleukin (IL)-6, IL-8 and the anti-inflammatory cytokine transforming growth factor (TGF) beta 1. The production of hydrogen peroxide (H2O2) released from PMN could also be raised by hyperoxia characterizing their reactivity.Supported by IFORES, University of Essen, Germany.

P269 REPROGRAMMING OF MURINE PERITONEAL CELLS BY ENDOTOXIN TOLERANCE

F. Ulrich Schade, Danute Pupjalis, and Daniela Plitzko. Surgical Research-Trauma Surgery, University Hospital Essen, Virchowstr. 171, 45147 Essen, Germany

"Endotoxin Tolerance" (ET) is induced in animals by injection of tiny amounts of lipopolysaccharide (LPS, endotoxin). ET protects against bacterial infections and ischemia-reperfusion injury. To get insight into the cellular mechanisms of ET different cellular components of the peritoneal cell (PC) populations of endotoxin tolerant and normal mice were studied regarding regulation of cytokine production. Mice were made tolerant by i.p. injection of LPS and peritoneal cells were prepared 4 days later. Only slight changes in the relative number of DCs, macrophages T-cells and PMNs were determined, the amount of B-cells was increased in PC from tolerant mice. To test the functional consequences of these changes, both populations were incubated in a mixed culture, stimulated with LPS and TNF was determined in the supernatant. PCs of tolerant mice suppressed the synthesis of TNF by PCs of normal mice (normal: 1534, tolerant: 127 normal/tolerant: 414 all: pg/ml). Results are presented showing that non B-cells-probably adherent cells-are responsible for the observed inhibitory effect.

Inflammation/Immunodysfunction Clinical Research

P270 IMMUNE RESPONSE IN WAR INJURIES WITH OR WITHOUT SECONDARY SEPSIS

Maja Surbatovic, Miodrag Jevtic, Sonja Radakovic, and Nikola Filipovic. Military Medical Academy, Belgrade, Serbia

Despite the advances made in prehospital and critical care medicine, mortality rate after blast and explosive trauma continues to rise. Severe war injuries and secondary sepsis complicated with multiple organ dysfunction syndrome (MODS) are leading causes of death in combat casualties with mortality rate exceeding 50%. Outcome is not determined only by the severity of trauma and/or infection, but also by intensity of immuno-inflammatory response, which is essential for host defense, but uncontrolled leads to the MODS. Aim of this study was to assess the prognostic value of interleukin (IL)-8, tumor necrosis factor (TNF)-alpha, IL-4 and IL-10 in combat casualties. Fifty six casualties with severe trauma (blast and explosive) who developed sepsis (mortality 56%) and 20 casualties with same severity of trauma without sepsis (mortality 20%) were enrolled. Fifty five casualties developed multiple organ dysfunction syndrome (MODS), 36 died. Blood was drown on the first day of trauma. Concentrations of IL-8, TNF-alpha, IL-4 and IL-10 were determined in plasma using ELISA assays. Mean values of IL-8 were 230-fold (p<0.01), IL-10 42-fold (p<0.01), TNF-alpha 17-fold (p<0.05) higher in trauma+sepsis group. Mean values of IL-8 were 60-fold, TNF-alpha 43.5-fold and IL-10 70-fold higher in MODS group (p<0.01). Mean values of IL-8 were 2.3-fold and IL-10 1.4-fold higher in non-survivors while mean values of TNF-alpha were 2.2-fold higher in survivors (p<0.01). IL-4 had no significance as a predictor of severity and outcome whatsoever. Further studies are needed to explain that. To our knowledge, next to nothing was done in research of cytokine response to combat trauma with or without sepsis in war time conditions.

P271 SERUM-PROTEOMICS CHRONICLES AFTER MONOTRAUMA OF THE UPPER ANKLE JOINT

M.R. Raum, G.C. Niemeijer, H. Roelofsen, G. van Dam, R.J. Vonk, and H.J. ten Duis. University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9700RB Groningen, Netherlands

Introduction:

The individual immune responds of organisms after trauma is still an unsolved problem for estimation of severe traumatized patients. A new approach is the protein-spectral-analyses (proteomics), whereby interpretations of protein-profiles are taking place.

Aim of this study was getting first experience with this method in a well-defined patient-collective.

Material and Methods:

In a time-period from October 2006 up to May 2007 7 patients with an isolated ankle-joint-fracture were included. In between the first 24 hours after trauma the first blood samples were drawn. On day 2, 4, 6, 13, 21, 27 and 90 after trauma additional samples were taken. The analysis took place by using the SELDI-TOF-MS-technique (surface-enhanced-laser-desorption-time of flight-mass-spectrometry).

Using cluster-analysis-software different protein-profiles were identified and compared with the clinical career of the patient.

Results:

Two clusters were identified with a quite homogenous time course. Molecules with a weight of 24911 Da, 13977 Da, 14877 Da und 11629 Da, formed cluster 1. In Cluster 2 molecules of 4514 Da, 6484 Da und 66206 Da were found. In a data-bank-research these molecular weights were attached to different proteins. Cluster 1 was acknowledged as different acute-phases-proteins (immuneglobuline kappa, interleukin 17, CRP, bone morphogenetic protein), whereas cluster 2 was quite unspecific. In detail-analysis per patient single clinical events could be related to protein-peaks.

Conclusion:

By using the proteomics-analysis different changes of protein-profiles after isolated ankle-fractures could be identified and be correlated with clinical events. We think this is the first step in learning more about this technique. In further research more complex pathologies will be analysed with the goal of identifying typical protein-profiles, which are related with high inflammation. This will help in estimating patient's traumaload and better planning of therapeutic regimes.

P273 DISTINCT CHARACTERISTICS OF TRAUMA PATIENTS' TOLEROGENIC DENDRITIC CELLS (TolDC) VERSUS IL-10 INDUCED TolDC

C. Miller-Graziano. Univ. of Rochester Med Ctr, Rochester, NY, USA

As circulating monocytes(MO) are the primary source of tissue DC during inflammation and infection, alteration in trauma patients'(pt) MO to DC differentiation resulting from altered circulating cytokines or endogenous mediators affects their immune activation versus immunosuppression balance. We showed the DC inhibitory mediator Thrombo-spondin-1 as increased in pt plasma after severe trauma as is also reported for IL-10. IL-10 both inhibits MO to DC differentiation and induces DC which subsequently inhibit fresh T cell(Tc) activation (TolDC). Our immunosuppress-ed trauma pts(Ispt) had inhibited MO to DC differentiation and a subset of TolDC. Here, trauma pts' TolDC were assess-ed for equivalence to invitro generated IL-10 TolDC. ISpt whose MO differentiated to TolDC inhibited allogenic Tc proliferation and cytokine levels equivalently to IL-10 indu-ced TolDC. Both ISpt and IL-10 TolDC had decreased CD-86 and CD40 levels versus immunocompetent pt(IComp) DC. Coinhibitory receptor expression varied between the TolDC types and from IComp pt's DC. Program cell death ligand 1(PD-L1), CD47(TSP-1 receptor), and ILT4 were assessed. Both IL-10 TolDC's and ISpts' TolDC's PD-L1 was significantly increased compared to IComp pt DCs' or controls' (PD-L1 IComp pt 14±5%, ISpt 42±5%, IL-10 DC 32±4). ILT4 increased only on IL-10 DC (IComp pt MFI 30±5, ISpt 35±4, IL-10 DC 65±6) while CD47 increased only on ISpts' TolDC (IComp pt DC 14±5, IS pt TolDC 42±5, IL-10 TolDC 10±2). Only ISpt TolDC had increased TSP-1 production (IS pt TolDC MFI 22±4, IComp pt DC 9±3, IL-10 TolDc 6±2) allowing auto inhibition through CD47-TSP-1 interaction. These data demonstrate IS trauma pts' TolDC as a unique subset not totally resulting from their increased circulating IL-10 levels.

P274 SIGNIFICANCE OF CHANGE IN T CELL-MEDIATED IMMUNITY AND ITS RELATIONSHIP WITH HMGB1 LEVELS IN SEVERELY BURNED PATIENTS

Yong-ming Yao, Ning Dong, and Zhi-yong Sheng. Burns Institute, First Hospital Affiliated to the Chinese PLA General Hospital, Beijing 100037, P.R.China

The purpose of this study was to investigate the significance of change in T cell-mediated immunity and its relationship with plasma high mobility group box-1 protein (HMGB1) levels in severely burned patients. 35 large area burned patients (>30% total body surface area) were included in this study, and they were divided into MODS group (n=13) and non-MODS group (n=22). The blood samples were collected on postburn days 1, 3, 5, 7, 14, 21, and 28. The plasma levels of HMGB1 were measured by ELISA, and T lymphproliferation response as well as production ability of IL-2 in peripheral blood were determined. In addition, the ratio of CD4+/CD8+T lymphocytes were detected by flow cytometry. Plasma HMGB1 levels were markedly elevated on postburn day 1 in severely burned patients, of them HMGB1 levels were significantly higher in MODS group than in non-MODS group (P<0.05). Lymphproliferation response and IL-2 production in peripheral blood, and the ratio of CD4+/CD8+T lymphocytes in MODS group were markedly lower than those in non-MODS group on postburn days 1, 14, 21, and 28 (P<0.05 or 0.01). Meanwhile, plasma HMGB1 levels were negatively correlated with T cellular immune function parameters, including lymphproliferation response, IL-2 release, and the ratio of CD4+/CD8+T lymphocytes in patients following major burns, respectively (P<0.05 or 0.01). Large area burns could lead to T cellular immune system dysfunction, which appears to be associated with the development of MODS. HMGB1, as an important late mediators of inflammation, might be involved in the pathogenesis of suppression of T cell-mediated immunity in the large area burned patients.

P275 SOLUBLE ENDOTHELIUM-SELECTIN (sE-SELECTIN) LEVELS CORRELATE WITH THROMBOMODULIN LEVELS IN CRITICALLY ILL PATIENTS

Tomoyuki Harada, Takahiro Terada, Munekazu Takeda, Ryuichi Moroi, Masaru Abe, Mizuho Namiki, Junji Yuzawa, and Arino Yaguchi. Tokyo Women's Medical University, Dept. Critical Care and Emergency Medicine

In our previous study, sE-selectin could predict the prognosis and the severity in critically ill patients. Furthermore, in septic patients, sE-selectin levels related to antithrombin III (AT III) activity. Thrombomodulin is an integral membrane protein expressed on the injured endothelial cells. We hypothesize thrombomodulin levels correlate with sE-selectin levels or AT III activity in critically ill patients. Twenty eight ICU patients (15 men, 13 women; age range 22-96 years [mean 55.3 ±20.1]) were included in this study. Serum thrombomodulin, sE-selectin, and AT III activity were measured on admission. Thrombomodulin assay was used by enztmeimmunoassay (Fujiyakuhinn Co, Saitama) (normal<4.5 FU/mL).SE-selectin assay was used by rapid latex immunonephelometry (Mitsubishi Kagaku Iatron Inc., Tokyo) (normal<29.7ng/mL) and AT III activity was using by chromogenics assay (Sysmex, Kobe) (normal: 80-130%). Thrombomodulin levels were compared with sE-selectin and AT III activity. Data were analyzed by Peason's correlation coefficient. Values are expressed as mean ± SD. A p<.05 was considered as statistically significant. There were 23 survivors and 5 non-survivors. Primary diagnoses on admission were 4 trauma patients, 17 non-septic patients and 7 septic patients. The SOFA score on admission was 4.9±3.3. Thrombomodulin level, sE-selectin level and AT III activity on admission were 5 ± 3, 31.0 ± 23.2 and 85.9 ± 18.7 respectively. There was no difference in thrombomudulin levels between survivors and non-survivors. Thrombomodulin levels was correlated with sE-selectin and AT III activity (r=0.65, r=0.42, p<0.05). In critically ill patients, thrombomodulin may be associated with sE-selectin and AT III.

P276 A PATIENT SPECIFIC STOCHASTIC MODEL FOR CD4 LEVEL PREDICTION IN HIV POSITIVE PATIENTS

Qi Mi, PhD1,2*, Shlomo Ta'asan, PhD4, and Ume Abbas, MD3. 1Department of Sports Medicine and Nutrition; 2Center for Inflammation and Regenerative Modeling; 3Division of Infectious Diseases, School of Medicine, University of Pittsburgh, Pittsburgh, U.S.A.; 4Department of Mathematical Sciences, Carnegie Mellon University, Pittsburgh, U.S.A. *email: [email protected]

Objectives:

HIV infection is the most destructive pandemics in recorded human history. It may lead to AIDS that result in death in most patients. The estimated number of AIDS related deaths from December 1981 to January 2006 is over 25 million. Highly Active Antiretroviral Therapies (HARRT) is the common treatment for HIV infected patients. It can greatly increase the patient's survival rate and reduce the morbidity. However, its cost is high, it has severe side effects and its availability in certain countries is limited. Initiation of HAART treatment is based on clinical assessment of CD4 cell count and viral load. Our goal is to develop a patient specific mathematical model that can assist clinicians in decision making. Due to limited availability of viral load in certain countries our models and predictions are based on CD4 level only.

Methods:

We have modeled patient specific CD4 cell count dynamics as an Ornstein-Uhlenbeck (OU) stochastic process with linearly declining mean value. This seems to fit the majority of patients in the MACS data set. Patient specific parameters are estimated from a few (about five) measurements using standard procedures. Prediction of future behavior is done using the model.

Results:

The model is used to obtain expected time for CD4 count to reach different levels, such as 350 cells/mm3 and 200 cells/mm3. In addition, risk for developing AIDS in individual patient can be estimated using the model. Visualization of the predicted CD4 cell count is given as a channel showing trend and range of future values, helping clinician assess patient condition.

Conclusions:

We developed a patient based stochastic model describing CD4 cell dynamics capturing the noise in a realistic manner. The model can be used to assist clinicians in decision making, in estimating risk for developing AIDS within a prescribed time, such as 1 year, 2 year, etc. These toolscan provide useful insights for clinicians in making decisions regarding antiretroviral therapies.

P277 CYTOKINES IN COLORECTAL CANCER PATIENTS

Edwin Bölke, Matthias Peiper, Christiane Matuschek, Stephan Gripp, Maximillian Pelzer and Wilfried Budach. University of Düsseldorf, Germany

Background:

IL-6, IL-8, TNF alpha and CRP are acute phase proteins. Their role in the development of colorectal cancer (CRC) has not been fully explained. The aim of our study was to analyze their character and explain their predictive implication.

Patient, Material and Methods:

IL-6, IL-8, TNFalpha and CRP plasma levels were measured in the daily routine morning in 30 CRC patients and the relationships between their elevations and both the clinicopathological aspects and clinical outcome of all patients were examined. Plasma concentrations of IL-8, IL-6 and TNFalpha were determined by enzyme-linked immunosorbent assay (DPC Biermann GmbH, Bad Nauheim, Germany) on the DPC-Biermann Immulite-analyser. CRP was determined in cell-free plasma using a Dade-Behring Dimension RXL analyzer with the C-Reactive-Protein-FlexTM reagent from Dade-Behring (Dade-Behring, Marburg, Germany).

Resultscolon;

Median IL-6, IL-8, TNFalpha and CRP levels were significantly elevated in CRC patients than in healthy volunteers. Elevated plasma levels of IL-6, IL-8, TNFalpha and CRP were linked with larger tumor volume. In addition elevated IL-6, IL-8 and CRP plasma levels were correlated with increased mortality.

Conclusion:

Plasma levels of IL-6, IL-8, TNFalpha and CRP are elevated in CRC patients. Pre-operative plasma levels of IL-6, IL-8 and CRP seem to be an indicator for the outcome of CRC patients. The clinical impact of IL-6, IL-8, TNFalpha and CRP in CRC needs to be further examined.

P278 CYTOKINES AND MACROPHAGE PHENOTYPES PREDICT VIRUS REACTIVATION AFTER SEVERE TRAUMA

T. Woehrle, H.-Y. Hsu, M. Weiss, L. von Müller, T.O. Joos, U.B. Brueckner, and E.M. Schneider. Dept. Anesthesiology and Virology, University of Ulm; NMI, Reutlingen, University Tuebingen, Germany

Human cytomegalovirus (hCMV) and herpes simplex virus-1 (HSV-1) have a high seroprevalence, thus constituting classical pathogens related to complications in immunocompromised patients. In this prospective study, 28 seropositive patients with septic shock after trauma were monitored for hCMV and HSV-1 reactivation. In addition blood leukocyte phenotypes and plasma cytokine concentrations were assessed on day 0, day 1, and day 3 of septic shock.

Twelve patients had reactivated hCMV and/or HSV-1 at the onset of septic shock (day 0) and were compared to the 16 patients without virus reactivation. CD56+CD16+NK cell numbers were similar in both groups, but CD64+, HLA-DR+MO populations with depressed differentiation (J. Immunol 2003,170:6355) were characteristic of the virus reactivation group. These patients also had lower levels of IFN-gamma (p<0.05). Concentrations of IL-15, IL-12(p70), and Eotaxin were found to be reduced with borderline significance. Reduced levels of IFN-gamma, IL-15, and IL-12(p70) despite CD56+CD16+activation and overexpression of CD64+HLA-DR+may indicate non-functional NK cells and impaired DC maturation, suggesting a cause for hCMV and HSV-1 reactivation and immune dysfunction in trauma patients.

P279