Secondary Logo

Journal Logo

Basic Science Aspects

Role of Hemorrhagic Shock in Experimental Polytrauma

Denk, Stephanie; Weckbach, Sebastian; Eisele, Philipp; Braun, Christian K.; Wiegner, Rebecca; Ohmann, Julia J.; Wrba, Lisa; Hoenes, Felix M.; Kellermann, Philipp; Radermacher, Peter; Wachter, Ulrich; Hafner, Sebastian; McCook, Oscar; Schultze, Anke; Palmer, Annette; Braumüller, Sonja; Gebhard, Florian§; Huber-Lang, Markus

Author Information
doi: 10.1097/SHK.0000000000000925



Besides modern surgical orthopedic damage control and evidence-based shock treatment algorithms, hemorrhagic shock (HS) frequently results in major organ complications and bad clinical outcome (1). HS has been proposed as a pathophysiological hub for the tissue damage-caused immune response (2), development of endotheliopathy and subsequent barrier failure, as well as for the serine protease system dysfunction (3–5). Furthermore, trauma-hemorrhage has been characterized as a driver for cellular apoptosis and a major contributor to mucosal immune dysfunction (6).

In our recently established hemodynamically stable polytrauma (PT) model in mice, highly standardized injuries of different body regions (closed traumatic head injury, blunt thorax trauma, soft tissue injury, and femur fracture) revealed supra-additive effects for particular injury combinations, especially when all injuries were combined, mimicking the clinical conditions of PT (7, 8). However, although challenged with major trauma impacts, the PT-induced organ damage in mice without HS scarcely reached the maximal inflammatory response or robust organ dysfunction found in severely injured PT patients with an injury severity score (ISS) of at least 25 early after trauma. Furthermore, acute traumatic coagulopathy has been reported in humans and rodents to significantly develop only in copresence of both tissue damage and HS (9). Thus, organ damage and inflammatory reactions after PT require closer discriminative characterization in regards to the presence or absence of an additional HS. Although murine trauma models that address more than one body region in combination with a subsequent well-defined (e.g., pressure-controlled) HS are rare (10, 11), a hemodynamically instable PT model seems essential to reliably simulate the clinical reality answering future pathophysiological and therapeutic questions while considering interspecies differences in the posttraumatic response (12). To assess “first-hit”-induced organ damage and to monitor the organ performance and the early pro- and anti-inflammatory response after trauma (13), multiple biomarkers have been proposed—in many cases with high sensitivity but low specificity values. Interleukin-6 (IL-6), for example, has been established as a rather reliable, although not organ-specific marker to evaluate initial overall organ injury (14). In the clinical daily routine, imaging procedures, such as whole-body computerized tomography scans, are currently the only established way to determine the organ-specific trauma load in a timely manner. In recent times, some novel markers have appeared on the horizon to evaluate hidden early organ damage and to reliably monitor and detect early secondary organ damage or development of inflammatory/infectious complications. Clara cell protein 16 (CC-16), neutrophil gelatinase-associated lipocalin (NGAL), liver-type fatty acid binding protein (L-FABP), or glial fibrillary acidic protein (GFAP) were proposed to estimate the extent of lung contusions (15), kidney damage (16), liver damage (17), and diffuse brain injury (18), respectively. However, so far it is rather unknown to what extent these proposed organ-specific markers are influenced by an additional HS in a complex PT setting.

Therefore, we hypothesized and present evidence that HS contributes within hours after PT to early (multiple) organ damage.


Animals and anesthesia

The study protocol for the murine PT model with HS was approved by the University Animal Care Committee and the Federal Authorities for animal research, Tuebingen, Germany (No. 1194). Furthermore, all experiments were performed in adherence to the National Institutes of Health Guidelines for the use of laboratory animals.

C57BL/6 mice aged 8 to 9 weeks (Jackson Laboratories, Bar Harbour, Maine) with a mean body weight of 25 g (±2.5 g) were randomly divided into the following groups with n = 8 animals/group: polytrauma (PT), hemorrhagic shock (HS), polytrauma + hemorrhagic shock (PT+HS), and untreated control animals (Ctrl). Mice were anaesthetized with 2.5% sevoflurane (Sevorane Abbott, Wiesbaden, Germany)/97.5% oxygen, which was continued during the trauma-hemorrhage procedure and during the whole observation period. For analgesia, 0.03 mg/kg buprenorphine was administrated by subcutaneous injection.

Induction of PT and HS

An overview about the experimental procedure is given in Figure 1A. PT was induced by the application of a blunt bilateral chest trauma (TXT), a traumatic brain injury (TBI), and a closed transverse femoral fracture (inclusive soft tissue injury) as previously described (8).

Fig. 1:
Murine polytrauma with and without hemorrhagic shock.

After the induction of PT, the shaved and cleaned left hind limb of the anaesthetized mouse was incised and a catheter (Föhr Medical Instruments, Seeheim/Ober-Beerbach, Germany) was inserted microsurgically into the femoral artery, allowing the monitoring of the blood pressure (blood pressure analyzer, DSI, St. Paul, Minn) and a controlled blood loss. Another incision was created along the ventral cervical skin, and a further catheter was inserted into the jugular vein enabling the resuscitation regime and the controlled infusion of catecholamines. For the induction of HS, mice were bled for 5 to 10 min to reach a mean blood pressure of 30 mmHg (±5 mmHg) which was kept stable for 60 min. After hemorrhage, mice were resuscitated via the jugular vein with the 4-fold volume of the drawn blood with balanced electrolyte solution (ionosterile) over 30 min. During the 2-h observation period after HS, animals were subjected to a preset protocol, adjusting anesthesia and norepinephrine support (0.01–0.12 μg/kg/min) (Sanofi, Frankfurt am Main, Germany) in a standardized manner to maintain a mean arterial blood pressure (MAP) of 50 mmHg. Four hours after PT, EDTA blood was drawn by cardiac puncture. Plasma was obtained by centrifugation (5 min at 500 × g and 4°C) and stored at −80°C until further analysis.

Preparation of BAL fluids

For the collection of the bronchoalveolar lavage (BAL), the trachea was dissected and cannulated, the left lung was clamped, and the right lung was flushed 3 times with 0.5 mL PBS containing a 1:1,000 broad spectrum protease inhibitor (Sigma-Aldrich, St. Louis, Mo). The BAL fluids were then centrifuged (10 min at 450 × g and 4°C) and the supernatant was stored at −80°C until analysis. The cell pellet was resuspended in 100 μL PBS. Thereof, 10 μL were mixed with crystal violet to determine the total cell number in a Neubauer chamber by light microscopy. The residual BAL cell suspension was subjected to cytospin preparation (Shandon Cytospin 3, Thermo Scientific, Dreieich, Germany). Cytospins were fixed and stained with Hemacolor rapid stainig kit (Merck, Darmstadt, Germany), and 300 cells for each experimental condition were differentially analyzed by light microscopy to determine the number of infiltrated neutrophils in the BAL.

Lung myeloperoxidase activity

For determination of lung myeloperoxidase (MPO) activity, lungs were homogenized (ULTRA-TURRAX, IKA, Staufen, Germany) in KH2PO4 buffer (Merck, Darmstadt, Germany) and incubated for 2 h at 60°C and centrifuged at 3,950 × g at room temperature for 15 min; 25 μL supernatant were mixed with 25 μL tetramethylbenzidine (Sigma) and 200 μL 0.002% H2O2 (Fluka, Deisenhofen, Germany). After an incubation time of 5 min at 37°C, the extinction was read at 450 nm (Tecan Sunrise Reader, Tecan, Crailsheim, Germany).

Protein assay

The total protein concentration in the plasma samples, BAL, and tissue homogenates of mice was quantified using a bicinchoninic acid Protein Assay Kit (Pierce, Rockford, Ill) according to the manufacturer's instructions.

ELISA analyses

The concentration of cytokines, danger-associated molecular patterns (DAMPs), and organ performance parameters in plasma and BAL were determined by sandwich enzyme-linked immunosorbent assay (ELISA). Due to dilution effects of the HS/resuscitation procedure, measured parameter concentrations were related to total protein concentration in the corresponding sample. The following ELISA kits were used according to the manufacturers’ recommendations: BD OptEIA Mouse IL-6 ELISA Set (BD Pharmingen, San Diego, Calif), high mobility group box 1 protein (HMGB1) ELISA Kit (IBL International, Hamburg, Germany), Mouse CC16 ELISA kit (Abbexa, Cambridge, UK), Mouse GFAP ELISA kit, Mouse Syndecan 1 ELISA kit, Mouse S100B ELISA kit, Mouse NGAL ELISA kit, and Mouse L-FABP ELISA kit (all Lifespan Biosciences Inc, Seattle, Wash).

Analysis of plasma creatinine

Fifty microliters of internal standard solution, consisting of 5 μg/mL of 2H3-creatinine (CDN isotopes, Pointe-Claire, Canada), were added to 10 μL of mouse plasma. After mixing, samples were equilibrated for 10 min at room temperature, deproteinized by addition of 500 μL acetonitrile and centrifuged at 13,000 × g. Twenty-five microliters of the supernatant were directly used for urea quantification. The residual sample was evaporated to dryness, reconstituted in 500 μL 0.01% formic acid, and extracted over an anion exchange column (Phenomenex, Aschaffenburg, Germany). After washing with water and methanol, creatinine was recovered with methanol/formic acid (9/1). For creatinine, the trimethylsilyl-derivative was prepared by adding 100 μL acetonitrile/N,O-Bis(trimethylsilyl)trifluoroacetamide (2/1) to the dry sample and heating to 80°C for 60 min.

Analyses were carried out on an Agilent 5890/5970 gas chromatography/mass spectrometry system, housing a MN Optima-5MS capillary column (Macherey-Nagel, Düren, Germany). Creatinine analysis was carried out by monitoring ions m/z 329 and 332 for analytes and internal standard. For quantification, six-point calibration curves were used (19).

Histological evaluation

Formalin-fixed lungs, livers, and kidneys from mice were dehydrated with ethanol and xylol and embedded in paraffin. Four micrometer sections were prepared followed by hematoxylin and eosin (H&E) staining. Slides were visualized using a Zeiss Axio Imager A1 microscope (Zeiss, Jena, Germany). Lung injury was assessed using a scoring system as described elsewhere (20) grading congestion of alveolar septae, intra-alveolar cell infiltrates, fibrin deposition, and alveolar hemorrhage. Ten fields per slide of four animals per group were evaluated.

Multiple organ edema assessed by the wet-dry organ weight ratio

In a separate set of experiments (n = 5 for all groups), the wet to dry weight (w/d) ratio of the lungs, kidneys, and liver was determined after measuring the freshly harvested organ and the corresponding residium after heating for 24 h in a gravity convection oven (at 60°C). The ratio was indicative of the water content reflecting the extent of organ edema.


Results are presented as mean ± SEM. For determination of significant differences between experimental means, datasets were analyzed by one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls test as a post hoc test. In case of nonparametric distribution, Kruskal–Wallis ANOVA on ranks was performed, followed by the Dunn Method. A P < 0.05 was considered as statistically significant.


Monitoring of the vital parameter after PT and HS

For the continuous monitoring of vital parameters, MAP, heart rate, and body temperature of traumatized mice were evaluated every 5 min from the end of instrumentation until sacrifice (Fig. 1A). The heart rate was reasonably stable at around 430 beats per minute and did not significantly differ between HS, PT, and PT+HS mice (data not shown). Due to the close temperature control with a feedback loop device, the core body temperature did also not show any significant differences between the animal groups (data not exhibited). The MAP was quite stable in PT mice and fluctuated at round 60 mmHg during the 3-h observation period (Fig. 1B). Strictly in accordance to the protocol, in HS and PT+HS mice, the controlled blood loss led to a MAP of 30 (±5) mmHg which was kept for 60 min. Of note, in HS animals a significantly larger volume of blood had to be drawn than in PT+HS animals (639 μL ± 39 vs. 470 μL ± 60, Supplemental Figure 1c, to reach the intended MAP. The resuscitation procedure restored the MAP to values of around 50 mmHg (Fig. 1B). However, PT+HS animals required earlier and significantly more catecholamines (norepinephrine) than mice with HS alone (Fig. 1C), illustrating some impact of an additional HS in polytraumatized mice.

PT/hemorrhage-induced systemic release of IL-6 and HMGB1

To find out whether the modelled trauma induced an early systemic inflammatory response, we determined the proinflammatory cytokine IL-6 and the DAMP HMGB1 in the plasma of mice 4 h after the induction of HS alone, PT alone, and PT plus HS. As depicted in Fig. 1D, HS alone already significantly induced the release of IL-6 which was further increased in PT animals. PT+HS resulted in the most pronounced release of IL-6. Likewise the nuclear DNA-binding protein HMGB1 revealed a similar increase after the HS, PT, and PT+HS (Fig. 1E); thereby, the HS seems to represent a major trigger for the release of HMGB1. These results indicate that HS significantly contributed to the systemic inflammatory reaction and the release of endogenous DAMPs already in the early phase after trauma.

PT-induced pulmonary damage is enhanced by an additional HS

For evaluation of the PT-induced tissue damage of specific organs, H&E-stained sections of lungs, livers, and kidneys were morphologically examined for classical signs of organ damage. Histology-based lung injury scoring of lung histological sections from trauma animals showed evidence of alveolar wall thickening, disruption of the alveolar architecture and intra-alveolar hemorrhage, which were significantly increased after PT+HS but not in the setting of HS or PT alone (Fig. 2, A and B). Furthermore, signs of lung edema were only developing in the case of PT+HS but not in HS or PT alone (Fig. 2C).

Fig. 2:
Lung injury after isolated and combined traumatic insults.

The lung epithelial injury marker CC16 was increased after the PT procedure alone (including a blunt chest trauma) and revealed a visible, significant increase by an additional HS (Fig. 3A). This was paralleled by markedly elevated HMGB1 levels in the BAL after PT and PT+HS (Fig. 3B). Furthermore, increased protein concentrations as indicators for pulmonary leakage were found in the BAL fluids of PT and PT+HS mice (Fig. 3C) even though the total protein concentration were expectedly to some extend compromised by the HS. Noteworthy, alveolar neutrophil infiltration as marker for acute lung injury was significantly increased after PT only in the case of an additional HS (Fig. 3D) with concomitantly higher MPO activity (data not shown).

Fig. 3:
Systemic and local evaluation of trauma-induced lung injury.

The liver offers resistance against very early remote organ damage after PT and HS

In present model, the liver represented a remote organ which was not directly damaged by the PT impact. In contrast to the lungs, the liver tissues did not show classical morphological changes after the different trauma insults even in presence of HS (Fig. 4A). Although the liver damage marker L-FABP did not show any significant differences among the groups during the first hours after trauma (Fig. 4B), some signs of edema were detectable in all trauma and HS groups (Fig. 4C). Overall, the liver seems not to be much affected as remote organ in present multiple injury model during the first hours after PT and HS.

Fig. 4:
Modest liver injury after trauma and/or hemorrhagic shock.

Functional changes of kidneys early after PT plus HS

Other remote organs are the kidneys, here without any directly inflicted trauma vector. Early after injury, several kidneys of the PT + HS animals displayed some reported signs of acute kidney injury, such as thinning of the epithelial layer, interstitial edema, tubular dilatation, an enhanced gap between the Bowman capsule and the capillary convolute, leukocyte infiltration, cytoplasmic vacuolization, and intraluminal shedding of cytoplasmic debris (Fig. 5A). However, the overall statistical analyses neither revealed any significant differences between the groups nor any kidney edema development (Fig. 5B).

Fig. 5:
Posttraumatic kidney damage.

In contrast to the absence of significant morphological changes early after the different trauma insults, there was a clear decline in renal function evidenced by enhanced plasma NGAL and creatinine concentrations as early as 4 h after trauma, especially in the PT + HS group (Fig. 5, C and D). Furthermore, staining of histological sections for the kidney injury molecule-1 (KIM-1) revealed a significantly enhanced staining pattern in all trauma and HS groups in comparison to the Ctrl group (Supplemental Figure 2,

Monitoring of the PT/hemorrhage-induced brain damage

To further assess the impact of the trauma/hemorrhage-induced organ injury, we determined reported organ damage-specific markers in the plasma of mice. For the detection of brain injury, we used the brain-specific astroprotein GFAP and the S100 calcium-binding protein B (S100B). S100B revealed the highest levels after HS even in the absence of TBI (Fig. 6A). In contrast, GFAP was significantly increased in mice that were subjected to TBI (PT and PT+HS), whereas no increase was found after HS alone (Fig. 6b), indicating GFAP as a more specific marker for brain injury.

Fig. 6:
Monitoring of trauma-induced brain injury and impairment of blood-organ barriers.

Enhancement of endothelial injury after experimental PT by HS

As maker for the integrity of the blood–organ barrier and the endothelium, we determined the appearance of the tight junction molecule claudin 5 and the glycocalyx component syndecan-1, respectively. Claudin 5 could be detected in mice after PT and PT+HS (Fig. 6C). In addition, shedding of syndecan-1 into the plasma was evidenced by a significant increase of syndecan-1 after PT+HS (Fig. 6D), suggesting an early trauma-hemorrhage-mediated endothelial barrier damage or dysfunction.


Multiple injuries expose the body to various pathogen- and danger-associated molecular patterns (PAMPs and DAMPs) resulting in an almost synchronic pro- and anti-inflammatory response (13). Although several murine PT models have been described in the literature (10), they are often not severe enough to fully mimic the situation of patients with an ISS at least 25, especially of those patients who suffer from an additional HS. Current data from the German trauma registry indicate that the most severely injured body regions (abbreviated injury scale [AIS] ≥ 3) in PT patients are allocated to the head (45.4%), thorax (45.2%), extremities (30.1%), and abdomen (12.0%) (TraumaRegister DGU of the German Trauma Society 2015). Based on these representative data, we have recently established corresponding highly standardized trauma impacts affecting the most relevant body regions (head, chest, extremities) with an AIS of approximately 3 to 4 (AIS femur = 3, AIS thorax = 3–4, AIS head = 3) integrated in a hemodynamically stable rodent model (8). This setting allowed us to investigate the impact of the individual tissue trauma to the overall inflammatory response in a severe PT model translating to an ISS of about 27 to 34. The traumatized animals showed a systemic early increase of key inflammatory mediators such as IL-6, MCP-1 and keratinocyte-derived chemokine after specific trauma combinations, especially after PT, along with increased pulmonary apoptotic events and neutrophil infiltration (8). However, in this setting the effect of an additional HS on the inflammatory response and early organ damage has not been investigated so far. Therefore, we combined our PT model with a standardized, pressure-controlled HS in order to establish a novel, clinically relevant hemodynamically instable PT+HS model. To exclude any iatrogenic-induced hypothermia which would significantly contribute to the lethal triad (hypothermia, acidosis, coagulopathy) or modify the inflammatory response after HS (21), we enforced a sufficiently tight feedback loop temperature control resulting in almost similar intergroup core temperature courses (data not shown). In HS mice, anesthesia had to be reduced after the HS and resuscitation phase based on a preset protocol to stabilize their hemodynamics. These animals additionally required catecholamines, which was higher in PT+HS than in the HS animals. This might be due to internal tissue bleeding after PT application and the associated reduction of the circulatory blood volume as well as the systemic inflammatory response, which might have resulted in increased capillary leakage. The impact of the HS on the release of DAMPs and the resulting inflammatory response was reflected by the plasma levels of HMGB1 and IL-6, respectively, which were already significantly increased after HS alone but showed by tendency an enhanced response in case of an underlying PT. In support, elevated plasma levels of HMGB1 have been reported in clinical and experimental HS studies and were associated with systemic inflammation and remote organ dysfunction (22, 23). Thus, one aim was to determine if and to what extent there was an organ-specific primary injury after HS, PT, and PT+HS and whether this could be adequately monitored. In PT patients, close monitoring of all organ systems in the intensive care unit (ICU) is well established and broadly included in the daily routine (e.g., ECG, continuous blood pressure monitoring, urine output, blood gas analysis, thromboelastometry), and some clinical scores were introduced to closely define organ function (e.g., sequential organ failure assessment score). However, only a few markers have been investigated and validated in defined clinical and experimental settings to specifically and reliably detect primary organ damage (24). To our knowledge, this is the first murine PT study in which the analysis of specific organ damage markers was used to detect specific trauma/hemorrhage-induced tissue damage. We found that S100B protein, a broadly proposed marker for brain injury (25), revealed significantly enhanced values already after HS alone, and failed to specifically discriminate the impact of TBI within the PT group. This finding was supported by a recent study demonstrating that severe hemorrhage in patients without TBI was associated with increased serum levels of S100B which was predictive for mortality (26). Thus, S100B seems to be a sensitive but rather unspecific biomarker for brain tissue injury. In contrast, GFAP appeared to be less confounded by HS conditions in present PT model and therefore may represent a better marker to assess primary brain damage after TBI. In accordance with our findings, in patients with TBI, serum GFAP correlated with Glasgow Coma Scale scoring and neuroradiological findings at hospital admission (18). Furthermore, persistent elevation on day 2 was predictive of increased mortality (27). Of note, any established organ-specific biomarker might be less reliable in settings of various trauma impacts. Thus, future studies should address the reliability in different standardized trauma combinations.

Trauma-induced primary lung injuries are often underestimated in patients even in areas of advanced CT scanning. Clinically, CC16 has been suggested as a potential damage marker for lung injury in PT patients based on a high correlation with the volume of contused lung parenchyma (15). The present study supports these findings since we detected significantly increased levels of CC16 after trauma/hemorrhage, which was associated with increased inflammatory mediators in the BAL fluids, neutrophil infiltration and lung edema as signs for substantial lung inflammation. Furthermore, studies have shown that CC16 serves to evaluate the integrity of the air–blood barrier (28), which might also be affected in present PT model as indicated by increased protein leakage into the lung. Although HS alone did not result in an increase in CC16 plasma levels, in the setting of PT the early lung damage was only significantly increased compared with control after an additional HS, indicating that HS might represent an amplifier of early pulmonary damage after PT. In contrast, the liver seems to be less affected in our model since no increase of the liver damage marker L–FABP (17) and no classical histological signs of a remote trauma/hemorrhage-induced liver damage were detectable.

Although in the present model neither the kidneys nor the liver were directly hit by the inflicted trauma vectors, markers for kidney damage (NGAL) (16), and renal dysfunction (creatinine) were increased after HS alone but markedly enhanced in the PT group with HS. HS might contribute to early remote kidney damage and dysfunction after PT presumably via prerenal shock pathomechanisms. This is in line with human studies, indicating that HS-mediated hypoperfusion and hypoxia contribute to tubular injury and development of acute kidney injury (AKI) (29). In mice and rats, renal ischemia-reperfusion injury induced a marked up-regulation of NGAL mRNA expression in ischemic injured kidneys within 3 h after injury (30), which can be related to increased NGAL levels in urine and blood (31). Of note, the present absence of consistent morphological signs of AKI might be well attributed to the early timing of the sampling after trauma.

In addition, the function of the endothelium as an “organ” seems to play a significant role after severe tissue trauma and degradation of the apical glycocalyx is involved in increased vascular permeability (32). In the present study, PT and HS alone resulted in an early slight shedding of the endothelial glycocalyx component syndecan-1 into the plasma, which was only significantly increased after PT+HS. These data together with the by tendency increased plasmatic levels of the tight junction protein claudin 5 are indicative of a trauma-hemorrhage-induced dysfunction of the endothelial integrity with likely subsequent barrier dysfunction. An early initiation of such a leakage syndrome with subsequent shift of intravasal volume into the interstitial space could be recently indicated in the hemodynamically stable murine PT model by the appearance of the tight junction molecule JAM-1 in the circulation (7). In addition, shedding of syndecan-1 into the circulation with its heparin-like moieties might further considerably contribute to the “autoheparinization” process with subsequent coagulopathy observed in patients after severe tissue trauma (33).

Although the present model led to a significant systemic inflammation and early organ damage and dysfunction, the present study has some limitations. Noninjured healthy mice were used as controls in accordance to healthy volunteers in most human studies (15, 16, 18). However, further experiments are required to determine the mid-term impact of continuous anesthesia and/or surgical instrumentation on the posttraumatic inflammatory reaction and tissue damage. The impact of an additional HS on the response after PT was often evident only by tendency. This might be due to the limited animal number in regard to the complexity of the model. Furthermore, the biomarker profiling was restricted to one time point since no multiple blood drawings were performed to prevent any confounders by further blood losses. The timing of the sample collection was chosen based on our previous studies revealing an increasing systemic inflammatory response within the first 6 h after PT (8), which is comparable to the early cytokine and chemokine storm after severe human trauma. Moreover, long-term outcome could not be assessed due to the severity of PT and HS which required ongoing anesthesia and mandatory intensive care support. In this regard, further attention should be paid on the anesthesia and the fluid resuscitation protocol as well as the breathing conditions in all PT models. In the present study, the animals were subjected to a preset protocol adjusting infused volume, anesthesia and catecholamine support in a standardized manner to maintain hemodynamics, but the animals were neither intubated nor ventilated. Here, future studies with lung protective ventilation protocols for PT and HS might further improve clinical translation potential and gain reliable data on respiratory function. Overall, the data have to be interpreted cautiously since the present results presumably depend on the respective severity of HS and tissue trauma; it is likely that the observed effects of an additional HS might have been even more pronounced in a less severe PT setting.

Taken together, the HS-induced aggravation of the early systemic inflammatory reaction and the solid organ damage as reflected by the evident changes of specific marker profiles may significantly contribute to the early onset of multiple organ failure as a rather common event in PT patients. Furthermore, the present model may rather realistically simulate the patient's situation during severe hemodynamically instable PT to investigate fundamental pathophysiological changes (such as development of edema) and innovative treatment strategies such as a recently published histone deacetylase inhibitor (34) or toll-like receptor inhibitors (35) before passing over to a large animal PT model and to clinical reality. The current findings also support the idea of HS as an important driver of specific early organ damage and the associated molecular danger response after trauma, and furthermore indicate markers to detect and monitor specific organ injury early after PT and HS.


1. Wutzler S, Maegele M, Wafaisade A, Wyen H, Marzi I, Lefering R. Risk stratification in trauma and haemorrhagic shock: scoring systems derived from the TraumaRegister DGU((R)). Injury 2014; 45 (Suppl. 3):S29–S34.
2. Lomas JL, Chung CS, Grutkoski PS, LeBlanc BW, Lavigne L, Reichner J, Gregory SH, Doughty LA, Cioffi WG, Ayala A. Differential effects of macrophage inflammatory chemokine-2 and keratinocyte-derived chemokine on hemorrhage-induced neutrophil priming for lung inflammation: assessment by adoptive cells transfer in mice. Shock 2003; 19 4:358–365.
3. Burk AM, Martin M, Flierl MA, Rittirsch D, Helm M, Lampl L, Bruckner U, Stahl GL, Blom AM, Perl M, et al. Early complementopathy after multiple injuries in humans. Shock 2012; 37 4:348–354.
4. Thuijls G, de Haan JJ, Derikx JP, Daissormont I, Hadfoune M, Heineman E, Buurman WA. Intestinal cytoskeleton degradation precedes tight junction loss following hemorrhagic shock. Shock 2009; 31 2:164–169.
5. Jenkins DH, Rappold JF, Badloe JF, Berseus O, Blackbourne L, Brohi KH, Butler FK, Cap AP, Cohen MJ, Davenport R, et al. Trauma hemostasis and oxygenation research position paper on remote damage control resuscitation: definitions, current practice, and knowledge gaps. Shock 2014; 41 (Suppl. 1):3–12.
6. Xu YX, Ayala A, Monfils B, Cioffi WG, Chaudry IH. Mechanism of intestinal mucosal immune dysfunction following trauma-hemorrhage: increased apoptosis associated with elevated Fas expression in Peyer's patches. J Surg Res 1997; 70 1:55–60.
7. Denk S, Wiegner R, Hones FM, Messerer DA, Radermacher P, Weiss M, Kalbitz M, Ehrnthaller C, Braumuller S, McCook O, et al. Early detection of junctional adhesion molecule-1 (JAM-1) in the circulation after experimental and clinical polytrauma. Mediators Inflamm 2015; 2015:463950.
8. Weckbach S, Hohmann C, Braumueller S, Denk S, Klohs B, Stahel PF, Gebhard F, Huber-Lang MS, Perl M. Inflammatory and apoptotic alterations in serum and injured tissue after experimental polytrauma in mice: distinct early response compared with single trauma or “double-hit” injury. J Trauma Acute Care Surg 2013; 74 2:489–498.
9. Frith D, Goslings JC, Gaarder C, Maegele M, Cohen MJ, Allard S, Johansson PI, Stanworth S, Thiemermann C, Brohi K. Definition and drivers of acute traumatic coagulopathy: clinical and experimental investigations. J Thromb Haemost 2010; 8 9:1919–1925.
10. Frink M, Andruszkow H, Zeckey C, Krettek C, Hildebrand F. Experimental trauma models: an update. J Biomed Biotechnol 2011; 2011:797383.
11. Lomas-Niera JL, Perl M, Chung CS, Ayala A. Shock and hemorrhage: an overview of animal models. Shock 2005; 24 (Suppl.):133–139.
12. Osterburg AR, Hexley P, Supp DM, Robinson CT, Noel G, Ogle C, Boyce ST, Aronow BJ, Babcock GF. Concerns over interspecies transcriptional comparisons in mice and humans after trauma. Proc Natl Acad Sci USA 2013; 110 36:E3370.
13. Xiao W, Mindrinos MN, Seok J, Cuschieri J, Cuenca AG, Gao H, Hayden DL, Hennessy L, Moore EE, Minei JP, et al. A genomic storm in critically injured humans. J Exp Med 2011; 208 13:2581–2590.
14. Frink M, van GM, Kobbe P, Brin T, Zeckey C, Vaske B, Krettek C, Hildebrand F. IL-6 predicts organ dysfunction and mortality in patients with multiple injuries. Scand J Trauma Resusc Emerg Med 2009; 1749.
15. Wutzler S, Lehnert T, Laurer H, Lehnert M, Becker M, Henrich D, Vogl T, Marzi I. Circulating levels of Clara cell protein 16 but not surfactant protein D identify and quantify lung damage in patients with multiple injuries. J Trauma 2011; 71 2:E31–E36.
16. Bolignano D, Donato V, Coppolino G, Campo S, Buemi A, Lacquaniti A, Buemi M. Neutrophil gelatinase-associated lipocalin (NGAL) as a marker of kidney damage. Am J Kidney Dis 2008; 52 3:595–605.
17. Relja B, Szermutzky M, Henrich D, Maier M, de Haan JJ, Lubbers T, Buurman WA, Marzi I. Intestinal-FABP and liver-FABP: novel markers for severe abdominal injury. Acad Emerg Med 2010; 17 7:729–735.
18. Lei J, Gao G, Feng J, Jin Y, Wang C, Mao Q, Jiang J. Glial fibrillary acidic protein as a biomarker in severe traumatic brain injury patients: a prospective cohort study. Crit Care 2015; 19362.
19. Smith-Palmer T. Separation methods applicable to urinary creatine and creatinine. J Chromatogr B Analyt Technol Biomed Life Sci 2002; 781 (1–2):93–106.
20. Matute-Bello G, Winn RK, Jonas M, Chi EY, Martin TR, Liles WC. Fas (CD95) induces alveolar epithelial cell apoptosis in vivo: implications for acute pulmonary inflammation. Am J Pathol 2001; 158 1:153–161.
21. Kutcher ME, Howard BM, Sperry JL, Hubbard AE, Decker AL, Cuschieri J, Minei JP, Moore EE, Brownstein BH, Maier RV, et al. Evolving beyond the vicious triad: differential mediation of traumatic coagulopathy by injury, shock, and resuscitation. J Trauma Acute Care Surg 2015; 78 3:516–523.
22. Yang R, Harada T, Mollen KP, Prince JM, Levy RM, Englert JA, Gallowitsch-Puerta M, Yang L, Yang H, Tracey KJ, et al. Anti-HMGB1 neutralizing antibody ameliorates gut barrier dysfunction and improves survival after hemorrhagic shock. Mol Med 2006; 12 (4–6):105–114.
23. Ombrellino M, Wang H, Ajemian MS, Talhouk A, Scher LA, Friedman SG, Tracey KJ. Increased serum concentrations of high-mobility-group protein 1 in haemorrhagic shock. Lancet 1999; 354 9188:1446–1447.
24. Furmaga W, Cohn S, Prihoda TJ, Muir MT, Mikhailov V, McCarthy J, Arar Y. Novel markers predict death and organ failure following hemorrhagic shock. Clin Chim Acta 2015; 44087–44092.
25. Raabe A, Kopetsch O, Woszczyk A, Lang J, Gerlach R, Zimmermann M, Seifert V. Serum S-100B protein as a molecular marker in severe traumatic brain injury. Restor Neurol Neurosci 2003; 21 (3–4):159–169.
26. Stamataki E, Stathopoulos A, Garini E, Kokkoris S, Glynos C, Psachoulia C, Pantziou H, Nanas S, Routsi C. Serum S100B protein is increased and correlates with interleukin 6, hypoperfusion indices, and outcome in patients admitted for surgical control of hemorrhage. Shock 2013; 40 4:274–280.
27. Nylen K, Ost M, Csajbok LZ, Nilsson I, Blennow K, Nellgard B, Rosengren L. Increased serum-GFAP in patients with severe traumatic brain injury is related to outcome. J Neurol Sci 2006; 240 (1–2):85–91.
28. Hermans C, Knoops B, Wiedig M, Arsalane K, Toubeau G, Falmagne P, Bernard A. Clara cell protein as a marker of Clara cell damage and bronchoalveolar blood barrier permeability. Eur Respir J 1999; 13 5:1014–1021.
29. Yu L, Seguro AC, Rocha AS. Acute renal failure following hemorrhagic shock: protective and aggravating factors. Ren Fail 1992; 14 1:49–55.
30. Mishra J, Ma Q, Prada A, Mitsnefes M, Zahedi K, Yang J, Barasch J, Devarajan P. Identification of neutrophil gelatinase-associated lipocalin as a novel early urinary biomarker for ischemic renal injury. J Am Soc Nephrol 2003; 14 10:2534–2543.
31. Han M, Li Y, Liu M, Li Y, Cong B. Renal neutrophil gelatinase associated lipocalin expression in lipopolysaccharide-induced acute kidney injury in the rat. BMC Nephrol 2012; 13:25.
32. Rahbar E, Cardenas JC, Baimukanova G, Usadi B, Bruhn R, Pati S, Ostrowski SR, Johansson PI, Holcomb JB, Wade CE. Endothelial glycocalyx shedding and vascular permeability in severely injured trauma patients. J Transl Med 2015; 13:117.
33. Ostrowski SR, Johansson PI. Endothelial glycocalyx degradation induces endogenous heparinization in patients with severe injury and early traumatic coagulopathy. J Trauma Acute Care Surg 2012; 73 1:60–66.
34. Liu Z, Li Y, Chong W, Deperalta DK, Duan X, Liu B, Halaweish I, Zhou P, Alam HB. Creating a prosurvival phenotype through a histone deacetylase inhibitor in a lethal two-hit model. Shock 2014; 41 2:104–108.
35. Korff S, Loughran P, Cai C, Lee YS, Scott M, Billiar TR. Eritoran attenuates tissue damage and inflammation in hemorrhagic shock/trauma. J Surg Res 2013; 184 2:e17–e25.

Experimental model; hemorrhagic shock; inflammatory response; organ damage; polytrauma

Supplemental Digital Content

Copyright © 2017 by the Shock Society