Different therapy strategies are currently used to restore cardiocirculatory homeostasis and subsequent sufficient cerebral oxygen supply during hemorrhagic shock. In past decades, patients with uncontrolled hemorrhagic shock were predominantly resuscitated with crystalloid and colloid solutions. While fluid management is established in controlled hemorrhagic shock, its use in uncontrolled hemorrhagic shock is under debate (1). Alternatively, small volume resuscitation with a hypertonic-hyperoncotic hydroxyethyl starch solution (HHS) may be beneficial (2), and delayed aggressive fluid resuscitation has been found to result in a survival benefit in penetrating truncal trauma (3). Arginine vasopressin (AVP) has been shown to decrease bleeding by shifting blood away from the splanchnic circulation towards the heart and the brain, resulting in an improved vital organ blood flow (4); therefore, AVP may be an effective vasopressor in the late phase of hemorrhagic shock (5).
As hemorrhagic shock can result in global cerebral ischemia (6), markers of cerebral metabolism and cell damage may be useful in evaluating therapy strategies. Intracerebral microdialysis is a sensitive, established technique for analyzing tissue biochemistry (7). However, the value of this technique in monitoring the effects of therapy on cerebral metabolism during hemorrhagic shock is unknown. Extracellular glucose, lactate, and the lactate/pyruvate ratio are reliable markers of tissue acidosis and cell ischemia, while elevated glycerol levels may indicate cell membrane damage (8).
Thus, the purpose of this study was to compare the effects of fluid therapy and the effects of AVP combined with HHS on cerebral perfusion pressure (CPP) and on markers of cerebral metabolism and cell damage in a porcine model of hemorrhagic shock. The hypothesis was that AVP/HHS would be superior to fluid resuscitation with respect to 1) restoring CPP, 2) maintaining cerebral metabolism, and 3) reducing the extent of secondary cerebral cell damage.
This project was approved by the Animal Investigation Committee of the University of Kiel, Schleswig-Holstein, and the animals were managed in accordance with the American Physiologic Society and institutional guidelines. This study was performed according to the Utstein-style guidelines (9) on 16 healthy pigs (German domestic pigs) of either gender ranging in age from 12 to 16 weeks and weighing 42–46 kg. Anesthesia was used in all surgical interventions, all unnecessary suffering was avoided, and research was terminated if unnecessary pain or fear resulted. The animals were fasted overnight, but had free access to water.
The pigs were premedicated with azaperone (neuroleptic drug; 8 mg kg−1 IM) and atropine (0.05 mg kg−1 IM) 1 h before surgery, and anesthesia was induced with a bolus dose of ketamine (2 mg kg−1 IM), propofol (1–2 mg kg−1 IV), and sufentanil (0.3 μg kg−1 IV). After endotracheal intubation during spontaneous respiration, the animals’ lungs were ventilated with a volume-controlled ventilator (Siemens SV 900C, Germany) with 30% oxygen at 20 breaths/min, and with a tidal volume adjusted to maintain normocapnia (Paco2 from 35 to 40 torr [4.7–5.3 kPa]). Anesthesia was maintained with a continuous infusion of propofol (6–8 mg kg−1 hr−1) and sufentanil (0.3 μg kg−1 hr−1) and muscle relaxation was provided by continuous infusion of pancuronium (0.2 mg kg−1 hr−1). Ringer’s solution (10 mL kg−1 hr−1) was administered continuously throughout the preparation phase to replace fluid loss during instrumentation. A standard lead II electrocardiogram was used to monitor cardiac rhythm; depth of anesthesia was judged according to blood pressure, heart rate (HR), and electroencephalography (Neurotrac, Engström, Munich, Germany). Four electroencephalographic electrodes were placed subcutaneously over the fronto-occipital regions bilaterally. If cardiovascular variables or electroencephalography indicated a reduced depth of anesthesia, additional propofol and sufentanil were given.
Intracranial pressure (ICP) was measured subdurally (Ventrix, Integra NeuroSciences, Plainsboro, NJ). For sampling of cerebral venous (cv) blood, a burr hole was drilled into the skull over the midline, and a 4F catheter was placed into the sagittal sinus. One 7F catheter was advanced transcutaneously via the femoral artery for measurement of mean aortic blood pressure (MAP) and withdrawal of arterial blood samples. Additionally, an 8.5F catheter was inserted transcutaneously into the superior caval vein in order to measure core temperature and to administer drugs; a 7.5F multilumen, flow-directed, fiberoptic pulmonary artery catheter (Edwards Swan Ganz Combo EDV Thermodilution Catheter, Baxter Laboratories, Irvine, CA) was placed in the pulmonary artery to measure cardiac output (CO). All catheters were flushed with isotonic saline containing 5 U mL−1 heparin at a rate of 3 mL hr−1 to prevent obstruction during the preparation phase. Body temperature was maintained between 38.0 and 39.0°C with a heating blanket. Each animal received an IV bolus of heparin (100 U kg−1) to prevent intracardiac clot formation. Ventilation was monitored using an inspired/expired gas analyzer that measured oxygen and end-tidal carbon dioxide (petCO2) (M-PRESTN; Datex-Ohmeda, Helsinki, Finland). Another skull burr hole (left hemisphere, 10 mm paramedian and 10 mm rostral of the coronary suture) was prepared to insert a microdialysis catheter into the cerebral cortex 10 mm below the dura mater (CMA 71; CMA/Microdialysis, Stockholm, Sweden). The catheter was perfused with isotonic Ringer’s solution at a flow rate of 1 μL min−1. The microdialysis samples were collected at intervals of 30 min to obtain a concentrated dialysate with a recovery rate of 70%. The concentrations of brain tissue glucose (Glu), lactate (La), pyruvate (Py), and glycerol (Gly) were analyzed using conventional enzymatic methods immediately after collection (CMA 600, CMA/Microdialysis, Stockholm, Sweden). The La/Py ratio was calculated. After instrumentation for cerebral and hemodynamic variables and a 60-min equilibration phase, a midline laparotomy was performed; propofol infusion was adjusted to 4 mg kg−1 hr−1, and infusion of Ringer’s solution was stopped before induction of shock. All variables were stable for a five-minute period before the experiment was started. Baseline (BL) cerebral (ICP, CPP, calculated by MAP minus ICP), and hemodynamic (MAP, HR, CO) variables were assessed. Arterial (a) and cv blood gases were obtained to analyze hemoglobin content (Hb), pH, partial pressure of oxygen (Po2), partial pressure of carbon dioxide (Pco2), base excess (BE), oxygen saturation (SO2), and La (ABL System 615; Radiometer Medical, Copenhagen, Denmark). According to the following formulas, different variables were calculated:
- Oxygen content (cO2) = Hb · 1.34 · SO2 + (0.003 · Po2)
- Systemic oxygen delivery (DO2) = CO · caO2
- Arterial–cerebral venous difference of oxygen content (cavDO2) = caO2 − ccvO2
- Cerebral oxygen extraction ratio (COER) = cavDO2/caO2
- Cerebral venous-arterial difference of partial pressure of carbon dioxide (p(cv-a)CO2) = pcvCO2 − Paco2
- Arterial–cerebral venous difference of pH [pH(a−cv)] = pHa − pHcv
The experiment was started with an incision (width, 12 cm; depth, 3 cm) across the right liver lobe, and subsequent finger fraction of the liver was performed. During the following first or “trauma” phase, blood was continuously removed from the abdominal cavity. When mean arterial blood pressure was <25 mm Hg, or HR declined progressively for more than 20% of its peak value, pharmacological support was provided to simulate a prehospital or “transport” phase. Arterial and cv blood gases were taken before pharmacological intervention.
At this time, 16 pigs were randomly assigned to receive either a combination of crystalloid (Ringer’s solution, 40 mL kg−1) and colloid (hydroxyethyl starch 130/0.4, 20 mL kg−1) solutions over 30 min (fluid group, n = 8), or AVP (AVP; Pitressin®, Parke-Davis, Karlsruhe, Germany; bolus of 10 U, and continuously 2 U kg−1 hr−1) combined with a bolus infusion of HHS (HyperHAES®, Fresenius, Bad Homburg, Germany; 4 mL kg−1 over 2 min; AVP/HHS group, n = 8), respectively. Fraction of inspired oxygen (FiO2) was increased to 1.0. During this second phase of uncontrolled hemorrhage, further blood loss was continuously monitored. Arterial and cv blood gases were taken after 5 and 30 min of therapy. Thirty minutes after drug administration, bleeding was controlled by manual compression of the liver, simulating emergency laparotomy. Continuous administration of crystalloid (Ringer’s solution, 10 mL kg−1 hr−1) and colloid (hydroxyethyl starch 130/0.4; 10 mL kg−1 hr−1) solutions was started. FiO2 was decreased to 0.5. All surviving animals were observed for one hour. Blood gas measurements were taken after 90 min of therapy.
After the experimental protocol was finished, the animals were killed with an overdose of propofol, sufentanil, and potassium chloride. All pigs were then subjected to necropsy to check for correct positioning of the catheters and for damage to internal organs.
A Kolmogorov–Smirnov test was used to test for Gaussian distribution. Two-way repeated measures analysis of variance (ANOVA) with post hoc Bonferroni correction was used to determine statistical significance of cerebral and hemodynamic variables, and blood gases between groups. One-way ANOVA was used to determine statistical significance within a group; values are expressed as mean ± sem. Correlation between Gly and La/Py ratio values was analyzed with Spearman’s rank correlation. Fisher’s exact test was used to compare survival rates between groups. Statistical significance was considered at P < 0.05.
Hemodynamic data at the experimental stages are presented in Table 1. Criteria for hemodynamic decompensation were reached after 38 ± 3 min in the fluid group and 38 ± 2 min in the AVP/HHS group, and total blood loss was 37 ± 4 and 39 ± 4 mL kg−1, respectively. After hemodynamic decompensation, AVP/HHS resulted in a faster and larger increase of MAP and CPP compared to fluid administration (P < 0.01; Table 1, Fig. 1). Thirty minutes after therapy, MAP and CPP did not differ between groups, but HR was increased in the AVP/HHS group (P < 0.01 vs fluid), and CO and DO2 were lower compared to fluid therapy (P < 0.01). Total blood loss further increased in the fluid group during therapy (56 ± 6 mL kg−1; P < 0.05), but did not change in the AVP/HHS group (40 ± 6 mL kg−1). All animals (8/8) of the AVP/HHS group survived, compared to 62.5% (5/8) of the fluid group (P = 0.20). Cerebral microdialysis revealed no significant differences in extracellular La, Py, Gly, Glu and the La/Py ratio between groups at any time. La, Gly, and the La/Py ratio increased during trauma in both groups compared with baseline (P < .001 for La, P < 0.05 for Gly, P < 0.05 for the La/Py ratio). At the end of the therapy phase, La, Gly, and the La/Py ratio decreased, but they remained elevated compared with BL (Table 2). Glycerol and the La/Py ratio were significantly (P < 0.001) correlated in both the fluid (r = 0.72) and AVP/HHS (r = 0.61) groups.
Changes in a and cv blood gases are shown in Table 3. Arterial and cv Hb were significantly lower in the fluid group compared with the AVP/HHS group (P < 0.01). In correlation with the increase of FiO2, Pao2 increased during the therapy phase (P < 0.001) and was comparable between groups. No significant changes of pcvO2 could be observed in the fluid group, but pcvO2 was significantly increased in the AVP/HHS group versus baseline (P < 0.01 for Th + 30 min, P < 0.001 for Th + 5 min; P < 0.01 vs fluid; Fig. 2).
In both groups, cavDO2, COER, p(cv-a)CO2, and pH(a−cv) (arterial–cerebral venous difference of pH) significantly increased with bleeding versus baseline (range P < 0.05 to P < 0.01). Compared with animals subjected to fluid, cavDO2, COER, and p(cv-a)CO2 decreased significantly after 5 min of AVP/HHS therapy (range P < 0.01 to P < 0.001). In both groups, COER reached BL values after 30 min of therapy, while cavDO2 remained decreased (P < 0.01 vs. BL; Table 3, Fig. 3). Lacv levels were not different from Laa in either group. Necropsy revealed correct position of all catheters, and no additional damage to internal organs.
This study was designed to evaluate the effects of fluid versus AVP/HHS administration on CPP and cerebral oxygenation, as well as on cerebral metabolism and cell damage, assessed with intracerebral microdialysis, in an animal model of hemorrhagic shock with near-fatal hypotension.
A comparison of hemodynamic variables revealed initially superior effects on MAP and CPP after AVP/HHS compared with fluid administration (Fig. 1). Within the therapy phase, a larger cumulating blood loss was observed in the fluid group compared with the AVP/HHS group. This was thought to be caused by effects of increased portal vein blood flow, high hydrostatic pressure on the wound, and dilution of platelets and coagulation factors, although this was not proven by laboratory data in our study. Furthermore, in contrast to placebo, fluid, or epinephrine treatment, vasopressin does not intensify hepatic hemorrhage due to a massive vasoconstriction of the truncal and mesenteric arteries (10). Based on its pharmacology, vasopressin may decrease bleeding by shifting blood away from the liver injury to the heart and brain, resulting in improved vital organ blood flow (4). Hemodilution resulted in a Hb level as low as 3.2 g dL−1 after 30 min of fluid therapy, which was significantly lower compared with that of the AVP/HHS group. However, the systemic DO2 in the fluid group was not compromised, and moreover, in contrast to AVP/HHS, systemic DO2 increased significantly over time with increasing CO. Increased systemic DO2 in the fluid group may not reflect sufficient cerebral DO2. At least in a model of traumatic brain injury, hemorrhagic hypotension further reduced cerebral oxygen delivery despite normalization of systemic oxygen delivery (11,12).
After the trauma phase, we found a comparable increase of cavDO2, COER, p(cv-a)CO2, and pH(a−cv) in both groups, indicating uncompensated cerebral hypoperfusion (13–15). Both therapy strategies were associated with a significant comparable increase of systemic and cv La, as well as the development of acidemia based on decreased pH and BE. Accumulation of La has been traditionally interpreted as a result of tissue hypoperfusion, hypoxia, anaerobic metabolism, or inadequate hepatic clearance (16). Although mean La levels at baseline were increased compared with physiological values of conscious pigs (17), the groups were comparable. We speculate that this might be explained by the observed large variability of values in our study, and the small number of animals in each group.
Both pcvO2 and cavDO2 may serve as indirect variables of global cerebral oxygen supply and demand. Thus, compared with fluid therapy, the elevated pcvO2 (Fig. 2) and decreased cavDO2 after 5 min of AVP/HHS administration (Table 3) may be interpreted as enhanced cerebral oxygenation, resulting in ongoing aerobic metabolism sufficient for the integrity of neuronal structures. Additionally, the COER reflecting the balance between cerebral oxygen delivery and cerebral oxygen consumption was found to be significantly lower after 5 min of AVP/HHS therapy (Fig. 3). AVP may improve cerebral oxygen delivery, and hyperemia may be the result due to enhanced cerebral blood flow followed by reduced oxygen extraction (13,18). In this context, AVP/HHS ensured short-term survival, while three of eight animals of the fluid group died. However, this study was not designed to show a survival difference.
In contrast to enhanced global cerebral oxygen supply after AVP/HHS administration, cerebral biochemical alterations assessed with intracerebral microdialysis indicated a mismatch between cerebral energy metabolism and supply of oxygen and Glu in surviving animals of either group. The extracellular cerebral La/Py ratio and Gly are most sensitive and specific in indicating cerebral ischemia (19). Additionally, a different range of information regarding substrate availability (Glu), redox state of the tissue (La/Py ratio), and cell membrane damage (Gly) can be obtained from intracerebral microdialysis. The observed impairment of cerebral aerobic metabolism in our study was significantly correlated with an increase in cerebral Gly level, reflecting degradation of neuronal cellular membranes in either group (8,20).
There are several possible interpretations for this discrepancy. AVP can cause vasodilatation in large cerebral arteries (21) and vasoconstriction in small cerebral blood vessels (22,23), which may cause relative shunting of blood flow within the cerebral cortex, and may result in redistribution of blood flow from the superficial cortex, a region of the brain known to be selectively vulnerable to ischemia and reperfusion. This phenomenon has already been observed with epinephrine (24). Additionally, the observed cell damage was most likely caused by cerebral hypoperfusion during the trauma phase, rather than various postshock treatment strategies. Our findings, therefore, emphasize the importance of early resuscitation, as cerebral cell damage begins with the onset of hemodynamic shock.
Some limitations to this study should be noted. First, long-term survival was not investigated because of the invasive cerebral monitoring; accordingly, the functional neurological outcome after restoration of spontaneous circulation was not evaluated. Second, different vasopressin receptors in pigs (lysine vasopressin) and humans (AVP) may result in a different hemodynamic response to exogenously administered AVP. However, the circulatory effects of AVP, as administered in the present investigation, may be even greater in humans than in pigs. Since we were unable to measure cerebral blood flow using radioactive microspheres, because of limitations posed by government regulations, we cannot comment on effects of drugs given throughout the study on cerebral blood flow. Furthermore, we do not know the effects of anesthesia, which may be neuroprotective. Cerebral oxygenation variables, such as pcvO2 and cavDO2, are dependent on FiO2, which was changed twice in this study. However, the study protocol was designed to reflect a realistic model of out-of-hospital hemorrhagic shock trauma management. Blinding the investigators to the drug administered was not possible because of the marked skin pallor after vasopressin administration and the methodological difference in volumes of administered fluids. Measurement of cerebral metabolism with a catheter placed in the cerebral cortex is a local method and may, therefore, underestimate global mismatch of cerebral oxygen demand and supply due to heterogeneity of the brain. Finally, the dural sinuses of the pig drain blood mostly from cerebral veins, and only partially from extracranial tissue (25). Therefore, it cannot completely be excluded that blood obtained from the sagittal catheter contained some blood of non-cerebral origin, as the cerebral cortex draining into the sinus was not isolated by removing much of the outer table of the skull in the vicinity of the catheter, as recommended by Michenfelder et al (26).
In conclusion, AVP/HHS showed initially superior effects on CPP and cerebral oxygenation in this model of hemorrhagic shock, but animals receiving AVP/HHS were comparable to surviving animals of the fluid group with respect to restoration of cerebral energy metabolism and the magnitude of secondary cell damage due to cerebral ischemia detected by intracerebral microdialysis. Our findings emphasize the importance of early resuscitation, and demonstrate that changes in cerebral energy metabolism are reversible, at least in part, after restoration of vital organ blood flow. Further studies should focus on the sensitivity and specificity of both La/Py ratio and Gly in predicting neurological outcome after hemorrhagic shock. Various approaches should assess the role of intracerebral microdialysis in the evaluation of neuroprotective therapeutic strategies.
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