CAN POLYETHYLENE GLYCOL-20K REPLACE ALBUMIN FOR PREHOSPITAL TREATMENT OF HEMORRHAGIC SHOCK WHEN FULL RESUSCITATION IS UNAVAILABLE?

ABSTRACT: A solution of high concentration albumin has been used for temporal volume expansion when timely resuscitation was unavailable after hemorrhagic shock. However, during prolonged hemorrhagic shock, cell edema and interstitial dehydration can occur and impede the volume expansion effect of albumin. Polyethylene glycol-20K (PEG) can establish an osmotic gradient from swollen cells to capillary lumens and thus facilitate capillary fluid shift and volume expansion. We hypothesized that with similar osmolality, 7.5% PEG elicits more rapid and profound compensatory responses after hemorrhagic shock than 25% albumin. Rats were randomized into three groups (n = 8/group) based on treatment: saline (vehicle), PEG (7.5%), and albumin (25%). Trauma was induced in anesthetized rats with muscle injury and fibula fracture, followed by pressure-controlled hemorrhagic shock (MAP = 55 mm Hg) for 45 min. Animals then received an intravenous injection (0.3 mL/kg) of saline, PEG, or albumin. MAP, heart rate, blood gases, hematocrit, skeletal muscle capillary flow, renal blood flow, glomerular filtration rate, urinary flow, urinary sodium concentration, and mortality were monitored for another 2 hours. Polyethylene glycol-20K and albumin both improved MAP, renal and capillary blood flow, and renal oxygen delivery, and decreased hyperkalemia, hyperlactatemia, hematocrit, and mortality (saline: 100% PEG: 12.5%; albumin: 38%) over saline treatment. Compared with albumin, PEG had a more rapid decrease in hematocrit and more profound increases in MAP, diastolic pressure, renal blood flow, glomerular filtration rate, and urinary flow. These results suggest that PEG may be a better option than albumin for prolonged prehospital care of hemorrhagic shock.

Effects of extremity trauma on physiological responses to hemorrhage in conscious rats.

Although physiological responses to hemorrhage are well-studied, hemorrhage is often accompanied by trauma, and it remains unclear how injury affects these responses. This study examined effects of extremity trauma on cardiorespiratory responses and survival to moderate (37%; H-37) or severe (50%; H-50) hemorrhage in rats. Transmitter and carotid catheter implantation and extremity trauma (fibular fracture and muscle injury) were conducted 2 wk, 24 h, and 90 min, respectively, before conscious hemorrhage. Mean arterial pressure (MAP) and heart rate (HR; via telemetry), and respiration rate (RR), minute volume (MV), and tidal volume (TV; via plethysmography) were measured throughout the 25 min hemorrhage and remainder of the 4 h observation period. There were four groups: 1) H-37, no trauma (NT; n = 17); 2) H-37, extremity trauma (T, n = 18); 3) H-50, NT (n = 20); and 4) H-50, T (n = 20). For H-37, during and after hemorrhage, T increased HR (P ≤ 0.031) and MV (P ≤ 0.048) compared with NT rats. During H-50, T increased HR (0.041) and MV (P = 0.043). After hemorrhage, T increased MV (P = 0.008) but decreased HR (P = 0.007) and MAP (P = 0.039). All cardiorespiratory differences between T and NT groups were intermittent. Importantly, both survival time (159.8 ± 78.2 min vs. 211.9 ± 60.3 min; P = 0.022; mean ± SD) and percent survival (45% vs. 80%; P = 0.048) were less in T versus NT rats after H-50. Trauma interacts with physiological systems in a complex manner and no single cardiorespiratory measure was sufficiently altered to indicate that it alone could account for increased mortality after H-50.NEW & NOTEWORTHY In both civilian and military settings, severe hemorrhage rarely occurs in the absence of tissue trauma, yet many animal models for the study of hemorrhage do not include significant tissue trauma. This study using conscious unrestrained rats clearly demonstrates that extremity trauma worsens the probability of survival after a severe hemorrhage. Although no single cardiorespiratory factor accounted for the increased mortality, multiple modest time-related cardiorespiratory responses to the trauma were observed suggesting that their combined dysfunction may have contributed to the reduced survival.

A novel animal model to study delayed resuscitation following traumatic hemorrhage.

A focus of combat casualty care research is to develop treatments for when full resuscitation after hemorrhage is delayed. However, few animal models exist to investigate such treatments. Given the kidney's susceptibility to ischemia, we determined how delayed resuscitation affects renal function in a model of traumatic shock. Rats were randomized into three groups: resuscitation after 1 h (ETH-1) or 2 h (ETH-2) of extremity trauma and hemorrhagic shock, and sham control. ETH was induced in anesthetized rats with muscle injury and fibula fracture, followed by pressure-controlled hemorrhage [mean arterial pressure (MAP) = 55 mmHg] for 1 or 2 h. Rats were then resuscitated with whole blood until MAP stabilized between 90 and 100 mmHg for 30 min. MAP, glomerular filtration rate (GFR), creatinine, blood gases, and fractional excretion of sodium (nFENa+) were measured for 3 days. Compared with control, ETH-1 and ETH-2 exhibited decreases in GFR and nFENa+, and increases in circulating lactate, creatinine, and blood urea nitrogen (BUN) before and within 30 min after resuscitation. The increases in creatinine, BUN, and potassium were greater in ETH-2 than in ETH-1, whereas lactate levels were similar between ETH-1 and ETH-2 before and after resuscitation. All measurements were normalized in ETH-1 within 2 days after resuscitation, with 22% mortality. However, ETH-2 exhibited a prolonged impairment of GFR, increased nFENa+, and a 66% mortality. Resuscitation 1 h after injury therefore preserves renal function, whereas further delay of resuscitation irreversibly impairs renal function and increases mortality. This animal model can be used to explore treatments for prolonged prehospital care following traumatic hemorrhage.NEW & NOTEWORTHY A focus of combat casualty care research is to develop treatment where full resuscitation after hemorrhage is delayed. However, animal models of combat-related hemorrhagic shock in which to determine physiological outcomes of such delays and explore potential treatment for golden hour extension are lacking. In this study, we filled this knowledge gap by establishing a traumatic shock model with reproducible development of AKI and shock-related complications determined by the time of resuscitation.

Extremity trauma exacerbates acute kidney injury following prolonged hemorrhagic hypotension.

BACKGROUND: The incidence of and mortality due to acute kidney injury is high in patients with traumatic shock. However, it is unclear how hemorrhage and trauma synergistically affect renal function, especially when timely volume resuscitation is not available. METHOD: We hypothesized that trauma impairs renal tolerance to prolonged hemorrhagic hypotension. Sprague-Dawley rats were randomized into six groups: control, extremity trauma (ET), hemorrhage at 70 mm Hg (70-H), hemorrhage at 55 mm Hg (55-H), ET + 70 mm Hg (70-ETH), and ET + 55 mm Hg (55-ETH). Animals were anesthetized, and ET was induced via soft tissue injury and closed fibula fracture. Hemorrhage was performed via catheters 5 minutes after ET with target mean arterial pressure (MAP) clamped at 70 mm Hg or 55 mm Hg for up to 3 hours. Blood and urine samples were collected to analyze plasma creatinine (Cr), Cr clearance (CCr), renal oxygen delivery (DO2), urinary albumin, and kidney injury molecule-1 (KIM-1). RESULTS: Extremity trauma alone did not alter renal hemodynamics, DO2, or function. In 70-H, CCr was increased following hemorrhage, while Cr, renal vascular resistance (RVR), KIM-1, and albumin levels remained unchanged. Compared with 70-H, ET + 70 mm Hg exhibited increases in Cr and RVR with decreases in CCr and DO2. In addition, ET decreased the blood volume loss required to maintain MAP = 70 mm Hg by approximately 50%. Hemorrhage at 55 mm Hg and ET + 55 mm Hg exhibited a marked and similar decrease in CCr and increases in RVR, Cr, KIM-1, and albumin. However, ET greatly decreased the blood volume loss required to maintain MAP at 55 mm Hg and led to 50% mortality. CONCLUSION: These results suggest that ET impairs renal and systemic tolerance to prolonged hemorrhagic hypotension. Thus, traumatic injury should be considered as a critical component of experimental studies investigating outcomes and treatment following hemorrhagic shock. LEVEL OF EVIDENCE: This is an original article on basic science and does not require a level of evidence.

Effects of ketamine analgesia on cardiorespiratory responses and survival to trauma and hemorrhage in rats.

Ketamine is the recommended analgesic on the battlefield for soldiers with hemorrhage, despite a lack of supportive evidence from laboratory or clinical studies. Hence, this study determined the effects of ketamine analgesia on cardiorespiratory responses and survival to moderate (37% blood volume; n = 8/group) or severe hemorrhage (50% blood volume; n = 10/group) after trauma in rats. We used a conscious hemorrhage model with extremity trauma (fibular fracture + soft tissue injury) while measuring mean arterial pressure (MAP), heart rate (HR), and body temperature (Tb) by telemetry, and respiration rate (RR), minute volume (MV), and tidal volume (TV) via whole body plethysmography. Male rats received saline (S) or 5.0 mg/kg ketamine (K) (100 µL/100 g body wt) intra-arterially after trauma and hemorrhage. All rats survived 37% hemorrhage. For 50% hemorrhage, neither survival times [180 min (SD 78) vs. 209 min (SD 66)] nor percent survival (60% vs. 80%) differed between S- and K-treated rats. After 37% hemorrhage, K (compared with S) increased MAP and decreased Tb and MV. After 50% hemorrhage, K (compared with S) increased MAP but decreased HR and MV. K effects on cardiorespiratory function were time dependent, significant but modest, and transient at the analgesic dose given. K effects on Tb were also significant but modest and more prolonged. With the use of this rat model, our data support the use of K as an analgesic in injured, hypovolemic patients.NEW & NOTEWORTHY Ketamine administration at a dose shown to alleviate pain in nonhemorrhaged rats with extremity trauma had only modest and transient effects on multiple aspects of cardiorespiratory function after both moderate (37%) and severe (50%) traumatic hemorrhages. Such effects did not alter survival.

Fentanyl impairs but ketamine preserves the microcirculatory response to hemorrhage.

BACKGROUND: Peripheral vasoconstriction is the most critical compensating mechanism following hemorrhage to maintain blood pressure. On the battlefield, ketamine rather than opioids is recommended for pain management in case of hemorrhage, but effects of analgesics on compensatory vasoconstriction are not defined. We hypothesized that fentanyl impairs but ketamine preserves the peripheral vasoconstriction and blood pressure compensation following hemorrhage. METHOD: Sprague-Dawley rats (11-13 weeks) were randomly assigned to control (saline vehicle), fentanyl, or ketamine-treated groups with or without hemorrhage (n = 8 or 9 for each group). Rats were anesthetized with Inactin (i.p. 10 mg/100 g), and the spinotrapezius muscles were prepared for microcirculatory observation. Arteriolar arcades were observed with a Nikon microscope, and vessel images and arteriolar diameters were recorded by using Nikon NIS Elements Imaging Software (Nikon Instruments Inc. NY). After baseline perimeters were recorded, the arterioles were topically challenged with saline, fentanyl, or ketamine at concentrations relevant to intravenous analgesic doses to determine direct vasoactive effects. After arteriolar diameters returned to baseline, 30% of total blood volume was removed in 25 minutes. Ten minutes after hemorrhage, rats were intravenously injected with an analgesic dose of fentanyl (0.6 µg/100 g), ketamine (0.3 mg/100 g), or a comparable volume of saline. For each drug or vehicle administration, the total volume injected was 0.1 mL/100 g. Blood pressure, heart rate, and arteriolar responses were monitored for 40 minutes. RESULTS: Topical fentanyl-induced vasodilation (17 ± 2%), but ketamine caused vasoconstriction (-15 ± 4%, p < 0.01). Following hemorrhage, intravenous ketamine did not affect blood pressure or respiratory rate, while fentanyl induced a slight and transient (<5 minutes, p = 0.03 vs. saline group) decrease in blood pressure, with a profound and prolonged suppression in respiratory rate (>10 minutes, with a peak inhibition of 57 ± 8% of baseline, p < 0.01). The compensatory vasoconstriction observed after hemorrhage was not affected by ketamine treatment. However, after fentanyl injection, although changes in blood pressure were transiently present, arteriolar constriction to hemorrhage was absent and replaced with a sustained vasodilation (78 ± 25% to 36 ± 22% of baseline during the 40 minutes after injection, p < 0.01). CONCLUSION: Ketamine affects neither systemic nor microcirculatory compensatory responses to hemorrhage, providing preclinical evidence that ketamine may help attenuate adverse physiological consequences associated with opioids following traumatic hemorrhage. Microcirculatory responses are more sensitive than systemic response for evaluation of hemodynamic stability during procedures associated with pain management.

Long-term consequences of abdominal aortic and junctional tourniquet for hemorrhage control.

BACKGROUND: Specialized tourniquets have been deployed to the battlefield for the control of junctional/pelvic hemorrhage despite limited knowledge concerning their safety and duration of use. This study investigated long-term effects of abdominal application of the abdominal aortic and junctional tourniquet (AAJT) in a swine survival model. METHODS: Anesthetized spontaneously air-breathing swine were subjected to bilateral femoral artery injuries and subsequent 40% hemorrhage. Further hemorrhage was controlled by applying the AAJT on the lower abdomen for 0 h (n = 2, controls), 1 h (n = 6), 1.5 h (n = 6), or 2 h (n = 3). Before tourniquet release, arterial injuries were repaired, and mechanical ventilation and rapid crystalloid fluid were provided for at least 5 min. Additional fluid and 500 mL autologous blood were transfused after restoring blood flow. Animals were recovered and their mobility and health monitored up to 2 wk. RESULTS: AAJT application occluded the infrarenal abdominal aorta and stopped bilateral groin hemorrhage with rapid reversal of hemorrhagic shock and improved cranial blood pressure. All animals including controls recovered overnight but regaining hind leg function varied among AAJT-treated groups. In contrast to 1 h AAJT-treated swine that recovered full mobility in 1 wk, 2 h animals developed persistent hind leg paraplegia concurrent with urinary retention and ischemic necrosis of lumber muscles and had to be euthanized 3 d after surgery. Half of the 1.5-h group also had to be euthanized early due to paraplegia, whereas the other half recovered motor function within 2 wk. CONCLUSIONS: The results of this animal study indicated that ischemic reperfusion injuries associated with abdominal application of the AAJT were time-dependent. To avoid permanent injuries, AAJT application on the abdomen to control a groin hemorrhage could not be longer than 1 h. This was consistent with recent instructions for application of this tourniquet on the abdomen in patients.

A novel rat model of extremity trauma for prehospital pain management research.

BACKGROUND: Pain management is important in prehospital care of patients with extremity trauma (ET). The goal of this study was to establish a rat model of ET for prehospital pain research and validate it using pain behaviors and analgesics. METHODS: Rats were anesthetized using isoflurane, and ET was induced in one hindlimb via clamping retrofemoral tissues for 30 seconds, followed by closed fibula fracture. Rats regained consciousness after ET. Pain responses in the injured hindlimb to thermal hyperalgesia (paw withdrawal latency [PWL]), mechanical allodynia (paw withdrawal pressure [PWP]), and weight bearing (WB) were determined before and 90 minutes after ET. Morphine (2 mg/kg), fentanyl (10 µg/kg), sufentanil (1 µg/kg), ketamine (5 mg/kg), or vehicle (saline) were then administered via intravenous (i.v.) injection, followed by PWL, PWP, and WB assessments at 10 minutes, 40 minutes, 80 minutes, and 120 minutes after analgesia. RESULTS: After ET, PWL, PWP, and WB were significantly decreased by 61 ± 4%, 64 ± 8%, and 65 ± 4%, respectively, compared with pre-ET values. These pain behaviors were maintained for 3 hours to 4 hours. Compared with the saline group, opioid analgesics significantly increased PWL for at least 80 minutes, with sufentanil exhibiting the highest analgesic effect. An increase in PWL was only observed at 10 minutes after ketamine. The PWP was transiently increased with opioid analgesics for 10 minutes to 40 minutes, but was not changed with ketamine. Weight bearing was improved with opioid analgesics for at least 2 hours, but only for up to 80 minutes with ketamine. CONCLUSION: Our ET model includes long bone fracture and soft tissue injury, but no fixation surgery, mimicking prehospital ET. Our model produces acute, steady, and reproducible trauma-related pain behaviors, and is clinically relevant regarding the pain behaviors and established responses to common analgesics. This model of acute pain due to ET is ideal for prehospital pain management research.

Do vented chest seals differ in efficacy? An experimental evaluation using a swine hemopneumothorax model.

OBJECTIVE: Airways compromise was the second leading cause of potentially preventable death among combat casualties. We investigated the ability of five Food and Drug Administration-approved nonocclusive chest seals (CSs) to seal a bleeding chest wound and prevent tension hemopneumothorax (HPTX) in a swine model. METHODS: Following instrumentation, an open chest wound was created in the left thorax of spontaneously air-breathing anesthetized pigs (n = 26; 43 kg). Autologous fresh blood (226 mL) was then infused into the pleural cavity to produce HPTX. The chest wounds were then sealed with CSs. The sealant strength and venting function of CSs were challenged by infusion of 50 mL more blood directly into the chest wound and incremental air injections into the pleural cavity. Tension HPTX was defined as intrapleural (IP) pressure equal to or more than +1 mm Hg and more than 20% deviation in physiologic measurements. RESULTS: An open chest wound with HPTX raised IP pressure (~ -0.7 mm Hg) and caused labored breathing and reductions in PaO2 and SvO2 (p < 0.01). Sealing the wounds with the CSs restored IP pressure, and improved breathing and oxygenation. Subsequent blood infusion into the wound and IP air injections produced CS-dependent responses. Chest seals with one-way valves (Bolin and SAM) did not evacuate the blood efficiently; pooled blood either detached the CSs from skin and leaked out (75%), or clotted and clogged the valve and led to tension HPTX (25%). Conversely, CSs with laminar venting channels allowed escape of blood and air from the pleural cavity and maintained IP pressure and oxygenation near normal levels. Success rates were 100% for Sentinel and Russell (6/6); 67% for HyFin (4/6); 25% for SAM (1/4); and 0% for Bolin (0/4) CSs (p = 0.002). CONCLUSION: The sealant and valve function of vented CS differed widely in the presence of bleeding chest wounds. Medics should be equipped with more effective CSs for treating HPTX in the field.

Relationship between Plasma Albumin Concentration and Plasma Volume in 5 Inbred Rat Strains.

Using the Evans Blue procedure, we previously found strain-related differences in plasma volumes in 5 inbred rat strains. Because albumin binds strongly with Evans blue, this protein is important in the Evans blue method of plasma volume determination. Therefore, we speculated that interstrain differences in plasma albumin concentration (PAC) could distort calculated plasma volumes. To address this concern, we used ELISA techniques to measure PAC in these inbred rat strains. In study A, the blood volume was measured by using Evans blue dye, and albumin was measured at the start of hemorrhage. In study B, blood volume was not measured, and albumin was measured twice, near the start and end of hemorrhage (approximately 14 min apart). Neither study revealed any interstrain differences in PAC, which decreased after hemorrhage in all 5 strains. No correlation was found between PAC and plasma volume, survival time, blood lactate, or blood base excess. Percentage changes in PAC during hemorrhage were greater in salt-sensitive compared with Lewis rats. Moreover, these percentage changes were associated with survival time in Fawn hooded hypertensive rats. Our data show that the plasma volumes we measured previously were not misrepresented due to variations in PAC.

Vented versus unvented chest seals for treatment of pneumothorax and prevention of tension pneumothorax in a swine model.

BACKGROUND: Unvented chest seals (CSs) are currently recommended for the management of penetrating thoracic injuries in the battlefield. Since no supporting data exist, we compared the efficacy of a preferred unvented with that of a vented CS in a novel swine model of pneumothorax (PTx). METHODS: An open chest wound was created in the left thorax of spontaneously air-breathing anesthetized pigs (n = 8). A CS was applied over the injury, then tension PTx was induced by incremental air injections (0.2 L) into the pleural cavity via a cannula that was also used to measure intrapleural pressure (IP). Both CS were tested on each pig in series. Tidal volume (V(T)), respiratory rate, IP, heart rate, mean arterial pressure, cardiac output, central venous pressure, pulmonary arterial pressure, venous and peripheral oxygen saturations (SvO2, SpO2) were recorded. Tension PTx was defined as a mean IP equal to or greater than +1 mm Hg plus significant (20-30%) deviation in baseline levels of the previously mentioned parameters and confirmed by chest x-ray study. PaO2 and PaCo2 were also measured. RESULTS: PTx produced immediate breathing difficulty and significant rises in IP and pulmonary arterial pressure and falls in V(T), SpO2, and SvO2. Both CSs returned these parameters to near baseline within 5 minutes of application. After vented CS was applied, serial air injections up to 2 L resulted in no significant change in the previously mentioned parameters. After unvented CS application, progressive deterioration of all respiratory parameters and onset of tension PTx were observed in all subjects after approximately 1.4-L air injection. CONCLUSION: Both vented and unvented CSs provided immediate improvements in breathing and blood oxygenation in our model of penetrating thoracic trauma. However, in the presence of ongoing intrapleural air accumulation, the unvented CS led to tension PTx, hypoxemia, and possible respiratory arrest, while the vented CS prevented these outcomes.

Arterial blood gases, electrolytes, and metabolic indices associated with hemorrhagic shock: inter- and intrainbred rat strain variation.

We have previously shown interstrain variation (indicating a genetic basis), and intrastrain variation in survival time after hemorrhage (STaH) among inbred rat strains. To assist in understanding physiological mechanisms associated with STaH, we analyzed various arterial blood measures (ABM; pH, Paco2, oxygen content, sodium, potassium, glucose, bicarbonate, base excess, total CO2, and ionized calcium) in inbred rats. Rats from five inbred strains (n = 8-10/strain) were catheterized and, ≈ 24 h later, subjected to a conscious, controlled, 47% hemorrhage. ABM were measured at the start (initial) and end (final) of hemorrhage. Inter- and intrainbred strain variations of ABM were quantified and compared, and correlations of ABM with STaH were determined. All final ABM values and some initial ABM values were different among strains. Most ABM changed (Δ) during hemorrhage, and these changes differed among strains (P <0.03). Some strain-dependent correlations (r ≥ 0.7; P ≤ 0.05) existed between ΔABM and STaH (e.g., BN/Mcwi, ΔK(+), r = -0.84). Dark Agouti rats (longest STaH) had the smallest ΔPaco2, ΔHCO3(-), and Δbase excess, and the highest final glucose. High coefficients of variation (CVs, >10%), strain-specific CVs, and low intraclass correlation coefficients (rI < 0.5) defined the large intrastrain ABM variation that exceeded interstrain variation for most ABM. These results suggest that some ABM (K(+), Paco2, glucose, oxygen content) could predict subsequent STaH in an inbred rat strain-dependent manner. We speculate that whereas genetic differences may be responsible for interstrain variation, individual-specific epigenetic processes (e.g., DNA methylation) may be partly responsible for both inter- and intrastrain ABM variation.

Cardiac mitochondrial proteomic expression in inbred rat strains divergent in survival time after hemorrhage.

We have previously identified inbred rat strains differing in survival time to a severe controlled hemorrhage (StaH). In efforts to identify cellular mechanisms and ultimately genes that are important contributors to enhanced STaH, we conducted a study to characterize potential differences in cardiac mitochondrial proteins in these rats. Inbred rats from three strains [Brown Norway/Medical College of Wisconsin (BN); Dark Agouti (DA), and Fawn Hooded Hypertensive (FHH)] with different StaH (DA = FHH > BN) were assigned to one of three treatment groups (n = 4/strain): nonoperated controls, surgically catheterized rats, or rats surgically catheterized and hemorrhaged 24 h postsurgery. Rats were euthanized 30 min after handling or 30 min after initiation of a 26 min hemorrhage. After euthanasia, hearts were removed and mitochondria isolated. Differential protein expression was determined using 2D DIGE-based Quantitative Intact Proteomics and proteins identified by MALDI/TOF mass spectrometry. Hundreds of proteins (791) differed among inbred rat strains (P ≤ 0.038), and of these 81 were identified. Thirty-eight were unique proteins and 43 were apparent isoforms. For DA rats (longest STaH), 36 proteins increased and 30 decreased compared with BN (shortest STaH). These 81 proteins were associated with lipid (e.g., acyl CoA dehydrogenase) and carbohydrate (e.g., fumarase) metabolism, oxidative phosphorylation (e.g., ubiquinol-cytochrome C reductase), ATP synthesis (F1 ATPase), and H2S synthesis (3-mercaptopyruvate sulfurtransferase). Although we cannot make associations between these identified mitochondrial proteins and StaH, our data do provide evidence for future candidate proteins with which to consider such associations.

Genetic influences on survival time after severe hemorrhage in inbred rat strains.

To find a genetic basis for differential ability to survive severe hemorrhage, we previously showed eightfold differences in survival times among inbred rat strains. We assumed that rat strains had similar normalized blood volumes (NBV; ml/100 g body wt). As NBV might vary among strains and constitute one genetic variable affecting survival time to hemorrhage, in experiment 1 of the current studies we first measured total blood volumes and calculated NBV in specific inbred rat strains (Brown Norway/Medical College of Wisconsin, BN; Dark Agouti, DA; Fawn Hooded Hypertensive, FHH; Lewis, LEW; and Dahl Salt-Sensitive, SS) previously found to be divergent in survival time. NBV differed by 20% (P < 0.01; BN > SS > FHH = LEW = DA) and had a heritability (h(2)) of 0.56. Hence, differential survival times in our previously published study might reflect strain-dependent differences in NBV. Then studies were conducted wherein rats were catheterized and, ∼24 h later, 47% of their blood volume was removed; these rats were observed for a maximum of 4 h. In experiment 2, blood volumes were measured the day prior to hemorrhage. Percent survival and survival time did not differ among strains. To obviate possible confounding effects of blood volume determination, in experiment 3 the average NBV for each strain was used to determine hemorrhage volumes. Percent survival (P < 0.01) and survival times (P < 0.001) were different with DA demonstrating the best (62.5%, 190 ± 29 min) and BN the worst (0%, 52 ± 5 min) survival responses. These data indicate that both blood volume and survival time after hemorrhage in rats are heritable quantitative traits, and continue to suggest that genetic assessment of these phenotypes might lead to novel therapeutics to improve survival to hemorrhage.

Life or death? A physiogenomic approach to understand individual variation in responses to hemorrhagic shock.

Severe hemorrhage due to trauma is a major cause of death throughout the world. It has often been observed that some victims are able to withstand hemorrhage better than others. For decades investigators have attempted to identify physiological mechanisms that distinguish survivors from nonsurvivors for the purpose of providing more informed therapies. As an alternative approach to address this issue, we have initiated a research program to identify genes and genetic mechanisms that contribute to this phenotype of survival time after controlled hemorrhage. From physiogenomic studies using inbred rat strains, we have demonstrated that this phenotype is a heritable quantitative trait, and is therefore a complex trait regulated by multiple genes. Our work continues to identify quantitative trait loci as well as potential epigenetic mechanisms that might influence survival time after severe hemorrhage. Our ultimate goal is to improve survival to traumatic hemorrhage and attendant shock via regulation of genetic mechanisms and to provide knowledge that will lead to genetically-informed personalized treatments.

Rat strains bred for low and high aerobic running capacity do not differ in their survival time to hemorrhage.

Hemorrhagic shock reflects low tissue perfusion that is inadequate to maintain normal metabolic functions. Often associated with this condition are impairments in cellular oxygen delivery and utilization. Rat strains divergent in their running endurance have been artificially selected over 12 generations. As these rats bred for high (HCR) vs low (LCR) aerobic running capacity have greater tissue O(2) utilization capacity and improved cardiovascular function, we hypothesized that HCR would be more tolerant (i.e., have greater survivability) to the global ischemia of hemorrhagic shock than LCR. To address this hypothesis, survival time to a severe-as substantiated by dramatic changes in plasma lactate, HCO(3), and base deficit-controlled hemorrhage was measured. Male rats were catheterized and, approximately 24 h later, an estimated >35% of the calculated blood volume was removed during a 26-min period while the rats were conscious and unrestrained. Rats were observed for 6 h or until death. Contrary to our hypothesis, survival time in HCR (220 +/- 63 min; n = 6) did not differ statistically (P = 0.46) from that in LCR (279 +/- 53 min; n = 7). Similarly, there were no statistical differences (P >or= 0.08) between rat lines in blood pH, lactate, HCO(3), and base deficit pre- or post-hemorrhage. In addition, few significant differences between lines in response to hemorrhage were detected by measures of cellular antioxidant status in heart, liver, or lung. Since animals with genetically greater tissue oxygen utilization capacity failed to show longer survival times, our results suggest that other mechanisms must play a more dominant role in determining survivability to hemorrhage under conditions of this hemorrhage.

Comparison of new hemostatic granules/powders with currently deployed hemostatic products in a lethal model of extremity arterial hemorrhage in swine.

BACKGROUND: HemCon bandage (HC) and QuikClot granules (QC) have been deployed for the past 5 years for treating external hemorrhage in combat casualties. We examined efficacy and initial safety of three new hemostatic granules/powders in a swine extremity arterial hemorrhage model that was 100% fatal with army standard gauze treatment. The new products were compared with the most advanced forms of HC and QC products. METHODS: Anesthetized pigs (37 kg, n = 46) were instrumented, splenectomized, and their femoral arteries were isolated and injured (6 mm arteriotomy). After 45 seconds free bleeding, a test agent [WoundStat (WS), super quick relief (SQR), Celox (CX)] or a control product [HC or QC bead bags (advanced clotting sponge plus)] was applied to the wounds and compressed with a large gauze for 2 minutes. Fluid resuscitation (colloid and crystalloid) was given and titrated to a mean arterial pressure of 65 mm Hg. Animals were observed for 180 minutes or until death. Computed tomography angiography was performed on survivors and tissue samples were collected form wounds for histologic examination. RESULTS: No differences were found in baseline measurements and pretreatment blood loss (17.4 mL/kg +/- 0.5 mL/kg, mean +/- SEM) among groups. Advanced clotting sponge plus testing was halted after six unsuccessful attempts (no hemostasis observed) whereas other agents were tested each in 10 animals. Stable hemostasis was achieved in 10 (WS), 7 (SQR), 6 (CX), and 1 (HC) subjects in each group, resulting in the recovery of mean arterial pressure and survival of the animals for 3 hours (p < 0.05, SQR or WS vs. HC). Posttreatment blood loss was significantly reduced with the use of the new agents (CX = 40 +/- 16.6, SQR = 34.5 +/- 16.3, WS = 9.5 mL/kg +/- 5.2 mL/kg) as compared with HC (85.6 mL/kg +/- 10 mL/kg, p < 0.05). The granular treated animals lived for 180 (WS), 164 +/- 8.2 (SQR) and 138 +/- 17.7 (CX) minutes, significantly (p < 0.05) longer than the HC (83.3 +/- 12 minutes) group. A significant (p < 0.05) rise in temperature (53.5 degrees C +/- 1.8 degrees C) over baseline (36.5 degrees C +/- 0.3 degrees C) was measured only in the wounds treated with SQR. Computed tomography images showed no blood flow through treated vessels. Histologic evidence indicated the least tissue damage with HC, moderate damage with WS and CX, and most damage including axonal necrosis with SQR. CONCLUSION: The new hemostatic agents are significantly more effective in treating arterial hemorrhage than currently deployed products. Among them, WS granules appear to be most efficacious, followed by SQR and CX powders. The clinical significance of tissue damage caused by these agents and any potential risk of embolism with procoagulant granular/powder products are unknown and warrant survival studies.

Is survival time after hemorrhage a heritable, quantitative trait?: an initial assessment.

Enhancing survival to hemorrhage of both civilian and military patients is a major emphasis for trauma research. Previous observations in humans and outbred rats show differential survival to similar levels of hemorrhage. In an initial attempt to determine potential genetic components of such differential outcomes, survival time after a controlled hemorrhage was measured in 15 inbred strains of rats. Anesthetized rats were catheterized, and approximately 24 h later, 55% of the calculated blood volume was removed during a 26-min period from conscious unrestrained animals. Rats were observed for a maximum of 6 h. Survival time was 7.7-fold longer in the longest-lived strain (Brown Norway/Medical College of Wisconsin; 306 +/- 36 min; mean +/- SEM) than in the shortest-lived strain (DA; 40 +/- 5 min; P < or = 0.01). Mean survival times for the remaining inbred strains ranged from 273 +/- 44 to 49 +/- 4 min (Dahl-Salt Sensitive > Brown Norway > Munich Wistar Fromter> Dahl-Salt Resistant > Copenhagen > Noble > Spontaneous-hypertensive > Lewis > BDIX > Fawn Hooded Hypertensive > FISCHER 344 > Black agouti > PVG). The variance in the hazard of death attributable to different strains was estimated to be 1.22 log-hazard units, corresponding to a heritability of approximately 48%. Graded and divergent survival times to hemorrhage in inbred rat strains are remarkable and suggest multiple genetic components for this characteristic. However, this interpretation of differential responses to hemorrhage may be confounded by potential strain-associated differences related to the surgical preparation. Identification of inbred strains divergent in survival time to hemorrhage provides the opportunity for future use of these strains to identify genes associated with this complex response.

A novel swine model for evaluation of potential intravascular hemostatic agents.

Because uncontrolled hemorrhage is a leading cause of battlefield mortality, finding an intravenous treatment that could assist endogenous clotting mechanisms is a major mission for military researchers. Evaluation of potential intravenous hemostatic agents requires both in vitro and in vivo tests. For in vivo evaluation, we have developed a novel swine model in which 1) bleeding times (BT) and coagulation function could be ascertained after multiple doses of hemostatic drug administration and 2) a subsequent exsanguinating injury could be performed in the same animal, yielding screening information regarding the effects of drug pretreatment on blood loss and survival. Transection of small mesenteric arteries and veins allowed for multiple and reproducible BT measures that correlated with coagulation function. Subsequent excision of defined areas of the liver produced bleeding predominantly from small vessels (diameter, less than 2 mm) and parenchyma while resulting in 62% mortality without the use of either heparinization or aggressive fluid infusion. This swine model allows for multiple, repeatable BT measures in the same animal in experiments already involving laparotomy. Such a model is well suited for terminal studies to test effects of multiple doses of the same drug or multiple drugs on BT and allows for multiple, easily visualized measures that permit enhanced repeatability. The liver injury provides for numerous small vessel lesions that could be amenable to closure by coagulation. Therefore, drugs or mechanisms that enhance coagulation and concomitantly decrease blood loss and increase survival time may be accurately evaluated in this new model.