Francoise Arnaud

Comparison of 10 hemostatic dressings in a groin transection model in swine.

BACKGROUND Major improvements have been made in the development of novel dressings with hemostatic properties to control heavy bleeding in noncompressible areas. To test the relative efficacy of different formulations in bleeding control, recently manufactured products need to be compared using a severe injury model. METHODS Ten hemostatic dressings and the standard gauze bandage were tested in anesthetized Yorkshire pigs hemorrhaged by full transection of the femoral vasculature at the level of the groin. Application of these dressings with a 5-minute compression period (at approximately 200 mm Hg) was followed with a subsequent infusion of colloid for a period of 30 minutes. Primary outcomes were survival and amount and incidence of bleeding after dressing application. Vital signs and wound temperature were continuously recorded throughout the 3-hour experimental observation. RESULTS These findings indicated that four dressings were effective in improving bleeding control and superior to the standard gauze bandage. This also correlated with increased survival rates. Absorbent property, flexibility, and the hemostatic agent itself were identified as the critical factors in controlling bleeding on a noncompressible transected vascular and tissue injury. CONCLUSIONS Celox, QuikClot ACS, WoundStat, and X-Sponge ranked superior in terms of low incidence of rebleeding, volume of blood loss, maintenance of mean arterial pressure >40 mm Hg, and survival.

Bovine polymerized hemoglobin (hemoglobin-based oxygen carrier-201) resuscitation in three swine models of hemorrhagic shock with militarily relevant delayed evacuation--effects on histopathology and organ function.

Objective:To test our hypothesis that hemoglobin-based oxygen carrier (HBOC)-201 resuscitation in hemorrhagic shock (HS) will not lead to increased organ injury and dysfunction. Design:Three swine HS models simulating military-relevant delayed evacuation: a) moderate controlled HS, b) severe controlled HS, and c) severe uncontrolled HS. Setting:Military research laboratory. Subjects:Swine. Interventions:Swine were anesthetized/intubated and instrumented. To induce HS, in two controlled hemorrhage experiments, 40% (moderate controlled HS) or 55% (severe controlled HS) of blood volume was withdrawn; in an uncontrolled HS experiment, the liver was crushed/lacerated. During a 4-hr “prehospital phase,” pigs were resuscitated with HBOC-201 (HBOC) or Hextend (HEX) or were nonresuscitated (NON). Upon “hospital arrival,” liver injury was repaired (severe uncontrolled HS), blood or saline was infused, hemodynamics were monitored, and blood was collected. Upon animal death and/or 72 hrs, necropsy was followed by histopathologic evaluation of organ injury (hematoxylin and eosin, electron microscopy) and immunohistochemistry of oxidative potential (3-nitrotyrosine). Significance (p < .05) was assessed by Kruskal-Wallis, analysis of variance/Bonferroni, and mixed procedure tests. Measurements and Main Results:Survival was significantly higher with HBOC than HEX only with severe uncontrolled HS (p = .002). Myocardial necrosis/fibroplasia, fluid requirements, cardiac output, and cardiac enzymes were generally similar or lower in HBOC than HEX pigs, but creatine kinase-MB (but not creatine kinase-MB/creatine kinase ratio) was higher with HBOC in moderate controlled HS. Alveolar/interstitial pulmonary edema was similar with HBOC and HEX, but Po2 was higher with HBOC in severe uncontrolled HS. Jejunal villar epithelial and hepatocellular necrosis were similarly minimal to moderate in all groups. Minimal biliary changes occurred exclusively with HBOC. Aspartate aminotransferase, lactate dehydrogenase, and alkaline phosphatase were generally higher with HBOC than HEX. Mild renal papillary injury occurred more frequently with HBOC, but consistent patterns for urine output, blood urea nitrogen, and creatinine, were not seen. The 3-nitrotyrosine staining intensity was not different. Conclusions:In comparison with hetastarch, HBOC-201 resuscitation of swine with HS increased survival (with severe HS), did not increase evidence of oxidative potential, and had histopathologic and/or functional effects on organs that were clinically equivocal (myocardium, lungs, hepatic parenchyma, jejunum, and renal cortex/medulla) and potentially adverse (hepatobiliary and renal papilla). The effects of HBOC-201-resuscitation in HS should be corroborated in controlled clinical trials.

Development of a Standard Swine Hemorrhage Model for Efficacy Assessment of Topical Hemostatic Agents

BACKGROUND The diverse information of efficacy of hemostatic products, obtained from different military laboratories using different models, has made it difficult to ascertain the true benefit of new hemostatic agents in military medicine. The aim of this study was to recommend a standard hemorrhage model for efficacy testing acceptable by most investigators in the field and avoid contradictory and duplicative efforts by different laboratories. METHODS The swine femoral artery injury model (6-mm arteriotomy) with some modifications was tested to standardize the model. The suggested modifications included no splenectomy, one-time treatment, 30 seconds free bleeding, and 5 L limit for fluid resuscitation. The model was tested with all or some of these modifications in four experimental conditions (n = 5-6 pigs per condition) using Combat Gauze (CG) as control agent. RESULTS The primary end points including blood pressure, blood loss, and survival rates were modestly changed in the four conditions. The second experimental condition in which bleeding was treated with a single CG with 3-minute compression produced the most suitable results. The average blood loss was 99 mL/kg, and hemostasis was achieved in one-third of the pigs, which led to matching survival rate. CONCLUSION A rigorous hemorrhage model was developed for future evaluation of new hemostatic agents and comparison with CG, the current standard of care. This model may not be suitable for testing every agent and some modifications may be necessary for specific applications. Furthermore, laboratory studies using this or similar models must be accompanied by operational testing in the field to confirm the efficacy and practical utility of selected agents when used on the battlefield.

Evaluation of coagulation stages of hemorrhaged swine: comparison of thromboelastography and rotational elastometry.

Thromboelastography (TEG) or rotational thromboelastometry (ROTEM) assesses blood viscoelastic properties and clotting kinetics that can be measured by Haemoscope TEG and Pentapharm ROTEM devices using slightly different methodologies. These devices were compared by measuring blood samples associated with various degrees of coagulopathy. Blood samples, collected from swine undergoing three types of severe injury and resuscitation protocol resulting in normal, hypercoagulopathy, and hypocoagulopathy, were assessed with TEG or ROTEM before the surgical procedures, and after injury, fluid resuscitation, and simulated hospital phase. Standard clotting parameters were compared by Students t-test at a significance of a P value less than 0.05. Regression analysis indicated a positive correlation between TEG and ROTEM for reaction time (R), clotting rate (K), and maximum amplitude (Ma) parameters. With samples of normal coagulation, R (440 ± 136 vs. 391 ± 73 s), K (99 ± 39 vs. 81 ± 20 s), and Ma (74 ± 4 vs. 69 ± 5 mm) were higher, whereas (α) (68 ± 8 vs. 75 ± 3 mm) was lower with TEG than ROTEM, respectively; a P value is less than 0.05. The magnitude of changes from baseline in hypercoagulable or hypocoagulable samples due to level of injury was equivalent with TEG and ROTEM indicating comparable use of the instruments. However, when samples were extremely hypocoagulopathic due to resuscitation fluid, the TEG values could not be readily determined. Overall, TEG readings were higher than ROTEM readings; this disparity between the two instruments was attenuated with hypercoaguable samples. Both devices yielded similar information regarding the status of coagulation related to trauma. Because of operating characteristics, the same instrument should be used for monitoring the same patient or study.

Immune effects of resuscitation with HBOC-201, a hemoglobin-based oxygen carrier, in swine with moderately severe hemorrhagic shock from controlled hemorrhage.

HBOC-201, a hemoglobin-based oxygen carrier, improved physiologic parameters and survival in hemorrhagic shock (HS) animal models. However, resuscitation from HS and the properties of different fluids influence immune responses. The aim of this study was to determine if HBOC-201 significantly alters immune function in traumatic HS. Anesthetized pigs underwent soft tissue injury, controlled hemorrhage of 40% of blood volume, and resuscitation with HBOC-201 or Hextend, or no resuscitation. Sequential whole-blood samples were collected for analyses of leukocyte differential (hematology analyzer), T-lymphocyte subsets (CD3+, CD4+, and CD8+) (FACS), lymphocyte adhesion marker CD49d (α4-integrin) expression (FACS), plasma cytokines-tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10-(ELISA), and lymphocyte apoptosis (annexin-V/propidium iodide staining) (FACS). Statistical analyses were performed by the mixed procedure. Total WBC counts decreased posthemorrhage in both resuscitation groups. Lymphocyte percentages decreased and PMN percentages increased around 4 h posthemorrhage in all groups. CD3 cells decreased in all groups, but CD4 and CD8 cells decreased only in the resuscitation groups. TNF-α levels were not detectable in any groups. IL-6 levels were similar across treatment groups (P > 0.05); however, IL-10 levels were higher in the HBOC group, as early as 1 h posthemorrhage (P = 0.04). Increases in lymphocytic CD49d expression levels and apoptosis occurred only in nonresuscitation and Hextend groups, respectively (P ≤ 0.01). In comparison with Hextend, HBOC-201 had no significant adverse or beneficial effects on immune function in this model of moderately severe HS in swine, suggesting that it may be safe as a resuscitation fluid in HS patients.

The Effects of Decreasing Low-molecular Weight Hemoglobin Components of Hemoglobin-based Oxygen Carriers in Swine With Hemorrhagic Shock

BACKGROUND Some hemoglobin-based oxygen carriers (HBOCs) improve outcome in animal models of hemorrhagic shock (HS) in comparison with standard asanguinous resuscitation fluids. Nevertheless, concern about intrinsic vasoactivity, linked in part to low-molecular weight (MW) hemoglobin (Hb), has slowed HBOC development. We assessed the impact of decreasing the low-MW Hb component of bovine HBOC on vasoactivity in severe HS. METHODS Anesthetized invasively monitored swine were hemorrhaged 55% blood volume and resuscitated with bovine HBOC containing 31% (31 TD [HBOC-301]), 2% (2 TD [HBOC-201]), or 0.4% (0.4 TD) low-MW Hb. Pigs received four 10 mL/kg infusions over 60 minutes, hospital arrival was simulated at 75 minutes, organ blood flow (BF) was evaluated by microsphere injection, and monitoring was continued for 4 hours followed by complete necrotic evaluation. RESULTS There were few differences between 2 TD and 0.4 TD. Thirty-one TD pigs had higher systemic and pulmonary blood pressure (BP), systemic vascular resistance index, and pulmonary artery wedge pressure, compared with 2 TD or 0.4 TD (p < 0.01); however, pigs in all groups had at least mildly elevated BP. Transcutaneous tissue oxygenation, base excess, and mixed venous oxygen saturation were similar across groups; lactate and methemoglobin were highest with 0.4 TD (p < 0.03). There were no group differences in BF. Over time, myocardial BF increased and hepatic BF decreased in all groups (for 31 TD, p < 0.05); renal BF was unchanged in all groups. There were no group differences in heart, lung, or liver histopathology, and survival. CONCLUSIONS Although purification from 31% to 2% low-MW Hb content significantly decreased vasoactive responses, further purification to 0.4% had no additional clinically measurable effects in severe HS. If further diminution in HBOC vasoactivity is desired for use in HS, additional technical approaches may be required.

Traumatic brain injury and severe uncontrolled haemorrhage with short delay pre-hospital resuscitation in a swine model

INTRODUCTION Unavailability of blood (and oxygen delivery) for pre-hospital resuscitation in haemorrhagic shock patients are major problems, supporting the importance for novel resuscitation strategies. In a combined polytrauma model of uncontrolled haemorrhage and traumatic brain injury (TBI) in swine, we investigated if pre-hospital administration of the haemoglobin based oxygen carrier HBOC-201 will improve tissue oxygenation and physiologic parameters compared to Lactated Ringers (LR) solution. MATERIALS AND METHODS Anaesthetised Yorkshire swine underwent fluid-percussion TBI and Grade III liver laceration. During a 30-min pre-hospital phase, the animals were resuscitated with a single infusion of HBOC-201, LR solution, or nothing (NON). Upon hospital arrival, the animals were given blood or normal saline as needed. Surviving animals were euthanised 6h post-injury. Cerebral blood flow was measured by microsphere injection, and pathology was assessed by gross observation and immunohistochemical analysis. RESULTS Mean TBI force (2.4±0.1atm) (means±standard error of the mean) and blood loss (22.5±1.7mL/kg) were similar between groups. Survival at the 6h endpoint was similar in all groups (∼50%). Cerebral perfusion pressure (CPP) and brain tissue oxygen tension were significantly greater in HBOC-201 as compared with LR animals (p<0.005). Mean arterial pressure (MAP) and mean pulmonary artery pressure (MPAP) were not significantly different amongst groups. Blood transfusion requirements were delayed in HBOC-201 animals. Animals treated with HBOC-201 or LR showed no immunohistopathological differences in glial fibrillary acidic protein (GFAP) and microtubule-associated protein 2 (MAP-2). Severity of subarachnoid and intraparenchymal haemorrhages were similar for HBOC and LR groups. CONCLUSION In this polytrauma swine model of uncontrolled haemorrhage and TBI with a 30-min delay to hospital arrival, pre-hospital resuscitation with one bolus of HBOC-201 indicated short term benefits in systemic and cerebrovascular physiological parameters. True clinical benefits of this strategy need to be confirmed on TBI and haemorrhagic shock patients.

Hematology patterns after hemoglobin-based oxygen carrier resuscitation from severe controlled hemorrhage with prolonged delayed definitive care.

BACKGROUND: The hemoglobin‐based oxygen carrier (HBOC‐201) resuscitation fluid improves outcome in hemorrhagic shock swine models with minimal coagulopathy. Herein, coagulation parameters were evaluated after resuscitation with HBOC‐201 after severe bleeding and prolonged delay to definitive care.

Sodium nitroprusside ameliorates systemic but not pulmonary HBOC-201-induced vasoconstriction: an exploratory study in a swine controlled haemorrhage model.

BACKGROUND Vasoconstriction is a side effect that may prevent the use of haemoglobin based oxygen carrier (HBOC) as blood substitute. Therefore, we tested the hypothesis that the NO donor, sodium nitroprusside (SNP), would mitigate systemic and pulmonary hypertension associated with HBOC-201 in a simple controlled haemorrhage swine model. METHODS After 55% estimated blood volume withdrawal through a venous catheter, invasively anesthetized and instrumented animals were resuscitated with three 10 ml/kg infusions of either HBOC-201 or Hextend (HEX) with or without 0.8 μg/kg/min SNP (infused concomitantly via different lines). Haemodynamics, direct and indirect measures of tissue oxygenation, and coagulation were measured for 2h. RESULTS Haemorrhage caused a state of shock manifested by hypotension and base deficit. HBOC-201 resuscitation resulted in higher systemic (p<0.0001) and pulmonary (p<0.002) blood pressure than with HEX. Elevation of systemic (p<0.0001) but not pulmonary (p>0.05) arterial pressure was attenuated by co-infusion of SNP, without significant group differences in haemodynamics, tissue oxygenation, platelet function, coagulation, methaemoglobin, or survival (p>0.05). CONCLUSION In swine with haemorrhagic shock, co-administration of the NO donor, SNP, effectively and safely reduces HBOC-201-related systemic but not pulmonary vasoactivity. Interestingly, co-administration of the vasodilator SNP with HEX had no deleterious effects in comparison with HEX alone.

Dose Response of Sodium Nitrite on Vasoactivity Associated with HBOC-201 in a Swine Model of Controlled Hemorrhage

Abstract Sodium nitrite (NaNO2) was evaluated in a 55% EBV hemorrhage swine model to mitigate the increased blood pressure due to HBOC-201. Animals were resuscitated by three 10ml/kg infusions of either HBOC-201 or Hextend with and without NaNO2. All vital signs, coagulation and blood chemistry were measured for 2hr. HBOC-201-vasoconstriction was attenuated only after the first 10.8μmol/kg NaNO2 infusion. Complete abolition was obtained with the highest 3 NaNO2 dose, but side effects were observed. There was no reduction in platelet function due to NaNO2. NaNO2 ability to reduce HBOC-201 vasoactivity was transient and 10.8μmol/kg NaNO2 seems an acceptable dose for further investigation.