Effects of sequential aeromedical evacuations following traumatic brain injury in swine.

INTRODUCTION: Traumatic brain injuries (TBI) represent a significant percentage of critical injuries in military conflicts. Following injury, wounded warfighters are often subjected to multiple aeromedical evacuations (AE) and associated hypobaria, yet the impact in TBI patients remains to be characterized. This study evaluated the impact of two consecutive simulated AEs in a fluid-percussion TBI model in swine to characterize these effects. METHODS: Following instrumentation, anesthetized Yorkshire swine underwent a frontal TBI via fluid-percussion. A hypobaric chamber was then used to simulate AE at simulated cabin pressure equivalent to 8000ft (hypobaria) in a 6 h initial flight on day 3, followed by a 9 h flight on day 6, and were monitored for 14 days. Animals in the normobaria group were subjected to the same steps at sea level while Sham animals in both groups were instrumented but not injured. Parameters measured included physiologic response, intracranial pressure (ICP), hematology, chemistry, and serum cytokines. Histopathology of brain, lung, intestine, and kidney was performed, as well as fluorojade staining to evaluate neurodegeneration. All animals were divided into sub-groups by block randomization utilizing a 2-way ANOVA to analyze independent variables. RESULTS: Survival was 100% in all groups. Physiologic parameters were largely similar across groups as well during both 6 and 9 h AE. Animals exposed to hypobaria in both the TBI and Sham groups had elevated heart rate (HR) during the 6 h flight (p<0.05). Three animals in the TBI hypo group demonstrated leukocytosis with histologic evidence of meningeal inflammatory response. Expression of serum cytokines was low across all groups. No significant neuronal degeneration was identified in areas away from the site of injury. CONCLUSION: Aeromedical evacuation in swine was not associated with significant differences in physiologic measures, cytokine expression or levels of neuronal degeneration. Histological examination revealed higher risk of meningeal inflammatory response and leucocytosis in swine exposed to hypobaria.

Histopathological Evidence of Multiple Organ Damage After Simulated Aeromedical Evacuation in a Swine Acute Lung Injury Model.

INTRODUCTION: Rapid aeromedical evacuation (AE) is standard of care in current conflicts. However, not much is known about possible effects of hypobaric conditions. We investigated possible effects of hypobaria on organ damage in a swine model of acute lung injury. METHODS: Lung injury was induced in anesthetized swine via intravenous oleic acid infusion. After a stabilization phase, animals were subjected to a 4 hour simulated AE at 8000 feet (HYPO). Control animals were kept at normobaria. After euthanasia and necropsy, organ damage was assessed by combined scores for hemorrhage, inflammation, edema, necrosis, and microatelectasis. RESULTS: Hemodynamic, neurological, or hematologic measurements were similar prior to transport. Hemodynamic instability became apparent during the last 2 hours of transport in the HYPO group. Histological injury scores in the HYPO group were higher for all organs (lung, kidney, liver, pancreas, and adrenal glands) except the brain, with the largest difference in the lungs (P < 0.001). CONCLUSIONS: Swine with mild acute lung injury subjected to a 4 hour simulated AE showed more injury to most organs and, in particular, to the lungs compared with ground transport. This may exacerbate otherwise subclinical pathology and, eventually, manifest as abnormalities in gas exchange or possibly end-organ function.

Brain oxygenation with a non-vasoactive perfluorocarbon emulsion in a rat model of traumatic brain injury.

OBJECTIVE: The aim of this study was to assess, in two experiments, the safety and efficacy of the PFC emulsion Oxycyte as an oxygen therapeutic for TBI to test the hypothesis that early administration of this oxygen-carrying fluid post-TBI would improve brain tissue oxygenation (Pbt O2 ). METHODS: The first experiment assessed the effects of Oxycyte on cerebral vasoactivity in healthy, uninjured rats using intravital microscopy. The second experiment investigated the effect of Oxycyte on cerebral Pbt O2 using the PQM in TBI model. Animals in the Oxycyte group received a single injection of Oxycyte (6 mL/kg) shortly after TBI, while NON animals received no treatment. RESULTS: Oxycyte did not cause vasoconstriction in small- (<50 µm) or medium- (50-100 µm) sized pial arterioles nor did it cause a significant change in blood pressure. Treatment with Oxycyte while breathing 100% O2 did not improve Pbt O2 . However, in rats ventilated with ~40% O2 , Pbt O2 improved to near pre-TBI values within 105 minutes after Oxycyte injection. CONCLUSIONS: Although Oxycyte did not cause cerebral vasoconstriction, its use at the dose tested while breathing 100% O2 did not improve Pbt O2 following TBI. However, Oxycyte treatment while breathing a lower enriched oxygen concentration may improve Pbt O2 after TBI.

Cerebral Microvascular and Systemic Effects Following Intravenous Administration of the Perfluorocarbon Emulsion Perftoran.

Oxygen-carrying perfluorocarbon (PFC) fluids have the potential to increase tissue oxygenation during hypoxic states and to reduce ischemic cell death. Regulatory approval of oxygen therapeutics was halted due to concerns over vasoconstrictive side effects. The goal of this study was to assess the potential vasoactive properties of Perftoran by measuring brain pial arteriolar diameters in a healthy rat model. Perftoran, crystalloid (saline) or colloid (Hextend) solutions were administered as four sequential 30 min intravenous (IV) infusions, thus allowing an evaluation of cumulative dose-dependent effects. There were no overall changes in diameters of small-sized (<50 µm) pial arterioles within the Perftoran group, while both saline and Hextend groups exhibited vasoconstriction. Medium-sized arterioles (50-100 µm) showed minor (~8-9%) vasoconstriction within saline and Hextend groups and only ~5% vasoconstriction within the Perftoran group. For small- and medium-sized pial arterioles, the mean percent change in vessel diameters was not different among the groups. Although there was a tendency for arterial blood pressures to increase with Perftoran, pressures were not different from the other two groups. These data show that Perftoran, when administered to healthy anesthetized rats, does not cause additional vasoconstriction in cerebral pial arterioles or increase systemic blood pressure compared with saline or Hextend.

Sanguinate's effect on pial arterioles in healthy rats and cerebral oxygen tension after controlled cortical impact.

Sanguinate, a polyethylene glycol-conjugated carboxyhemoglobin, was investigated for cerebral vasoactivity in healthy male Sprague-Dawley rats (Study 1) and for its ability to increase brain tissue oxygen pressure (PbtO2) after controlled cortical impact (CCI) - traumatic brain injury (TBI) (Study 2). In both studies ketamine-acepromazine anesthetized rats were ventilated with 40% O2. In Study 1, a cranial window was used to measure the diameters of medium - (50-100µm) and small-sized (<50µm) pial arterioles before and after four serial infusions of Sanguinate (8mL/kg/h, cumulative 16mL/kg IV), volume-matched Hextend, or normal saline. In Study 2, PbtO2 was measured using a phosphorescence quenching method before TBI, 15min after TBI (T15) and then every 10min thereafter for 155min. At T15, rats received either 8mL/kg IV Sanguinate (40mL/kg/h) or no treatment (saline, 4mL/kg/h). Results showed: 1) in healthy rats, percentage changes in pial arteriole diameter were the same among the groups, 2) in TBI rats, PbtO2 decreased from 36.5±3.9mmHg to 19.8±3.0mmHg at T15 in both groups after TBI and did not recover in either group for the rest of the study, and 3) MAP increased 16±4mmHg and 36±5mmHg after Sanguinate in healthy and TBI rats, respectively, while MAP was unchanged in control groups. In conclusion, Sanguinate did not cause vasoconstriction in the cerebral pial arterioles of healthy rats but it also did not acutely increase PbtO2 when administered after TBI. Sanguinate was associated with an increase in MAP in both studies.

Perfluorocarbon NVX-108 increased cerebral oxygen tension after traumatic brain injury in rats.

BACKGROUND: Hypoxia is a critical secondary injury mechanism in traumatic brain injury (TBI), and early intervention to alleviate post-TBI hypoxia may be beneficial. NVX-108, a dodecafluoropentane perfluorocarbon, was screened for its ability to increase brain tissue oxygen tension (PbtO2) when administered soon after TBI. METHODS: Ketamine-acepromazine anesthetized rats ventilated with 40% oxygen underwent moderate controlled cortical impact (CCI)-TBI at time 0 (T0). Rats received either no treatment (NON, n=8) or 0.5 ml/kg intravenous (IV) NVX-108 (NVX, n=9) at T15 (15 min after TBI) and T75. RESULTS: Baseline cortical PbtO2 was 28±3 mm Hg and CCI-TBI resulted in a 46±6% reduction in PbtO2 at T15 (P<0.001). Significant differences in time-group interactions (P=0.013) were found when comparing either absolute or percentage change of PbtO2 to post-injury (mixed-model ANOVA) suggesting that administration of NVX-108 increased PbtO2 above injury levels while it remained depressed in the NON group. Specifically in the NVX group, PbtO2 increased to a peak 143% of T15 (P=0.02) 60 min after completion of NVX-108 injection (T135). Systemic blood pressure was not different between the groups. CONCLUSION: NVX-108 caused an increase in PbtO2 following CCI-TBI in rats and should be evaluated further as a possible immediate treatment for TBI.

The effect of the perfluorocarbon emulsion Oxycyte on platelet count and function in the treatment of decompression sickness in a swine model.

Decompression from elevated ambient pressure is associated with platelet activation and decreased platelet counts. Standard treatment for decompression sickness (DCS) is hyperbaric oxygen therapy. Intravenous perfluorocarbon (PFC) emulsion is a nonrecompressive therapy being examined that improves mortality in animal models of DCS. However, PFC emulsions are associated with a decreased platelet count. We used a swine model of DCS to study the effect of PFC therapy on platelet count, function, and hemostasis. Castrated male swine (n = 50) were fitted with a vascular port, recovered, randomized, and compressed to 180 feet of sea water (fsw) for 31 min followed by decompression at 30 fsw/min. Animals were observed for DCS, administered 100% oxygen, and treated with either emulsified PFC Oxycyte (DCS-PFC) or isotonic saline (DCS-NS). Controls underwent the same procedures, but were not compressed (Sham-PFC and Sham-NS). Measurements of platelet count, thromboelastometry, and coagulation were obtained 1 h before compression and 1, 24, 48, 96, 168 and 192 h after treatment. No significant changes in normalized platelet counts were observed. Prothrombin time was elevated in DCS-PFC from 48 to 192 h compared with DCS-NS, and from 96 to 192 h compared with Sham-PFC. Normalized activated partial thromboplastin time was also elevated in DCS-PFC from 168 to 192 h compared with Sham-PFC. No bleeding events were noted. DCS treated with PFC (Oxycyte) does not impact platelet numbers, whole blood clotting by thromboelastometry, or clinical bleeding. Late changes in prothrombin time and activated partial thromboplastin time associated with PFC use in both DCS therapy and controls warrant further investigation.

Use of recombinant factor VIIa (rFVIIa) as pre-hospital treatment in a swine model of fluid percussion traumatic brain injury.

CONTEXT: Recombinant factor VIIa (rFVIIa) has been used as an adjunctive therapy for acute post-traumatic hemorrhage and reversal of iatrogenic coagulopathy in trauma patients in the hospital setting. However, investigations regarding its potential use in pre-hospital management of traumatic brain injury (TBI) have not been conducted extensively. AIMS: In the present study, we investigated the physiology, hematology and histology effects of a single pre-hospital bolus injection of rFVIIa compared to current clinical practice of no pre-hospital intervention in a swine model of moderate fluid percussion TBI. MATERIALS AND METHODS: Animals were randomized to receive either a bolus of rFVIIa (90 µg/kg) or nothing 15 minutes (T15) post-injury. Hospital arrival was simulated at T60, and animals were euthanized at experimental endpoint (T360). RESULTS: Survival was 100% in both groups; baseline physiology parameters were similar, vital signs were comparable. Animals that received rFVIIa demonstrated less hemorrhage in subarachnoid space (P = 0.0037) and less neuronal degeneration in left hippocampus, pons, and cerebellum (P = 0.00009, P = 0.00008, and P = 0.251, respectively). Immunohistochemical staining of brain sections showed less overall loss of microtubule-associated protein 2 (MAP2) and less Flouro-Jade B positive cells in rFVIIa-treated animals. CONCLUSIONS: Early pre-hospital administration of rFVIIa in this swine TBI model reduced neuronal necrosis and intracranial hemorrhage (ICH). These results merit further investigation of this approach in pre-hospital trauma care.

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 (alpha4-integrin) expression (FACS), plasma cytokines-tumor necrosis factor (TNF)-alpha, 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-alpha 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 < or = 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.

Physiological evaluation of a rabbit kidney perfused with VS41A.

The current report compares the renal physiological impact of a standard vitrification solution, VS41A, as measured by normothermic blood perfusion, to the physiological effects of VS4, a related but more dilute vitrification solution previously shown to be consistently compatible with life support function of transplanted rabbit kidneys. VS41A, which allows survival of only about half of the kidneys perfused with it, also appeared to be more damaging than VS4 based on in vitro functional indices and histology in one rabbit kidney so evaluated.

WBC reduction in cryopreserved RBC units.

BACKGROUND: WBC reduction of blood components by filtration is widely practiced to decrease the incidence of alloimmunization. Freezing RBCs reduces the WBC load but is insufficient to achieve the currently recommended US limit of 5 x 10(6) cells per unit. STUDY DESIGN AND METHODS: Blood units were WBC reduced by filtration or by buffy-coat (BC) removal and then frozen in the presence of a high-glycerol concentration. The count of residual WBCs was determined by flow cytometry after deglycerolization. RESULTS: Without WBC reduction, the total number of WBCs present after freezing and thawing was 11.5 +/- 9.2 x 10(6) WBCs per unit (n = 18). Particulate residues from monocytes and neutrophils that were detected in the remaining cell populations were positive for CD66b, CD3, CD14, and CD41. Removal of 40 mL of BC at the time of blood collection lowered the number of WBCs after freezing and deglycerolization to 1.9 +/- 1.20 x 10(6) per unit (n = 11). Similar results were obtained when only 20 mL of BC was removed using a modified blood-bag design. Unfiltered RBC units that were stored for 15 days at 4 degrees C after BC removal contained fewer than 5 x 10(6) WBCs after deglycerolization. Units WBC reduced by filtration before freezing had no detectable WBCs after thawing and washing (n = 14) and did not contain particulate residues. Filtration after deglycerolization was effective in reducing the WBC count below 10(6), although some debris was still present. CONCLUSION: RBC freezing alone will not reduce residual counts to recommended levels. However, initial removal of BC can provide an economical alternative to WBC filtration for cryopreserved units. Units that were not WBC reduced before freezing can be filtered after deglycerolization when needed.

Normothermic blood perfusion of isolated rabbit kidneys. III. In vitro physiology of kidneys after perfusion with Euro-Collins solution or 7.5 M cryoprotectant (VS4).

Cryopreservation of solid organs might be possible using a mixture of cell-permeable agents, cryoprotectants (CPA), which are designed to completely preclude ice crystal formation during cooling to cryogenic temperatures. The effects of a specific prototype solution (VS4) were evaluated by normothermic blood perfusion in vitro. Rabbit kidneys were divided into three groups: untreated controls ( n=7), Euro-Collins (EC)-perfused controls ( n=6) and VS4 (49%, w/v) CPA-perfused kidneys ( n=7). After a 2-h blood perfusion, five of the seven CPA-perfused kidneys developed polyuria (0.21 mlxmin(-1)xg(-1)) relative to untreated controls (0.07 mlxmin(-1)xg(-1)) or EC-perfused kidneys (0.06 mlxmin(-1)xg(-1)), owing to the lower reabsorption of water (34.3%), Na(+) (34.2%) and glucose (35.6%). Furthermore, two kidneys were non-functional with virtually no urine production. Reduced tubular function was associated with reduced oxygen consumption (3.6 versus 2.3 versus 2.0 micromolexmin(-1)xg(-1) for controls, EC- and CPA-perfused kidneys, respectively) and increased weight gain (17% versus 20% versus 30%, respectively) after blood perfusion. Therefore, the current results provide insight into both the physiological effects of VS4 and the limits of reversibility of renal pathophysiological states. Our results also indicate that in vitro monitoring of oxygen consumption and weight gain of perfused organs could be used as predictors of renal function.