Richard Michael McCarron

Hemorrhaged animals have benefited from resuscitation with the hemoglobin-based oxygen carrier (HBOC-201). Co-infusion of nitric oxide (NO) via separate intravascular lines is effective in attenuating HBOC-induced elevation of blood pressure. We tested whether nitroglycerin (NTG) and HBOC-201 can be packaged together as a single drug for resuscitation. Since NTG binds easily to plastics such as polyvinylchloride, we assessed the stability of this combination in oxygen barrier double-layer ethylene-vinyl alcohol/polyolefin bags over a 30-day period. Outcome measures indicative of the stability of HBOC/NTG were reported as changes in levels of hemoglobin (Hb), methemoglobin (MetHb), NTG, and nitrite over time. Individual tightly sealed small aliquots of HBOC/NTG were prepared under nitrogen and analyzed in a timely fashion from 0 to 30 days using hematology instruments, HPLC, FPLC, and chemiluminescence. The level of NTG in the HBOC/NTG mixture was reduced significantly over time whereas it was stable in control mixtures of NTG/saline. The level of total Hb in the HBOC/NTG and HBOC/saline mixtures remained stable over time. MetHb formed and increased to 6% up to day 1 and then slowly decreased in the HBOC/NTG mixture whereas it remained unchanged in the HBOC/saline mixture. Nitrite was produced in the HBOC/NTG group upon mixing, was increased at day 1, and then became undetectable. The reaction between HBOC-201 and NTG occurring upon mixing and developing over time in polyolefin bags makes the long-term storage of this mixed combination inappropriate.

Background/aims Hemoglobin-based oxygen carriers (HBOCs) have been previously studied as resuscitation fluids. Due to HBOCs specific molecular conformation, hemoglobin (Hb) and methemoglobin (MetHb) determination is not always possible with automated apparatus. A practical technique was designed that allows simultaneous reading of MetHb and Hb in small volume samples. Methods A spectrophotometric method for measuring low levels of MetHb and Hb from limited volume samples was developed using a 96-well-plate by downsizing the Evelyn-Malloy and Drabkin methods. Either blood or buffer samples were spiked with one of five HBOCs (HBOC-201, M101, MP4CO-NP, Sanguinate and Oxyvita C). After treatment with cyanides, the samples were read at 540, 630, and 680 nm, and Hb and MetHb results were compared to certificate-of-analysis. Results Hb levels ranging from 0.2 to 2.8 g/dl were detected accurately with the 96-well-plate method with HBOC-201. Similarly, this method accurately measured Hb from either plasma or buffer samples containing any of the HBOCs. The MetHb plasma samples with HBOC-201 were also in agreement with ABL results (R = 0.99719). MetHb from all HBOCs in buffer measured with this method was comparable to reference but the accuracy was compromised for HBOCs in blood. Conclusions A useful 96-well-plate method of measuring HBOCs’ Hb was designed for small-volume plasma samples. It was accurate for measuring MetHb from samples, that contained M101, MP4CO-NP, Sanguinate, and Oxyvita C diluted in buffer. This well-plate method allows reading of batch samples, multiple replicates, and using small volumes to accommodate limited animal blood collection which would not be otherwise detected by automated instrumentation.

The use of hemoglobin-based oxygen carriers (HBOC) as oxygen delivering therapies during hypoxic states has been hindered by vasoconstrictive side effects caused by depletion of nitric oxide (NO). OxyVita C is a promising oxygen-carrying solution that consists of a zero-linked hemoglobin polymer with a high molecular weight (~17 MDa). The large molecular weight is believed to prevent extravasation and limit NO scavenging and vasoconstriction. The aim of this study was to assess vasoactive effects of OxyVita C on systemic blood pressures and cerebral pial arteriole diameters. Anesthetized healthy rats received four intravenous (IV) infusions of an increasing dose of OxyVita C (2, 25, 50, 100 mg/kg) and hemodynamic parameters and pial arteriolar diameters were measured pre- and post-infusion. Normal saline was used as a volume-matched control. Systemic blood pressures increased (P ≤ 0.05) with increasing doses of OxyVita C, but not with saline. There was no vasoconstriction in small (<50 µm) and medium-sized (50–100 µm) pial arterioles in the OxyVita C group. In contrast, small and medium-sized pial arterioles vasoconstricted in the control group. Compared to saline, OxyVita C showed no cerebral vasoconstriction after any of the four doses evaluated in this rat model despite increases in blood pressure.

Blast-related traumatic brain injury (TBI) is a common cause of injury in the military operations in Iraq and Afghanistan. How the primary blast wave affects the brain is not well understood. The aim of the present study was to examine whether blast exposure affects the cerebral vasculature in a rodent model. We analyzed the brains of rats exposed to single or multiple (three) 74.5 kPa blast exposures, conditions that mimic a mild TBI. Rats were sacrificed 24 hours or between 6 and 10 months after exposure. Blast-induced cerebral vascular pathology was examined by a combination of light microscopy, immunohistochemistry, and electron microscopy.

There is a dearth of knowledge about the effects of long range aero-medical evacuation on injured organs, as well as an emerging published database suggesting clinically significant adverse effects of hypobaria on even healthy tissues. Cabin pressure is equivalent to an altitude around 8,000ft. at which inspired oxygen is sufficient to maintain blood oxygen saturation above 90% in a healthy individual. In combat casualties with multiple injuries this could however compromise oxygen delivery and result in hypoxemia. Additionally, increase in altitude with concomitant decrease in atmospheric pressure allows gas expansion in body cavities. The volume of trapped gas expands by approximately 35% from sea level to an altitude of 8,000 feet. This can expose already vulnerable patients to severe complications. In light of this, a thorough investigation of the effects of hypobaria in clinical settings simulating the most important injury patterns encountered by combat casualties is necessary to optimize treatment efficacy and safety. During the first year of this project, the Naval Medical Research Center (NMRC) designed and built a chamber that can be de-pressurized to atmospheric pressure at 8,000ft. Chamber construction has been completed and swine experiments have been initiated. The NMRC Center for Hypobaric Experimentation, Simulation and Testing (CHEST) provides a unique platform for evaluation of long-range evacuation effects on physiology and therapeutic interventions in military relevant large animal combat injury models, which may contribute to optimization of combat casualty care and evacuation guidelines.

The long-term monitoring of intracranial pressure (ICP) is important for the management of acute and chronic neuropathological conditions which include head injury, traumatic brain injury, hydrocephalus, etc. In this study, we developed an implantable device for measuring ICP over long periods of time in an animal model of blast-induced brain injury. The performance of the device was first evaluated in vitro and subsequently utilized to measure ICP in rats exposed to blast overpressures. The effects of blast-induced brain injury on ICP were measured for six days. A significant difference was observed between the injured group and the nonexposed control group. ICP in injured animals showed a biphasic transient increase; an immediate increase within the first 1-3 h and a more gradual elevation occurring two days after the blast. The ability to monitor changes of ICP continuously over long periods after brain injury and during the course of treatment may improve the prognosis after injury and can also serve as a tool in determining the therapeutic effectiveness of new drugs.

Background: Blast-induced neurotrauma (BINT) is the signature life threatening injury of current military casualties. Neuroinflammation is a key pathological occurrence of secondary injury contributing to brain damage after blast injury. We have recently demonstrated that blast-triggered complement activation and cytokine release are associated with BINT. Here, we evaluated if administration of the complement inhibitor recombinant human decay-accelerating factor (rhDAF) is beneficial on neuroinflammation and neurodegeneration in a rat model of moderate BINT. Administration of rhDAF after exposure to moderate blast overpressure (BOP, 120 kPa) mitigated brain injury characterized by neuronal degeneration. rhDAF treatment reduced complement hemolytic activity at 3 hours and tissue complement deposition at 3, 24, and 48 hours as well as systemic and local cytokine release at 24 hours post BOP. Furthermore, rhDAF protected blood–brain barrier (BBB) integrity and reduced cytotoxic edema. Interaction between complement cleavage component, C3a and C3a receptor and tau phosphorylation were also attenuated in rhDAF treated animals at 3 and 24 hours after BOP. These novel findings suggest early complement targeted inhibition as a new therapeutic strategy to decrease neuroinflammation and neurodegeneration after blast TBI. Result: Administration of rhDAF after exposure to moderate blast overpressure (BOP, 120 kPa) mitigated brain injury characterized by neuronal degeneration. rhDAF treatment reduced complement hemolytic activity at 3 hours and tissue complement deposition at 3, 24, and 48 hours as well as systemic and local cytokine release at 24 hours post BOP. Furthermore, rhDAF protected blood–brain barrier (BBB) integrity and reduced cytotoxic edema. Interaction between complement cleavage component, C3a and C3a receptor and tau phosphorylation were also attenuated in rhDAF treated animals at 3 and 24 hours after BOP. Conclusion: These novel findings suggest early complement targeted inhibition as a new therapeutic strategy to decrease neuroinflammation and neurodegeneration after blast TBI.

Blast-related traumatic brain injury (TBI) has been a significant cause of injury in the military operations of Iraq and Afghanistan, affecting as many as 10-20% of returning veterans. However, how blast waves affect the brain is poorly understood. To understand their effects, we analyzed the brains of rats exposed to single or multiple (three) 74.5 kPa blast exposures, conditions that mimic a mild TBI.

Mild traumatic brain injury (mTBI) resulting from exposure to improvised explosive devices (IEDs) has fueled a requirement to develop animals models that mirror this condition using exposure to blast overpressure (BOP). En route to developing a model of repeated exposure to BOP we sought to initially characterize the effects of acute BOP exposure in rodents, focusing specifically on the levels of BOP exposure that produced clinical mTBI symptoms. We first measured BOP effects on gross motor function on a balance beam. Separate groups of unanesthetized rats were exposed (in different orientations) to 36.6, 74.5, and 116.7 kPa BOP exposure inside a pneumatically driven shock tube. Results demonstrated that rats exposed to 116.7 kPa demonstrated transient alterations or loss of consciousness indicated by a transient loss of righting and by increased latencies on the balance beam. The 116.7 kPa exposure was the threshold for overt pathology for acute BOP exposure with approximately 30% of rats presenting with evidence of subdural hemorrhage and cortical contusions. All animals exposed to 116.7 kPa BOP manifested evidence of significant pulmonary hemorrhage. Anterograde memory deficits were observed in rats exposed to 74.5 kPa facing the BOP wave and rats exposed to 116.7 kPa in the lateral (side) orientation. We next assessed repeated exposure to either lateral or frontal 36.6 kPa BOP in anesthetized rats, once per day for 12 days. Results showed that repeated exposure in the frontal, but not side, orientation to the BOP wave produced a transitory learning deficit on a Morris water maze task as shown by significantly longer latencies to reach the submerged platform in the second and third blocks of a four block session. Implications of these data are discussed in relation to the manifestation of mTBI in military personnel exposed to IEDs. Finally, we suggest that there are multiple types of long-term brain injury from blast exposure.