Charles Auker

Relationship between protein nitration and oxidation and development of hyperoxic seizures

Recent studies have implicated nitric oxide (NO*) as a mediator of CNS hyperbaric O2 (HBO2) toxicity. One mechanism by which NO* may contribute to HBO2-induced brain toxicity involves a neurotoxic, pro-oxidative action of NO* via the formation of the potent oxidant peroxynitrite (ONOO-). The present study compares: (a) the formation of protein nitrotyrosine as a marker of ONOO- accumulation and (b) protein oxidation as an indicator of reactive oxygen species production during HBO2 exposure. Rats were exposed to 5 atm 100% O2 to pre-convulsive exposure or until the occurrence of electroencephalographic (EEG) seizures. After exposures, brains were analyzed for protein nitrotyrosine (NT) and protein carbonyl measurement by Western blot and for superoxide dismutase (SOD) activity by NBT assay. The results show a significant increase in protein NT, exceeding control level by several fold. There was only a slow and non-significant increase in the quantity of oxidized proteins during the pre-convulsive phase of HBO2 exposure. Levels of both protein NT and protein carbonyls were significantly (p<0.05) elevated after seizures. Total SOD activity was not changed during preconvulsive exposures, but was significantly (p<0.05) elevated post-seizures. The specific neuronal nitric oxide synthase (NOS) inhibitor, 7-nitroindazole (7-NI), significantly reduced the increases in seizure-induced protein NT and protein carbonyl and at the same time very effectively (p<0.05) delayed onset of HBO2 seizures. Pre-seizure increases in protein NT might indicate its role in the mechanism of HBO2-induced brain toxicity. This is supported by the observed capacity of 7-NI to inhibit tyrosine nitration and increase time to seizure.

Microarray Analysis of Gene Expression in Rat Cortical Neurons Exposed to Hyperbaric Air and Oxygen

To gain a global view of the genomic response of neurons to normobaric and hyperbaric hyperoxic stress, we performed a microarray analysis of gene expression after exposure to varying levels of partial oxygen pressures. Rat neurons were exposed to normobaric hyperoxia, hyperbaric (2, 4, and 6 atmosphere absolute) air or hyperbaric O2. We identified 183 genes significantly altered (increased or decreased ≥1.5-fold) in response to pressure and/or oxidative stress. Among them, 17 genes changed in response to all exposure conditions. More genes were altered in response to hyperbaric air than hyperbaric O2. The altered genes included factors associated with stress responses, transport/neurotransmission, signal transduction, and transcription factors. The results may serve as guidance for selection of biomarkers of hyperoxia and hyperbaric O2 response and provide a starting point for further studies to investigate the global molecular mechanisms underlying hyperbaric oxidative stress.

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.

Advances in Intracranial Pressure Monitoring and Its Significance in Managing Traumatic Brain Injury.

Intracranial pressure (ICP) measurements are essential in evaluation and treatment of neurological disorders such as subarachnoid and intracerebral hemorrhage, ischemic stroke, hydrocephalus, meningitis/encephalitis, and traumatic brain injury (TBI). The techniques of ICP monitoring have evolved from invasive to non-invasive—with both limitations and advantages. Some limitations of the invasive methods include short-term monitoring, risk of infection, restricted mobility of the subject, etc. The invasiveness of a method limits the frequency of ICP evaluation in neurological conditions like hydrocephalus, thus hampering the long-term care of patients with compromised ICP. Thus, there has been substantial interest in developing noninvasive techniques for assessment of ICP. Several approaches were reported, although none seem to provide a complete solution due to inaccuracy. ICP measurements are fundamental for immediate care of TBI patients in the acute stages of severe TBI injury. In severe TBI, elevated ICP is associated with mortality or poor clinical outcome. ICP monitoring in conjunction with other neurological monitoring can aid in understanding the pathophysiology of brain damage. This review article presents: (a) the significance of ICP monitoring; (b) ICP monitoring methods (invasive and non-invasive); and (c) the role of ICP monitoring in the management of brain damage, especially TBI.

Fluid resuscitation of uncontrolled hemorrhage using a hemoglobin-based oxygen carrier: effect of traumatic brain injury.

ABSTRACT Animal models of combined traumatic brain injury (TBI) and hemorrhagic shock (HS) suggest a benefit of hemoglobin-based oxygen carrier (HBOC)–based resuscitation, but their use remains controversial, and little is known of the specific effects of TBI and high-pressure (large arterial injury) bleeding on resuscitation. We examine the effect of TBI and aortic tear injury on low-volume HBOC resuscitation in a swine polytrauma model and hypothesize that HBOC-based resuscitation will improve survival in the setting of aortic tear regardless of the presence of TBI. Anesthetized swine subjected to HS with aortic tear with or without fluid percussion TBI underwent equivalent limited resuscitation with HBOC, lactated Ringer’s solution, or HBOC + nitroglycerine (vasoattenuated HBOC) and were observed for 6 h. There was no independent effect of TBI on survival time after adjustment for fluid type, and there was no interaction between TBI and resuscitation fluid type. However, total catheter hemorrhage volume required to reach target shock blood pressure was less with TBI (14.0 mL · kg−1 [confidence interval, 12.4–15.6 mL · kg−1]) versus HS only (21.0 mL · kg−1 [confidence interval, 19.5–22.5 mL · kg−1]), with equivalent lactate accumulation. Traumatic brain injury did not affect survival in this polytrauma model, but less hemorrhage was required in the presence of TBI to achieve an equivalent degree of shock suggesting globally impaired cardiovascular response to hemorrhage in the presence of TBI. There was also no benefit of HBOC-based fluid resuscitation over lactated Ringer’s solution, contrary to models using liver injury as the source of hemorrhage, considering wound location is of paramount importance when choosing resuscitation strategy.

Intravenous Perfluorocarbon After Onset of Decompression Sickness Decreases Mortality in 20-kg Swine

INTRODUCTION Decompression sickness (DCS) occurs when bubbles form due to pressure decreases with severity ranging from trivial to fatal. Standard treatment requires a hyperbaric chamber, not likely to be available at remote sites or during a disabled submarine escape or rescue. Alternative (non-recompressive) treatments are needed. Intravenous administration of emulsified perfluorocarbons (PFCs) enhances oxygen delivery to, and inert gas removal from, tissues. Swine studies show PFCs administered with supplemental oxygen before symptom onset can decrease DCS incidence. We used a swine model to test whether PFC plus supplemental oxygen could improve outcome when infused after DCS symptom onset. METHODS After rapid decompression from 31 min at 200 fsw (7.06 ATA) animals were observed for signs of DCS. Upon DCS onset animals received 100% 02 and were randomized to receive either saline or PFC. Oxygen administration was continued for 1 h and the primary outcomes of mortality and/or abnormal gait were noted 24 h after surfacing. RESULTS PFC significantly improved survival, with 18/25 (72%) PFC treated animals and 13/29 (45%) saline treated animals alive at 24 h post-exposure. Objective measures of stance/gait trended toward improvement; spinal cord lesions correlated with severity of stance/gait abnormalities. CONCLUSION PFC administered after DCS onset improved survival in this 20-kg swine model. Further study into the mechanisms of benefit and delayed DCS therapy are warranted.

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.

Effects of Top-Loading a Zero-Link Bovine Hemoglobin, OxyVita, on Systemic and Microcirculatory Variables

This study was designed to test the effect of top-load infusions of increasing doses of two versions of the novel, high molecular weight hemoglobin-based oxygen carrier, OxyVita and OxyVita C solution ([Hb] = 6 g/dL), on mean arterial pressure (MAP), arteriolar diameter, and tissue oxygenation. Experiments were carried out on 18 anesthetized male Sprague-Dawley rats in which microcirculatory observations were made on the spinotrapezius muscle. Intravenous infusions of four increasing doses of the OxyVita solutions (2, 22, 230, and 780 mg/kg) were made for each group, and a separate group of animals was used for volume control. Tissue oxygenation was measured as interstitial fluid (ISF) PO2 using phosphorescence quenching microscopy. Increasing doses of either OxyVita solution or Lactated Ringers solution (LRS, volume control) were associated with increasing MAP. For LRS infusions, MAP returned to baseline between each incremental dose injected, whereas there was an incomplete return for either of the OxyVita solutions. ISF PO2 for OxyVita was significantly lower than that for either LRS or OxyVita C, whereas ISF PO2 for OxyVita C was never statistically different from LRS. There were no significant changes in arteriolar diameters for LRS and either of the OxyVita solutions.

Effects of N-Acetyl-L-Cysteine and Hyaluronic Acid on HBOC-201- Induced Systemic and Cerebral Vasoconstriction in the Rat

Hemoglobin-based oxygen carrier-201 (HBOC) was developed as a resuscitative fluid but concerns exist over potentially adverse vasoconstriction. This study evaluated whether concurrent IV (intra venous) N-acetyl-L-cysteine (NAC) or hyaluronic acid (HA) would attenuate HBOC-associated vasoconstriction, assessed by systemic blood pressures and cerebral pial microvasculature, when administered to healthy, anesthetized rats. Rats (8-9/group) received a 30 min infusion of 3 ml/kg HBOC, HBOC plus 600 mg/kg NAC (HBOC/NAC), HBOC plus 1.5 mg/kg HA (HBOC/HA) or 3 ml/kg Albumin. Mean (MAP) and systolic (SBP) blood pressures, blood chemistries and cerebral pial vessel diameters were measured at baseline, end of infusion, and intermittently for an additional 90 min. HBOC caused immediate and sustained increases in SBP and MAP (35.3 ± 3.6 and 29.1 ± 2.5 mm Hg peak increases above baseline, respectively; mean ± SEM) and immediate but progressive vasoconstriction (11 µm maximum reduction) in medium-sized (50-100 µm) pial arterioles. When NAC was co-administered, blood pressure changes were attenuated and vessel changes were abolished. Similar trends were noted with co-administration of HA but were not statistically different from HBOC-alone. Small-sized (< 50 µm) pial vessels and blood parameters showed no differences from baseline or among groups. No adverse clinical signs were observed. We demonstrated that it is possible for adjuvant drugs to reduce the vasoconstriction associated with HBOC-201. Coinfusion of the anti-oxidant NAC mitigated HBOC-201-associated increases in blood pressures and vasoconstriction in medium-sized cerebral pial vessels. The drag-reducing polymer HA may be more effective at a higher dose as a similar but non-significant trend was observed.