Edward D. Hall

Clinical Trials in Head Injury

Secondary brain damage, following severe head injury is considered to be a major cause for bad outcome. Impressive reductions of the extent of brain damage in experimental studies have raised high expectations for cerebral neuroprotective treatment, in the clinic. Therefore multiple compounds were and are being evaluated in trials. In this review we discuss the pathomechanisms of traumatic brain damage, based upon their clinical importance. The role of hypothermia, mannitol, barbiturates, steroids, free radical scavengers, arachidonic acid inhibitors, calcium channel blockers, N-methyl-D-aspartate (NMDA) antagonists, and potassium channel blockers, will be discussed. The importance of a uniform strategic approach for evaluation of potentially interesting new compounds in clinical trials, to ameliorate outcome in patients with severe head injury, is proposed. To achieve this goal, two nonprofit organizations were founded: the European Brain Injury Consortium (EBIC) and the American Brain Injury Consortium (ABIC). Their aim lies in conducting better clinical trials, which incorporate lessons learned from previous trials, such that the succession of negative, or incomplete studies, as performed in previous years, will cease.

Central nervous system trauma and stroke. I. Biochemical considerations for oxygen radical formation and lipid peroxidation.

The generation of oxygen radicals and the process of lipid peroxidation have become a focus of attention for investigators in the fields of central nervous system (CNS) trauma and stroke (e.g., ischemia). Considering our level of understanding of free radical and lipid peroxidation chemistry, absolute proof for their involvement in the pathophysiology of traumatic and ischemic damage to the CNS has been meager. While direct, unequivocal evidence for the participation of free radicals and lipid peroxidation as primary contributors to the death of neuronal tissue waits to be established, numerous recent studies have provided considerable support for the occurrence of free radical and lipid peroxidation reactions in the injured or ischemic CNS. In addition, the pharmacological use of antioxidants and free radical scavengers in the treatment of experimental CNS trauma and ischemia has provided convincing, although indirect evidence, for the involvement of oxygen radicals and lipid peroxidation in these conditions. The intent of this and its companion paper is to review: 1) the biochemical processes which may give rise to free radical reactions in the CNS, 2) the environment of the ischemic cell as it may affect the generation of oxygen radicals and the catalysis of lipid peroxidation reactions, 3) the evidence for the involvement of free radical mechanisms in CNS trauma and ischemia, and 4) the pathophysiological consequences of these phenomena.

Central nervous system trauma and stroke. II. Physiological and pharmacological evidence for involvement of oxygen radicals and lipid peroxidation.

The previous article outlined the biochemical basis and evidence for the occurrence of oxygen radical generation and lipid peroxidation during the acute phase of central nervous system (CNS) trauma or stroke (ischemic and hemorrhagic). The identification of oxygen radicals and lipid peroxidation as important pathophysiological mediators of trauma or stroke-induced neural degeneration, rather than simply epiphenomena, depends upon the successful demonstration of their association with actual secondary physiological and structural degenerative events. Moreover, their significance in the pathophysiology of CNS trauma or stroke must be supported by experimental observations that pharmacological antagonism of either oxygen radical generation and/or lipid peroxidation results in a therapeutic effect (i.e., interruption of secondary nervous tissue degeneration). Indeed, recent investigations have provided compelling evidence for the view that oxygen radical-mediated processes play a key pathophysiological role during the acute phase of CNS trauma or stroke. Furthermore, their pharmacological manipulation may serve as an avenue for therapeutic attempts aimed at limiting neural degeneration and improving neurological recovery.

Neuroprotection and Acute Spinal Cord Injury: A Reappraisal

SummaryIt has long been recognized that much of the post-traumatic degeneration of the spinal cord following injury is caused by a multi-factorial secondary injury process that occurs during the first minutes, hours, and days after spinal cord injury (SCI). A key biochemical event in that process is reactive oxygen-induced lipid peroxidation (LP). In 1990 the results of the Second National Acute Spinal Cord Injury Study (NASCIS II) were published, which showed that the administration of a high-dose regimen of the glucocorticoid steroid methylprednisolone (MP), which had been previously shown to inhibit post-traumatic LP in animal models of SCI, could improve neurological recovery in spinal-cord-injured humans. This resulted in the registration of high-dose MP for acute SCI in several countries, although not in the U.S. Nevertheless, this treatment quickly became the standard of care for acute SCI since the drug was already on the U.S. market for many other indications. Subsequently, it was demonstrated that the non-glucocorticoid 21-aminosteroid tirilazad could duplicate the antioxidant neuroprotective efficacy of MP in SCI models, and evidence of human efficacy was obtained in a third NASCIS trial (NASCIS III). In recent years, the use of high-dose MP in acute SCI has become controversial largely on the basis of the risk of serious adverse effectsversus what is perceived to be on average a modest neurological benefit. The opiate receptor antagonist naloxone was also tested in NASCIS II based upon the demonstration of its beneficial effects in SCI models. Although it did not a significant overall effect, some evidence of efficacy was seen in incomplete (i.e., paretic) patients. The monosialoganglioside GM1 has also been examined in a recently completed clinical trial in which the patients first received high-dose MP treatment. However, GM1 failed to show any evidence of a significant enhancement in the extent of neurological recovery over the level afforded by MP therapy alone. The present paper reviews the past development of MP, naloxone, tirilazad, and GM1 for acute SCI, the ongoing MP-SCI controversy, identifies the regulatory complications involved in future SCI drug development, and suggests some promising neuroprotective approaches that could either replace or be used in combination with high-dose MP.

21-Aminosteroid lipid peroxidation inhibitor U74006F protects against cerebral ischemia in gerbils.

U74006F (21-[4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl]-16 alpha-methylpregna-1,4,9(11)-triene-3,20-dione, monomethane sulfonate) is a novel and potent inhibitor of central nervous system tissue lipid peroxidation that is devoid of classical steroid hormonal activities. Its possible efficacy in attenuating postischemic mortality and neuronal necrosis was examined in gerbils following 3-hour unilateral carotid artery occlusion. Male Mongolian gerbils received two intraperitoneal injections of either vehicle or U74006F (3 or 10 mg/kg), the first injection 10 minutes before and the second injection at the end of the 3-hour ischemic episode. In an initial series of experiments, vehicle-treated gerbils displayed 60.9% (14 of 23) survival 24 hours after ischemia, which decreased to 34.8% (8 of 23) at 48 hours. In contrast, the 10 mg/kg U74006F-treated group showed 86.7% (13 of 15) survival at 24 hours (p less than 0.15 vs. vehicle) and 80.0% (12 of 15) survival at 48 hours (p less than 0.02). In a second series, neurons in the hippocampal CA1 subfield and the medial and lateral cerebral cortex were counted in gerbils surviving 24 hours after unilateral carotid artery occlusion. Comparison of neuronal densities in the ischemic hemisphere with those in the contralateral nonischemic hemisphere revealed significant neuronal preservation in all three brain regions of 10 mg/kg i.p. x 2 U74006F-treated gerbils. Our results show that U74006F can improve survival and attenuate neuronal necrosis in a severe brain ischemia model.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein oxidative damage in a transgenic mouse model of familial amyotrophic lateral sclerosis.

Abstract: The Gly93→Ala mutation in the Cu,Zn superoxide dismutase (Cu,Zn‐SOD) gene (SOD1) found in some familial amyotrophic lateral sclerosis (FALS) patients has been shown to result in an aberrant increase in hydroxyl radical production by the mutant enzyme that may cause oxidative injury to spinal motor neurons. In the present study, we analyzed the extent of oxidative injury to lumbar and cervical spinal cord proteins in transgenic FALS mice that overexpress the SOD1 mutation [TgN(SOD1‐G93A)G1H] in comparison with nontransgenic mice. Total protein oxidation was examined by spectrophotometric measurement of tissue protein carbonyl content by the dinitrophenylhydrazine (DNPH) assay. Four ages were investigated: 30 (pre‐motor neuron pathology and clinical disease), 60 (after initiation of pathology, but pre‐disease), 100 (∼50% loss of motor neurons and function), and 120 (near complete hindlimb paralysis) days. Protein carbonyl content in 30‐day‐old TgN(SOD1‐G93A)G1H mice was twice as high as the level found in age‐matched nontransgenic mice. However, at 60 and 100 days of age, the levels were the same. Then, between 100 and 120 days of age, the levels in the TgN(SOD1‐G93A)G1H mice increased dramatically (557%) compared with either the nontransgenic mice or transgenic animals that overexpress the wild‐type human Cu,Zn‐SOD [TgN(SOD1)N29]. The 100–120‐day increase in spinal cord protein carbonyl levels was confirmed by sodium dodecyl sulfate‐polyacrylamide gel electrophoretic separation and western blot immunoassay, which enabled the identification of heavily oxidized individual proteins using a monoclonal antibody against DNPH‐derivatized proteins. One of the more heavily oxidized protein bands (14 kDa) was identified by immunoprecipitation as largely Cu,Zn‐SOD. Western blot comparison of the extent of Cu,Zn‐SOD protein carbonylation revealed that the level in spinal cord samples from 120‐day‐old TgN(SOD1‐G93A)G1H mice was significantly higher than that found in age‐matched nontransgenic or TgN(SOD1)N29 mice. These results suggest that the increased hydroxyl radical production associated with the G93A SOD1 mutation and/or lipid peroxidation‐derived radical species (peroxyl or alkoxyl) causes extensive protein oxidative injury and that the Cu,Zn‐SOD itself is a key target, which may compromise its antioxidant function.

Sex Differences in Postischemic Neuronal Necrosis in Gerbils

Twenty-four hour postischemic neuronal necrosis was compared in male vs. female Mongolian gerbils subjected to a 3-h period of severe incomplete hemispheric ischemia produced by unilateral carotid occlusion. The incidence of stroke-prone males was 42.9% versus 26.7% for the females. Among the stroke-prone animals, the males displayed significantly greater neuronal necrosis at 24 h after ischemia compared to the females in the cerebral cortex and CA, region of the hippocampus. In the CA, region of the stroke-prone males, only 2.0% of the normal neuronal population remained by 24 h compared to 36.8% in the stroke-prone females (p < 0.02). In the cerebral cortex, the males had only 19.9% of normal versus 58.2% in the females (p < 0.05). In a second series of mechanistic experiments, no differences in cortical blood flow (CBF) were disclosed between preselected male and female stroke-prone animals before, during, or for 2 h after ischemia. As with the CBF, the extent of cortical extracellular hypocalcia during ischemia did not differ significantly. However, the degree of postischemic recovery of cortical extracellular calcium was significantly better in the females from 30 min to 2 h after reperfusion. In the same experiments, hemispheric vitamin E levels were measured at the 2 h time point as an index of postischemic brain lipid peroxidation. No difference in baseline vitamin E levels was observed between male and female sham-operated gerbils. In the males subjected to 3 h of ischemia plus 2 h of reperfusion, the hemispheric vitamin E decreased by 43.5% compared to the sham-operated males. In contrast, the females displayed only a 4.2% decline (p < 0.05 versus males). Previous studies showing the protective efficacy of antioxidants in this model have suggested an important role of oxygen radical-induced lipid peroxidation. Thus, it is proposed that the lesser ischemic vulnerability of females may be based upon an antioxidant effect of endogenous estrogen. Indeed, estrogen was found to be a more potent inhibitor of iron-catalyzed lipid peroxidation in brain tissue than vitamin E.

Antioxidant therapies for traumatic brain injury

SummaryFree radical-induced oxidative damage reactions, and membrane lipid peroxidation (LP), in particular, are among the best validated secondary injury mechanisms in preclinical traumatic brain injury (TBI) models. In addition to the disruption of the membrane phospholipid architecture, LP results in the formation of cytotoxic aldehyde-containing products that bind to cellular proteins and impair their normal functions. This article reviews the progress of the past three decades in regard to the preclinical discovery and attempted clinical development of antioxidant drugs designed to inhibit free radical-induced LP and its neurotoxic consequences via different mechanisms including the O2·− scavenger Superoxide dismutase and the lipid peroxidation inhibitor tirilazad. In addition, various other antioxidant agents that have been shown to have efficacy in preclinical TBI models are briefly presented, such as the LP inhibitors U83836E, resveratrol, curcumin, OPC-14177, and lipoic acid; the iron chelator deferoxamine and the nitroxide-containing antioxidants, such as α-phenyl-tert-butyl nitrone and tempol. A relatively new antioxidant mechanistic strategy for acute TBI is aimed at the scavenging of aldehydic LP byproducts that are highly neurotoxic with “carbonyl scavenging” compounds. Finally, it is proposed that the most effective approach to interrupt posttraumatic oxidative brain damage after TBI might involve the combined treatment with mechanistically complementary antioxidants that simultaneously scavenge LP-initiating free radicals, inhibit LP propagation, and lastly remove neurotoxic LP byproducts.

Pathophysiology of spinal cord trauma

This article reviews the pathophysiology of spinal cord injury. The focus is on the role of post-traumatic membrane lipid changes, including lipid hydrolysis with enzymatic lipid peroxidation (ie, eicosanoid production) and nonenzymatic, free radical-induced lipid peroxidation in the secondary autodestruction of injured spinal cord tissue. A speculative etiopathogenesis of secondary injury is presented in an attempt to explain the importance and order of the pathophysiologic events that result in tissue death and the apparent effectiveness of diverse pharmacologic agents in the treatment of experimental spinal cord injury.

Time Course of Post-Traumatic Mitochondrial Oxidative Damage and Dysfunction in a Mouse Model of Focal Traumatic Brain Injury: Implications for Neuroprotective Therapy

In the present study, we investigate the hypothesis that mitochondrial oxidative damage and dysfunction precede the onset of neuronal loss after controlled cortical impact traumatic brain injury (TBI) in mice. Accordingly, we evaluated the time course of post-traumatic mitochondrial dysfunction in the injured cortex and hippocampus at 30 mins, 1, 3, 6, 12, 24, 48, and 72 h after severe TBI. A significant decrease in the coupling of the electron transport system with oxidative phosphorylation was observed as early as 30 mins after injury, followed by a recovery to baseline at 1 h after injury. A statistically significant (P < 0.0001) decline in the respiratory control ratio was noted at 3 h, which persisted at all subsequent time-points up to 72 h after injury in both cortical and hippocampal mitochondria. Structural damage seen in purified cortical mitochondria included severely swollen mitochondria, a disruption of the cristae and rupture of outer membranes, indicative of mitochondrial permeability transition. Consistent with this finding, cortical mitochondrial calcium-buffering capacity was severely compromised by 3h after injury, and accompanied by significant increases in mitochondrial protein oxidation and lipid peroxidation. A possible causative role for reactive nitrogen species was suggested by the rapid increase in cortical mitochondrial 3-nitrotyrosine levels shown as early as 30 mins after injury. These findings indicate that post-traumatic oxidative lipid and protein damage, mediated in part by peroxynitrite, occurs in mitochondria with concomitant ultrastructural damage and impairment of mitochondrial bioenergetics. The data also indicate that compounds which specifically scavenge peroxynitrite (ONOO) or ONOO−derived radicals (e.g. ONOO− + H+ → ONOOH → †NO2 + †OH) may be particularly effective for the treatment of TBI, although the therapeutic window for this neuroprotective approach might only be 3 h.