Attenuation of acute mitochondrial dysfunction after traumatic brain injury in mice by NIM811, a non-immunosuppressive cyclosporin A analog.

Following traumatic brain injury (TBI), mitochondrial function becomes compromised. Mitochondrial dysfunction is characterized by intra-mitochondrial Ca(2+) accumulation, induction of oxidative damage, and mitochondrial permeability transition (mPT). Experimental studies show that cyclosporin A (CsA) inhibits mPT. However, CsA also inhibits calcineurin. In the present study, we conducted a dose-response analysis of NIM811, a non-calcineurin inhibitory CsA analog, on mitochondrial dysfunction following TBI in mice, and compared the effects of the optimal dose of NIM811 (10 mg/kg i.p.) against an optimized dose of CsA (20 mg/kg i.p.). Male CF-1 mice were subjected to severe TBI utilizing the controlled cortical impact model. Mitochondrial respiration was assessed from animals treated with either NIM811, CsA, or vehicle 15 min post-injury. The respiratory control ratio (RCR) of mitochondria from vehicle-treated animals was significantly (p<0.01) lower at 3 or 12 h post-TBI, relative to shams. Treatment of animals with either NIM811 or CsA significantly (p<0.03) attenuated this reduction. Consistent with this finding, both NIM811 and CsA significantly reduced lipid peroxidative and protein nitrative damage to mitochondria at 12 h post-TBI. These results showing the ability of NIM811 to fully duplicate the mitochondrial protective efficacy of CsA supports the conclusion that inhibition of the mPT may be sufficient to explain CsA's protective effects.

The novel calpain inhibitor SJA6017 improves functional outcome after delayed administration in a mouse model of diffuse brain injury.

A principal mechanism of calcium-mediated neuronal injury is the activation of neutral proteases known as calpains. Proteolytic substrates for calpain include receptor and cytoskeletal proteins, signal transduction enzymes and transcription factors. Recently, calpain inhibitors have been shown to provide benefit in rat models of focal head injury and focal cerebral ischemia. The present study sought to investigate, in experiment 1, the time course of calpain-mediated cytoskeletal injury in a mouse model of diffuse head injury by measuring the 150- and 145-kDa alpha-spectrin breakdown products (SBDP). Secondly, in experiment 2, we examined the effect of early (20 min postinjury) administration of the novel calpain inhibitor SJA6017 on functional outcome measured 24 h following injury and its effect on posttraumatic alpha-spectrin degradation. Lastly, in experiment 3, we examined the effect of delayed (4 or 6 h postinjury) administration of SJA6017 on 24-h postinjury functional outcome. In experiment 1, isoflurane-anesthetized male CF-1 mice (18-22 g) were subjected to a 750 g-cm weight drop-induced injury and were sacrificed for SBDP analysis at postinjury times of 30 min, and 1, 2, 6, 24 and 48 h (plus sham). In experiments 2 and 3, mice were injured as described, and delivered a single tail vein injection of either SJA6017 (0.3, 1, or 3 mg/kg) or vehicle (administered immediately, 4 or 6 h postinjury [3 mg/kg]). Functional outcome was evaluated in both studies, and, in experiment 2, 24-h postinjury assessment of SBDPs was determined. Following injury, the level of SBDP 145 was significantly different from sham at 24 and 48 h in cortical and at 24 h in the hippocampal tissues and at 48 h in the striatum. Immediate postinjury administration of SJA6017 resulted in a dose-related improvement in 24-h functional outcome (p < 0.05 at 3 mg/kg). Significance was maintained after a 4-h delay of the 3 mg/kg, but was lost after a 6-h delay. Despite improvement in functional outcome at 24 h, SJA6017 did not reduce spectrin breakdown in cortical or hippocampal tissues. These results support a role for calpain-mediated neuronal injury and the potential for a practical therapeutic window for calpain inhibition following traumatic brain injury. However, measurements of regional spectrin degradation may not be the most sensitive marker for determining the effects of calpain inhibition.

A comparison of long-term functional outcome after 2 middle cerebral artery occlusion models in rats.

BACKGROUND AND PURPOSE: Proven behavioral assessment strategies for testing potential therapeutic agents in rat stroke models are needed. Few studies include tasks that demand higher levels of sensorimotor and cognitive function. Because behavioral outcome and rate of recovery vary among ischemia models, there is a need to characterize and compare performance on specific tasks across models. METHODS: To this end, sensorimotor and cognitive deficits were assessed during a 5-week period after either permanent proximal middle cerebral artery occlusion (pMCAO) or permanent distal middle cerebral artery occlusion combined with a 90-minute occlusion of both common carotid arteries (dMCAO/tCCAO) in Sprague-Dawley rats. The EBST, hindlimb and forelimb placing, and cylinder tests were given at regular intervals postinjury to assess sensorimotor function. Cognitive function was assessed with a multitrial water navigation task. RESULTS: pMCAO, which caused both striatal and cortical damage, produced persistent sensorimotor and cognitive deficits. Limb placing responses and postural reflexes were impaired throughout the month of testing. A persistent bias for using the ipsilateral forelimb for wall movements in the cylinder test was observed as well as a bias for landing on the opposite forelimb. pMCAO rats were also impaired in the water navigation task. dMCAO/tCCAO, which caused only cortical damage, produced similar sensorimotor deficits, but these were greatly diminished by 2 weeks after injury. No impairment was found for water tank navigation. Correlations between forelimb placing (both models), water navigation performance (pMCAO model), and sensorimotor asymmetry (dMCAOtCCAO model) and infarct volume were observed. CONCLUSIONS: Based on the range of functions affected and stability of observed deficits, the pMCAO model appears to be preferable to the dMCAO/tCCAO model for use in assessing therapeutic agents for stroke.

Pharmacological treatment of acute spinal cord injury: how do we build on past success?

Most acute spinal cord injuries (SCI) do not involve complete transection of the spinal cord; typically, a rim of white matter survives. The potential for neurological recovery depends on optimal preservation of the ascending and descending white matter axons and their normal myelination. Pharmacologic strategies focus on the control of secondary injury processes, primarily lipid peroxidation (LP), and the salvage of as much white matter as possible. The first effective neuroprotective agent was methylprednisolone (MP), a glucocorticosteroid that in high doses improves neurological recovery in animals and humans following acute SCI. Tirilazad is a more targeted non-glucocorticoid LP inhibitor that has been shown to be neuroprotective and has fewer side effects than MP. Future SCI therapy is likely to encompass various neuroprotective agents, including inhibitors of LP, inhibitors of the nitric oxide-derived reactive oxygen species peroxynitrite, inhibitors of calpain (which is responsible for degrading the spinal cord cytoskeleton), and inhibitors of post-traumatic apoptosis of neurons and myelin-forming oligodendroglia. In addition, neuroprotective strategies will eventually be followed by neurorestorative agents that stimulate the plasticity of surviving neural pathways, and will be used in conjunction with other neurorestorative therapies like cell transplantation and gene therapy techniques.

Measurement of oxygen radicals and lipid peroxidation in neural tissues.

An important role for oxygen radical-mediated neuronal damage has been implicated in a number of acute and chronic neurodegenerative disorders. Particular interest has centered upon oxygen radical-induced, iron-catalyzed lipid peroxidation (LP) as the principal mechanism of the neuronal injury associated with oxygen radicals. Thus, there has been a growing interest in methods for monitoring increased oxygen radical levels as an index of oxidative stress as well as markers of LP-associated oxidative injury in a number of in vitro and in vivo model systems. This unit provides a detailed description of the salicylate trapping method for the measurement of the most highly reactive oxygen radical, the hydroxyl radical, as well as several direct or indirect methods for assessment of cellular LP in either cell cultures or in in vivo models.

LC-MS/MS detection of peroxynitrite-derived 3-nitrotyrosine in rat microvessels.

3-Nitrotyrosine (3NT) is used as a biomarker of nitrative pathology caused by peroxynitrite (PN), myeloperoxidase (MPO)-, and/or eosinophil peroxidase (EPO)-dependent nitrite oxidation. 3NT measurements in biological materials are usually based on either antibody staining, HPLC detection, or GC detection methodologies. In this report, a procedure is described for the measurement of 3NT and tyrosine (TYR) by LC-MS/MS that is simple, direct, and sensitive. Though highly specialized in its use as an assay, LC-MS/MS technology is available in many research centers in academia and industry. The critical assay for 3NT was linear below 100 ng/ml and the limit of detection was below 100 pg/ml. Regarding protein digested samples, we found that MRM was most selective with 133.1 m/z as the daughter ion. In comparison, LC-ECD was 100 times less sensitive. Basal levels of 3NT in extracted digests of rat brain homogenate were easily detected by LC-MS/MS, but were below detection by LC-ECD. The LC-MS/MS assay was used to detect 3NT in rat brain homogenate that was filtered through a 180 micron nylon mesh. Three fractions were collected and examined by phase contrast microscopy. The mass ratio (3NT/TYR) of 3NT in fractions of large vessel enrichment, microvessel enrichment, and vessel depletion was 0.6 ng/mg, 1.2 ng/mg, and 0.2 ng/mg, respectively. Ultimately, we found that the basal 3NT/TYR mass ratio as determined by LC-MS/MS was six times greater in microvessel-enriched brain tissue vs. tissue devoid of microvessels.

Gender differences in acute CNS trauma and stroke: neuroprotective effects of estrogen and progesterone.

Increasing evidence has demonstrated striking sex differences in the pathophysiology of and outcome after acute neurological injury. Lesser susceptibility to postischemic and posttraumatic brain injury in females has been observed in experimental models. Additional evidence suggests this sex difference extends to humans as well. The greater neuroprotection afforded to females is likely due to the effects of circulating estrogens and progestins. In fact, exogenous administration of both hormones has been shown to improve outcome after cerebral ischemia and traumatic brain injury in experimental models. The neuroprotection provided by periinjury administration of these hormones extends to males as well. The mechanisms by which estrogen and progesterone provide such neuroprotection are likely multifactorial, and probably depend on the type and severity of injury as well as the type and concentration of hormone present. Both genomic and nongenomic mechanisms may be involved. Estrogen's putative effects include preservation of autoregulatory function, an antioxidant effect, reduction of A beta production and neurotoxicity, reduced excitotoxicity, increased expression of the antiapoptotic factor bcl-2, and activation of mitogen activated protein kinase pathways. It is hypothesized that several of these neuroprotective mechanisms can be linked back to estrogen's ability to act as a potent chemical (i.e., electron-donating) antioxidant. Progesterone, on the other hand, has a membrane stabilizing effect that also serves to reduce the damage caused by lipid peroxidation. In addition, it may also provide neuroprotection by suppressing neuronal hyperexcitability. The following review will discuss experimental and clinical evidence for sex differences in outcome after acute brain trauma and stroke, review the evidence implicating estrogens and progestins as mediators of this neuroprotection following acute neurological injury, and finally, address the specific mechanisms by which these hormones may protect the brain following acute neurological injury.

4-Hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) inhibits peroxynitrite-mediated phenol nitration.

Peroxynitrite (PN), a very reactive oxidant formed by the combination of superoxide and nitric oxide, appears to play a role in producing tissue damage in a number of inflammatory conditions. Pharmacological scavenging and decomposition of PN within these areas has therapeutic value in several tissue injury models. Recently, we have been interested in nitroxide free radical-containing compounds as possible scavengers of PN decomposition products. Nitroxides can undergo redox reactions to the corresponding hydroxylamine anion or oxoammonium cation in biological systems as shown by its ability to react with superoxide, leading to the formation of hydrogen peroxide and molecular oxygen. We found that 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) inhibits PN-mediated nitration of phenolic compounds in the presence of a large molar excess of PN, suggesting a catalytic-like mechanism. In these experiments, Tempol inhibited PN-mediated nitration over the pH range of 6.5-8.5. This inhibition was specific for nitration and had no effect on hydroxylation. After the inhibition of PN-mediated nitration, Tempol was recovered from the reaction mixtures unmodified. In addition, Tempol was effective in protecting PC-12 cells from death induced by SIN-1, a PN-generating compound. The exact mechanism of Tempol's interaction with PN is not clear; however, we propose that an intermediate in this reaction may be a nitrogen dioxide radical-Tempol complex. This complex could react with water to form either nitrite or nitrate, or with a phenol radical to produce nitrophenol or nitrosophenol products and regenerate the nitroxide.

Ovariectomy exacerbates and estrogen replacement attenuates photothrombotic focal ischemic brain injury in rats.

BACKGROUND AND PURPOSE: We previously reported the infarct volumes in female spontaneously hypertensive rats (SHR) to be significantly smaller than those in male SHR. The purpose of the present study was to determine whether estrogen is responsible for the sex difference in ischemic vulnerability in SHR. METHODS: In experiment 1, 1 week (short-term) or 4 weeks (long-term) after the ovariectomy (OVX), female SHR (5 months old) were randomly subjected to photothrombotic occlusion of the middle cerebral artery, and the infarct volumes were determined. In experiment 2, the rats were randomly assigned to 3 groups (ie, the sham-ovariectomized, ovariectomized, and estrogen replacement groups). In the replacement group, estradiol valerate (200 microgram/kg) was subcutaneously injected once a week after the OVX. Four weeks after the OVX or sham-OVX, all rats were subjected to middle cerebral artery occlusion. Changes in regional cerebral blood flow were determined by laser-Doppler flowmetry. RESULTS: In experiment 1, the infarct volume produced 1 week after the OVX was not different from that of the sham-ovariectomized group. In contrast, the infarct volume produced 4 weeks after the OVX was significantly larger than that of the sham-ovariectomized group (82.4+/-11.6 versus 54.5+/-16.0 mm(3), P=0.0058). In experiment 2, estradiol replacement after the OVX was observed to attenuate the infarct volume compared with the ovariectomized group (55.6+/-18.8 versus 78.5+/-21.0 mm(3), P=0.0321). The degrees of regional cerebral blood flow reduction did not differ among the sham-ovariectomized, ovariectomized, and estrogen replacement groups. CONCLUSIONS: Chronic estrogen depletion was thus found to increase the infarct size, which was attenuated by estradiol replacement. These findings indicate that estrogen contributes to the sex difference in ischemic vulnerability and that endogenous estrogen also has a neuroprotective effect against ischemic brain damage.

Estrogen-related gender difference in survival rate and cortical blood flow after impact-acceleration head injury in rats.

While a number of laboratories have begun to examine gender differences in outcome following experimental stroke, little is known about the relative response of male and female brains to traumatic injury. In the following series of experiments, we used the Marmarou impact-acceleration head injury model (with a 500-g, 1.5-m weight drop) to compare the pathophysiological responses of male and female rats to closed-head injury. Cortical blood flow (CBF; laser-doppler flowmetry), mean arterial blood pressure (MAP), blood gas levels, blood pH, and body temperature were measured preinjury and at regular intervals postinjury. Acute survival was assessed 1 h after injury. The role of estrogen in the observed gender differences was assessed by examining these physiological measures after injury in ovariectomized females, with or without 17beta-estradiol replacement, and in intact males, with or without exogenous 17beta-estradiol administration. In the first experiment, significantly more females (100%) survived the acute injury period (60 min) after injury than did males (72%). Survival appeared related to the magnitude and persistence of the posttraumatic drop in MAP. In a second experiment, females showed a less dramatic reduction in and better recovery of CBF than males. The gender difference in CBF was paralleled to some degree by differences in the pattern of MAP changes after injury. Differences in body weight, blood gas levels, or blood pH did not account for the gender difference in CBF. Postinjury CBF was higher in female and male rats given 2 weeks of daily 17beta-estradiol injections prior to injury compared to those given the vehicle only. However, 17beta-estradiol administration did not alter MAP, suggesting that the gender difference in CBF was not strictly due to MAP changes. Our findings suggest that estrogen plays a role in maintaining adequate cerebral perfusion in the acute period following closed-head injury. This protective mechanism may underlie the gender difference in acute survival observed in this study, and may help explain observations of better outcome in females than in males after brain injury. We conclude that CBF preservation is one mechanism by which estrogen is neuroprotective following traumatic brain injury. We hypothesize, based upon known effects of estrogen, that the beneficial microvascular effects of estrogen most likely involve a combination of endothelial nitric oxide synthase induction and an antioxidant effect.

Peroxynitrite scavengers for the acute treatment of traumatic brain injury.

Recent evidence has suggested that the superoxide and nitric oxide-derived reactive oxygen species peroxynitrite (ONOO-) may play a significant role in the acute pathophysiology of brain injury. One pharmacological mechanism by which ONOO(-)-mediated damage might be interrupted is by the administration of scavenging compounds such as the thiol-containing compound penicillamine. In the present study, we examined the ability of either penicillamine (Pen) or the more brain penetrable penicillamine methyl ester (PenME) (0.01, 0.1, 1.0 or 10.0 mg/kg i.v. 5 min post-injury) to improve the early (1 hr) neurological recovery (grip score) of male CF-1 mice after a severe (900 g-cm; 50 g x 18 cm) injury. Pen produced a dose-related improvement in grip score. At 1.0 mg/kg, a +112% improvement was observed compared to vehicle-treated mice, and at 10.0 mg/kg, the increase was +168% (both, p < 0.05). PenME more potently improved the 1-hr grip score, but the magnitude of the optimal effect (+96% at 0.1 mg/kg; p < 0.02) was no greater than that observed with Pen, which largely remains in the cerebral microvasculature. These results are consistent with a role of ONOO- in acute head injury, but suggest that microvascular scavenging may be of primary therapeutic importance during the early post-traumatic period.

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

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 (approximately 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.

Infant rat model of the shaken baby syndrome: preliminary characterization and evidence for the role of free radicals in cortical hemorrhaging and progressive neuronal degeneration.

Infants subjected to repeated episodes of violent shaking develop brain damage characterized by intracranial hemorrhage and progressive cortical atrophy. We have developed an animal model that mimics this pathological state and investigated its etiology and treatment. Anesthetized male rats, 6 days of age, were subjected to one episode of shaking per day for 3 consecutive days. Separate groups of rats were sacrificed 1 h postinjury on the third day of shaking for HPLC quantification of cortical .OH and vitamin E levels, and histological assessment of cortical hemorrhaging. Additional groups were sacrificed 7 or 14 days postinjury to demonstrate progressive neuronal degeneration via cortical wet weight comparisons. In comparison to noninjured shams, the results indicated that cortical vitamin E and .OH levels rose 53.7% (p < 0.005) and 457.1% (p < 0.001), respectively, in shaken infant rats. Brain histologies revealed a moderate-to-severe degree of cortical hemorrhaging in these animals 1 h postinjury. By 7 and 14 days postinjury, there was a 13.3% and 28.7% (p < 0.0001 vs. sham) loss of cortical tissue in shaken infants, respectively, indicating progressive neuronal degeneration. Treatment with 10 mg/kg (ip) of the 21-aminosteroid antioxidant, tirilazad mesylate, 10 min before and 2 h after each episode of shaking, resulted in a 53.1% attenuation of cortical .OH levels and a 34.9% decrease in brain hemorrhaging (p < 0.05 vs. vehicle). Tirilazad treatment did not, however, significantly effect cortical vitamin E concentrations at 1 h postinjury or the extent of progressive neuronal degeneration at either 7 or 14 days postinjury. The present animal model mimics the brain pathology seen in abused children. Our observation that tirilazad mesylate, an antioxidant-lipid peroxidation inhibitor, significantly reduces cortical .OH levels and brain hemorrhaging in shaken infant rats supports a role for oxygen radicals in the pathophysiology of this type of CNS injury. The failure of tirilazad to block progressive cortical degeneration suggests that mechanisms other than free radicals may be of prime importance in the mediation of this aspect of the pathology.

Tirilazad widens the therapeutic window for riluzole-induced attenuation of progressive cortical degeneration in an infant rat model of the shaken baby syndrome.

Our infant rat model of traumatic subarchnoid hemorrhage combines violent shaking and hypoxia to produce subdural hemorrhaging and progressive cortical degeneration similar to that seen in victims of the shaken baby syndrome. Anesthetized, 6-day-old male rats were subjected to one episode of shaking under hypoxic conditions. Brain histologies revealed moderate-to-severe cortical hemorrhaging at 48 h postinjury and progressive cortical degeneration, as indicated by a 15.3% and 20.2% reduction in cortical wet weight, at 7 and 14 days postinjury, respectively. The purpose of the present study was to assess the effects of two antioxidant lipid peroxidation inhibitors (tirilazad mesylate and PNU-101033E), and the glutamate release inhibitor (riluzole), upon the brain pathology seen in this model. A significant, 54.3-75.3%, reduction in cortical hemorrhaging was observed in rats that were treated with a total of three doses of tirilazad (10 mg/kg, i.p.): 10 min before or 5-30 min after injury, and again at 2 and 24 h postinjury (p < 0.01 vs. vehicle). However, treatment with tirilazad or the more potent, brain-penetrating pyrrolopyrimidine, PNU-101033E (10 min before plus 2, 24, 48, and 72 h after), did not attenuate the progressive cortical degeneration typically seen at 14 days postinjury. These results suggest that free radicals play an important role in the pathophysiology of secondary brain hemorrhaging due to shaking + hypoxia, but may not be critical in the mediation of the subsequent neurodegeneration. Rather, glutamate neurotoxicity may be a key factor here. This is suggested by our observation that the glutamate release inhibitor, riluzole, significantly reduced cortical degeneration when it was administered up to 1 h postinjury in the present model. Specifically, the cortical wet weights of rats treated with 8 mg/kg riluzole (i.p.) 10 min before or 1 h after shaking + hypoxia (and again at 24 h postinjury) were 95.3% and 97.4% of noninjured controls, respectively, at 14 days postinjury (p < 0.02 vs. vehicle). Riluzole treatment beyond 1 h (e.g., 2 or 4 h postinjury) did not reduce the neurodegeneration. Lastly, we attempted to demonstrate that the therapeutic window for riluzole-induced attenuation of cortical degeneration could be extended beyond 1 h through the use of combination therapy. In this experiment, rat pups were treated with 10 mg/kg tirilazad (i.p.) at 30 min postinjury followed by 8 mg/kg riluzole (i.p.) at 4 and 24 h postinjury. At 14 days postinjury, the cortical wet weights of these rats were 94.5% of noninjured controls, thus demonstrating significant neuroprotection (p < 0.05 vs. vehicle) and a widening of the therapeutic window from 1 to 4 h in length. These results suggest that early attenuation of free radical-induced lipid peroxidation may slow down the biochemical cascade of events related to glutamate-induced excitotoxicity and, in doing so, prolong the time during which a glutamate release inhibitor, such as riluzole, is effective.

Mutant CuZn superoxide dismutase in motor neuron disease.

CuZn superoxide dismutase (CuZn SOD) is one of several antioxidant enzymes that defend the cell against damage by oxygen free radicals. Mutations of the SOD1 gene encoding CuZn SOD are found in patients with familial amyotrophic lateral sclerosis (FALS), a progressive and fatal paralytic disease that is caused by the death of motor neurons in cortex, brainstem and spinal cord. The disease can be reproduced in transgenic mice by expression of mutant human CuZn SOD. Recent studies both in vitro and in vivo suggest that the effect of mutation is to enhance the generation of oxygen radicals by the mutant enzyme. Thus, mutation converts a protective, antioxidant enzyme into a destructive, prooxidant form that catalyses free radical damage to which motor neurons are selectively vulnerable. Recent studies of neuroprotective agents in the FALS model show that inhibition of oxidative mechanisms (copper chelation therapy, dietary antioxidants, and coexpression of bcl-2) delays disease onset but does not extend disease duration. In contrast, inhibition of glutamatergic or apoptotic mechanisms (riluzole, gabapentin, and coexpression of glutamatergic or apoptotic mechanisms (riluzole, gabapentin, and coexpression of an inhibitor of caspase-1) has no effect on disease onset but extends survival by increasing the duration of symptomatic disease. Thus, neuroprotective agents differentially target the processes underlying disease initiation and propagation.

Comparative neuroprotective properties of the benzodiazepine receptor full agonist diazepam and the partial agonist PNU-101017 in the gerbil forebrain ischemia model.

Recent studies have demonstrated the neuroprotective properties of the novel imidazoquinoline benzodiazepine receptor partial agonist, PNU-101017, in the gerbil forebrain ischemia model. The compound effectively reduces delayed post-ischemic (5 min bilateral carotid occlusion) hippocampal CA1 neuronal degeneration even when its administration is withheld until 4 h after reperfusion and the effect is unrelated to hypothermia. The purpose of the present study was to determine the comparative abilities of PNU-101017 versus the full agonist diazepam to attenuate post-ischemic CA1 damage. Male gerbils were treated either 30 min before ischemia induction or immediately after reperfusion with an initial dose of PNU-101017 (30 mg/kg i.p.) or diazepam (10 mg/kg i.p.) with a second dose being given at 2 h after reperfusion. Possible hypothermic effects of either compound were prevented by external heating. In vehicle (0.05 N HCl)-treated gerbils, the loss of hippocampal CA1 neurons at 5 days was 85%. PNU-101017 pretreatment reduced the loss to 50% (p<0.05 vs. vehicle) whereas pretreatment with diazepam attenuated damage to only 17% (p<0.001 vs. vehicle). Delaying treatment with PNU-101017 until just after reperfusion still resulted in a reduction in CA1 degeneration statistically that was indistinguishable from that seen with pretreatment. In contrast, diazepam post-treatment did not significantly decrease CA1 neuronal loss. These results suggest that a benzodiazepine receptor partial agonist may have greater neuroprotective practicality than a full agonist for the treatment of global cerebral ischemia. The mechanistic basis for this difference may relate to the partially pro-excitatory neuronal response to endogenous GABA before and after neuronal insult.

Relationship of oxygen radical-induced lipid peroxidative damage to disease onset and progression in a transgenic model of familial ALS.

Transgenic mice that overexpress a mutated human CuZn superoxide dismutase (SOD1) gene (gly93-->ala) found in some patients with familial ALS (FALS) have been shown to develop motor neuron disease, as evidenced by motor neuron loss in the lumbar and cervical spinal regions and a progressive loss of voluntary motor activity. The mutant Cu,Zn SOD exhibits essentially normal dismutase activity, but in addition, generates toxic oxygen radicals as a result of an enhancement of a normally minor peroxidase reaction. In view of the likelihood that the manifestation of motor neuron disease in the FALS transgenic mice involves an oxidative injury mechanism, the present study sought to examine the extent of lipid peroxidative damage in the spinal cords of the TgN(SOD1-G93A)G1H mice over their life span compared to nontransgenic littermates or transgenic mice that overexpress the wild-type human Cu,Zn SOD (TgN(SOD1)N29). Lipid peroxidation was investigated in terms of changes in vitamin E and malondialdehyde (MDA) levels measured by HPLC methods and by MDA-protein adduct immunoreactivity. Four ages were investigated: 30 days (pre-motor neuron pathology and clinical disease); 60 days (after initiation of pathology, but predisease); 100 days (approximately 50% loss of motor neurons and function); and 120 days (near complete hindlimb paralysis). Compared to nontransgenic mice, the TgN(SOD1-G93A)G1H mice showed blunted accumulation of spinal cord vitamin E and higher levels of MDA (P < 0.05 at 30 and 60 days) over the 30-120 day time span. In the TgN(SOD1)N29 mice, levels of MDA at age 120 days were significantly lower than in either the TgN(SOD1-G93A)G1H or nontransgenic mice. MDA-protein adduct immunoreactivity was also significantly increased in the lumbar spinal cord at age 30, 100, and 120 days, and in the cervical cord at 100 and 120 days. The results clearly demonstrate an increase in spinal cord lipid peroxidation in the FALS transgenic model, which precedes the onset of ultrastructural or clinical motor neuron disease. However, the greatest intensity of actual motor neuronal lipid peroxidative injury is associated with the active phase of disease progression. These findings further support a role of oxygen radical-mediated motor neuronal injury in the pathogenesis of FALS and the potential benefits of antioxidant therapy.

U74389G prevents vasospasm after subarachnoid hemorrhage in dogs.

OBJECTIVE: Oxygen-derived free radicals may contribute to vasospasm after the rupture of an intracranial aneurysm through direct vasoconstricting effects occurring within the arterial wall or, secondarily, by causing lipid peroxidation in the subarachnoid erythrocytes with secondary induction of vasoconstriction. U74389G is a potent inhibitor of lipid peroxidation and a scavenger of oxygen-derived free radicals. This study determined the relative contributions of oxygen-derived free radicals and lipid peroxidation to vasospasm in the double-hemorrhage dog model. METHODS: Sixteen dogs underwent baseline (Day 0) cerebral angiography and induction of subarachnoid hemorrhage by two injections of blood into the cisterna magna 2 days apart. They were randomized to receive drug vehicle (n=8) or U74389G (n=8, 3 mg/kg of body weight/d) intravenously. Drug administration and end point analysis were blinded. The end points were angiographic vasospasm, as assessed by comparison of angiograms obtained before and 7 days after subarachnoid hemorrhage, and the levels of malondialdehyde and salicylate hydroxylation products (dihydroxybenzoic acids) in cerebrospinal fluid and of malondialdehyde in subarachnoid blood clots and basilar arteries 7 days after hemorrhage. RESULTS: Comparisons within groups of Day 0 and Day 7 angiograms and between groups of angiograms obtained at Day 7, showed significant vasospasm in animals in the vehicle group (mean+/-standard error, 51%+/-4) but not in the U74389G group (25%+/-11, P < 0.05, unpaired t test). High-pressure liquid chromatographic assays of malondialdehyde and dihydroxybenzoic acids in cerebrospinal fluid, subarachnoid blood clots, and basilar arteries showed no significant differences between groups. CONCLUSION: The significant prevention of vasospasm by U74389G without change in levels of indicators of free radical reactions suggests that the effect of the drug is related to other processes occurring in the arterial wall and that cerebrospinal fluid levels of oxygen radicals and lipid peroxides are not useful markers of vasospasm.