Roger G. Boulu

The neuroprotective effect of a nitric oxide inhibitor in a rat model of focal cerebral ischaemia

Recent data showed that glutamate toxicity in primary cortical cultures is mediated by nitric oxide. In order to investigate the effect of inhibition of NO synthase on focal cerebral ischaemia in rats, we studied the histological consequences of a middle cerebral artery (MCA) occlusion after post‐operative treatment with NG‐nitro‐l‐arginine methyl ester, an inhibitor of nitric oxide synthase. We found a significant reduction of cortical (−43%) and striatal (−25%) necrotic volumes induced by MCA occlusion, indicating that NO synthesis plays an important role in the neurotoxic cascade leading to neuronal damage after focal cerebral ischaemia in rats.

Nitric oxide: an endogenous anticonvulsant substance.

IN the present study, we examine the involvement of the L-arginine-nitric oxide pathway in seizure activity termination. Convulsions were induced reproducibly by intracerebroventricular administration of N-methyl-D-aspartate to conscious mice. The duration of the seizure activity was increased by inhibition of the NO-pathway or by intracerebroventricular injection of methylene blue, an inhibitor of guanylate cyclase activity. This increased duration in seizure activity was reversed by co-administration of L-arginine or by intracerebroventricular injection of guanosine 3‘:5’ cyclic monophosphate (cGMP). These results suggest that nitric oxide produced in response to NMDA receptor activation leads to an increase in cGMP which induces the seizure activity termination.

Mechanisms Involved in the Neuroprotective Activity of a Nitric Oxide Synthase Inhibitor During Focal Cerebral Ischemia

Abstract: We have reported previously that posttreatment with NG‐nitro‐L‐arginine methyl ester (L‐NAME), an inhibitor of the nitric oxide synthase, reduced the volume of cortical and striatal infarct induced by middle cerebral artery occlusion in rats. In the present study, we investigated the mechanisms by which L‐NAME (3 mg/kg i.p.) is neuroprotective in this model of cerebral ischemia. First, we have shown the reversal of the neuroprotective effect of L‐NAME by a coinjection of L‐arginine. Second, in order to determine by which mechanism nitric oxide exacerbates neuronal damage produced by focal cerebral ischemia, we studied the effect of the inhibition of nitric oxide synthase by L‐NAME on the histological consequences of a focal injection of N‐methyl‐D‐aspartate (NMDA) in the striatum, and on the striatal overflow of glutamate and aspartate induced either by K+ depolarization or by focal cerebral ischemia. We have found that L‐NAME treatment reduced the excitotoxic damage produced by NMDA injection. By using microdialysis, we have shown that the K+‐ and the ischemia‐induced glutamate efflux was reduced by 52 and 30%, respectively, after the L‐NAME treatment. These results indicate that nitric oxide synthesis induced by the NMDA receptor overstimulation is one of the major events leading to neuronal damage. One possible mechanism by which nitric oxide may contribute to the excitotoxic process is by facilitating the ischemia‐induced glutamate overflow.

Extracellular Glutamate During Focal Cerebral Ischaemia in Rats: Time Course and Calcium Dependency

Abstract: The time course of changes in extracellular glutamic acid levels and their Ca2+ dependency were studied in the rat striatum during focal cerebral ischaemia, using microdialysis. Ischaemia‐induced changes were compared with those produced by high K+‐evoked local depolarization. To optimize time resolution, glutamate was analysed continuously as the dialysate emerged from the microdialysis probe by either enzyme fluorimetry or biosensor. The Ca2+ dependency of glutamate changes was examined by perfusing the probe with Ca2+‐free medium. With normal artificial CSF, ischaemia produced a biphasic increase in extracellular glutamate, which started from the onset of ischaemia. During the first phase lasting ∼10 min, dialysate glutamate level increased from 5.8 ± 0.9 µM· min−1 to 35.8 ± 6.2 µM where it stabilized for ∼3 min. During the second phase dialysate glutamate increased progressively to its maximum (82 ± 8 µM), reached after 55 min of ischaemia, where it remained for as long as it was recorded (3 h). The overall changes in extracellular glutamate were similar when Ca2+ was omitted from the perfusion medium, except that the first phase was no longer detectable and, early in ischaemia, extracellular glutamate increased at a significantly slower rate than in the control group (2.2 ± 1 µM· min−1; p < 0.05). On the basis of these data, we propose that most of the glutamate released in the extracellular space in severe ischaemia is of metabolic origin, probably originating from both neurons and glia, and caused by altered glutamate uptake mechanisms. Comparison with high K+‐induced glutamate release did not suggest that glutamate “exocytosis,” early after middle cerebral artery occlusion, was markedly limited by deficient ATP levels.

Protective effect of melatonin in a model of traumatic brain injury in mice

ABSTRACT: The pineal hormone melatonin has recently been shown to exert neuroprotective activity in a variety of experimental neuropathologies in which free radicals are involved. This neuroprotective effect has been attributed to the antioxidant properties of melatonin. Considering that free radicals also play a deleterious role in traumatic brain injury (TBI), the purpose of the present study was to determine whether melatonin would have a beneficial effect in this pathology. Head injury was induced in mice and the neurological deficit was evaluated at 24 hr by a grip test. In this model, the free radical scavenger, α‐phenyl‐tert‐butyl‐nitrone (2 ± 100 mg/ kg, i.p.) given 5 min and repeated at 4 hr after TBI was neuroprotective. Melatonin (1.25 mg/kg, i.p.) given 5 min and repeated at 1,2, and 3 hr after head trauma also significantly reduced the neurological deficit. This beneficial effect was not due to melatonin‐induced hypothermia since repeated treatment with melatonin did not modify the colonic temperature of mice. This study shows that melatonin exerts a beneficial effect on the neurological deficit induced by traumatic brain injury in mice. The mechanisms of this neuroprotection remains to be established, and more particularly, the contribution of the antioxidant activity of melatonin.

Effect of riluzole on focal cerebral ischemia in rats

The effects of riluzole, a putative inhibitor of glutamate release, on the histological and neurobehavioral consequences of middle cerebral artery occlusion were tested in Sprague-Dawley rats. Neurobehavioral studies (neurological examination, passive avoidance task) were carried out with sham-operated and occluded rats. Riluzole 4 and 8 mg/kg administered 30 min after occlusion reduced (P < 0.01) the cortical infarct (respectively 94 +/- 12 mm3 and 73 +/- 15 mm3 versus 139 +/- 8 mm3 for control rats). Striatum necrosis was not modified by the low dosage (46 +/- 3 mm3 versus 49 +/- 3 mm3 for control rats), whereas the high dosage increased it (61 +/- 3 mm3, P < 0.05). The ischemia-induced neurological and memory impairments were not improved by riluzole. Our results indicate that a drug depressing glutamatergic neurotransmission without blocking the glutamate receptors exerts anti-ischemic activity. Moreover, the results highlight the need for carrying out histological and neurobehavioral studies in parallel in this model.

Striatal Protection Induced by Lesioning the Substantia Nigra of Rats Subjected to Focal Ischemia

Abstract: Unilateral 6‐hydroxydopamine lesion of the substantia nigra reduced the volume of striatal necrosis and suppressed the increase in extracellular glutamate concentration in the striatum induced by middle cerebral artery occlusion in rats. These results indicate that the dopaminergic nigrostriatal pathway is highly involved in the vulnerability of the striatum to ischemia and suggest that glutamate‐dopamine interactions may play a key role in the striatal ischemic insult.

Calcium-independent NO-synthase activity and nitrites/nitrates production in transient focal cerebral ischaemia in mice.

1 The temporal changes in constitutive NO‐synthase (cNOS) and in calcium‐independent NO‐synthase activities were studied in mice subjected to 2 h of transient focal cerebral ischaemia. The changes in brain nitrites/nitrates (NOx) content were also studied. 2 NOS activities were measured by the conversion of L‐[14C]‐arginine to L‐[14C]‐citrulline. Brain NOx contents were investigated by the Griess colourimetric method. 3 cNOS activity in the infarcted cortical area was significantly reduced after 6 h of reperfusion and this activity remained attenuated for up to 10 days after ischaemia. A calcium‐independent NOS activity began to increase 48 h after reperfusion, reached a maximum at 7 days and returned to baseline at 10 days. 4 There was a significant increase of brain NOx content beginning after 3 days of reperfusion. This increase was maximal at 7 days and returned to baseline at 10 days. 5 Thus, ischaemia followed by recirculation leads to a rapid, prolonged drop in cNOS activity in the infarcted cortex. There is also a substantial appearance of calcium‐independent NOS activity in the later phase of transient ischaemia, leading to an important increase of NOx production.

Dose- and time-dependence of L-NAME neuroprotection in transient focal cerebral ischaemia in rats.

In this study the effect of the dose and administration time of NG‐nitro‐l‐arginine methyl ester (l‐NAME), an NO‐synthase inhibitor, in a model of transient focal cerebral ischaemia in rats was investigated. Two injections of l‐NAME were given, of 1, 3 and 10 mg kg−1, 5 min and 3 h after the onset of ischaemia. None of the doses gave any striatal neuroprotection, but 1 and 3 mg kg−1l‐NAME reduced the infarcted volume in the cortex (by 26%, P<0.01 for 1 mg kg−1 and 21%, P<0.05 for 3 mg kg−1), whereas 10 mg kg−1 had no neuroprotective effect. Single injections of l‐NAME 1 mg kg−1, given 5 min or 3 h after ischaemia onset, had similar neuroprotective effects on the cortical infarction as did the repeated injections. l‐NAME 1 mg kg−1 given 3, 6 or 9 h after ischaemia induction reduced the cortical infarct volume by 19% (P<0.01) when given 3 h after ischaemia, by 21% (P<0.01) when given at 6 h, and by 16% (P<0.5) when given at 9 h, but had no neuroprotective activity when given 12 h after ischaemia. Thus a low dose of l‐NAME is neuroprotective in a model of transient focal ischaemia, with a wide therapeutic window, much larger than that found for MK‐801.

Deleterious poly(ADP-ribose)polymerase-1 pathway activation in traumatic brain injury in rat

Traumatic brain injury produces nitric oxide and reactive oxygen species. Peroxynitrite, resulting from the combination of nitric oxide and superoxide anions, triggers DNA strand breaks, leading to the activation of poly(ADP-ribose)polymerase-1. As excessive activation of this enzyme induces cell death, we examined the production of nitrosative stress, the activation of poly(ADP-ribose)polymerase-1, and the role of this enzyme in the outcomes of traumatic brain injury produced by fluid percussion in rats. Immunohistochemistry showed that 3-nitrotyrosine, an indicator of nitrosative stress, and poly(ADP-ribose), a marker of poly(ADP-ribose)polymerase-1 activation, were present as early as 30 min post-injury, and that persisted for 72 h. The poly(ADP-ribose)polymerase inhibitor, 3-aminobenzamide, at 10 and 30 mg/kg, significantly improved the neurological deficit, with a 60% reduction in the brain lesion volume and inhibition of poly(ADP-ribose)polymerase-1 activation. Thus, poly(ADP-ribose)polymerase-1 is involved in the neurological consequences of traumatic brain injury and may be a promising therapeutic target in clinical treatment of acute brain trauma.