Michel Plotkine

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.

NMDA Receptor Activation Inhibits α-Secretase and Promotes Neuronal Amyloid-β Production

Acute brain injuries have been identified as a risk factor for developing Alzheimers disease (AD). Because glutamate plays a pivotal role in these pathologies, we studied the influence of glutamate receptor activation on amyloid-β (Aβ) production in primary cultures of cortical neurons. We found that sublethal NMDA receptor activation increased the production and secretion of Aβ. This effect was preceded by an increased expression of neuronal Kunitz protease inhibitory domain (KPI) containing amyloid-β precursor protein (KPI-APP) followed by a shift from α-secretase to β-secretase-mediated APP processing. This shift is a result of the inhibition of the α-secretase candidate tumor necrosis factor-α converting enzyme (TACE) when associated with neuronal KPI-APPs. This KPI-APP/TACE interaction was also present in AD brains. Thus, our findings reveal a cellular mechanism linking NMDA receptor activation to neuronal Aβ secretion. These results suggest that even mild deregulation of the glutamatergic neurotransmission may increase Aβ production and represent a causal risk factor for developing AD.

Short Therapeutic Window for MK-801 in Transient Focal Cerebral Ischemia in Normotensive Rats

The present study investigates the role of N-methyl-D-aspartate (NMDA) receptors in a model of transient focal cerebral ischemia in normotensive rats. The left middle cerebral artery and both common carotid arteries were occluded for 60 min. Preliminary studies indicated that this gave reproducible infarctions of the cortex and striatum. These infarctions were the result of severe ischemia followed by complete reperfusion after clamp removal, as showed by striatal tissue Po2 monitoring. Microdialysis indicated that glutamate concentration increased immediately after occlusion and returned to the baseline value 40 min after clamp removal. MK-801 (1 mg kg−1 i.v.), an antagonist of the NMDA glutamatergic receptor, reduced the cortical infarct volume by 29% (p < 0.001) and the striatal infarct volume by 14% (p < 0.05) when given just prior to ischemia, but had no neuroprotective activity when given 30 min after the onset of ischemia. This short therapeutic window for MK-801 suggests that NMDA receptors play only a transient role in reversible focal ischemia in rats.

Caspase inhibition after neonatal ischemia in the rat brain.

Caspase-3 has been identified as a key protease in the execution of apoptosis and appears to be an important downstream event after hypoxia-ischemia in the immature brain. The efficacy of a pan-caspase inhibitor, boc-aspartyl-(Ome)-fluoromethyl-ketone (BAF), was tested in a model of unilateral focal ischemia with reperfusion in 7-day-old rats. The BAF inhibitor was given intraperitoneally 5 minutes before reperfusion via the carotid artery. This procedure reduced the activity of caspase-3 by 79% but did not induce a significant reduction in infarct volume (23.8 ± 7.5% versus 30.1 ± 6.4%). Animals were distributed in two populations. One population exhibited an infarct, whereas the other appeared to be fully protected. BAF-treated animals exhibiting an infarct mostly displayed necrotic cell death, whereas apoptotic nuclei were observed in untreated or vehicle-treated animals. Repeated dose of BAF (5 minutes before and 9 hours after reperfusion) did not also provide benefit after neonatal ischemia, although a general trend to reduce lesion was observed (20.5 ± 3.7% versus 34.4 ± 5.9%). These findings raise critical questions about the use of peptide ketone apoptotic inhibitors in improving histopathologic outcomes after neonatal stroke.

Inhibition of glutamate release in rat hippocampus by kynurenic acid does not protect CA1 cells from forebrain ischemia

We assessed the effect of a broad spectrum glutamatergic receptor antagonist, kynurenic acid (500 mg/kg) on ischemia-induced hippocampal glutamate release and neuronal damage. Kynurenic acid significantly decreased glutamate release during ischemia but had no effect on the hippocampal lesion. Some protection was observed in the cortex and in the striatum. These data suggested that the extracellular accumulation of glutamate during forebrain ischemia does not play a major role in the hippocampus.

Enhancement of endogenous excitatory amino acids by theophylline does not modify the behavioral and histological consequences of forebrain ischemia.

The neuroprotective role of endogenous adenosine during forebrain ischemia elicited by 4-vessel occlusion in rats was assessed using the adenosine antagonist, theophylline (32 mg/kg). Despite an increase in the release of glutamate in the hippocampus during ischemia, theophylline did not alter the neurological and histological outcomes. These results indicate that endogenous adenosine does not act as an endogenous neuroprotector by modulating glutamate release in this model.

Competitive NMDA receptor blockers reduce striatal glutamate accumulation in ischaemia

Our previous studies have shown that kynurenic acid, a broad-spectrum antagonist of excitatory amino acid receptors, depressed the ischaemia-induced accumulation of glutamate and aspartate in rat striatum. In the present experiments we examined the effect of two competitive N-methyl-D-aspartate (NMDA) receptor antagonists on striatal extracellular glutamate concentrations induced by a 30 min 4-vessel occlusion ischaemia in rats. Local perfusion with 2-amino-5-phosphonovalerate (AP5; 300 microM) and with 2-amino-7-phosphonoheptanoate (AP7; 300 microM), using a microdialysis fibre markedly reduced the ischaemia-induced increase in glutamate concentrations. These results indicate that, during forebrain ischaemia the NMDA receptor type mediates glutamate and aspartate accumulation in rat striatum.

Effect of kynurenic acid on the ischaemia-induced accumulation of glutamate in rat striatum.

We examined the effect of kynurenic acid, a broad spectrum antagonist of excitatory amino acid receptors, on striatal extracellular glutamate and aspartate accumulation induced by a 30 min forebrain ischaemia in rats. Kynurenic acid, given systemically (500 mg kg-1, i.p.) or administered in situ through the dialysis probe (10 mM), markedly depressed the ischaemia-induced increase in glutamate and aspartate concentrations. These results indicate that, during forebrain ischaemia, local glutamate receptors play a major role in glutamate and aspartate accumulation in the striatum. Ischaemia-induced increase in extracellular concentrations of these excitatory amino acids may be due in part to a positive glutamatergic feedback loop via activation of NMDA and/or non-NMDA receptors.

Down-regulation of striatal enkephalinergic (PPA) messenger RNA without prior apoptotic features following reversible focal ischemia in rat

In order to determine whether striatal enkephalinergic neurons were affected by reversible focal ischemia, we have investigated the expression of the preproenkephalin (PPA) messenger by in situ hybridization (ISH) combined with TUNEL staining to display apoptosis in the same rat brain sections. Our data demonstrated a massive reduction of the number of PPA-mRNA containing neurons concomitant with the emergence of apoptotic cells. However, double-labeled neurons (ISH- and TUNEL-positive cells) were not detected, suggesting that either disruption of mRNA precedes DNA fragmentation or ischemia leads to a long lasting reduction of mRNA(s) without damage.