The pyrimidine-derivative, BW1003C87, protects CA1 and striatal neurons following transient severe forebrain ischaemia in rats. A microdialysis and histological study.

The four vessel occlusion model of severe transient global ischaemia in Wistar rats has been used to study changes in the extracellular concentration of amino acids in hippocampus and striatum during and after ischaemia. We have investigated the effect of a pyrimidine derivative. BW1003C87 [5-(2,3,5-trichlorophenyl) pyrimidine-2,4-diamine 1.1 ethanesulphonate], 10 or 20 mg/kg, administered before or after ischaemia, on the amino acid accumulation and the pathological outcome. BW1003C87, 10 or 20 mg/kg, given intraperitoneally 20 min prior to ischaemia significantly reduces the extracellular accumulation of the amino acid in hippocampus and in striatum. BW1003C87, 10 or 20 mg/kg, injected 20 min prior to and 4 h after ischaemia protects against the hippocampal (CA1) and the striatal lesions. Administration of BW1003C87, 20 mg/kg, at 0 and 4 h or at 2 and 6 h post-ischaemia, significantly reduces CA1 lesions whereas no significant protection is seen in the striatum. These data indicate that BW1003C87 is able to inhibit the extracellular accumulation of amino acids during severe forebrain ischaemia. The compound is also a potent neuroprotector in this model (in which N-methyl-D-aspartate receptor antagonists fail to protect CA1 neurons). The effect of BW1003C87 on ischaemic glutamate release may contribute to protection in the striatum; it cannot explain the neuroprotection in CA1 since delayed administration is still highly protective. An action on voltage-sensitive sodium channels may contribute to the effects on amino acid release and to the cerebroprotective effect.

Effect of NO synthase inhibition on NMDA- and ischaemia-induced hippocampal lesions.

We assessed the effect of NG-Nitro-L-arginine methylester (L-NAME), an inhibitor of nitric oxide synthase, on the hippocampal lesions induced either by the focal injection of N-methyl-D-aspartate (NMDA) or (s)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (s-AMPA) or by 10 min of severe forebrain ischaemia (4-vessel occlusion), in the rat. We find that L-NAME, 20 or 40 mg kg-1, selectively decreases NMDA-induced CA1 lesions while it has no effect on AMPA toxicity. L-NAME, 20 mg kg-1, does not decrease hippocampal damage induced by ischaemia. These results suggest that NO contributes to NMDA toxicity and support data indicating that NMDA receptor antagonists fail to protect against hippocampal CA1 lesions in the 4-vessel occlusion model.

Cerebroprotective effect of BW619C89 after focal or global cerebral ischaemia in the rat.

BW619C89 (4-amino-2-(4-methylpiperazin-l-yl)-5-(2,3,5-trichlorophenyl) pyrimidine) was evaluated for cerebroprotection after focal or global ischaemia. BW619C89, as the mesylate dihydrate salt, 20 mg kg-1, i.v. for 10 min immediately, or with a 1 h delay after permanent middle cerebral artery occlusion in Fischer rats reduces cortical infarct volume (visualized with (2,3,5-triphenyltetrazolium) by 49% (p < 0.05) or by 61% (p < 0.001) and improves neurological deficit. Administration of BW619C89 with a 2 h delay is ineffective. BW619C89, given i.p. 0 and 4 h after 20 min of transient bilateral common carotid artery occlusion in vertebral artery-occluded Wistar rats reduces glutamate release and neuronal cell loss in the hippocampal CA1 sector (p < 0.01) and striatum (p < 0.05). BW619C89 resembles BW1003C87 (5-(2,3,5-trichlorophenyl)-2,4-diaminopyrimidine) in inhibiting veratrine-induced glutamate release and protecting against ischaemic brain damage.

Biphasic transforming growth factor-beta production flanking the pro-inflammatory cytokine response in cerebral trauma.

We determined the time-course of the production of transforming growth factor-beta (TGF-beta) after fluid-percussion injury using a bioassay. Biophasic production of TGF-beta composed mainly of TGF-beta 2 was detected in the ipsilateral cortex, with a first peak 30 min and a second peak 48 h after the lesion, flanking the transient production of tumor necrosis factor-alpha and interleukin-6 occurring between 5 and 18 h after trauma. This temporal pattern suggested that TGF-beta plays alternatively a pro- and anti-inflammatory role in the regulation of the brain cytokine network in response to injury, providing an endogenous mechanism for the control of the inflammatory reaction in traumatic brain injury.

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.

Neuroprotection afforded by a combination of eliprodil and a thrombolytic agent, rt-PA, in a rat thromboembolic stroke model.

In the present study, we have assessed the efficacy of eliprodil, a neuroprotective agent which blocks both the modulatory polyamine site of the NMDA receptor and neuronal voltage-sensitive calcium channels, alone or in combination with the thrombolytic agent, rt-PA, in a rat embolic stroke model using a neurological score and the volume of the infarct as endpoints. Embolization was induced by intracarotid injection of an arterial blood clot. Eliprodil, administered at the dose of 1 mg/kg, iv. 10 min and 2 h 30 after embolization, reduced the neurological deficit by 54% (P < 0.01) and the total volume of the brain lesion by 49%. Thrombolysis with rt-PA (2.5 mg/kg, as a 30 min iv infusion beginning 1 h after embolization) decreased the neurological deficit by 48% (P < 0.05) and the size of the total infarct by 55% (P < 0.05). Combined therapy greatly improved the degree of neuroprotection as assessed by neurological and histological outcomes (70% (P < 0.001) and 89% (P < 0.01) neuroprotection, respectively). These results demonstrate that the administration of a neuroprotective drug (eliprodil) or a thrombolytic agent (rt-PA) similarly reduce the volume of brain damage and the neurological deficit in a rat embolic stroke model. Combined cytoprotective therapy and thrombolysis markedly improved the degree of neuroprotection and may, thus, represent a valuable approach for the treatment of stroke in humans.

Effects of an A1 adenosine receptor agonist on the neurochemical, behavioral and histological consequences of ischemia.

Untreated rats and rats given the A1 receptor adenosine agonist, R-phenylisopropyladenosine (R-PIA), were subjected to four vessel ischemia. The effect of R-PIA on hippocampal amino acid release, hippocampal neuronal damage, exploratory behavior, learning capacity and global neurological score were evaluated. R-PIA decreased by half the glutamate released during ischemia and improved the global neurological scores 3, 24, 48, 78 h and 7 days after ischemia. But R-PIA had no effect on taurine/GABA release (during ischemia), hippocampal neuronal damage (7 days post-ischemia), exploratory behavior (48 h post-ischemia) or learning capacity (7 days post-ischemia). Thus, a decrease in glutamate release by R-PIA is not systematically correlated with an improvement of histological damage or learning capacity. Reduced glutamate release is not therefore a sufficient criterion on which to evaluate the neuroprotective capacity of a drug.

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.

Kynurenic acid antagonizes hippocampal quinolinic acid neurotoxicity: behavioral and histological evaluation.

In the present study, we evaluate the ability of kynurenic acid to protect hippocampal neurons from the neurotoxicity of the N-methyl-D-aspartate (NMDA) agonist quinolinic acid. Bilateral intrahippocampal injection of quinolinic acid (120 nmol) led to severe behavioral disturbances and total loss of hippocampal neurons. Intrahippocampal co-injection of kynurenic acid (360 nmol) completely prevented cell loss and behavioral disturbances. However, the protection was incomplete when kynurenic acid was intraperitoneally injected (500 mg/kg, repeated during 4 days). These above results indicate that kynurenic acid can antagonize the neuronal degeneration mediated by excessive stimulation of NMDA receptors in vivo.

Concomitant increases in the extracellular concentrations of excitatory and inhibitory amino acids in the rat hippocampus during forebrain ischemia.

The extracellular concentrations of aspartate, glutamate, glutamine, taurine and gamma-aminobutyric acid in the hippocampus were determined during and after forebrain ischemia (4-vessel model) in the unanaesthetized rat. Ischemia led to a large increase in both inhibitory (taurine and gamma-aminobutyric acid) and excitatory amino acids (aspartate, glutamate). These results suggest that in this model, as previously proposed in other models of ischemia, the large increase of inhibitory amino acids could counterbalance the excitotoxicity due to aspartate and glutamate.

Increased neurosteroids synthesis after brain and spinal cord injury in rats.

We studied the effect of brain and spinal cord injury induced by fluid-percussion on the local synthesis of neurosteroids as measured by a gas-chromatographic/mass-spectrometric method. In the nervous system of sham operated rats i.v. infusion of pregnenolone (PREGN)-sulfate results in a 2-4 fold increase in PREGN, progesterone (PROG), 5alpha-dehydroprogesterone (5alpha-DHP) and 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha5alpha-THP, allopregnanolone) concentrations, as compared to vehicle treated rats. When PREGN-sulfate was infused 1, 3 or 7 days after brain or spinal cord injury it was observed a large time-dependent increase of PROG, 5alpha-DHP and 3alpha5alpha-THP levels in the peri-focal but not in the focal site. This increase in neurosteroids content may be due essentially to the glial cells hyperplasia in the peri-focal area and to an activation of the pathways involved in the metabolism of PREGN-sulfate to PROG, 5alpha-DHP and 3alpha5alpha-THP.

Effect of the non-NMDA receptor antagonist GYKI 52466 on the microdialysate and tissue concentrations of amino acids following transient forebrain ischaemia.

The effect of the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466) on ischaemia-induced changes in the microdialysate and tissue concentrations of glutamate, aspartate, and gamma-aminobutyric acid (GABA) was studied in rats. Twenty minutes of four-vessel occlusion resulted in a transient increase in microdialysate levels of glutamate, aspartate, and GABA in striatum, cortex, and hippocampus. Administration of GYKI 52466 (10 mg/kg bolus + 10 mg/kg/60 min intravenously starting 20 min before onset of ischaemia) inhibited ischaemia-induced increases in microdialysate glutamate and GABA in striatum without affecting the increases in hippocampus or cortex. Twenty minutes of four-vessel occlusion resulted in immediate small decreases and larger delayed (72 h) decreases in tissue levels of glutamate and aspartate. Transient increases in tissue levels of GABA were shown in all three structures at the end of the ischaemic period. At 72 h, after the ischaemic period, significantly reduced GABA levels were observed in striatum and hippocampus. GYKI 52466, given under identical conditions as above, augmented the ischaemia-induced decrease in striatal tissue levels of glutamate and aspartate, without significantly affecting the decreases in hippocampus and cortex. Twenty minutes of ischaemia resulted in a large increase in microdialysate dopamine in striatum. GYKI 52466 failed to inhibit this increase. Kainic acid (500 microM infused through the probe for 20 min) caused increases in microdialysate glutamate and aspartate in the striatum. GYKI 52466 (10 mg/kg bolus + 10 mg/kg/60 min) completely inhibited the kainic acid-induced glutamate release. In conclusion, the action of the non-NMDA antagonist, GYKI 52466, in the striatum is different from that in the cortex and hippocampus. The inhibition by GYKI 52466 of ischaemia-induced and kainate-induced increases in microdialysate glutamate concentration in the striatum may be related to the neuroprotection provided by GYKI 52466 in this region.

Reduced postischemic expression of a glial glutamate transporter, GLT1, in the rat hippocampus.

Perturbations of the synaptic handling of glutamate have been implicated in the pathogenesis of brain damage after transient ischemia. Notably, the ischemic episode is associated with an increased extracellular level of glutamate and an impaired metabolism of this amino acid in glial cells. Glutamate uptake is reduced during ischemia due to breakdown of the electrochemical ion gradients across neuronal and glial membranes. We have investigated, in the rat hippocampus, whether an ischemic event additionally causes a reduced expression of the glial glutamate transporter GLT1 (Pines et al. 1992) in the postischemic phase. Quantitative immunoblotting, using antibodies recognizing GLT1, revealed a 20% decrease in the hippocampal contents of the transporter protein, 6 h after an ischemic period lasting 20 min induced by four vessel occlusion. In situ hybridization histochemistry with 35S labelled oligonucleotide probes or digoxigenin labelled riboprobes directed to GLT1 mRNA showed a decreased signal in the hippocampus, particularly in CA1. This reduction was more pronounced at 3 h than at 24 h after the ischemic event. We conclude that the levels of GLT1 mRNA and protein show a modest decrease in the postischemic phase. This could contribute to the delayed neuronal death typically seen in the hippocampal formation after transient ischemia.