Torben Bruhn

Chapter 7 Glutamate receptor transmission and ischemic nerve cell damage:evidence for involvement of excitotoxic mechanisms

Publisher Summary Cerebral ischemia has been studied in a number of animal models, mainly with rats. Although the most popular ones differ somewhat with respect to the neuropathological changes, often a hierarchy within the vulnerable cells can be identified. The development of morphological changes (acidophilia, eosinophilia) takes from a few hours to four days or longer. More severe ischemia results in an earlier occurring eosinophilia and vice versa, the so-called maturation phenomenon. In studying the influence of the excitatory (glutamatergic) input on the ischemic neuronal damage, the use of short ischemia is necessary. Neuropathological examination some days after an episode of cerebral ischemia reveals that a number of cell types are involved. The hematoxylin–eosin stain or acid–fuchsin stain are standard for the detection of eosinophilic neurons, but the Fink-Heimer impregnation is more suitable to show both dead nerve cell bodies as well as their axons and dendrites, and is the superior method if the distribution of ischemic damage must be mapped.

Ischemia induced changes in expression of the astrocyte glutamate transporter GLT1 in hippocampus of the rat.

Changes in cellular uptake of glutamate following transient cerebral ischemia is of possible importance to ischemia induced cell death. In the present study, we employed in situ hybridization and immunohistochemistry to investigate the influence of cerebral ischemia on expression of mRNA and protein of the astrocyte glutamate transporter GLT1, and of glial fibrillary acidic protein. Different subfields of CA1 and CA3 of the rat hippocampus were studied at various time-points after ischemia (days 1, 2, 4, and 21). In CA1, GLT1-mRNA was decreased at all time-points after ischemia except from day 2, whereas in CA3, decreases were seen only on day 1. Expression of GLT1-protein in CA1 was unchanged during the initial days after ischemia, but decreased markedly from day 2 to 4. In CA3, GLT1-protein increased progressively throughout the observation period after ischemia. Following the degeneration of CA1 pyramidal cells, a positive correlation between the number of CA1 pyramidal cells and expression of either GLT1-mRNA or -protein was evident selectively in CA1. Increases in expression of mRNA and protein of glial fibrillary acidic protein were present from day 2, most notable in CA1. The present data provide evidence that expression of GLT1 in CA1 of the hippocampus is not decreased persistently before the degeneration of CA1 pyramidal cells, but is downregulated in response to loss of these neurons. Since the reduction in GLT1 expression evolved concomitantly with the degeneration of CA1 pyramidal cells, it may contribute to the severity of CA1 pyramidal cell loss. A progressive postischemic increase in GLT1 expression in CA3 may be linked to the resistance of CA3 neurons to ischemic cell damage.

Effect of phenylsuccinate on potassium- and ischemia-induced release of glutamate in rat hippocampus monitored by microdialysis

The extracellular concentration of glutamate in rat hippocampus during physiological conditions, elevated extracellular K+ and global ischemia was followed by microdialysis and subsequent determination of glutamate by HPLC. The effect of phenylsuccinate, an inhibitor of the mitochondrial dicarboxylate carrier, was studied. It was found that while phenylsuccinate had no effect on the extracellular glutamate concentration during perfusion under physiological and ischemic conditions, the potassium-induced increase in the extracellular glutamate concentration was totally blocked by phenylsuccinate. Ischemia led to a pronounced glutamate overflow. The finding that phenylsuccinate could inhibit potassium-induced glutamate release into the extracellular space but not that induced by ischemia suggests that glutamate released under these conditions originates from different pools. Since glutamate released by a depolarizing concentration of potassium is likely to originate primarily from the transmitter pool, the ischemia-induced glutamate overflow may primarily be released from both the transmitter and the metabolic pool. This is compatible with the previous finding that phenylsuccinate specifically prevents biosynthesis of transmitter glutamate leaving the metabolic glutamate pool unaffected.

Secretory phospholipase A2 potentiates glutamate-induced rat striatal neuronal cell death in vivo.

The secretory phospholipases A2 (sPLA2) OS2 (10, 20 and 50 pmol) or OS1, (50 pmol) purified from taipan snake Oxyuranus scutellatus scutellatus venom, and the excitatory amino acid glutamate (Glu) (2.5 and 5.0 micromol) were injected into the right striatum of male Wistar rats. Injection of 10 and 20 pmol OS2 caused no neurological abnormalities or tissue damage. OS2 (50 pmol) caused apathy and circling towards the injection side. Histology revealed an infarct at the injection site. Injection of 50 pmol OS1 showed very little or no signs of neurotoxicity. Injection of 2.5 micromol Glu caused no tissue damage or neurological abnormality. After injection of 5.0 micromol Glu, the animals initially circled towards the side of injection, and gradually developed generalized clonic convulsions. These animals showed a well demarcated striatal infarct. When non-toxic concentrations of 20 pmol OS2 and 2.5 micromol Glu were co-injected, a synergistic neurotoxicity was observed. Extensive histological damage occurred in the entire right hemisphere, and in several rats comprising part of the contralateral hemisphere. These animals were apathetic in the immediate hours following injection, with circling towards the side of injection in the following days. Thus, OS2 greatly potentiates glutamate excitoxicity in vivo.

Limbic seizure-induced changes in extracellular amino acid levels in the hippocampal formation: a microdialysis study of freely moving rats.

Limbic seizure‐activity was induced by injecting kainic acid into the amygdala of rats. Extracellular levels of amino acids were monitored by microdialysis in the hippocampus. No changes were detected in the levels of glutamate and aspartate. The level of glycine also remained unchanged, whereas GABA showed an increase of approximately 35%. The level of glutamine decreased by approximately 30%, and that of serine by approximately 20%. The results indicate that increased turnover may exist in the glutamate transmitter pool. In addition, impairment of GABA‐release seems not to be a pathogenetic factor in seizure‐induced hippocampal neuron loss. It is concluded that even during sustained seizure‐activity, the extracellular level of glutamate, is maintained within narrow limits. A proposed index for excitatory neurodegeneration, glutamate x glycine/GABA, was found to be decreased in this seizure model. We therefore suggest that seizure‐induced neuron death is not reflected by alterations in the extracellular levels of glutamate and aspartate, thought to act as direct neurotoxins.

The effect of an AMPA antagonist (NBQX) on postischemic neuron loss and protein synthesis in the rat brain

Two groups of rats were subjected to 12 min of global cerebral ischemia and 6 days recirculation using the four-vessel occlusion model with hypotension and then treated with the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) antagonist NBQX [2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo (F) quinoxa-linedione (Honoré et al. 1988). One group was used for routine and quantitative histology and immunostaining for glial fibrillary acidic protein (GFAP). The second group was subjected to autoradiographic studies of regional cerebral protein synthesis, with special emphasis on the hippocampus, the frontal cortex, and the thalamus. It was found that neuroprotective treatment with NBQX normalized cerebral protein synthesis rate (CPSR) in all investigated regions 6 days after ischemia. In untreated ischemic animals CPSR was normalized in all regions except for the CA3 and thalamus, where it had increased by 29% and 41%, respectively. Treatment of controls with NBQX had no effect on CPSR after 6 days. The histological investigations revealed that NBQX did not protect vulnerable cells in the dentate hilus and the reticular thalamic nucleus (RTN). In these regions reactive astrocytosis visualized by GFAP immunostaining was equally pronounced in both ischemic and NBQX-treated animals, and most neurons in the RTN were eosinophilic. The 80–100% pyramidal neuron loss in CA1 was accompanied by a high degree of reactive astrocytosis, whereas the NBQX-treated animals showed no signs of astrocytosis in this region. The ischemic CA1 pyramidal layer was also massively invaded by microglia. Together these observations strongly suggest that the quantitatively normal protein synthesis in this region after ischemia must be attributed to these glial cell populations.

Evidence for formation of hydroxyl radicals during reperfusion after global cerebral ischaemia in rats using salicylate trapping and microdialysis

Systemic administration of salicylate (SA) to rats (100 mg kg-1 i.p. ) was used as an in vivo trap of hydroxyl radicals (.OH). In the brain SA reacts with hydroxyl radicals to form the stable adducts 2, 3- and 2,5 dihydroxybenzoic acid (DHBAs) which can thus be taken as an index of .OH formation. The DHBAs were recovered by intracerebral microdialysis in hippocampus or striatum and quantified by high pressure liquid chromatography (HPLC) with electrochemical detection. There were no peaks corresponding to 2,5-DHBA or 2,3-DHBA in the chromatograms from rats not receiving SA. A basal level of 2,5-DHBA was seen in the dialysates from all animals given SA whereas 2, 3-DHBA was not detected. In one group of rats generation of free oxygen radicals was induced in the striatum by adding Fe2+ and ascorbate to the perfusion fluid to test the sensitivity of the system. Addition of Fe2+ ascorbate to the perfusion fluid induced a significant 7-fold increase in 2,5-DHBA that gradually returned to baseline after removal of Fe2+/ascorbate. In two other groups the microdialysis probes were implanted in either the striatum or the hippocampus and the animals were subjected to 20 min of four-vessel occlusion + hypotension (4-VOH). Significant reductions in 2,5-DHBA were detected during ischaemia followed by significant increases of 5-fold and 3-fold in the striatum and hippocampus, respectively, beginning immediately upon reperfusion and lasting for the remainder of the observation period (160 min).

Regional cerebral protein synthesis after transient ischemia in the rat: Effect of the AMPA antagonist NBQX

Normothermic rats with 12 min, complete cerebral ischemia were treated with the AMPA antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo (F) quinoxalinedione (NBQX) [10], which prevents CA1 pyramidal neuron loss. Twenty hours after ischemia, cerebral protein synthesis rate (CPSR) was measured autoradiographically using [35S]methionine. Ischemia caused a 38% decrease of CPSR in CA1, and postischemic treatment with NBQX caused a 66% decrease in this region. Also treatment with NBQX alone resulted in a decrease (22% in CA1) of the CPSR. Since some evidence exists that the neuroprotective effect of NBQX is related to blockade of the fast AMPA-mediated transmission, the further decrease of the postischemic CPSR in CA1 could be a mere side effect.

In vivo cellular uptake of glutamate is impaired in the rat hippocampus during and after transient cerebral ischemia: A microdialysis extraction study

Using microdialysis in CA1 of the rat hippocampus, we studied the effect of transient cerebral ischemia on in vivo uptake and on extracellular levels of glutamate during, and at different time points after ischemia. 3H‐D‐aspartate (test substance), and 14C‐mannitol (reference substance), were added to the dialysis perfusate, and the cellular extraction of 3H‐D‐aspartate was calculated from scintillation analysis of fractionated dialysate samples. The extraction of 3H‐D‐aspartate was studied both in a tracer like condition with a perfusate concentration of 0.2 μM, and in a condition of high saturation level, with 1.0 mM D‐aspartate added to the perfusate. In between radioisotope perfusions, dialysate was sampled for analysis of amino acid content by HPLC. During ischemia, extraction of 3H‐D‐aspartate (0.2 μM) declined to a maximum reduction of 68%. In the hours after ischemia, extraction of 3H‐D‐aspartate (0.2 μM) was decreased by 32%. In the days after ischemia, there was a progressive decline in extraction of 3H‐D‐aspartate (1.0 mM), reaching a reduction of 89% on Day 4 after ischemia. Extracellular glutamate remained at control levels at all time points after ischemia. The present study is the first to investigate uptake of glutamate in the intact rat brain in relation to cerebral ischemia. Evidence is provided that uptake of Glu is restrained during ischemia, and that in the hours after ischemia, the extracellular turnover of glutamate is decreased. In the course of the days after ischemia, degeneration of CA1 pyramidal cells occurs concomitantly with a progressive decline in glutamate transport ability, possibly of pathogenetic importance to CA1 pyramidal cell loss. J Neurosci. Res. 66:1118–1126, 2001.

Evidence for increased cellular uptake of glutamate and aspartate in the rat hippocampus during kainic acid seizures: A microdialysis study using the `indicator diffusion' method

Using a newly developed technique, based on microdialysis, which allows cellular uptake of glutamate and aspartate to be studied in awake animals, we investigated uptake of glutamate and aspartate in the hippocampal formation of rats during limbic seizures induced by systemical administration of kainic acid (KA). With [14C]mannitol as an extracellular reference substance, the cellular extraction of the test substance [3H]D-aspartate was measured at different stages of seizure-activity. The results were compared to those obtained in a sham operated control group. During severe generalized clonic seizures, the extraction of [3H]D-aspartate was increased by 17%. The increase in uptake of [3H]D-aspartate was accompanied by a 24% increase in the extracellular level of aspartate, as obtained by conventional microdialysis. No significant changes were observed in the extracellular level of glutamate. The results indicate that during KA-induced seizures, uptake of glutamate and aspartate is increased, possibly aimed at maintaining the extracellular homeostasis of these two excitatory amino acids.