NMDA receptor antagonism, but not AMPA receptor antagonism attenuates induced ischaemic tolerance in the gerbil hippocampus

Abstract

Recent studies have shown that a brief `pre-conditioning' ischaemic insult reduces the hippocampal cell death caused by a subsequent more severe test insult. In the present studies, we have examined the effects of the non-competitive NMDA receptor antagonist ((5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine, MK-801) a competitive NMDA receptor antagonist, LY202157, AMPA receptor antagonist ((3S,4aR,6R,8aR)-6-[2-(1(2)H-tetrazole-5-yl)]decahydroisoquinoline-3-carboxylic acid, LY293558), a non-competitive AMPA receptor antagonist ((−)-1-(4-amino-phenyl)-4-methyl-7,8-methylenedioxy-4,5-dihydro-3-acetyl-2,3-benzodiazepine, LY300164), and a mixed NMDA/AMPA receptor antagonist, LY246492, in a gerbil model of ischaemic tolerance. Ischaemic tolerance was induced by subjecting gerbils to a 2-min `pre-conditioning' ischaemia (bilateral carotid occlusion) 2 days prior to a 3-min test ischaemia. The effects of MK-801 (2 mg/kg i.p.), LY293558 (20 mg/kg i.p., followed by 4×10 mg/kg at 3 h intervals), LY300164 (4×10 mg/kg i.p. at 1 h intervals), LY246492 (40 mg/kg i.p., followed by 4×20 mg/kg i.p. at 3 h intervals) and LY202157 (30 mg/kg i.p., followed by 4×15 mg/kg i.p. at 2 h intervals) were then examined in this model. Initial dosing commenced 30 min prior to the 2-min `pre-conditioning' ischaemia. Results indicated that a 2-min `pre-conditioning' ischaemia produced ischaemic tolerance in all cases. The non-competitive NMDA receptor antagonist, MK-801, produced a significant (P<0.01) reduction in the induced tolerance, while the competitive NMDA receptor antagonist, LY202157, also attenuated (P<0.05) the induction of tolerance. In contrast, two AMPA receptor antagonists (LY293558 and LY300164) and a mixed NMDA/AMPA receptor antagonist (LY246492) had no effect on the induction of tolerance. These results suggest that NMDA receptor activation, but not AMPA receptor activation is involved in the phenomenon of ischaemic tolerance.

Introduction

Transient global cerebral ischaemia in gerbils produces a selective pattern of neuronal damage (Kirino, 1982; Kirino and Sano, 1984; Crain et al., 1988). Five minutes of bilateral carotid artery occlusion produces severe damage in the CA1 pyramidal cell layer of the hippocampus (Kirino and Sano, 1984). The damage in the CA1 pyramidal cells develops slowly, starting 2 days after occlusion, with almost total destruction of the cells being observed 4 days post-occlusion. This phenomenon has been termed `delayed neuronal death' (Kirino, 1982). The exact mechanisms of damage remain to be fully elucidated, but several mechanisms (activation of voltage-gated calcium channels, excitotoxicity, free radicals, mitochondria and apoptosis) appear to be involved (Boxer and Bigge, 1997; Del Zoppo et al., 1997). The excessive increase of glutamate in the synaptic cleft following ischaemia is thought to play a critical role in the development of neuronal damage (Butcher et al., 1990). Several studies have indicated that many compounds acting at excitatory amino acid receptors have beneficial effects against cerebral ischaemia (Park et al., 1988, Park et al., 1992; Bullock et al., 1990, Bullock et al., 1994; Sheardown et al., 1990). Many of the early studies demonstrated that NMDA receptor antagonists are neuroprotective in animal models of global and focal cerebral ischaemia (Simon et al., 1984; Park et al., 1988, Park et al., 1992; McCulloch, 1992). Other studies have focused on the neuroprotective actions of AMPA receptor antagonists in animal models of global (Sheardown et al., 1990, Sheardown et al., 1993; Lodge et al., 1996; O'Neill et al., 1998) and focal (Bullock et al., 1994; Gill, 1994; Gill and Lodge, 1995; Gill et al., 1992; Graham et al., 1996; Yatsugi et al., 1996; Shimizu-Sasamata et al., 1998) cerebral ischaemia.

In 1991, Kirino et al. reported that a 2-min `pre-conditioning' occlusion, 2 days prior to a 5-min occlusion, led to a significant reduction in hippocampal cell loss compared with animals subjected to 5-min ischaemia alone. The authors also reported that this brief ischaemia caused an increased in 70 kDa heat shock protein and that this may render the neurones more tolerant to subsequent metabolic stress (Kirino et al., 1991). More recent studies have indicated that this tolerance is lost if the second ischaemia is carried out 4 weeks after the first brief ischaemia (Chen et al., 1994). However, if a second `pre-conditioning' ischaemia is carried out before the second 5-min ischaemia, at 4 weeks, the neurones are protected indicating that ischaemic tolerance can be induced repeatedly in gerbil hippocampal neurones (Chen et al., 1994). Other studies have shown that transient forebrain ischaemia also protects against subsequent focal cerebral ischaemia in rats (Matsushima and Hakim, 1994).

The mechanism of this induced tolerance is not clear, but brief periods of ischaemia alter gene expression and protein synthesis. Several studies have indicated an increase in heat shock proteins (Kirino et al., 1991) and other studies have shown regional increases in apoptotic gene expression (Chen et al., 1996). Further studies have reported that (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) attenuates the production of HSP-72 in the CA1 neurones and inhibits the induction of tolerance to ischaemia in the gerbil (Kato et al., 1992). However, the same study reported that anisomysin (a protein synthesis inhibitor) reduced HSP-72 synthesis, but failed to inhibit the induction of tolerance.

We wanted to further evaluate the role of NMDA receptor blockade on induced ischaemic tolerance and investigate if AMPA receptor antagonists, which provide greater protection in global ischaemia, also inhibited the induction of tolerance at neuroprotective doses. Therefore, in the present studies, we have examined the effects of a non-competitive NMDA receptor antagonist (MK-801), a competitive NMDA receptor antagonist (LY202157), a non-competitive AMPA receptor antagonist ((−)-1-(4-amino-phenyl)-4-methyl-7,8-methylenedioxy-4,5-dihydro-3-acetyl-2,3-benzodiazepine, LY300164), a competitive AMPA receptor antagonist ((3S,4aR,6R,8aR)-6-[2-(1(2)H-tetrazole-5-yl)]decahydroisoquinoline-3-carboxylic acid, LY293558) and a mixed NMDA/AMPA rece-t6r antagonist (LY246492) in a gerbil model of ischaemic tolerance.