Influence of ketamine on the neuronal death caused by NMDA in the rat hippocampus

Neuropharmncology Vol.

34, No. 4, pp. 411417, 1995 Copyright 0 1995 Elswier Science Ltd Printed-in Great Britain. All rights reserved 0028-3908/95 $9.50 + 0.00

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Influence of Ketamine on the Neuronal Death Caused by NMDA in the Rat Hippocampus G. J. LEES Department of Psychiatry and Behavioural Science, School of Medicine, University of Auckland, Auckland, New Zealand (Accepted 12 December 1994) Summary--The protection provided by ketamine against the neuronal cytotoxicity of NMDA was investigated and compared wilth that provided by dizocilpine (MK 801). A massive anaesthetic dose of ketamine (180 mg/kg) was required for substantial protection (about 70%) of rat dorsal hippocampal neurons. Protection was markedly decreased if the ketamine was given in three divided doses of 60 mg/kg over a period of 2 hr, rather than as a bolus injection of 180 mg/kg. A lower dose (60 mg/kg i.p.) gave no protection when given 10 min prior to NMDA, but some protection (up to 30%) was found when administration was delayed for l-2 hr. After 3 hr, ketamine at this dose did not protect. In comparison, the toxicity of NMDA was reduced by about 70% by prior treatment with dizocilpine at 1 mg/kg, and completely eliminated at 10 mg/kg. The lack of protection when ketamine at 60 mg/kg was administered prior to NMDA may be due to a proconvulsant action of ketamine, as diazepam in the presence but not in the absence of ketamine significantly reduced the toxicity of NMDA. However, there was no behavioural or histological evidence of increased seizure activity in the presence of ketamine. Neuroprotectant effects may prevail with massive anaesthetic doses of ketamine or when diffusion has reduced the concentration of NMDA. The heroic doses of ketamine required for protection diminish its attractiveness as a potential anti-ischaemic agent. KeywordsNMDA,

excitotoxins,

ketamine,

NMDA

glutamate

antagonists,

diazepam,

hippocampus.

1993) and by an ischaemic episode (Meldrum et al., 1987; Church et al., 1988; Jensen and Auer, 1988; Marcoux et al., 1988; Ridenour et al., 1991). Ketamine has a very short half-life in the brain (White et al., 1976; De Sarro and De Sarro, 1993), and hence some of the inconsistencies could be due to the dose used and the length of time for which neuroprotective concentrations remain. In contrast, consistent protective effects have been found when in vitro conditions are used (Rothman et al., 1987; Choi et al., 1988; Takadera et al., 1990). Here the concentration of ketamine remains relatively constant over the course of the experiment. There is evidence that delayed administration of NMDA antagonists can also reduce the size of the lesions both in vim and in vitro (Foster et al., 1988; Bakker and Foster, 1991; Keilhoff et al., 1991; Massieu et al., 1993; Pellegrini and Lipton, 1993); the antagonist might thus have to be present for some time after the initial insult for maximum protection to be observed. This study was undertaken to determine the optimum conditions for protection by ketamine against the neuronal cytotoxicity of NMDA and to compare its effects with that of another non-competitive NMDA antagonist, dizocilpine (MK 801).

The contribution of glutamate and its analogues to the neuronal death occurring in experimental animal models of human neurological diseases is becoming well established [for recent reviews see Choi and Rothman (1990), Olney (1990), Leees (1993) and Meldrum (1993)]. The neuronal cytotoxicity is mediated mainly by interactions at ionotropic glutamate receptors. Of these, the N-methyl-D-aspartate (NMDA) receptor subtype appears to be of prime importance although the role of interactions at non-NMDA receptors is becoming increasingly recognized (e.g. Sheardown et al., 1990; Xue et al., 1994). Ketamine is a non-competitive NMDA antagonist (Anis et al., 1983; Won,g et al., 1986) which is of interest due to its clinical usage as a dissociative anaesthetic. Hence, its pharmacology and acceptability for human use is well known, and if effective against the toxicity of NMDA agonists would have major advantages over newer and lesser known drugs. Studies in vivo on the protective effects of ketamine have given inconsistent results. Both no (or mi:nimal) and substantial protective effects have been found against the lesions produced in vivo by NMDA agonists (Lees, 1987, 1989a; Beal et al., 1988; Keilhoff et al., 1990, 1991; Massieu et al., 411

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METHODS

One microlitre of NMDA (10 mM dissolved in 0.9% saline) was injected a rate of 0.4 pl/min into each dorsal hippocampus of Wistar rats ( 190-2 10 g), anaesthetized with l-1.5% halothane + 0.4 l/min OZ. Bilateral injections were given in order to reduce the number of rats used. At least one animal was injected with NMDA each session in order to confirm that there was no drift in its toxicity over time. The experiments were approved by Animal Ethics Committee of the University of Auckland, and all care was given to minimize any suffering by the animal. Behavioural evidence of seizure activity was present for up to 4 hr, but no other abnormal behaviour was detected after this time. Ketamine at doses of I5 or 60 mg/kg i.p. were injected 10 min before NMDA, and at various times subsequently. When ketamine at 180 mg/kg was used, no other anaesthetic was necessary. Injection conditions and coordinates have been described previously (Lees, 1992). Only the results from animals in which the tip of the cannula penetrated below the hippocampal fissure were included in the analysis. Dizocilpine was injected at doses of 1 and 10 mg/kg i.p. 15 min before NMDA. Diazepam (15 mg/kg i.p., 15 min before injection of NMDA) was used in some experiments to suppress seizure activity. It was injected as a 30 mg/ml solution in dimethylsulphoxide (DMSO). Behavioural evidence of seizure activity was assessed continuously over a period of 4 hr using the scale of Millan et al. (1986). Four days subsequently, the animals were deeply anaesthetized with pentobarbital (50 mg) and perfused transcardially with 4% formaldehyde in 0.1 M phosphate-buffered saline. Frozen sections (30 pm) were cut and stained for Nissl substance with thionine. Neuronal death was defined as the absence of all neuronal staining. Condensed basophilic neurons were not included. Some animals were sacrificed at earlier time points (6 and 24 hr) in order to detect evidence for early seizure-related injury to neurons in the brain. These brain sections were stained with a 0.1% solution of acid fuschin which stains both reversibly and irreversibly injured neurons following seizure activity (Lees, 1989b; Chang and Baram, 1994). Neuronal loss in the dorsal hippocampus was determined by a 40-point scoring system in which the overall extent of loss was estimated for each of the neuronal subgroups [subiculum, CAl, CA2, CA3, the polymorphic interneurons of the hilar region (PHR), and the dentate granule cell layer] (Lees, 1992). From these scores, a global estimate of neuronal damage over the whole of the dorsal hippocampus was made by summing the damage scores for the individual neuronal groups, using a correction factor to account for the area occupied by each neuronal group (Lees and Leong, 1994). The hilar interneurons were not included in this calculation, as they are sparse in number in relationship to the area they occupy. Statistical correlations for these values were by Mann-Whitney U.

Neuronal death outside the dorsal hippocampus (distal toxicity) was detected by an absence of Nissl-stained neurons. Regions examined for neuronal loss included the piriform and entorhinal cortices, the claustrum/ enteropiriform cortex, the thalamic nuclei, amygdala and medial and lateral geniculate nuclei. Of the 145 rats tested, 4 were found to contain distal lesions in other limbic regions. These included 2 out of 21 animals treated with NMDA alone, 1 out of 16 treated with NMDA and ketamine (60 mg/kg injected prior to NMDA) and 1 out of 10 treated with NMDA and ketamine at 180 mg/kg. Animals containing distal lesions were not included in the analyses, as the presence of these lesions indicated that substantial seizure activity had occurred (Ben-Ari, 1985; Olney et al., 1986). This by itself resulted in damage to the hippocampus over and above that caused by the direct toxicity of NMDA. For example, an animal treated with ketamine at 180 mg/kg which contained distal damage showed about a 75% destruction of the area occupied by pyramidal and granule cells in the hippocampus, compared with about a 12% destruction in the remaining 9 animals similarly treated with this dose of ketamine, but whose brains did not contain distal lesions. NMDA was obtained from Sigma, diazepam from Serva Feinbiochemica GMBH Co. Ketalar (Parke-Davis) was used as the source of ketamine. Dizocilpine was a gift from Merk, Sharp & Dohme. RESULTS NMDA (10 nmol) caused a sub-maximal lesion in the dorsal hippocampus, affecting the pyramidal neurons of the CAl, CA2 and CA3 regions, the dentate granule cells and the interneurons of the hilar region (Fig. 1). A moderate dose of ketamine (60 mg/kg, i.p. 10 min prior to NMDA) completely failed to prevent NMDA toxicity (Fig. 2). However, if the administration of this dose of ketamine was delayed for l-2 hr after NMDA the size of the lesion was significantly reduced in comparison to ketamine treatment given prior to NMDA. No effect of ketamine was found if the injection was delayed for 3 hr or if it was given in 4 divided doses of 15 mg/kg over 3 hr. Ketamine was only effective in blocking the majority of the lesion produced by NMDA when used as a bolus dose of 180 mg/kg i.p. (Fig. 3). If treatment with this dose of ketamine was delayed for 2 hr, or given in three divided doses of 60 mg/kg over a period of 2 hr, then its protective effect was markedly reduced. The long-acting noncompetitive NMDA antagonist dizocilpine (10 mg/kg, i.p.) reduced the size of the lesion to the background levels obtained with saline injections (Fig. 3). A lower dose of dizocilpine (1 mg/kg) gave a substantial protection with about a 70% reduction in the size of the lesion. Protection of all neuronal groups was found with either the high dose of ketamine or dizocilpine (Fig. 1). The possibility that concurrent administration of ketamine might alter seizure activity induced by NMDA prompted the use of diazepam as an anticonvulsant.

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Ketamine and NMDA-induced neuronal death Co-administration of diazepam reduced the toxicity of NMDA in the presenc:e of ketamine by about 40% (Fig. 2). Diazepam had no effect on NMDA toxicity in the absence of ketamine, in spite of behavioural evidence for seizures. Typically, animals treated with NMDA gave a short burst (up to 5 mim) of seizure activity 15-20 min following the injection. This ranged from rapid circling behaviour to full tonic+clonic convulsions. These bursts of seizure activity lasted for up to 45 min. For the next hour thereafter, only a very mild expression of seizure activity was observed, comprising gustatory movements, scratching and body and head shakes. Between 2-3 hr later, approximately 50% of the animals had a further short period of seizure activity (of about 2 min) where the animals reared, had forelimb clonus and in some cases, fell. Most animals demonstrated periods of head nodding and head and/or body tremor. By 4 hr, no further evidence of seizures was noted. Animals pretreated with ketamine (60 mg/kg) had milder symptoms initially, delayed with respect to latency (up to 30 min) and with briefer periods of motor seizures. By 2 hr, these animals exhibited the same symptoms as those treated with NMDA alone.

Animals sacrificed between 6-24 hr later were examined for histological evidence of injury. Neurons in the hippocampus were stained with acid fuschin at these times (stronger staining at 24 hr than at 6 hr). At 6 hr, Nissl staining revealed very pyknotic neurons in the core of the lesion.Surroundingthisarea,shrunken,basophilic“spiky” neurons with prominent dendritic staining were observed. Bothtypesofinjuredneuronsstainedwithacidfuschin,but the pyknotic neurons stained more intensely. The neuropil was also stained. By 24 hr, Nissl staining was lost, but Nissl-negative neurons staining with acid fuschin were still present. Outside the lesioned area, neurons were normal and failed to stain with acid fuschin. In limbic areas of the brain outside the dorsal hippocampus, faint staining of neurons with acid fuschin was observed at 6 hr, but not at 24 hr. The pattern of staining in the hippocampus and elsewhere in the brain was not altered in the presence of ketamine. DISCUSSION

The ability of competitive and non-competitive antagonists at NMDA receptors to protect neurons from damage following injection of glutamate agonists or after

60 -

T

SUB

CA1

PHR

CA2

DEN

AREA

Fig. 1. Neuronal loss in the dorsal hippocampus produced by the intrahippocampal injection of 10 nmol NMDA. Size of lesion with NMDA alone (O), n = 19; or in the presence of ketamine at 180 mg/kg ( q), n = 11; or MK 801 (dizocilpine) art 10 mg/kg (@), n = 10; saline control (m), n = 8. Significant differences in the cytotoxicity of NMDA over all neuronal groups were found in the presence of both ketamine and dizocilpine compared with damage produced. by NMDA alone (P < 0.05, Dunnett’s t-test). For individual neuronal groups, significant differences were found at P < 0.05(*)or P < 0.005(**)(r-test). SUB, subiculum; PHR, polymorphic interneurons of the hilar region; DEN, inner and outer blades of the granule cells of the dentate gyrus.

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T I

TREATMENT

Fig. 2. Neuronal cytotoxicity of NMDA after treatment with diazepam or ketamine (60 mg/kg i.p.) given at different times. Numbers in parentheses refer to the number of animals tested for each treatment. **Significant differences in cytotoxicity compared with damage produced by NMDA in rats given ketamine (60 mg/kg, i.p. 10 min prior to NMDA) (P < 0.05, Mann-Whitney U).

pathological events such as ischaemia or status epilepticus has been evaluated in many studies. Of the non-competitive inhibitors which bind within the ion channel of the NMDA receptor, dizocilpine is the most potent whereas ketamine is one of the least active (Anis et al., 1983; Wong et al., 1986). The protective effects of dizocilpine against the neurotoxicity of NMDA found here are in complete agreement with most previous investigations carried out in vivo (Woodruff et al., 1987; Foster et al., 1987, 1988), although Massieu et al. (1993) found that a dose of only 1 mg/kg prevented neuronal loss in the rat striatum. On the other hand, the use of ketamine as a neuroprotective agent in vivo has met with variable success (Meldrum et al., 1987; Church et al., 1988; Jensen and Auer, 1988; Marcoux et al., 1988; Clifford et al., 1990; Ridenour et al., 1991; Lees, 1992). These current results may help to explain why. Persistent efforts with ketamine have been made as its long acceptance as a clinically effective anaesthetic has meant that its pharmacology and safety is well understood.

Only a high anaesthetic dose of ketamine (180 mg/ kg) given as a bolus injection immediately before NMDA was found to have a major influence on the neuronal cytotoxicity of NMDA. A lower dose (60 mg/kg) given immediately before NMDA gave no protection whatsoever, yet the same lower dose given after a delay of 1 hr decreased the size of the lesion by about 30%. Delayed administration of NMDA antagonists are known to protect against NMDA toxicity, indicating an ongoing pathological activation of NMDA receptors (Foster et al., 1988; Bakker and Foster, 1991; Keilhoff et al., 1991; Massieu et al., 1993; Pellegrini and Lipton, 1993). Against ischaemic damage, massive doses of ketamine are also required for protection (Marcoux et al., 1988), and generally speaking no or only minor protection by ketamine is observed at doses of around 50 mg/kg or less (Church et al., 1988; Jensen and Auer, 1988; Ridenour et al., 1991); but note the contrary results of Meldrum et al. (1987).

Ketamine and NMDA-induced neuronal death

g

415

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5: k! 2

20-

s

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0-r

TREATMENT

Fig. 3. Effect of dizocilpine and high doses of ketamine (180 mg/kg) on the neuronal cytotoxicity of NMDA. Numbers in parentheses refer to the number of animals tested for each treatment. *Significant differences in cytotoxicity compared with damage produced by NMDA alone (P < 0.05, Mann-Whitney U).

The failure of prior administration of ketamine at 60 mg/kg to protect against NMDA toxicity is surprising. For example, Church et al. (1988) have estimated that ketamine at 20 mg/kg should block NMDA responses. It is, however, consistent with previous results obtained with ibotenate; although here ketamine substantially potentiated the toxicity (Lees, 1989a). One explanation of these results is that ketamine has opposing actions on NMDA toxicity. A proconvulsant action with moderate doses of ketamine could potent&e NMDA toxicity, but prevent it at higher anaesthetic doses. Proconvulsant and epileptiform activity can be induced by ketamine (Mori et al., 1971; Manohar et al., 1972; Ferrer-Allado et al., 1973; Myslobodsky et al., 1981; Nakao et al., 1993). Also consistent with this explanation is that diazepam significantly reduced the size of the lesion induced by NMDA in the presence of prior administration of ketamine, but not in its absence. Against this explanation is that while NMDA causes intense seizure activity (Zaczek et al., 1981), no behavioural evidence for a potentiation of this activity by ketamine could be detected. Nevertheless, it should be noted that NMDA

ion channel blockers fail to prevent electrogenic seizures while blocking the behavioural manifestations (Fariello et al., 1989). This would explain the relatively milder motor seizure activity found in the presence of ketamine. Intense seizure activity could result in additional neuronal damage. Indeed, in this investigation, a few animals (3% of all NMDA-treated rats) contained lesions in limbic areas other than at the site of injection. All animals in which limbic damage was found outside the hippocampus contained far larger lesions in the hippocampus (causing a neuronal loss of between 60-80%). Yet in the remaining 97% of animals, only transient acid fuchsin staining was found at 6 hr (but not at 24 hr), suggesting a very mild reversible distal injury. This pattern was found whether or not ketamine was used as an component of the anaesthetic. Acid fuschin has been used to detect early and completely reversible neuronal injury after seizures (Chang and Baram, 1994). Thus, the convulsive actions of NMDA only occasionally persist for long enough to cause neuronal death in brain regions outside the area of injection and ketamine fails to increase this distal toxicity.

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These results would suggest that ketamine does not have much potential as an anti-ischaemic agent under conditions where activation of NMDA receptors contributes to the brain pathology. It is possible that delayed administration of moderate doses might have some effect. This is in fact the most likely situation for drug administration after a traumatic event. Acknowledgements-This

research was supported by a grant from the New Zealand Neurological Foundation. The technical help of Dr W. Leong and the statistical analysis by Dr G. Elkind are gratefully acknowledged.

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