Protection by an Adenosine Analogue against Kainate-Induced Extrahippocampal Neuropathology

ISSN 0306-3623/98 $19.00 1 .00 PII S0306-3623(97)00455-2 All rights reserved

Gen. Pharmac. Vol. 31, No. 2, pp. 233–238, 1998 Copyright  1998 Elsevier Science Inc. Printed in the USA.

Protection by an Adenosine Analogue against Kainate-Induced Extrahippocampal Neuropathology D. G. MacGregor,1 D. I. Graham,2 P. A. Jones1 and T. W. Stone1* 1 Division of Neuroscience and Biomedical Systems, West Medical Building, and 2Department of Neuropathology, University of Glasgow, Glasgow G12 8QQ, Scotland

ABSTRACT. 1. The glutamate analogue kainic acid produces neuronal damage in the central nervous system. We have reported that analogues of adenosine, such as R-N6-phenylisopropyladenosine (R-PIA) can, at doses as low as 10 mg/kg IP, prevent the hippocampal damage that follows the systemic administration of kainate. The present work was designed to examine purine protection against kainate in extrahippocampal regions by using histological methods. 2. The results show that R-PIA, at a dose of 25 mg/kg IP in rats, can protect against the neuronal damage caused by kainate in the basolateral amygdaloid nuclei, the pyriform cortex and around the rhinal fissure. This protection could be prevented by the simultaneous administration of the A1 adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine, confirming that the protection involved adenosine A1 receptors. No protection was observed in the posterior amygdaloid nuclei or the entorhinal cortex, suggesting the absence of relevant adenosine receptors or a different mechanism of excitotoxicity. gen pharmac 31;2:233–238, 1998.  1998 Elsevier Science Inc. KEY WORDS. Kainic acid, purines, adenosine, neuroprotection, neurodegeneration INTRODUCTION Evidence from a variety of sources indicates the involvement of excitatory amino acids in several central nervous system disorders in which the most prominent feature is neuronal degeneration. The neuronal damage resulting from focal cerebral ischemia, for example, can be prevented by agents that block the neurotoxic effects of amino acid agonists applied into the central nervous system. (Gill et al., 1987; Park et al., 1988; Simon et al., 1984). The glutamate analogue kainic acid originally attracted much interest because of its ability to produce axon-sparing lesions in the brain (Coyle et al., 1978), raising the possibility that it might be a useful tool in the study of neuronal communication. In addition, kainate is able to cross the blood–brain barrier, causing neurotoxicity after parenteral administration (Heggli and Malthe-Sorensen, 1982; Lothman and Collins, 1981; Schwob et al.. 1980). In particular, parts of the limbic system exhibit a high sensitivity to kainateinduced damage. The nature and pattern of damage resemble that seen to follow some forms of ischemia (Liu et al., 1996) or the repeated convulsive seizures seen in temporal lobe epilepsy (Ben-Ari et al., 1981; Coyle, 1987; Lothman and Collins, 1981; Schwarcz et al., 1984). It has also been remarked that the regional pattern of kainate-induced neuronal damage is similar to that seen in Alzheimer’s disease (Altar and Baudry, 1990). Similarities between kainate and ischemic neuropathology are consistent with the view that, during episodes of cerebral ischemia, the tissue hypoxia results in a release of endogenous glutamate and aspartate (Benveniste et al., 1984; Butcher et al., 1990; Hagberg et al., 1985), which can then activate N-methyl-d-aspartate (NMDA) and non-NMDA receptors to cause further excitation and transmitter release. This cycle of events may develop and persist for many hours. One approach to breaking the depolarization–release cycle *To whom correspondence should be addressed. Received 19 June 1997.

may be to suppress amino acid release by agents such as adenosine analogues. We have reported that the systemic administration of (R)-N6-phenylisopropyladenosine (R-PIA) can prevent the hippocampal damage produced by systemically injected kainate (MacGregor and Stone, 1993; MacGregor et al., 1993). This prevention occurs at doses as low as 10 mg/kg IP and can be demonstrated with R-PIA injection as long as 3 hr after the administration of kainate. The neurotoxicity produced by kainate in these latter studies was assessed by using the binding of the peripheral benzodiazepine site ligand PK11195. That study also was limited to the hippocampus. We have now investigated the extent of protection in regions other than the hippocampus by using histological methods. MATERIALS AND METHODS Male Wistar rats, 190–220 g, were injected intraperitoneally with drugs in a volume not exceeding 1 ml/kg. Kainic acid (10 mg/kg) and 8-cyclopentyl-1,3-dipropylxanthine (CPX) (50 mg/kg) were dissolved in saline, and R-PIA (25 mg/kg) in methanol. In all cases, vehicles were used as control injections and all animals were pretreated with clonazepam (0.2 mg/kg IP) 10 min before kainate injection to prevent the complications introduced by convulsive seizures. We have previously shown that this dose of clonazepam does not modify the extent of in vivo kainate-induced neuronal damage (MacGregor and Stone, 1993). The animals were killed 7 days after the injections.

Tissue fixation After 7 days, animals were given an overdose of sodium pentobarbitone (5 ml of 60 mg/ml), and perfusion was fixed with 40% formaldehyde, glacial acetic acid, absolute alcohol in the ratio 1:1:8. v/v (FAM) (Brown and Brierley, 1972). The animals were placed in the supine position and heparinized (1,000 IU/kg) after thoractomy and cannula insertion into the ascending aorta through the left ventri-

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cle. Physiological saline was infused for 5–10 sec followed immediately by 200 ml of FAM fixative. After infusion, the rats were decapitated and the heads stored and fixed at 48C for at least 12 hr. The brain was then removed. The hindbrain was detached by a cut through the midbrain, and the cerebral hemispheres were cut into five coronal slices, each 2-mm thick, and the cerebellum into two slices perpendicular to the folia on the dorsal surface of each hemisphere. Bilateral blocks of brain were embedded in paraffin wax and sections 7–8-mm thick were stained by hematoxylin and eosin and by a method combining cresyl violet and Luxol fast blue. The sections were examined by conventional light microscopy initially by one of us (D.I.G) without prior knowledge of the animal’s history, and the extent of neuronal damage throughout the brain was charted on line diagrams. The regions of brain examined were: (1) the pyriform cortex— this area was examined at the level of the lateral and ventrolateral orbital cortex, interaural level 11.7 mm (Paxinos and Watson, 1986); (2) the entorhinal cortex, which was examined at the level of interaural line 3.4 mm; (3) the rhinal fissure at interaural line 9.7 mm; and (4) the amygdaloid nuclei, which were examined both at the level of the the amygdalopyriform transition and the posteromedial cortical amygdaloid nucleus (interaural line 3.4 mm) and at the level of the basolateral nuclei (interaural line 5.7 mm). RESULTS Kainate-induced behaviors were evident within 30 min of the injection and were similar to those described previously (MacGregor et al., 1993; Schwob et al., 1980; Sperk et al., 1983) and included wet dog shakes, hind-limb abduction, Straub tail and excessive salivation. These behaviors were observed in all animals receiving kainate, irrespective of the treatment received. The administration of clonazepam substantially reduced the occurrence of tonic–clonic seizure activity. After 7 days, animals treated with kainate alone exhibited marked motor hyperactivity and sensitivity to touch. Animals showed no significant loss of weight over the 7-day recovery period.

Neuropathology Adequate perfusion was achieved in all animals, as shown by the lack of blood in the cerebral blood vessels and both firmness and pallor of the brain, good neuronal morphology and the absence of cytological artefacts (the “dark cell” and “hydropic cell”) (Brown and Brierley, 1972; Cammermyer, 1961).

FIGURE 1. Summary of the distribution of kainate-induced extrahippocampal neuronal damage. The figure shows diagrammatic sections of rat brain illustrated at approximate coronal levels (a) 111.7, (b) 110.7, (c) 19.7, (d) 18.08, (e) 17.2, (f) 15.7, (g) 13.4, (h) 12.2 relative to the interaural line (Paxinos and Watson, 1986). The hatched areas indicate the white matter areas and the ventricles. The continuously shaded areas indicate damage in a typical animal and include the pyriform cortex (PC), entorhinal cortex (EC), rhinal fissure (RF), medial thalamus (MT) and amygdaloid nuclei (AN). The hippocampus has been left unshaded for clarity because different regions of the hippocampus show different susceptibilities to kainate.

CONTROL-SALINE GROUP. No histological abnormalitites were seen in any of the six animals within this group. KAINATE GROUP. Evidence of neuronal damage was present in 10 of the 11 specimens in this group.In the ten damaged specimens, the distribution of lesions was similar and symmetrical. The line diagrams of Figure 1 summarize the areas showing signs of damage in a typical instance of kainate pathology. Principal sites of damage were the subfrontal cortex including the pyriform cortex, the cortex related to the rhinal fissure and the cortex of the inferomedial quadrant of each frontal lobe immediately in front of the genu of the corpus callosum, the entorhinal cortex, all nuclei of the amygdaloid complex, the dorsomedial thalamus, and the nuclei of each septum—principally medial but also lateral groups. The hippocampus was affected in all animals but to a variable extent. In none of the animals was there any involvement of the basal ganglia and, in two of the animals, changes were present in the upper brain stem (in one, there were foci of neuronal damage in the

periaqueductal grey matter and, in the other, there was extensive change within each substantia nigra). In none of the animals was there damage in the cerebellum. Affected neurons were more angular in shape and showed loss of nuclear detail and shrinkage, and the cell contents were often severely contracted, yielding a residual halo around the perimeter of the somata. The cytoplasm showed marked eosinophilic staining with loss of Nissl substance. A number of neurons stained densely with cresyl violet but were not argyrophilic. There was marked swelling of dendrites. In the least severely affected areas, such as the neocortex and thalamic regions, neuronal damage was restricted to single neurons or small clusters of neurons separated by normal cells; in the most severely affected areas, the entorhinal cortex and amygdaloid nuclei, neuronal changes were observed in a larger proportion of the neurons (Figs. 2c and 3c), the associated neuropile of which

Kainate Neuropathology

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FIGURE 2. Kainate damage and R-PIA protection around the rhinal fissure in Nissl-stained sections of rat brain. (a) line diagram of a coronal section of the forebrain at interaural line 19.7 mm. RF5rhinal fissure. (b)-(d) photomicrographs of the rhinal fissure: (b) illustrates a control vehicle-treated animal; the nerve cells have round to oval shaped nuclei with distinct nucleoli, while the neuropil is compact and evenly stained. (c) illustrates a kainate-treated animal; here a few normal neurones are apparent (vertical arrow) while most are disorganised, many appearing necrotic with distorted nuclei and eosinophilic cytoplasm (horizontal arrows). The neuropil shows microvacuolation. (d) illustrates an animal treated with kainate plus R-PIA; the cells appear normal. had a loose texture and stained only lightly with eosin. Apart from slight swelling of astrocytes, no changes were present in glia. KAINATE–R-PIA GROUP. At a dose of 25 mg/kg, R-PIA fully protected against kainate-induced damage in the basolateral amygdaloid nuclei, the pyriform cortex and rhinal fissure in 8 of 11 animals in this group. In these animals, the cell groups appeared normal, with no evidence of loss of structure or organization, with a normal number of neurons and no sign of reactive gliosis. The protection is illustrated for the rhinal fissure (Fig. 2d) and the basolateral amygdala (Fig. 3d) (see Methods for precise locations). The more posteriorly located regions examined—the entorhinal cortex at interaural line 3.4mm, the posteromedial cortical amygdaloid nucleus and the amygdalopyriform transition—showed a degree of damage comparable to that found in the kainate animals, with no apparent protection. KAINATE/R-PIA/CPX GROUP. None of the four animals treated with the combination of kainate, R-PIA (25 mg/kg) and CPX (50 mg/kg) was histologically normal. Damaged neurons were present in all the hippocampi, especially in the CA3 region, indicating some blockade by CPX of the neuroprotective activity of R-PIA.

DISCUSSION

Regional distribution The kainate treatment produced signs of damage in a number of subcortical areas, most notably the hippocampus, entorhinal cortex, septum, dorsomedial thalamus and all nuclei of the amygdaloid complex. As reported in previous studies, damaged neurons ap-

peared dark and shrunken; the regional distribution was entirely consistent with that described in earlier histological studies (Heggli and Malthe-Sorensen, 1982; Schwob et al., 1980; Sperk et al., 1983) and parallels results obtained when the degree of neuronal activation is assessed directly by using the 2-deoxyglucose method (Lothman and Collins, 1981). The distribution is also consistent with the view that systemic kainate reproduces the pattern of neuronal damage occurring in some human epileptic conditions (Ben-Ari, 1985; Du et al., 1993). The use of clonazepam was introduced in previous studies because interest in peripheral benzodiazepine binding was limited to the hippocampus, and it had been reported that diazepam could suppress behavioral seizures and distant neuronal damage while having much less, if any, inhibitory effect on the hippocampal toxicity of kainate (Ben-Ari et al., 1979; Chastain et al., 1987; Heggli and MaltheSorensen, 1982). The suppression of seizures by nonbenzodiazepine compounds such as dizocilpine (Planas et al., 1995) or phenobarbitone (Sutula et al., 1992) does not prevent signs of neuronal injury in the amygdala and hippocampal CA3 region. In the present study, however, clonazepam did not prevent neuronal damage in any brain region, despite the almost total suppression of overt seizure behavior. Although the explanation for this finding is unclear, it is possible that the profile of action of diazepam, used in earlier studies, differs from that of clonazepam. Certainly, the dose of clonazepam used here is about one-tenth to one-hundredth the dose of diazepam, yet it substantially reduced the incidence of seizures. Clonazepam may thus have a higher ratio of antiseizure to neuroprotective activity. Alternatively, the difference may simply be due to our deliberate

236 choice of the minimum dose of clonazepam needed to suppress behavioral seizures, a consideration that was not evident in earlier studies, which typically used from 1 to 10 mg/kg of diazepam.

Protection by R-PIA The protection by R-PIA of kainate-induced neurotoxicity supports our previous work with PK11195 (MacGregor and Stone, 1993; MacGregor et al., 1993) and extends the previous work by revealing protection in a number of nonhippocampal regions. The result is consistent with studies from other laboratories that have demonstrated protection by adenosine analogues against toxin- or excitotoxin-induced damage (Arvin et al., 1989; Connick and Stone, 1989; Finn et al., 1991) or against ischemic damage (Miller and Hsu, 1992; Rudolphi et al., 1992; Tominaga et al., 1992; von Lubitz et al., 1989). The ability to protect may even extend to agents which do not directly activate purine receptors but that elevate the levels of endogenous adenosine (Dux et al., 1990; Parkinson et al., 1994). The role of changes in body temperature in mediating protection against excitotoxins and ischemia is highly controversial [see Welsh and Harris (1991) and Welsh et al. (1990)]. Although a fall in body temperature appears to afford a degree of neuroprotection, it is unlikely that this effect contributes to the protection seen here with R-PIA, because we have previously recorded body temperature in animals receiving kainate and R-PIA and have found that, at the doses employed here, any fall in temperature is transient and does not achieve statistical significance. It is clear from studies in which the destruction of afferent path-

D. G. MacGregor et al. ways reduces kainate-induced excitation and neurodegeneration that intact terminals are required for kainate excitotoxicity (Heggli and Malthe-Sorensen, 1982; Kohler et al., 1978; McGeer et al., 1978; Nadler and Cuthbertson, 1980; Okazaki et al., 1988). This requirement may result from a presynaptic action of kainate to evoke the release of other compounds, such as glutamate and aspartate (Connick and Stone, 1986; Ferkany et al., 1982; Jacobson and Hamberger, 1985; Lehmann et al., 1983; Virgili et al., 1986), that are primarily responsible for the subsequent degeneration or it may indicate the need for such presynaptic factors to exert a permissive action on the effects of kainate. Adenosine and its analogues are known to act at A1 nucleoside receptors to suppress the release of a variety of neurotransmitters including glutamate (Corradetti et al., 1984; Fastbom and Fredholm, 1985), acetylcholine (Brown et al., 1990; Spignoli et al., 1984), dopamine (Chowdhury and Fillenz, 1991; Michaelis et al., 1979) and peptides. This activity could therefore lead to the blockade of amino acid release induced by kainate. Arguing against a mechanism based on inhibiting glutamate release are recent studies reporting that the extracellular concentrations of glutamate do not correlate with the degree of neuronal damage in the hippocampus (Mitani et al., 1992), but it should be emphasized that these studies involved ischemic damage. On the other hand, it has been shown that the systemic administration of R-PIA or an adenosine uptake inhibitor can suppress the elevation of glutamate levels in brain which occurs during ischemia (Cantor et al., 1992; Heron et al., 1994). It is certainly relevant that other agents that can suppress the release of excitatory amino acids, such

FIGURE 3. Kainate damage and R-PIA protection in the basolateral amygdaloid nucei in Nissl-stained sections of rat brain. (a) line diagram of a coronal section of the forebrain at the level of the amygdaloid nuclei. AN5basolateral amygdaloid nuclei. (b)-(d) photomicrographs of the AN. (b) illustrates a control vehicle-treated animal; the nerve cells have regular nuclei embedded in an evenly staining neuropil. (c) illustrates a kainate-treated animal; here there is a mixture of normal neurons (vertical arrow) and cells which have undergone necrosis with contracted, darkly staining nuclei and eosinophilic cytoplasm in a background neuropil that is pale and vacuolated and includes dying cells stained pink with eosin and haematoxylin (horizontal arrows). (d) illustrates an animal treated with kainate plus R-PIA; the majority of cells appear normal. Calibration bar5100 mm.

Kainate Neuropathology as the pyrimidine derivative BW1002C87 (5-2,3,5-trichlorophenyl)pyrimidine-2,4-diamine-1,10-ethanesulfonate), and k opiate receptor agonists are able to prevent neuronal damage in studies of ischemia (Graham et al., 1993; Ochoa et al., 1992). However, it is almost certain that other mechanisms contribute to the protective activity of adenosine on cells, because it is able to protect nonneuronal tissues such as heart and lung from ischemic injury (Zhao et al., 1993). Adenosine has also been shown to induce hyperpolarization of neurons in the hippocampus (Ameri and Jurna, 1991) and striatum (Stone, 1991; Thompson et al., 1992; Trussel and Jackson, 1985); and it is possible that such a direct inhibition of neuronal activity could contribute to neuroprotection. One of the most important findings of the present work is that R-PIA can produce protection in some but not all regions of the brain. It is possible that this difference is due to the absence of adenosine receptors in the resistant areas or that the mechanisms of excitotoxicity are different in these regions. These observations would be important to pursue because they may provide clues to the mechanisms of neuronal damage, the role of amino acids and other agents in cell death and the mechanisms of protection by purines. In summary, this study demonstrated the neuroprotective activity of the purine analogue R-PIA against neurotoxicity induced by systemic injections of kainic acid in some regions of the brain. Protection is mediated through the A1 subtype of adenosine receptor. In view of the similar pathology of kainate damage and that resulting from repeated seizures in humans, it would appear that purines may represent a promising avenue for the development of clinically useful neuroprotectant agents and that further comparative study of those brain regions sensitive or resistant to purine protection may provide valuable information on the mechanisms of excitotoxicity and neuroprotection. D. G. M. was supported by a Glasgow University Scholarship. The project was supported by the Scottish Home and Health Department, the W. A. Cargill Trust and Tenovus, Scotland.

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