Ascorbate attenuates the systemic kainate-induced neurotoxicity in the rat hippocampus

BRAIN RESEARCH ELSEVIER

Brain Research 727 (1996) 133-144

Research report

Ascorbate attenuates the systemic kainate-induced neurotoxicity in the rat hippocampus D . G . M a c G r e g o r ~ M . J . H i g g i n s ~~ P . A . J o n e s ~ W . L . M a x w e l l b M . W . W a t s o n ~ D.I. G r a h a m ~ T . W . S t o n e ~'* ;' West Medical Building, University ~?[Glasgow, Glasgow G]2 8QQ, UK ~' Anatomv Laboratories, Dit~ision of Neuroscience and Biomedical Systems, Unil'ersiO" of Glasgow, Glasgow G I 2 ~'QQ, UK " Department c?(Neuropatholog)', Unit~ersiO' qfGlaseow, Glasgow GI2 8QQ, UK

Accepted 4 April 1996

Abstract

The neuronal damage induced by systemic administration of kainic acid reproduces the cellular and regional pattern of damage produced by repeated seizures. The ability of kainic acid to induce lipid peroxidation, and the ability of free radical inhibitors to prevent ischaemically-induced cell death, has led us to examine the possible role of free radicals in kainate-induced injury. Ascorbic acid was able |o reduce kainate-induced damage of the rat hippocampus, measured by means of the gliotic marker ligand [3H]PKI 1195. Ascorbate was significantly effeclive at doses of 30 mg kg- ~ and above, with total protection against kainate at 50 mg kg ~. Histologically, ascorbate at 50 mg kg-~ was able to prevent kainate-induced neuronal loss in the hippocampal CA1 and CA3a cell layers. The antioxidant was also effective when administered simultaneously with, or 1 h before the kainate. Protection was also obtained by allopurinol, 175 mg kg t and by oxypurinol, 40 mg kg--t. Ascorbate did hOt modify synaptically evoked potentials or long-term potentiation in hippocampal slices, ruling out any blocking activity at glutamate receptors. It is concluded that the neuronal damage produced by systemically administered kainate involves the formation of free radicals. Kevwords: Kainic açid: Ascorbic acid; Vitamin C: Allopurinol: Free radical; Neuroprotection: Neurodegeneration

1. Introduction

The naturally occurring neurotoxin, kainic acid, is a potent agonist at an excitatory amino acid receptor subtype in the CNS and, like the structurally related domoic acid, it tan cross the blood-brain barrier and cause neuronal death [2,26,27,35,57,63]. The areas sensitive to systemically administered kainate and domoate differ from those areas affected by middle cerebral artery occlusion in that the hippocampus, amygdala, septum, entorhinal cortex and the midline hypothalamus are damaged by the glutamate analogues, with the striatum spared from damage [57,63]. lndeed the pattern of damage induced by systemically administered kainate approximates to that seen following repeated temporal lobe seizures [6,12,19,56,57] and in Alzheimer's disease [2], leading to the use of systemic kainate as a model for the neuropathology associated with these disorders.

Corresponding author. Fax: (44) ( 141 ) 330-4100. 0006-8993/96/$15.()0 Published bv Elsevier Science B.V. I'H S 0 0 0 6 - ~ 9 9 ~ ( o 6 ) 0 0 3 6 2 - 9

Current models of kainate neurotoxicity support the hypothesis that the main cause of neurotoxicity is the activation of presynaptic kainate receptors and the release of endogenous glutamate [10,11,13,18,49]. The released glutamate then acts postsynaptically on non-NMDA receptors to depolarise the membrane and relieve the voltage-dependent block by magnesium of NMDA receptors which can then contribute to the neuronal damage. The overstimulation of N M D A receptors has been implicated in the mediation of neurotoxicity caused by neurotoxins and ischaemia-related insults [9]. However, kainate is also thought to mediate damage partly through an indirect mechanism which may involve the overproduction of free radicals. Dykens et al. [15] reported that kainate toxicity in cell cultures resembled that seen in reperfusion injury in vivo, a form of tissue damage believed t o b e caused by free radicals, while Facchinetti et al. [17] reported that the use of the xanthine oxidase inhibitor allopurinol attenuated the damage in offactory cortex caused by intracerebral injections of kainate.

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I). G. ~4a "Gr«çoret l./Brain Re~war«h727 ~/ 996 ) 133-144

If such a scheme is indeed involved in the neurotoxic activity of kainate, then naturally-occurring free radical scavengers, sucb as ascorbate (vitamin C) should diminish the neuronal damage. We have therefore used the systemic kainate model of neurotoxicity [38,39] to examine this possibility and have compared the activity of ascorbate with allopurinol and oxypurinol.

2. Materials and methods All binding and electrophysiological experiments employed 8-week-old maie Wistar rats, 190-220 g, whilst the histological experiments employed 10 week old male Wistar rats, 250-280 g. AIl animals were kept under standard conditions. 2.1. NeurotoxiciO,

Animais were injected intraperitoneally with drugs in a volume not exceeding 1 ml kg -~, for all but the 175 mg kg-~ allopurinol dose. Kainic acid and ascorbate were dissolved in saline. Allopurinol 10 mg ml ~, was suspended in 2% Tween-80 in saline, pli 6. Oxypurinol was dissolved in DMSO, and administered at t 0 h and /24 la. Clonazepam was administered as Rivotril for Injection (Roche). In all cases the corresponding vehicles were used as control injections. In all experiments animals were pretreated with clonazepam (0.2 mg kg ~) i.p. l0 rein prior to kainate injection at t o. As reported in an earlier paper, this dose does hOt prevent either the behavioural effects of kainate or the neuronal damage produced by kainate, but does prevent the occurrence of tonic-clonic seizures [39]. Animais were killed 7 days after the injections and the brains removed for analysis. Rectal temperatures were recorded every 0.5 h for 2 h following injections, and then every hour for a total of 6 b. There was no significant change in temperature between any of the groups as assessed by ANOVA. 2.2. Tissue p r e p a r a t i o n

The method used was that of Eshleman and Murray [ 16] for the preparation of P2 membranes, as modified by MacGregor and Stone [38]. Tbe animals were killed by stunning followed by cervical dislocation. The hippocampi were removed and homogenised in 5 ml ice-cold 0.27 M sucrose (pli 7.4) with a Braun Homogeniser, 10 strokes × 500 rpm. The homogenate was brought to 20 ml with sucrose solution and was then stored at - 2 0 ° C for 2 - 4 h. After completing the preparation of tissue from a group of animals the samples were defrosted and centrifuged for 10 rein at 4°C, 1000 × g (IEC DPR 6000 centrifuge). The pellet was discarded and the supernatant centrifuged for 20 rein at 4°C, 16000 × g (Sorval RC 5B Refrigerated Superspeed Centrifuge, SS 34 Rotor). After this step the super-

natant was discarded and the pellet resuspended in 20 ml ice-cold 50 mM Tris-HCl buffet (pli 7.8) and then centrifuged for 20 rein at 4°C, 30000 X g (Sorval RC 5B). The new pellet was resuspended in 5 ml Tris-HC1 buffer and stored at -20°C, normally for no more than 24 la. On the day of the assay the samples were defrosted and centrifuged l'or 20 rein at 4°C 30000 × g, and the supernatant discarded. The pellet was homogenised in 5 ml Tris-HC1 buffer, 7 strokes × 1500 rpm, brought to 20 ml with Tris-HC1 buffer, and stored on ice until needed, this being the P2 mitochondrial membrane fraction. 2.3. [-¢H]PK11195 a s s a y

Ail assays were performed on ice and samples were incubated for 60 rein. The assay was performed in duplicate for both nonspecific and total binding. The volume of the assay chamber was 2 ml, and contained 5 gl [3H]PKllI95, 5 ~1 cold ligand, with 500 gl P» membranes and the volume brought to 2 ml with Tris-HC1 buffer. Final assay conditions were 1.75 nM [3H]PK11195 in etbanol (0.25% final), 0.25% DMSO +_ 10 ~M PK11195 and 100-150 p~g protein. The assay samples were w~rtexed at the start of their incubation and every 20 rein before filtration. The incubation was terminated by vacuum filtration, with all of the sample being filtered through prewetted Whatman G F / C glass filters using a Millipore 12-well 1225 Sampling Manifold. Filters were washed twice with 12 ml ice-cold Tris-HCl buffer and vacuum dried, before being placed in scintillation vials in 5 ml Ecoscint scintillation fluid. The samples were left overnight and counted using a Packard 2000 Scintillation Counter for d.p.m. Protein concentrations were measured using the Lowry method [36], following solubilisation with 0.25 M NaOH and with bovine serum albumin as the standard. 2.4. E l e « t r o p h y s i o l o g y

For the examination of the effect of ascorbate on synaptic transmission, the glutamatergic Schaffer collateral/CAl pyramidal cell pathway in the hippocampal slice was used. Slices were prepared 450-t~m-thick in artificial cerebrospinal fluid (in mM: KH2PO 4 2.2, KC1 2, NaHCO 3 25, NaC1 115, CaC12 2.5, MgSO 4 1.2, glucose 10) saturated with 95% 0 2 / 5 % CO 2. For electrical recording slices were superfused at 30°C. Population spikes were recorded in the stratum pyramidale in response to submaximal orthodromic stimulation in the stratum radiatum. With the population spike at 50% maximal long-terre potentiation (LTP) was induced (three bursts of 4 stimuli at 200 Hz, each burst 200 ms apart) in the presence or absence of 4 mM ascorbate (n = 6 for both). Ascorbate was superfused for 15 rein before measurements were taken, and 15 rein were allowed after returning to control medium, befi)re recovery size was measured.

D.G. MacGregor et al. / Brain Resear«h 72 7 (1996) 133-144

135

2.5. Histolog3

2.6. Data analysis

The animais were treated as above. Two groups of animais were used. The first group comprised the kainate controls (n = 7), and these animals were given 0.2 mg kg-i clonazepam i.p. 10 min prior to 10 mg kg-i kainic acid and 1 ml k g ~ saline i.p. The second group was the ascorbate group (n = 4), and these animais were given 0.2 mg kg i clonazepam 10 min prior to 10 mg kg -t kainic acid and 50 mg kg J ascorbate, ail i.p. The animais were then left to recover. After 7 days all the animais in the groups were given an overdose of sodium pentobarbitone (5 ml of 60 mg ml ~) and perfusion fixed with 40% formaldehyde, glacial acetic acid, absolute alcohol in the ratio 1:1:8, v / v / v (FAM), using the method described by Brown and Brierley [8]. Briefly the animais were placed in the supine position and heparinised (1000 IU kg ~). A thoracotomy was pert'ormed, and a cannula introduced into the ascending aorta via the left ventricle. Physiological saline was infused into the animal at mean arterial blood pressure for 5 - 1 0 s after incising the right atrium. This was followed immediately by 200 ml of FAM fixative at the same pressure. After infusion the rats were decapitated and the head was stored and fixed at 4°C for at least 12 h. The brain was then removed and the left cerebral hemispheres marked with indelible ink. The hindbrain was detached by a cut through the midbrain and the cerebral hemispheres were cut into rive coronal slices, each 2 mm thick. The brainstem was cut at right angles toits long axis into slices 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 secfions 7-8 >m thick were stained by haematoxylin and eosin and by a method combining cresyl violet and Luxol fast blue. An 8>m section was selected at the rostrocaudal level of the habenular nuclei and examined by convenfional light microscopy at ×400 magnification by three observers (P.A.J., D.G.M. and T.W.S.) unaware of the Ireatment received by the animal. The left hippocampus was scored for percentage damaged/dead neurones in the oeil layer. AI this time point, 7 days after kainate injection, lhe majority of cells appeared to be either healthy, with pale blue cytoplasm and a darker but clear, large nucleus and no evidence of vacuolation, or dead, with pink-staining cytoplasm. Relatively few cells were in an intermediate stage, retaining blue staining but with small, very dark, shrunken nucleus and evidence of intracellular or pericellular vacuolation. The hippocampus was examined at five sites, corresponding to the CAl, CA2, CA3a, CA3b and CA4 regions, and damage scored on a 0 - 1 0 point scale, with () representing an absence of detectable cell Ioss, and 10 indicating the absence of any normal pyramidal neurones in the field of view (a score of 5 thus indicating an estimate that 50% of the neurones were damaged).

Specific binding was calculated in absolute terms and as percent same day control to minimise day to day variations. Ail values are mean _+ S.E.M. Statistical significance was assessed by ANOVA with Student-Newman-Keuls post hoc test for the binding and histological experiments, and paired Student's t-test for the electrophysiological experiments, significance being considered when P < 0.05.

2.7. Materials [3H]PKl 1195 (specific activity = 80-86 Ci/mmol) was purchased from DuPont/NEN (Stevenage, Herts, UK), kainic acid and ascorbate from Sigma Chemical Co. (Poole, Dorset, UK), allopurinol and oxypurinol from R B I / S E M A T Technical (St. Albans, Hertfordshire, UK). Rivotril (clonazepam for injection) was purchased from Roche Laboratories. Unlabelled PK11195 was a girl from Pharmuka Laboratories.

2.8. Kainate / ascorbate interaction The stability of kainic and ascorbic acids when present together in aqueous solution was examined by ;H-NMR spectroscopy. The ~H spectra at 200 MHz were obtained with D20 solutions containing 0.1 mM of kainic a n d / o r ascorbic acids and 0.1 mM sodium chloride at 20°C. The spectra of kainic acid and ascorbic acid were first obtained separately. It was possible to assign separate, well-resolved multiplets to each of the ten types of nonequivalent protons in deuterated kainic acid, with the exception of those for 3- and 4-H, which overlapped. The low-field position of the signais for 2-H and 5-H, confirmed the zwitterionic structure of kainate. The spectrum of ascorbate similarly showed separate multiplets for 4and 5-H and for 6-H,. The spectrum of a mixture was essentially a superimposition of the two individual spectra, with no serious overlap of signais for the two species. Spectra were run al several time points up to 5 days, with no apparent change in this pattern. There was, therefore, no indication that ascorbate was able to reduce kainate in solution or induce the formation of dihydrokainate.

3. Results In ail cases, the kainate-injected animais displayed behavioural disturbances during the first 6 h after injection similar to those reported previously (wet dog shakes, hind-limb abduction, Straub tail, excessive salivation) [39,57,63]. The pretreatment with clonazepam did not prevent these behaviours. Similarly, as we have reported previously [38,39] this low dose of clonazepam (0.2 m g / k g ) had no effect on the extent of hippocampal data-

I).G. MacGregor et al./Brain Research 727 (1996) 133 144

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Fig. 1. Histograms showing the dose response relationship of ascorbate protection against kainate-induced neuronal damage. The ordinate indicates fmol of [3H]PKI 1195 b o u n d / m g protien. Animals were injected with 0.2 mg kg i clonazepam 10 min before kainate 10 mg kg t and a s c o r b a t e / s a l i n e injection. P2 membranes prepared as in Section 2. Columns indicate m e a n + S.E.M. (n = 3 8). Veh, vehicles injected; KA, kainate; ASC, ascorbate. * P < 0 . 0 5 , * * * P < 0.001 versus kainate group.

Fig. 3. Effect of increasing doses of allopurinol on kainate toxicity. The ordinate indicates fmol of [3H]PKI 1195 binding per mg protein. Animais were injected with clonazepam (0.2 mg kg i ) 10 rein before kainate 10 mg kg l. Allopurinol or vehicle (2% Tween-80 in 0 . 9 ç NaC1) ,sas injected with kainate (t o) and 6 h later 06). Pe membranes prepared as in Section 2. Columns indicate mean + S.E.M. Veh, vehicles injected; KA, kainate; ALLO, altopurinol. * P < 0.05, ~ *~ P < 0.001 versus kainate group. + P < 0.05, +ŒEEP < 0.01 versus control (vehicles) group.

age although it prevented almost all seizure activity [39]. Neither allopurinol nor ascorbate appeared to modify the behavioural events.

reduction in the kainate-induced [~H]PKll195 binding with an estimated EDs0 of 12.9 mg kg I and maximal protection at about 50 mg kg ] (Fig. 1). The ascorbate gave a significant reduction in [3H]PKI 1195 binding at 30, 50 and 100 mg kg l.

3.1. Binding studies

Previous studies [38,39] showed that systemic kainate administration causes a significant increase in [3H]PK11195 binding to hippocampal P2 membranes, and in the present study kainate caused a 3-fold increase in [3H]PK11195 binding compared to saline injection (250.95 _+22.89 fmol (mg protein) i ( n = 6 ) and 81.5_+5.11 fmol (mg protein) -] (n = 8) respectively, P < 0.001). Ascorbate, administered at t0 produced a dose-dependent

3.2. Time course

In the time course experiments, ascorbate 50 mg kg was administered as a single injection either before or after kainate injection at time t 0. Ascorbate only gave significant protection when administered 1 h before, or simultaneously with, kainate injection, but not when given 2 h before kainate (Fig. 2). Ascorbate treatment I h after

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Fig. 4. Dose response curve of oxypurinol induced neuprotection. Rats were pretreated with 0.2 mg kg ~ clonazepam 10 rein prior fo kainate, 10 mg kg t administration i.p. Animais were then given either 1 ml kg DMSO or oxypurinol i.p. at t¢~ h and t 2 4 h " Animais were left fur 7 days and the hippocampal P~ membranes prepared as in Section 2. Columns indicate means_+ S.E.M. (n = 4-8). D, DMSO: KA, kainate: Oxyp, oxypurinol; S, saline. ~ P < 0.005, * * P _< 0.01 versus k a i n a t e / D M S O group ( A N O V A and Student-Newman-Keuls post hoc test).

D.G. Ma«Gregor et al. / Brain Research 727 (1996) 133 144

kainate administration resulted in an intermediate mean level of [3H]PKIl195 binding but this was not significantly different from kainate treatment (Fig. 2).

When testing the activity of allopurinol against the toxicity produced by kainate injected directly into the

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brain, Facchinetti et al. [17] administered the allopurinol 0.5 h before the kainate. To allow for the delay in kainate penetration into the CNS when administered intraperitoneally, therefore, allopurinol was administered simultaneously with the kainate injection in the present study. Allopurinol at 17.5 or 75 mg kg J did not alter significantly the kainate-induced elevation in [3 H]PK11195 binding relative to the control kainate binding for this series of experiments (245.31 + 13.67 fmol (mg protein) J, n = 4), but it was effective at the dose of 175 mg kg--~ (Fig. 3).

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Fig. 5. Effect of ascorbate on hippocampal slices. Top: effect of ascorbate I mM, 4 mM or 10 mM on population spike size in the hippocampal slice. Slices were prepared as in Section 2. Points correspond to means + S.E.M. Ascorbate caused no change in the size of the population spike

after I mM (n = 6). 4 mM (n = 6) or 10 mM (n = 3) ascorbate perfusion. The small decrease seen after 10 m M is due to the change in pli caused by the ascorbate and was not significant. Bottom: effect of 4 mM ascorbate on the induction and early maintenance of LTP in the hippocampal slice. Slices were prepared as in Section 2 before perfusion with 4 mM ascorbate for 20 min (t 9 rein--/29 rein) with LTP initiated at time t25 mm" The slices were allowed to recover in normal ACSF and were stimulated fl)r the next 110 rein. 4 mM ascorbate ( n = 6 slices) (filled circles) did hot significantly alter either the induction or the maintenance of LTP compared to control slices (n = 6 slices) (open circles). Points correspond to means _+ S.E.M.

The oxypurinol vehicle, DMSO, did not alter either the basal or the kainate-induced elevation in [3H]PKll195 binding to hippocampal P2 membranes. Kainate caused a significant ( P < 0.01) increase in basal binding (35.48 _+ 2.87 fmol (mg protein) -f ( n = 4 ) in the presence or absence of D M S O (212.36 _+ 61.23 ( n = 4) and 235.6 + 5.9 (n = 4) fmol (mg protein) -I). There was no significant difference between the kainate groups (Fig. 4). Oxypurinol was able to attenuate the k a i n a t e / D M S O induced elevation in a dose dependent manner, with maximal protection seen at 40 mg kg-~ oxypurinol (48.72 + 8.49 fmol (mg protein)-I (n = 7), P < 0.01 versus kainate/DMSO). The 10 mg kg- i dose of oxypurinol also significantly attenuated the elevation by kainate (84.05 _+ 36.51 fmol (mg protein) -1 (n = 7. P < 0.05) (Fig. 4). Neither of the other two doses of oxypurinol (1 or 3 mg k g - ~) were significantly different from the k a i n a t e / D M S O group (Fig. 4).

3.5. Electrophysiology Population spikes were recorded in the hippocampal stratum pyramidale in response to submaximal orthodromic stimulation in the stratum radiatum.

D.G. Ma«Gregor et al. fl Brain Resear«h 727 (1996) 133-144

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Fig. 7. Photomicrographs of hippocampal regions of a rat treated kainic acid. a: CA 1; b: CA2; c: CA3a; d: CA3b; e: CA4. There is severe disruption of the CAl and CA3a layers with shrunken, darkly staining ce|ls with condensed media (compare with Fig. 8). Remaining layers in this animal show lesser amounts of damage. Scale bar = 100 Ixm.

D.G. Ma«Gre~or et al./Brain Research 727 (1996) 133 144

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140

D.G. MaçGregor et al. / BraiJz Research 727 ( 1990 ) 133-144

Ascorbate at 1, 4 or 10 mM caused no significant changes in the population spike size (n = 6, 6 and 3 respectively). Long-terre potentiation (LTP) was induced with three bursts of 4 stimuli at 200 Hz (each burst 200 ms apart) with the population spike at 50% maximal,in the presence or absence of 4 mM ascorbate (n = 6). Ascorbate did not significantly alter either the induction or the time course of LTP when followed for a period of 120 rein (Fig. 5).

3.6. Histology 3.6.1. Hippocampal damage Kainate/clonazepam treatment resulted in similar damage to the CAl and CA3a subregions of the hippocampus, with low level disruption in the CA2, CA3b and CA4 (Fig. 6). Affected neurones were more angular in shape, 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. There was marked swelling of associated dendrites, appearances being typically those seen in 'dendrosomatotoxic' but 'axon sparing' lesions. In the least severely affected areas neuronal damage was restricted to single neurones or small clusters of neurones separated by normal cells; in the most severely affected areas neuronal changes were observed in a larger proportion of the neurones, the associated neuropile of which had a loose texture and stained only lightly with eosin. Apart from slight swelling of astrocytes no changes were apparent in the morphology of glia, although they were substantially more numerous, as expected from the raised [3 H]PKI 1195 binding. In those areas where all or most of the neurones were affected, the endothelium of some of the small blood vessels was prominent. Ascorbate (50 mg k g - ~) was able to attenuate the mean damage to less than 20% in ail the hippocampal areas. However, this protection only attained statistical significance in the CAl and CA3a regions [20] ( P < 0.01 for both regions, Fig. 6). Examples of the kainate-induced damage and their prevention by ascorbate are illustrated in Figs. 7 and 8.

3.6.2. Other brain regions Kainate produced damage in several areas of brain in addition to the hippocampus. Principal sites of involvement 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, each amygdaloid nucleus, and the nuclei of each septum principally medial but also lateral groups. In contrast, of eight ascorbate-treated animals only two had damage in areas outside the hippocampus. In one case

the damage was restricted to the amygdala and the entorhinal cortex, whilst the other had damage to the thalamus and cortical regions rostral to the hippocampus. In none of these regions was the damage quantified.

4. Discussion Kainic acid has attracted special attention as a model of neuropathological damage since, after systemic administration, it produces a highly region-specific pattern of neuronal loss. Cells are killed preferentially in the amygdala, medial thalamus, septum, entorhinal and pyriform cortices and the hippocampus. While the existing literature indicates consistently that the CA2 region of the hippocampus is relatively resistant to seizures [29] and that the induction of seizures causes damage preferentially in area CAl, the sensitivity of other hippocampal areas to kainate seems to depend on several factors, particularly the route of administration. Thus, Nadler et al. [47] and Kohler et al.[30] found greatest damage in areas CA3 and CA4, while Sperk et a1.[63] reported the major effect in CAl and Schwob et a1.[57] found similar degrees of damage in CA1 and CA3. Out results also indicate a similar degree of damage in these latter areas, though the CA3 region clearly exhibits a differential response in the CA3a and CA3b subregions (as defined by Franck and Scwartzkroin [20]), the latter being relatively resistant to kainate. Our results clearly indicate that preventing seizures with a low dose of clonazepam (0.2 m g / k g ) does not prevent the induction of hippocampal damage. The damage observed here can therefore be attributed to excitotoxicity due to kainate itself rather than to the indirect activation of excitatory neurones by seizure activity. This resuh is consistent with several previous studies in which diazepam was used as the anticonvulsant. Fuller and Olney [21] reported only a partial protection by a large dose of 20 m g / k g diazepam and noted that in respect of the damage still seen in these animais "the site most often affected was the hippocampus". Similarly, Heggli and Maltbe-Sorensen [26] observed that diazepam given at 4 m g / k g every 30 rein for 6 h did not prevent kainate damage in the hippocampus even though it reduced damage elsewhere probably caused by electrical activity. There are some similarities here with ischaemically-induced brain damage, which consistently causes the greatest damage in area CAl [19,28,53,56]. Interestingly, Magloczky and Freund [41] bave recently noted that unilateral intrahippocampal injections of kainate result in a preferential loss of CA3 neurones ipsilaterally and of CAl neurones contralaterally, leading to the suggestion that CA3 Ioss is a direct consequence of the action of kainate on the dense population of receptors found here, while thc CA1 loss is indirect, probably produced by seizure activity. The present results, with substantial damage in area CA1 even in the absence of overt seizures, argues against

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this interpretation (though gross behavioural observation cannot exclude the occurrence of electrographic seizure activity). Rather, we would suggest that the data of Magloczky and Freund [41] could best be explained by the results and hypothesis of Balchen et al. [4]. In that study 10 m g / k g kainate was found to produce a loss of CAl and CA3 neurones, as also seen in the present work. Higher doses of kainate were round to damage CA3 cells much more severely than CA1, leading to the proposal that, since innervation of CA 1 is known tobe necessary for excitotoxicity, the rapid death of CA3 cells would prevent damage to the CAl cells to which they would project. A similar situation may well obtain in the work of Magloczky and Freund [41] due to the relatively high local concentrations of kainate achieved by direct injection into the hippocampus.

Antagonists acting at kainate receptors can prevent neuronal death occurring after experimental cerebral or local ischaemia in animais [14,51] and can prevent oeil death produced by glutamate on neuronal cultures. One possibility is that kainate damage is mediated by free radicals. Frec radicals are likely to produce cell damage by inducing lipîd peroxidation in the cell membranes [60]. Lipid peroxidation results from the attack by hydroxyl radicals ( O H ) on phospholipid side chains in cell membranes. This initiates a chain reaction in which the disruption of one molecule generates another free radical; a large section of membrane can thus be destroyed by a single, initiating free radical. Several studies bave implicated free radicals in central neurodegeneration after different types of insult [50,60] and have led to proposais for free radical involvement in neurodegenerative disorders such as Alzheimer's disease [25]. Free radicals are produced during experimentally induced seizures, fatty acids representing a major probable source [3]. Free radicals and lipid peroxidation are produced during brain ischaemia and reperfusion, as demonstrated by salicylate trapping or electron spin trapping studies in vivo [23,64] and protection against ischaemia is afforded by the free radical metabolising enzyme superoxide dismutase [29]. Free radicals are also formed when kainic acid is administered to animals [65], resulting in a five-fold enhancement of lipid peroxidation [7,54]. Suppression of free radical activity or formation can reduce tissue damage [51,55]. Neuronal damage in cell cultures tan be prevented by the antioxidant o~-tocopherol (a component of vitamin E), and a number of new drugs are being developed as antioxidants or free radical scavengers wbich reduce neuronal damage by amino acids in culture or after ischaemia [24,37,70]. Kainic acid could induce free radical formation as a result of its elevation of intracellular calcium [40]. This is known to activate proteases and other degradative enzymes which promote the conversion of xanthine dehydrogenase to xanthine oxidase (XO), and hence initiates the production of the superoxide radical. Recently Oury et al. [48]

141

reported that 0 2 can react directly with nitric oxide (NO) to form peroxynitrite anions ( O N O O ) which is protonated at cellular pli to form peroxynitrous acid (HONO0). Peroxynitrous acid rapidly decomposes by homolytic cleavage to form hydroxyl radicals. The possible importance of at least the early part of this scheme in kainate toxicity is supported by the observation that oxypurinol, and to a lesser extent allopurinol, a potent inhibitor of xanthine oxidase [62], tan prevent neuronal damage in the present model. Previous work has demonstrated neuro-protection by allopurinol following local injections of kainate or middle cerebral artery occlusion [17,67]. Phillis and co-workers have found that oxypurinol is a more powerful neuroprotectant than allopurinol, and that the dose used in the present study can prevent frec radical formation following either traumatic brain injury or middle cerebral artery occlusion [34,52,65]. In addition, oxypurinol can afford protection against the morphological and biochemical consequences of cerebral ischaemia. AIthough data are not available to assess the extent of xanthine oxidase inhibition in the brain in the present experiments, itis important to note that doses h)wer than those used here (10 m g / k g ) produced a complete blockade of xanthine oxidase activity in the canine heart [31]. It should be emphasised, however, that xanthine oxidase activity in brain is reported t o b e very Iow or undetectable by some groups, and we cannot exclude the possibility thal allopurinol and oxypurinol are, in the present study, acting as free radical scavengers. However formed, free radicals would be able to disturb neuronal function either non-specifically, by the induction of lipid peroxidation, or by enhancing amino acid mediated excitotoxicity, this being the result of a reduction of cellular uptake [68], an increased release [22] of excitatory amino acids, or an enhancement of calcium influx [46]. 4.1. As«orbi« a«id

It is against this background that the neuroprotective properties of ascorbate must also be viewed. Several previous studies have demonstrated protection by ascorbate against cell damage induced by ischaemia and repeffusion [33,58] levodopa [44] or glutamate applied to cultured neurones [42]. We have now demonstrated that systemically administered ascorbate tan afford protection against systemic kainate in vivo, as assessed in both ligand binding and histological studies, with NMR studies of mixed solutions excluding any direct chemical interaction between these two agents. Ascorbate is unable to cross the blood-brain barrier at the level of the brain capillaries [61], but can enter the neuropil by a two-stage saturable system. The first system is an active transporter in the choroid plexus [59-61], and tbe second system is at the level of the plasma membrane [59,69]. These two systems concentrate ascorbatc, with an estimated CSF concentration of 0.2 mM and a neuropil

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D.G. MacGreçor et al./Brain Research 727 ¢1996) 133 144

concentration of 1.4 mM (to be compared to an estimated plasma concentration of 0.05 mM [60]). However, within the brain, the ascorbate levels are not homogeneous, with the highest levels being found in the amygdala, hippocampus and the hypothalamus, in humans. If 50 mg kg ~ ascorbate was administered to an animal, and provided there was no change in the ratios of ascorbate in the different pools, this would result in a 7-fold increase in the brain concentration. The rime window of protection by ascorbate is considerably shorter than that reported for other protectants such as dizocilpine or R-phenylisopropyladenosine [38,39]. This may reflect the fact that ascorbate is rapidly taken up from the extracellular space as revealed by the brief increases of extracellular concentrations induced by glutamate administration [9]. The protective activity of ascorbate in the present study may be the result of its acting as a free radical scavenger or as an antagonist at amino acid receptors (NMDA or non-NMDA). Ascorbate bas been shown to reduce ionic currents activated by NMDA, possibly by acting at the redox site associated with the receptor [1,43,66]. To assess the importance of this action we have examined the effect of ascorbate on LTP in the hippocampal slice. This phenomenon was selected because i t i s known to involve activation of NMDA receptors to the extent that an increase of postsynaptic calcium levels occurs [40]. This was felt to be of more relevance to the present work on neuronal damage, in which a fise of intracellular calcium has also been implicated, than a simple examination of ascorbate on NMDA-induced neuronal depolarisation. The lack of effect of ascorbate on LTP, even at 4 mM, or on normal population spikes, effectively rules out any direct antagonist activity at synaptically activated NMDA or n o n - N M D A sites sufficient to accourir for neuroprotection. It is most likely, therefore, that ascorbate is acting as a free radical scavenger in affording protection against kainate. In a previous study Beal et al [5] reported that neither ascorbate nor allopurinol could attenuate the heutonal damage produced by the NMDA receptor agonist quinolinic acid (see Ref. [64]). This result suggests that both ascorbate and allopurinol are acting in the present work a t a point between activation of kainate receptors and the subsequent recruitment of NMDA receptors. An earlier study by Miyamoto et al. [45] was among the first to demonstrate that a free radical scavenger (idebenone) could protect against kainate neurotoxicity. This view is complicated by the recent work of LafonCazal et al. [32], who have claimed that N M D A receptor stimulation but hOt kainate can result in the formation of superoxide anions in cultured cerebellar neurones. The sequence of events involving kainate receptors, NMDA receptors and free radical formation must therefore remain unclear, although it is possible that the results of LafonCazal et al. [32] reflect the existence of a different balance of N M D A and kainate receptor populations in cultured

cerebellum. The conclusions of the present paper are em tirely consistent with direct evidence in vivo or using freshly dissociated cells that kainate can increase the amount of lipid peroxidation [7,54,65]. The parallels between the amount of lipid peroxidation in the CNS and the amount of ischaemically-induced damage [23,24,60] also emphasise the utility of systemic kainate as a model for the study of protection against neuronal damage mediated by free radical mechanisms.

Acknowledgements D.G.M. was supported by a University of Glasgow Postgraduate Scholarship and grants from Tenovus Scotland and the W.A. Cargill Trust, Glasgow. We are grateful to Prof. G.W. Kirby, Department of Chemistry, for performing the NMR analysis.

References [1] Aizenman, E., Hartnett, K.A. and Reynolds. l.J., Oxygen free radicals regulate NMDA receptor function via a redox rnodulatorysite, Neuro,. 5 (1990) 841 846. [2] Altar, C.A. and Bau&y, M., Systemic injection of kainic acid: gliosis in olfactory and limbic brain regions quantified with [3H]PKI1195 binding autoradiography, Exp. Neurol., 1(/9 (1990) 333-341. [3] Armstead, W.M.. Mirro, R., Lelfler, C.W. and BusÖa, D.W., Cerebrai superoxide anion generationduring seizures in newbornpigs, ./. Cereb. Blood Flow Metab., 9 (1989) 175 179. [4] Balchen, T., Berg, M. and Diemer, N.H., A paradnx after systemic kainate injection in rats - lesser damage of hippocampal CAl neurons after higher doses. Neuros«i. Leu., 163 (1993) 151- 154. [5] Beal, M.F,. Kowall, N.W., Swartz, K.J., Ferrante, R.J. and Martin, J.B., Systemic approaches to modifying quinolinic acid striatal lesions in rats. J. Neuros«i., 8 (1988) 3901-3908. [6] Ben-Arî, Y., Tremblay, E., Riche, D., Ghilini, G. and Naquet, R., Electrographic clinical and pathological alterations following systemic administration of kainic acid, bicuculine nr pentetrazole: metabolic mapping using the deoxyglucose method with special referencc to tbe pathology of epilepsy, Neuroscien«e, 6 (1981) 1361 1391. [7] Bnse. R., Schnell, C.L., Pinsky, C. and Zitko, V.. Effects of excitotoxms on free radical indices in mouse brain, Toxicol. Lett., 60 (1992) 211 219. [8] Brown, A.W. and Brierley,J.B., Anoxic-ischaemiccell change in rai brain. Light microscopic and fine structural observations, ,/. N«urol. Soi., 16 (1972) 59-84. [9] Cammack, J., Ghasemzadeh, B. and Adams. R.N., The pharmacoIogical profile of glutamate-evokedascorbic acid effiux measured by in vivo electrochemistry, Bram Res., 565 (1991) 17-22. [10] Chni, D.W. and Rothman, S.M.. The role of glutamatc neurotoxicity in hypoxic ischaemic neuronal death, Anm«. Re«. N«uros«i., 13 (1990) 171 182. Il I] Connick.J.H. and Stone. T.W.. The effects of quinolinic,kainic and beta-kainic acids on the release of endogenous glutamate from rat brain slices, Bio«hem. Pharmacol., 35 (1986) 3631-3635. [12] Coyle. J.T., Kainic acid: insights into excitatory mechanismcausing selective neuronal degredation. In Sele«til'e N«uronal Death (Ciba Foundation Symposium 126), Wiley, Chichester, 1987, pp. 186-2(13.

D.G. MacGregor et al. / Brain Resear«h 727 (1996) 133 144

[13] Coyle. J.T. and Puttfarcken, P., Oxidative stress, glutamate and neurodegenerative disorders, Science, 262 (t993) 689-695. [14] Diemer. N.H., Johansen, F.F. and Jorgensen, M.B., NMDA and non-NMDA antagonists in global cerebral ischaemia, Stroke, 21 (Suppl. 3) (1990) 39-42. [15] Dykens, J.A., Stern. A. and Trenkner, E., Mechanisms of kainate toxicity fo cellular neurones in vitro as analogous to reperfusion tissue injury, ,/. Neurochem., 49 (1987) 1222-1228. [16] Eshleman, A.J. and Murray, T.F., Differential binding properties of the peripheral-type benzodiazepine ligands [3H]-PKI 1195 and [~H]Ro 05-4864 in mmt and mouse brain membranes, J. Neurosci., 53 (1989) 494-502. [17] Facchinetti, F.. Virgili, M., Contestabile, A. and BarnabeL O., Antagonists of the NMDA receptor and allopurinot protect the olfactory cortcx but not the striatum after intracerebral injection of kainic acid, Brain Res.. 585 (1992) 330-334. [18] Ferkany, J.W.. Zaczek, R, and Coyle, J.T., Kainic acid stimulates excitatory amino acid neurotransmitter release at presynaptic receptors, Nature. 298 (1982) 757-759. [19] Franck, J.E. and Roberts, D.L.. Combined kainate and ischaemia produce "mesial temporal sclerosis', N«uros«i. Leu., 118 (1990) 159-163. [20] Franck. J.E. and Schwartzkroin, P.A.. lmmature rabbit hippocampus is damaged by systemic but not intraventricular kainic acid injection, Der. BramR«~.. 13(1984) 219 227. [21] Fuller, T.A. and Olney, J.W., Only certain anticonvulsants protect against kainate neurotoxicity. Neu~wbehal'. Toxi«ol. T«ratol. 3 ( 1981 ) 355-3~M. [22] Gihnan. S.C., Bonnet, M.J. and Pellmar, T.C., Free radicals enhance basal release of l)-[XH]-aspartate from cerebral cortical synaptosomes, .I. Neuroehem.. 62 (1994) 1757 1763. [23] Hall, E.D., Andrus, P.K., AIthaus, J,S. and von Voigtlander, P.F., Hydroxyl radical production and lipid peroxidation parallels selective post-ischaemic vulnerability in gerbil brain, J. Neutws«i. Re~'., 34(1993) 107 112. [24] Hara. H. and Kogure, K., Prevention of hippocampus neuronal damage in ischaemic gerbils by a novel lipid peroxidation inhibitor (quinazoline derivative), ,I. Pharma«ol. Ærp. Ther., 255 (1990) 906 910. [25] Harman, I).. Free radical theory of aging: a hypothesis on pathogenesis of senile dementia of the Alzheimer type, Age, 16 (1993) 23 30. [26] Heggli, D.A. and Malthe-Sorensen, D., Systemic injection of kainic acid: effect on neurotransmitter markers in pyriform cortex, amygdaloid complex and hippocampus and protection by cortical lesioning and anticonvulsants, Neutws«ience, 7 (1982) 1257-1264. [27] Heggli. D.A.. Aamodt, A, and Malthe-Sorensen, D., Kainic acid neurotoxicity: effect of systemic injection on neurotransmitter markers in different brain regions, Brain Res., 230 (1981) 253-262. [28] Kirino, T.. Tamura, A. and Sano, K.. Selective vulnerability of the hippocampus to ischemia - reversible and irreversible types of ischemic oeil damage. Pro:,,. Brain Res., 63 (1985) 39-58. [29] Kitagawa. K.. Matsumoto, M.. Oda, T., Ninobe, M., Hata, R., Handa. N.. Fukunaga, R., lsaka,Y,, Kimura, K.. Maeda, H., Mikoshiba. K. and Kamada, T.. Free radical generation during brief period of cerebral ischaemia may trigger delayed neuronal death, Neuro.scietl««, 35 (1990) 551 558. [30] Kohler. C., Schwarcz, R. and Fuxe. K., Hippocampal lesions indicate differcnces between the excitotoxic properties of acidic amino acids. Bram Res., 175 (1979) 366 371. [31] Kuzmin, A.I., "Fskîtish,Ali, O.V., Serebryakova, V.I., Kapelko, V.I. and Mztiorova, I.V.. Allopurinol: kinetics, inhibition of xanthine oxidase activitv and protectivc effect in ischaemic-repeffused canine heart as studîed by cardiac microdialysis, J. Cardioz:as«. Pharrna«ol., 25 (1995) 564 571. [32] Laton-Cazal. M.. Pietri, S., Culcasi, M. and Bockaert, J., NMDA-

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48] [49]

[50]

[51]

[52]

143

dependent superoxide production and neurotoxicity, Nature. 364 (1993) 535 537. Lagerwall, K., Daneryd, P., Schersten, T. and Soussi, B., In vivo P31 NMR evidence of the salvage effect of ascorbate on the postischaemic reperfused rat skeletal muscle, Lf/i, Soi., 56 (1~)94) 389 397. Lin, Y. and Phillis, J.W., Deoxycoformycin and oxypurinol: protection against focal ischemic brain injury in the rat, Brain Re~'.. 571 ( 1991 ) 272-280. l_othman, E.W. and Collins. R.C., Kainic acid induced limbic seizures: metabolic, behavioural, electroencephalographic and neu ropathological correlates, Brain Res., 218 ( 1981 ) 299-318. Lowry, O.H., Rosebrough, N.J., Farr. A.L. and Randall, R.A.. Protein measurements with the folin phenol reagent. J. Biol. ('hem., 193 (1951) 265-275. Lysko, P.G., Lysko, K.A., Yue, T.L., Webb, C.L., Gu, J.[,. and Feuerstein, G., Neuroprotective effects of Carvedilol, new antihypertcnsive agent, in cultured rat cerebellar neurons and in gerbil global brain ischaemia. Stroke, 23 (1992) 1630-1636. MacGregor, D.G. and Stone, T.W., Inhibition by thc adenosmc analogue, (R-)-N%phenylisopropyladenosinc, of kainic acid neurotoxicity in rat hippocampus after systemic administration, Br. ,/. Pharmacol., 109 (1993) 316-321. MacGregor, D.G., Miller, W.J. and Stone. T.W., Mediation of thc neuroprotective action of R-phenylisopropyladenosine through a centrally located adenosine AI receptor. Br. ,I. Pharma«ol., I lO (1993) 470-476. MacDermott, A.B., Mayer, M.L..Westbrook, G.L., Smith. S..l. and Barker. J.L., NMDA receptor activation increases cytoplasmic calcium concentrations in cultured spinal tord neurones. Nature, 321 (1986) 519 522. Magloczky, Z and Freund, T.F., Selectivc neuronal death in the contralateral hippocampus following tnfilateral kainale injections into the CA3 subfield, Neuros«iem'e, 56 (1993) 317 336. Majewska, M.D. and Bell, J.A.. Ascorbic acid protecls neurons from injury induced by glutamate and NMDA, 3,'euroRel~orl. I (1990) 194 196. Majewska. M.D., Bell, J.A. and London. E.D., Rcgulation of thc NMDA receptor by redox phenomena: inhibimry role of ascorbate, Brain Res.. 537 (1990)328-332. Mena, M.A.. Pardo, B., Paino, C.L. and Yebenes..I.G.D.. Lc,,odopa toxicity in foetal rat midbrain neurones in cuhurc: modulation by ascorbic acid, NeuroRepotT, 4 (1993) 438-440. Miyamoto, M. and Coyle. J.T,, ldebenone attenuates neumnal degeneration induced by intrastriatal injections of excitotoxins. Exp. Neurol., 108 (1990) 38-45. Munns, P.[,. and Leach, K.L., Two novel antioxidants, U74006F and U78517F, inhibit oxidant-stimulated calciuna influx, fr«e Radical Biol. Med., 18 (1995) 467-478. Nadler, J.V., Perry, B.W., Gentry, C. and Cotman, C.W., Degeneration of hippocampal CA3 pyramidal cells induced by intraventricular kainic acid. J. Comp. N«ulwl., 192 (1980) 333 359. Oury, T.D., Pinatodosi, C.A. and Crapo, J.D.. Cold-induced brain edema in mice..I. Biol. Chem.. 268 (1993) 15394- 15398. Palmer, A.M.. Reiter, C.T. and Botscheller. M., Comparison of the release of exogenous anhd endogenous excitatory amino acids rioto rat cerebral cortex, Ami. NY A«ad. SeL (1992) 648, 361 364. Pazdernik. T.L., Layton, M.. Nelson, S,R. and Samson, F.E., The osmotic/calcium stress theory of brain damage: arc l'ree radicals involved?. Neuro«hem. Res., 17 (1992) 11 --21. Peruche, B, and Krieglstein, J., Mechanisms of drug actîons against neuronal damage caused by ischaemia an overview. Progr. Neuropsychopharm, Biol. Psychiat., 17 (1993) 21 7(1. Phillis. J.W. and Sen, S., Oxypurinol attenuates hydroxyl radical production during ischemia/reperfusion injury of the rat cerebral cortex: an ESR study, BrainR«ç.,628(l~193) 309 312.

144

D.G. MacGreçor et al./Brain Resear«h 727 (1996) 133-144

[53] Pulsinelli, W.A., Brierley, J.B. and Plum, F. (1982) Temporal profile of neuronal damage in a model of transient forebrain ischemia, Ann. Neurol., 11,491-498. [54] Puttfarcken, P.S., Getz, R.L. and Coyle, J.T., Kainic acid induced lipid peroxidation - protection with butylated hydroxytoluene and U78517F in primary cultures of cerebellar granule cells, Brain Res., 624 (1993) 223-232. [55] Saija, A., Princi, P., Pisani, A., Lanza, M., Scalese, M., Aramnejad, E., Ceserani, R. and Costa, G., Protective effect of glutathione on kainic acid induced neuropathological changes in rat brain, Gen. Pharmacol., 25 (1994) 97-102. [56] Schwarcz, R., Foster, A.C., French, E.D., Whetsell, W.O. and Kohler, C., II. Excitotoxic models for neurodegenerative disorders, Li[e Sci., 35 (1984) 19-32. [57] Schwob, J.E., Fuller, T., Price, J.L. and Olney, J.W., Widespread patterns of neuronal damage following systemic or intracerebral injections of kainic acid: a histological study, Neuroscience, 5 (1980) 991-1014. [58] Sciamanna, M.A. and Lee, C.P., Ischaemia-reperfusion-induced injury of forebrain mitochondria and protection by ascorbate, Arch. Biochem. Biophys., 305 (1993) 215-224. [59] Sharma, S.K., Johnstone, R.M. and Quastel, J.H., Active transport of ascorbic acid in adrenal cortex and brain cortex in vitro and the effects of ACTH and steroids, Can. J. Biochem. Physiol., 41 (1963) 597-604. [60] Siesjo,B.K., Agardh, C.-D. and Bengtsson, F., Free radicals and brain damage, Cerebrot,asc. Brain Metab. Re~,., 1 (1989) 165-211. [61] Spector, R., Penetration of ascorbic acid from cerebrospinal fluid into brain, Exp. Neurol., 72 (1981). 643-653.

[62] Spector, T., Oxypurinol as an inhibitor of xanthine oxidase-catalyzed production of superoxide radical, Biochem. Pharmacol., 37 (1987) 349 352. [63] Sperk, G.. Lassmann, H., Baran, H., Kish, S.J., Seitelberger, F. and Hornykiewicz, O., Kainic acid induced seizures: neurochemical and histopathological changes, Neuroscien«e, 10 (1983) 1301 1315. [64] Stone, T.W., The neuropharmacology of quinolinic and kynurenic acids, Pharmacol. Ret'., 45 (1993) 309-379. [65] Sun, A.Y., Cheng, Y., Bu, Q. and Oldfield, F. the biochemical mechanisms of the excitotoxicity of kainic acid - free radical formation, Mol. Chem. Neuropathol., 17 (1992) 51-63. [66] Tauck. I).L., Redox modulation of NMDA receptor mediated synaptic activity in the hippocampus, NeuroReport, 3 (1992) 781-784. [67] Taylor. M.D., Palmer, G.C. and Callahan, A.S., Protective action of methylprednisolone, allopurinol and indomethacin against stroke induced damage to adenylate cyclase in gerbil hippocampus, Stroke, 15 (1984) 329-335. [68] Voherra, A., Trotti, D. and Racagni, G., Glutamate uptake is inhibited by arachidonic acid and oxygen radicals via two distinct and additive mechanisms, Mol. Pharmacol., 446 (1994) 986 992. [69] Wilson. J.. Ascorbic acid uptake by a high-affinity sodium-dependent mechanism in cultures rat astrocytes, J. N«uroch«m., 53 (1989) 1064-1071. [70] Yue, T.L., McKenna, P.J., Gu, J.L., Cheng, H.Y., Ruffolo, R.R. and Feuerstein, G.Z., Carvedilol, a new antihypertensive agent, prevents lipid peroxidation and oxidative injury to endothelial cells, l~vpertension, 22 (1993) 922-928.