European Journal of Pharmacology 390 Ž2000. 295–298 www.elsevier.nlrlocaterejphar

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Nimesulide limits kainate-induced oxidative damage in the rat hippocampus Eduardo Candelario-Jalil ) , Hussam H. Ajamieh, Susana Sam, Gregorio Martınez, ´ Olga S. Leon Fernandez ´ ´ Center for Research and Biological EÕaluation, Institute of Pharmacy and Food Sciences (CIEB-IFAL), UniÕersity of HaÕana, HaÕana, Cuba Received 13 December 1999; accepted 21 December 1999

Abstract Kainate induces a marked expression of cyclooxygenase-2 after its systemic administration. Because cyclooxygenase-2 activity is associated to the production of reactive oxygen species, we investigated the effects of nimesulide, a selective cyclooxygenase-2 inhibitor, on kainate-induced in vivo oxidative damage in the rat hippocampus. A clinically relevant dose of nimesulide Ž6 mgrkg, i.p.. was administered three times following kainate application Ž9 mgrkg, i.p... After 24 h of kainate administration, the drastic decrease in hippocampal glutathione content and the significant increase in lipid peroxidation were attenuated in nimesulide-treated rats, suggesting that the induction of cyclooxygenase-2 is involved in kainate-mediated free radicals formation. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Kainate; Excitotoxicity; Cyclooxygenase-2; Nimesulide; Oxidative stress; Hippocampus

1. Introduction Kainate, a pyrrolidine excitotoxin isolated from the seaweed Digenea simplex, is a potent neuroexcitatory drug, which after intracerebral or systemic injection leads to generalized limbic seizures in rats ŽBen Ari, 1985.. Kainate-induced seizures are accompanied by severe neuronal damage predominantly in the hippocampus and amygdalarpiriform cortex ŽHeggli and Malthe-Sorenssen, ¨ 1982.. The pattern of damage induced by systemically administered kainate approximates to that seen following repeated temporal lobe seizures ŽBen Ari, 1985. and cerebral ischemia–reperfusion ŽDykens et al., 1987.. Therefore, kainate provides a valuable tool with which to model some features of ischemic damage and of the injury induced by repeated epileptic seizures ŽSperk et al., 1985.. Current models of kainate toxicity support the hypothesis that the main cause of neurotoxicity is the activation of presynaptic kainate receptors and the release of endogenous glutamate ŽFerkany et al., 1982.. The overstimulation of glutamate receptors has been implicated in the media-

tion of injury caused by neurotoxins and ischemia-related insults ŽChoi and Rothman, 1990.. Further, kainate is also thought to mediate damage partly through an indirect mechanism, which may involve the overproduction of reactive oxygen species. The generation of free radicals appears to be pivotal in kainate neurotoxicity ŽCheng and Sun, 1994; Carriedo et al., 1998.. It was recently found that kainate induces a marked expression of cerebral cyclooxygenase-2 mRNA and protein following its systemic administration ŽSanz et al., 1997; Hashimoto et al., 1998; Sandhya et al., 1998., but the link between kainate-mediated cyclooxygenase-2 induction and free radicals formation has not been clearly defined. In order to investigate the possibility that the induction of cyclooxygenase-2 is involved in kainate neurotoxicity, we examined the effects of nimesulide, a selective cyclooxygenase-2 inhibitor, on kainate-induced in vivo oxidative damage in the rat hippocampus. 2. Materials and methods 2.1. Drugs

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Corresponding author. Apartado Postal 6079, Havana City 10600, Cuba. Tel.: q53-7-219-531r219-536; fax: q53-7-336-811. E-mail address: [email protected] ŽE. Candelario-Jalil..

Kainate was purchased from Sigma ŽSt. Louis, MO, USA.. Nimesulide was kindly provided by Gautier-Bago´

0014-2999r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 1 4 - 2 9 9 9 Ž 9 9 . 0 0 9 0 8 - 5

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E. Candelario-Jalil et al.r European Journal of Pharmacology 390 (2000) 295–298

Laboratories ŽBuenos Aires, Argentina.. The Bioxytech LPO-586 kit for lipid peroxidation was obtained from Oxis International ŽPortland, OR, USA.. All other reagents were of the highest quality available.

2.7. Glutathione (GSH) assay Hippocampal glutathione content was measured spectrophotometrically in the deproteinized samples according to the procedure of Ellman Ž1959..

2.2. Animals

2.8. Protein assay

Male S.D. rats ŽCENPALAB, Havana, Cuba. with a body weight of 200–240 g were used for the experiments. The animals were housed in groups of four per cage in a room with controlled 12:12 lightrdark cycle and ad libitum food and water.

Total protein concentration was determined using the Coomassie Blue method ŽSpector, 1978. with bovine serum albumin as standard.

2.3. Experimental design Four groups of rats Ž n s 8 each. were prepared: Ž1. Kainate Ž9 mgrkg, i.p., in saline.; Ž2. nimesulide Ždissolved in polyvinylpyrrolidone ŽPVP. in the relation 1:4. was administered Ž6 mgrkg, i.p.. immediately and after 1 and 2 h following kainate application Žas in group 1.; Ž3. saline q nimesulide and Ž4. saline q PVP Žas the control group.. 2.4. EÕaluation of behaÕioral changes The behavior of the animals was evaluated during 4 h after injection of kainate according to the following rating scale ŽSperk et al., 1985.: 0: normal, rare wet dog shakes, no convulsions; 1: intermediate number of wet dog shakes, rare focal convulsions affecting head and extremities, staring and intense immobility; 2: frequent wet dog shakes, frequent focal convulsions Žno rearing or salivation.; 3: frequent wet dog shakes, progression to more severe convulsions with rearing and salivation Žbut without falling over.; 4: continuous generalized seizures Žrearing, falling over., salivation; 5: generalized tonic–clonic seizures, death within 4 h in status epilepticus. 2.5. Sample preparation After 24 h, animals were anesthetized with diethyl ether and perfused intracardially with ice-cold saline. Brains were quickly removed and frozen at y208C until assay; the hippocampus was dissected on a cold stage before freezing. Hippocampi from treated rats were homogenized in 20 mM Tris–HCl buffer ŽpH 7.4. and centrifuged for 10 min at 12,000 = g. The supernatant was collected and immediately tested for lipid peroxidation and glutathione content. 2.6. Lipid peroxidation assay Lipid peroxidation was assessed by measuring the concentration of malonaldehyde and 4-hydroxyalkenals using the Bioxytech LPO-586 kit. The assay was conducted according to the manufacturer’s instructions.

2.9. Statistical analysis Data are expressed as mean values " SD Žstandard deviations.. Results were analyzed by one-way analysis of variance ŽANOVA.. If the F values were significant, the Students–Newman–Keuls post-hoc test was used to compare groups. Statistical significance was accepted for P 0.05 and P - 0.01. 3. Results 3.1. BehaÕior The most characteristic behavioral changes observed after i.p. injection of kainate Ž9 mgrkg. were, in order of appearance, strong immobility, increased incidence of wet dog shakes and, initially, focal convulsions. Within 40–60 min after kainate administration, the convulsive activity progressed to generalized limbic seizures. Although the intensity of these symptoms showed considerable inter-individual variation, the average symptom rating in both kainate-treated groups reached a value of 3. Nimesulide did not modify the kainate-induced behavioral changes. 3.2. Effects of nimesulide on kainate-mediated oxidatiÕe damage After 24 h, the systemic administration of kainate Ž9 mgrkg, i.p.. produced a drastic decrease in hippocampal GSH levels Ž0.987 " 0.06 mmolrg tissue. as compared to those in control group Ž1.316 " 0.03 mmolrg tissue; P 0.01.. Nimesulide Ž6 mgrkg, i.p., administered three times. partially prevented the dramatic decrease in GSH of kainate-challenged rats Ž1.182 " 0.03 mmolrg tissue; P 0.05. as shown in Table 1. Table 1 Effects of nimesulide ŽNIM. on depletion of hippocampal glutathione induced by kainate Values are mean"SD. Treatment

GSH Žmmolrg tissue.

Control Kainate KainateqNIM NIM

1.316"0.033 0.987"0.058 a 1.182"0.030 b 1.314"0.020

a b

Significantly different from control Ž P - 0.01.. Significantly different from kainate and control Ž P - 0.05..

E. Candelario-Jalil et al.r European Journal of Pharmacology 390 (2000) 295–298

Fig. 1. Effect of kainate ŽKA, 9 mgrkg, i.p.. and administration of nimesulide ŽNIM, 6 mgrkg, i.p., three times. on levels of malonaldehyde ŽMDA. and 4-hydroxyalkenals Ž4-HDA. in the rat hippocampus. U P UU 0.05 and P - 0.01.

On the other hand, malonaldehyde and 4-hydroxyalkenals, as an index of lipid peroxidation, increased by 75% in kainate-treated group Ž6.45 " 0.13 nmolrmg protein. as compared with control group Ž3.69 " 0.24 nmolrmg protein; P - 0.01.. Kainate-mediated lipid peroxidation was attenuated in nimesulide-administered rats as shown in Fig. 1 Ž4.68 " 0.09 nmolrmg protein; P - 0.01.. The administration of nimesulide without kainate did not alter neither GSH nor malonaldehyde and 4-hydroxyalkenals levels.

4. Discussion The ability of kainate to induce oxidative damage has been well documented in the literature ŽCheng and Sun, 1994; Carriedo et al., 1998.. It is generally accepted that overactivation of excitatory amino acid receptors triggers marked intracellular Ca2q rises and consequent oxygen radical production. Multiple events may be elicited by kainate-induced cytosolic Ca2q accumulation. Activation of calpains may convert xanthine dehydrogenase to xanthine oxidase with the simultaneous formation of free radicals. Further, Ca2q-dependent nitric oxide synthase activation may also contribute to kainate-mediated neuronal damage ŽCheng and Sun, 1994.. Moreover, the release of arachidonic acid following Ca2q-triggered phospholipase A 2 activation plays an additional role in kainate-induced free radicals formation ŽCheng and Sun, 1994.. It has been demonstrated that inhibition of arachidonic acid metabolism protects against kainate-induced seizures and neurotoxicity ŽBaran et al., 1994.. Neuronal cyclooxygenase-2 expression is detected in certain regions of the rat brain, mainly the hippocampus and cortex under physiological conditions ŽBreder et al., 1995. and can be markedly induced under certain stimuli

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and insults, including seizures ŽAdams et al., 1996. and ischemia ŽPlanas et al., 1995; Nogawa et al., 1997.. Although the factors responsible for the cytotoxicity of cyclooxygenase-2 have not been clearly defined, it is likely that one of the mechanisms is related to production of reactive oxygen species, which are formed by the peroxidase step of the cyclooxygenase reaction. Cyclooxygenase-2 enzymatic activity can also mediate tissue damage by producing proinflammatory prostanoids ŽSeibert et al., 1995.. As previously reported, systemic administration of kainate is able to increase prostaglandin formation and this event may be partially responsible for the tissue damage seen after kainate or the consequences of it ŽBaran et al., 1987.. The effects of nimesulide have not been previously studied using the systemic kainate model of neurotoxicity. Our present results showed that nimesulide, a selective cyclooxygenase-2 inhibitor, partially protected against kainate-induced oxidative damage. These results are consistent with previous reports, which provide strong evidence that cyclooxygenase-2 is implicated in excitotoxicity and neuronal death following brain insults such as ischemia–reperfusion ŽNogawa et al., 1997; Nakayama et al., 1998.. In summary, the present study demonstrated that the administration of a clinically relevant dose of the cyclooxygenase-2 inhibitor nimesulide limited kainate-induced oxidative damage in the rat hippocampus. This finding may suggest that cyclooxygenase-2-derived reactive metabolites are involved in kainate excitotoxicity. On the other hand, nimesulide did not modify kainate-induced seizures. This may indicate that the effects of nimesulide are not sufficient to attenuate the kainate-mediated symptoms.

References Adams, J., Collac¸o-Moraes, Y., de Belleroche, J., 1996. Cyclooxygenase-2 induction in cerebral cortex: an intracellular response to synaptic excitation. J. Neurochem. 66, 6–13. Baran, H., Heldt, R., Hertting, G., 1987. Increased prostaglandin formation in rat brain following systemic application of kainic acid. Brain Res. 404, 107–112. Baran, H., Vass, K., Lassmann, H., Hornykiewicz, O., 1994. The cyclooxygenase and lipoxygenase inhibitor BW755C protects rats against kainic acid-induced seizures and neurotoxicity. Brain Res. 646, 201– 206. Ben Ari, Y., 1985. Limbic seizure and brain damage produced by kainic acid: mechanisms and relevance to human temporal lobe epilepsy. Neuroscience 14, 375–403. Breder, C.D., Dewitt, D., Kraig, R.P., 1995. Characterization of inducible cyclooxygenase in rat brain. J. Comp. Neurol. 355, 296–315. Carriedo, S.G., Yin, H.Z., Sensi, S.L., Weiss, J.H., 1998. Rapid Ca2q entry through Ca2q-permeable AMPArkainate channels triggers marked intracellular Ca2q rises and consequent oxygen radical production. J. Neurosci. 18, 7727–7738.

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Cheng, Y., Sun, A.Y., 1994. Oxidative mechanisms involved in kainateinduced cytotoxicity in cortical neurons. Neurochem. Res. 19, 1557– 1564. Choi, D.W., Rothman, S.M., 1990. The role of glutamate neurotoxicity in hypoxic ischemic neuronal death. Annu. Rev. Neurosci. 13, 171–182. Dykens, J.A., Stern, A., Trenkner, E., 1987. Mechanisms of kainate toxicity to cerebellar neurons in vitro is analogous to reperfusion tissue injury. J. Neurochem. 49, 1222–1228. Ellman, G.J., 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70–77. Ferkany, J.W., Zaczek, R., Coyle, J.T., 1982. Kainic acid stimulates excitatory amino acid neurotransmitter release at presynaptic receptors. Nature 298, 757–759. Hashimoto, K., Watanabe, K., Nishimura, T., Iyo, M., Shirayama, Y., Minabe, Y., 1998. Behavioral changes and expression of heat shock protein hsp-70 mRNA, brain-derived neurotrophic factor mRNA, and cyclooxygenase-2 mRNA in rat brain following seizures induced by systemic administration of kainic acid. Brain Res. 804, 212–223. Heggli, D.E., Malthe-Sorenssen, D., 1982. Systemic injection of kainic ¨ acid: effect on neurotransmitter markers in piriform cortex, amygdaloid complex and hippocampus and protection by cortical lesioning and anti-convulsants. Neuroscience 7, 1257–1264. Nakayama, M., Uchimura, K., Zhu, R.L., Nagayama, T., Rose, M.E., Stetler, R.A., Isakson, P.C., Chen, J., Graham, S.H., 1998. Cyclooxygenase-2 inhibition prevents delayed death of CA1 hippocampal

neurons following global ischemia. Proc. Natl. Acad. Sci. U.S.A. 95, 10954–10959. Nogawa, S., Zhang, F., Ross, M.E., Iadecola, C., 1997. Cyclooxygenase-2 gene expression in neurons contributes to ischemic brain damage. J. Neurosci. 17, 2746–2755. Planas, A.M., Soriano, M.A., Rodrıguez-Farre, ´ ´ E., Ferrer, I., 1995. Induction of cyclooxygenase-2 mRNA and protein following transient focal ischemia in the rat brain. Neurosci. Lett. 200, 187–190. Sandhya, T.L., Ong, W.Y., Horrocks, L.A., Farooqui, A.A., 1998. A light and electron microscopic study of cytoplasmic phospholipase A 2 and cyclooxygenase-2 in the rat hippocampus after kainate lesions. Brain Res. 788, 223–231. Sanz, O., Estrada, A., Ferrer, I., Planas, A.M., 1997. Differential cellular distribution and dynamics of HSP70, cyclooxygenase-2, and c-FOS in the rat brain after transient focal ischemia or kainic acid. Neuroscience 80, 221–232. Seibert, K., Masferrer, J., Zhang, Y., Gregory, S., Olson, G., Hauser, S., Leahy, K., Perkins, W., Isakson, P., 1995. Mediation of inflammation by cyclooxygenase-2. Agents Actions Suppl. 46, 41–50. Spector, T., 1978. Refinement of the Coomassie Blue method of protein quantification. Anal. Biochem. 86, 142–146. Sperk, G., Lassmann, H., Baran, H., Seitelberger, F., Hornykiewicz, O., 1985. Kainic acid-induced seizures: dose-relationship of behavioral, neurochemical and histopathological changes. Brain Res. 338, 289– 295.

Nimesulide limits kainate-induced oxidative damage in ...

administered three times following kainate application 9 mgrkg, i.p. . After 24 h of ..... and electron microscopic study of cytoplasmic phospholipase A and. 2.

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