Indian Journal of Experimental Biology Vol. 47, November 2009, pp. 880-892 Neuroprotective effect of MK-801 against intra-striatal quinolinic acid induced behavioral, oxidative stress and cellular alterations in rats Harikesh Kalonia1, Puneet Kumar1, Bimla Nehru2 & Anil Kumar1,* 1Pharmacology Division, University Institute of Pharmaceutical Sciences, UGC Centre for Advanced Studies, Panjab University, Chandigarh 160 014, India 2 Department of Biophysics, Panjab University, Chandigarh 160014, India Received 22 February 2009; revised 22 June 2009 Huntington’s Disease (HD) is a common neurodegenerative disorder characterized by motor disturbances, subcortical dementia and psychiatric disturbances. Pathogenesis of HD revolves so far around excitatory amino acids as the primary cause of neuronal loss. However, number of recent reports suggests the involvement of excitotoxicity and oxidative damage. In the present study, first the dose of quinolinic acid that mimics the symptoms of HD was standardized and then the neuroprotective effect of MK-801 (noncompetitive NMDAr antagonist) was evaluated against intrastriatal quinolinic acid induced behavioral, oxidative stress and cellular alterations in rats. A single unilateral (ipsilateral striatum) injections of quinolinic acid (100, 200 and 300 nM) were made in to striatum. Animals were tested for motor functions using actophotometer and rotarod apparatus. Quinolinic acid (300 nM) significantly reduced the body weight and caused motor in-coordination and produced oxidative damage in the cortex and striatum as indicated by raised lipid peroxidation, nitrite concentration, depletion of superoxide dismutase, catalase and different glutathione levels. Beside, quinolinic acid (300 nM) significantly altered the mitochondrial enzymes complex levels and caused histopathological alterations in the striatum. MK-801(0.02, 0.04, 0.08 mg/kg, ip) treatment significantly improved body weight, behavioral alterations (locomotor activity and rotarod performance) and attenuated oxidative damage and mitochondrial enzymes complex dysfunction. Besides, MK-801 treatment significantly reversed histopathological alterations in striatum. The results suggest antioxidant and neuroprotective action of MK-801 against the quinolinic acid induced Huntington’s like behavioral, oxidative stress and cellular alterations in rats. Keywords: Glutathione, Huntington’s disease, Mitochondrial dysfunction, MK-801, Oxidative Stress, Quinolinic acid, Striatum Huntington’s disease (HD) is a common this metabolite (5-10-fold in early stage HD patients), neurodegenerative disorder characterized by together with 3-hydroxykynurenine (3-HK), dementia, choreiform movements1 and degeneration (kynurenine metabolite) increased reactive oxygen of basal ganglia (caudate putamen) including cortex. species (ROS) formation in the striatum and cortex of Beside, irritability, obsessions, compulsions, HD brains. These findings explain the role of the hallucinations, altered family dynamics and decreased QUIN in HD pathogenesis and similarity with clinical executive functions in HD patients2,3. Presently, there symptoms7. QUIN is an endogenous tryptophan is no suitable drug treatment available for the metabolite that causes excitotoxicity by acting on N- effective management of this disease. Pathogenesis of methyl-D-aspartate receptor (NMDAr) subtype8,9. As HD has not been fully understood so far. However, a heterocyclic amino acid derived from L-tryptophan, key mechanism of neuronal death revolves around QUIN is a precursor of NAD+ at the kynurenine excitotoxicity, impaired energy metabolism, pathway5. QUIN causes excitotoxicity by stimulating neuroinflammation and oxidative damage4. NMDAr, causing membrane depolarization and Quinolinic acid (QUIN) (2, 3-pyridine calcium influx in the neurons, which in turn, results in decarboxylic acid) has been reported to produce HD activation of proteases, lipases, generation of free like symptoms as observed in HD patients5,6. Recent radicals, constitutive nitric oxide synthase (NOS), findings reinforced the consideration that the levels of disruption of mitochondrial oxidative phosphorylation and enhanced brain lipid peroxidation8,10-13. In ________________ addition, alterations in the number of NMDAr have *Correspondent author also been suggested to be other components of the Telephone: +91-172-2534106 Fax: +91-172-2541142 glutamatergic transmission, involved as causative 3 E-mail: [email protected] factors in HD pathogenesis . KALONIA et al.: MK-801 AGAINST QUINOLINIC ACID INDUCED ALTERATIONS IN RATS 881 MK-801 [(+)-5-methyl-10, 11-dihydro-5H- Intrastriatal administration of QUIN — Animals dibenzo-cyclo-hepten-5, 10-imine maleate] is a potent were anesthetized with thiopental sodium (45mg/kg, ip). selective and noncompetitive NMDA receptor The surface of the skull was exposed by making incision antagonist14. Upon systemic administration, MK-801 on the scalp. QUIN injections were made into the right prevents damage of striatal and hippocampal neurons striatum by means of a 28-gauge stainless steel needle caused by direct injection of NMDA15. MK-801, non- attached to a Hamilton syringe. Injections were made via competitive NMDA receptor antagonist has been a l-2 mm diameter hole made in the skull using a small reported to enhance locomotor activity in rodents15. hand drill at anterior +1.7mm; lateral±2.7mm; ventral- Non competitive NMDA antagonist affects 4.8mm from bregma and dura as described in Paxinos locomotion by increasing dopamine (DA) turnover in and Watson20. QUIN was injected in a volume of 4 µl mesolimbic structures and through NMDA receptor delivered over a period of 2 min, and injection needle antagonism. MK-801 inhibits the uptake of dopamine. was left in place for another 1.5 min to allow diffusion MK-801 is a highly selective for NMDA recognition of the injected drug solution. site and shows low affinity for the DA uptake site. Therefore, MK-801-induced hyperlocomotion is Behavioral assessments mediated, at least in part, by NMDA receptor Body weight change — The body weight of antagonism. Studies reported the protective action of animals was recorded before the start of drug the MK-801 in the various neurodegenerative treatment (before QUIN administration) and on last psychological disorders such as depression, epilepsy, day of the study thereafter behavioral quantification. ischemia, alzheimers etc16-19. Aim of the present study Assessment of gross behavioral activity was to evaluate the antioxidant and neuroprotective (Locomotor activity) — The locomotor activity was action of MK-801 (NMDAr antagonist) against the assessed by using actophotometer (IMCORP, India) st th th st QUIN induced HD like behavioral, oxidative stress on day 1 , 7 , 14 and 21 . Animals were placed and cellular alterations in the rats. individually in the activity chamber for 3 min as a habituation period before making actual motor Materials and Methods activity tasks monitoring for next 5 min. Total activity Animals — Male Wistar rats (132) bred in Central (horizontal and vertical) were expressed as counts per Animal House, Panjab University, Chandigarh and 5 min as described by Kulkarni21. The apparatus was weighing between 220-250 g were used. The animals placed in a darkened, light and sound attenuated and were kept under standard laboratory conditions, ventilated testing room during the assessment. maintained on 12-h light/dark cycle and had free access Rotarod activity — All animals were evaluated for to food and water. Animals were acclimatized to motor coordination and balance on rotarod test on 1st, 7th, laboratory conditions before the test. All the experiments 14th and 21st day after QUIN injection. The animals were were performed between 9.00 and 17.00 hrs. The given a prior training session before actual recording on experimental protocol was approved by Institutional rotarod apparatus (IMCORP, Ambala, India) to Animal Ethics Committee and conducted according to acclimatize. Rats were placed on the rotating rod with a the Indian National Science Academy Guidelines for the diameter of 7 cm (speed 25 rpm). The cut off time use and care of experimental animals. (90 sec) was fixed and each rat performed three separate Drug and treatment schedule — QUIN (Sigma trials at 5 min interval as described by Kulkarni21. Chemical, India) was dissolved in normal saline and administered unilaterally in the striatum (4 µl). Biochemical assessments Animals were divided into following 11 groups, of Dissection and homogenization — On 21st day, 12 animals each: Group-1 naïve (without surgery); animals were sacrificed by decapitation immediately group-2 sham (surgery without intra striatal injection) after behavioral assessments. The brains were received normal saline; Group 3-5 received dissected and cerebellum discarded. Cortex and intrastriatal QUIN 100, 200 and 300 nM single striatum were separated by putting on ice and then injection respectively; Group 6-8 received MK-801 weighted separately. A 10% (w/v) tissue homogenate (0.02, 0.04 and 0.08 mg/kg, ip). However, MK-801 was prepared in 0.1 M phosphate buffer (pH 7.4). The (0.02, 0.04 and 0.08 mg/kg, ip) treatment against homogenate was centrifuged at 10,000 g for 15 min intrastriatal QUIN 300 nM administration was and aliquots of supernatant was separated and used considered as group 9-11 respectively. for biochemical estimations. 882 INDIAN J EXP BIOL, NOVEMBER 2009 Lipid peroxidation assay — The quantitative Complex-I (NADH dehydrogenase activity) — was measurement