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Neuroprotective Effect of MK-801 Against Intra-Striatal Quinolinic Acid Induced Behavioral, Oxidative Stress and Cellular Alterations in Rats

Neuroprotective Effect of MK-801 Against Intra-Striatal Quinolinic Acid Induced Behavioral, Oxidative Stress and Cellular Alterations in Rats

Indian Journal of Experimental Biology Vol. 47, November 2009, pp. 880-892

Neuroprotective effect of MK-801 against intra-striatal quinolinic acid induced behavioral, 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 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 ) 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 peroxidation, nitrite concentration, depletion of superoxide dismutase, catalase and different levels. Beside, quinolinic acid (300 nM) significantly altered the mitochondrial 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 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 ( 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 . 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 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 derived from L-tryptophan, key mechanism of neuronal death revolves around QUIN is a precursor of NAD+ at the kynurenine excitotoxicity, impaired energy , pathway5. QUIN causes excitotoxicity by stimulating neuroinflammation and oxidative damage4. NMDAr, causing membrane depolarization and Quinolinic acid (QUIN) (2, 3- calcium influx in the , which in turn, results in decarboxylic acid) has been reported to produce HD activation of , lipases, generation of free like symptoms as observed in HD patients5,6. Recent radicals, constitutive synthase (NOS), findings reinforced the consideration that the levels of disruption of mitochondrial oxidative and enhanced 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 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 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 (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 of in striatum and measured spectrophotometrically (UV- Pharmaspec cortex was performed according to the method of 1700 Shimadzu, Japan) by the method of King and Wills22. The amount of malondialdehyde (MDA) was Howard30. The method involves catalytic oxidation of measured by reaction with thiobarbituric acid at NADH to NAD+ with subsequent reduction of 532 nm using spectrophotometer (UV- Pharmaspec cytochrome C. 1700 Shimadzu, Japan). Complex-II (Succinate dehydrogenase (SDH) Estimation of nitrite — The accumulation of nitrite in activity) — was measured spectrophotometrically the striatum and cortex, an indicator of the production of (UV- Pharmaspec 1700 Shimadzu, Japan) according to 31 nitric oxide (NO), was determined with a colorimetric King . The method involves oxidation of succinate by assay with Greiss reagent (0.1% N-(1-naphthyl) an artificial electron acceptor, potassium ferricyanide. ethylenediame dihydrochloride, 1% sulfanilamide and MTT assay — MTT, a pale yellow substrate produces 2.5% phosphoric acid) as described by Green23. a purple product when incubated with live cells. The number of viable cells/well is directly proportional to the Estimation of catalase — Catalase activity was production of product (purple), which follows assayed by method of Luck24, wherein the breakdown of solublization with DMSO, can be measured32,33. The H O was measured at 240 nm using spectrophotometer 2 2 absorbance of the resulting medium was measured by an (UV- Pharmaspec 1700 Shimadzu, Japan). ELISA reader at 580 nm wavelength. Glutathione estimation — Reduced glutathione Mitochondrial complex-IV (Cytochrome oxidase) (GSH) in striatum and cortex was estimated according 25 assay — Cytochrome oxidase activity was assayed to the method described by Ellman . Total according to the method of Sotocassa in brain glutathione was assayed by the method of Zahler and 34 26 mitochondria . Cleland . Oxidized glutathione (GSSG) was quantified by subtracting the value of glutathione Histological examination reduced from total glutathione. The brains were fixed in 10% formalin solution ratio of reduced glutathione/oxidized glutathione prior to embedding in paraffin wax. The tissue was (GSH/GSSG) was also calculated. then processed and sectioned (10 μm thick) using a Superoxide dismutase activity (SOD) — Superoxide rotary microtome. The sections were H & E dismutase activity was assayed according to the method (hematoxylin and eosin stain) stained. The slides were of Kono27 wherein reduction of nitrazobluetetrazolium then mounted with DPX (Mixture of 10 g distyrene (NBT) was inhibited by the superoxide dismutase is 80, 5 ml dibutyl phthalate and 35 ml ) and measured at 560 nm using spectrophotometer viewed under the light microscope (higher-power (UV- Pharmaspec 1700 Shimadzu, Japan). views, × 400) and photographed by using a digital Protein estimation — Protein estimation was done camera (Nikon, Japan). by Lowry’s method28 using bovine serum albumin as Statistical analysis — One specific group of twelve standard. (n = 12) animals was assigned to a specific drug treatment. All the values were expressed as means ± Mitochondrial complex estimation SE. The data were analyzed using one way analysis of Isolation of rat brain mitochondria — Rat brain variance (ANOVA) and two way ANOVA followed mitochondria were isolated by the method of Berman by Tukey’s test. In all the tests, criterion for statistical 29 and Hastings . The brain regions were homogenized significance was P<0.05. in isolation buffer with EGTA (215 mM Mannitol, 75 mM sucrose, 0.1% BSA, 20 mM HEPES, 1 mM Results EGTA, pH 7.2). Homogenate was centrifuged at Body weight — There was no significant change in 13000 g for 5 min at 4ºC. Pellet was resuspended in the initial and final body weight of sham group as isolation buffer with EGTA and spun again at 13000 g compared to naïve groups. However, QUIN for 5 min. The resulting supernatant was transferred to (100, 200 and 300 nM) treatment significantly new tubes and topped off with isolation buffer with decreased body weight as compared to sham and EGTA and again spun at 13,000 g for 10 min. Pellet naive group. MK-801 (0.02, 0.04 and 0.08 mg/kg, ip) containing pure mitochondria was resuspended in per se, did not produce any significant effect on the isolation buffer without EGTA. body weight. MK-801 (0.04 and 0.08 mg/kg) KALONIA et al.: MK-801 AGAINST QUINOLINIC ACID INDUCED ALTERATIONS IN RATS 883

significantly improved the body weight in QUIN compared naïve groups. QUIN 100 nM did not treated animals. However lower dose of MK-801 produce any significant change in the MDA level in (0.02 mg/kg) did not produce any significant effect as cortex and striatum. QUIN 200 and 300 nM compared to QUIN 300 treated animals (Fig. 1). significantly increased MDA levels in striatum and Locomotor activity — There was no significant cortex, as compared to the sham group. MK-801 change in the locomotor activity of sham group as (0.04 and 0.08 mg/kg) significantly attenuated MDA compared naïve groups. QUIN 100 and 200 nM did levels in the QUIN (300 nM) treated animals. While not significantly affect locomotor activity while MK-801 (0.02, 0.04 and 0.08 mg/kg, ip) per se, did 300 nM leads to a significant increase in the not affect lipid peroxidation levels as compared to locomotor activity on day 7. On day 14th and 21st day, naïve and sham group (Table 3). QUIN 300 nM significant decreased the locomotor Nitrite concentration — There was no significant activity as compared to naïve and sham group. difference in the nitrite levels of the naïve, sham and MK-801 at doses of 0.04 and 0.08 significantly QUIN 100 nM. QUIN 200 and 300 nM leads to a improved the locomotor activity on 7th, 14th and 21st significant increase in nitrite levels in rat striatum and day in the QUIN (300 nM) treated animals. While cortex, as compared to the sham group. MK-801 MK-801 (0.02, 0.04 and 0.08 mg/kg, ip) per se, did (0.02, 0.04 and 0.08 mg/kg) per se did not produce not have significant effect on the locomotor activity any effect on the nitrite levels. While the MK-801 (Table 1). (0.04 and 0.08 mg/kg) significantly lowered the nitrite Rotarod activity — There was no significant levels in the QUIN (300 nM) treated animals (Table 3). change in the fall off time of sham group as compared Redox ratio — Redox ratio was not significantly to naive group. QUIN (100, 200) did not affect fall off altered in the naïve, sham and QUIN 100 treated time on day 7, while on day 14 and 21 there was a groups. Intrastriatal, QUIN 200 and 300 nM significant decrease in fall off time. However, QUIN significantly depleted reduced glutathione (GSH), 300 nM treatment significantly decreased fall off time redox ratio (GSH/GSSG) and increased oxidized as compared to naïve and sham group. MK-801 glutathione levels in rat brain striatum and cortex, as (0.04 and 0.08 mg/kg) significantly improved the compared to sham group. While there was no rotarod performance. MK-801 (0.02, 0.04 and significant effect on the total glutathione levels (GSSG) 0.08 mg/kg, ip) per se, did not have significant effect as compared to sham group. MK-801 (0.02, 0.04 and on rotarod performance (Table 2). 0.08 mg/kg) per se did not have any significant effect Lipid peroxidation — There was no significant on redox ratio. While MK-801 at doses of 0.04 and increase in the MDA level of sham group as 0.08 significantly reversed the levels of GSH, redox

Fig. 1 — Effect of MK-801 on QUIN induced reduction in the Body weight [Values are expressed Mean ± SE (% age of sham). P<0.05 as compared to asham, bQUIN (100), cQUIN (200),dQUIN (300), eMK 801 (0.02), fMK 801 (0.04) in the respective region of the brain. (ANOVA followed by Tukey test). NS Not significant] 884 INDIAN J EXP BIOL, NOVEMBER 2009

ratio (GSH/GSSG) and oxidized glutathione levels in 0.04 and 0.08 significantly increased the levels of the QUIN treated animals (Table 4). catalase and SOD levels in the QUIN treated animals Catalase and SOD level — There was no (Table 3). significant decrease in the catalase and SOD level of Mitochondrial complexes (I, II and IV) — sham group as compared to naïve groups. QUIN QUIN 100 nM did not produce any significant 100 nM did not produce any significant change in the decrease in the enzyme complexes levels as compared catalase and SOD level in cortex and striatum. Intra- to sham and naïve group. QUIN 200 and 300 nM, lead striatal administration of QUIN 200 and 300 nM leads to a significant decrease in mitochondrial complex-I, to a significant decrease in catalase and SOD levels in II and IV levels in rat brain striatum and cortex as rat brain Striatum and cortex as compared to the sham compared to the sham group. MK-801 0.04 and group. MK-801 (0.02, 0.04 and 0.08 mg/kg), per se 0.08 mg/kg, significantly increased various did not have significant effect on catalase and SOD mitochondrial enzymes complex levels in the QUIN levels, at any of the dose. While MK-801 at doses of (300 nM) treated animals, while the MK-801 (0.02, Table 1 — Effect of MK-801 on QUIN induced alterations in the locomotor activity [Values (percentage of sham) are mean ± SE from 12 animals in each group]

Counts/5min (Locomotor activity, % age of sham) Treatment Day 1 Day 7 Day 14 Day 21 Naive 100.00±9.91 98.89±10.83 93.46±8.08 93.59±9.32 Sham 100.00±11.90 101.51±12.50 95.97±10.83 87.55±8.49 QUIN 100 100.00±12.23 105.56±18.16 78.09±12.22 66.67±9.46 QUIN 200 100.00±11.90 108.04±16.49 75.00±5.22 56.58±3.42 QUIN 300 100.00±10.99 127.12±12.20a 68.57±11.37 a 49.01±8.44 a MK 801 (0.02) 100.00±11.78 102.04±12.15 96.55±10.52 94.64±6.87 MK 801 (0.04) 100.00±7.73 101.30±9.29 96.88±8.94 97.53±8.49 MK 801 (0.08) 100.00±11.21 101.12±12.50 100.87±8.61 102.24±6.09 QUIN 300 + MK 801 (0.02) 100.00±9.17 102.67±9.88NS 78.76±10.04NS 61.29±3.26NS QUIN 300 + MK 801 (0.04) 100.00±12.02 100.00±13.44NS 90.38±5.76NS 78.10±5.36b,c QUIN 300 + MK 801 (0.08) 100.00±14.69 98.31±14.69NS 95.76±13.63b,c 94.15±6.54b,c,d P<0.05 as compared to asham, bQUIN (100), cQUIN (200), dQUIN (300), eMK 801 (0.02), fMK 801 (0.04) in the respective region of the brain. (ANOVA followed by Tukey test). NS Not significant.

Table 2 — Effect of MK-801 on QUIN induced altered the rotarod activity (fall off time) [Values (percentage of sham) are mean ± SE from 12 animals in each group]

Fall off time (% age of sham) Treatment Day 1 Day 7 Day 14 Day 21

Naive 100.00±1.23 110.53±1.20 113.16±1.10 116.58±1.11 Sham 100.00±1.17 105.41±1.36 110.81±1.10 116.22±0.87 QUIN 100 100.00±1.46 98.86±2.38NS 94.32±2.37a 89.77±0.89a QUIN 200 100.00±1.64 95.35±1.70NS 81.40±1.00a,b 75.58±1.00a,b QUIN 300 100.00±1.43 86.25±1.30a 74.75±1.52a,b,c 51.25±1.52a,b,c MK 801 (0.02) 100.00±1.19 102.70±1.28 106.76±1.10 109.46±1.11 MK 801 (0.04) 100.00±1.03 109.33±1.26 106.67±1.10 112.00±0.87 MK 801 (0.08) 100.00±1.11 109.72±1.26 109.72±1.10 118.06±0.87 QUIN 300 + MK 801 (0.02) 100.00±1.23 93.59±1.70NS 83.33±1.10NS 56.41±1.26NS QUIN 300 + MK 801 (0.04) 100.00±2.31 101.22±2.28d,e 91.46±2.37d,e 80.49±0.89d,e QUIN 300 + MK 801 (0.08) 100.00±2.93 105.95±2.28d,e,f 101.19±3.03d,e,f 96.43±1.10d,e,f P<0.05 as compared to asham, bQUIN (100), cQUIN (200), dQUIN (300), eMK 801 (0.02), fMK 801 (0.04) in the respective region of the brain. (ANOVA followed by Tukey test). NS Not significant. KALONIA et al.: MK-801 AGAINST QUINOLINIC ACID INDUCED ALTERATIONS IN RATS 885

Table 3 — Effect of MK-801 on the MDA, nitrite, catalase and SOD levels of brain cortex and striatum [Values (percentage of sham) are mean ± SE from 12 animals in each group]

Group MDA levels Nitrite levels Catalase levels SOD levels Cortex 94.15±3.45 97.33±2.43 104.13±2.23 103.45±2.20 Naive Striatum 96.97±2.79 94.92±3.33 101.68±2.89 104.82±2.22 Cortex 100.00±6.74 100±9.09 100±6.09 100±3.74 Sham Striatum 100±4.39 100±15.00 100±1.68 100±2.59 Cortex 113.30±0.47NS 118.18±2.56 NS 96.05±3.73NS 98.40±1.08NS QUIN 100 Striatum 143.92±1.37a 178.46±1.72 a 85.21±1.98a 77.98±1.69a Cortex 129.07±4.40a,b 154.55±1.96a,b 81.72±2.63a,b 83.49±1.14a,b QUIN 200 Striatum 160.97±1.13a,b 224.62±2.74a,b 48.49±4.05a,b 61.56±1.82a,b Cortex 148.22±1.44a,b,c 148.67±3.40a,b,c 64.51±3.33a,b,c 68.58±2.41a,b,c QUIN 300 Striatum 200.45±1.13a,b,c 295.76±1.98a,b,c 29.64±3.53a,b,c 30.92±1.41a,b,c Cortex 102.84±2.56 98.67±2.86 101.88±2.34 98.28±1.96 MK 801 (0.02) Striatum 97.58±2.34 96.61±2.73 99.33±2.46 100.00±2.33 Cortex 105.32±3.34 99.33±2.53 104.89±2.32 100.00±2.54 MK 801 (0.04) Striatum 100.15±2.45 98.31±2.25 100.34±2.22 102.41±2.55 Cortex 106.74±2.67 100.00±2.67 109.40±2.23 106.90±3.12 MK 801 (0.08) Striatum 103.18±2.54 100.00±2.12 101.68±1.99 107.23±1.87 Cortex 137.20±3.57NS 145.33±2.87NS 56.39±3.34NS 60.34±2.23NS QUIN 300 + MK 801 (0.02) Striatum 189.86±3.42 NS 258.98±3.34 NS 38.41±2.12 NS 49.78±1.67 NS Cortex 111.17±4.12d,e 124.00±3.67d,e 61.65±3.32d,e 82.76±2.78d,e QUIN 300 + MK 801 (0.04) Striatum 140.70±2.45d,e 205.08±2.98d,e 60.96±1.98d,e 77.11±3.43d,e Cortex 97.60±2.54d,e,f 116.00±2.34d,e,f 89.10±2.88d,e,f 89.66±2.65d,e,f QUIN 300 + MK 801 (0.08) Striatum 104.84±2.32d,e,f 151.99±2.18d,e,f 98.20±1.88d,e,f 108.43±2.42d,e,f

P<0.05 as compared to asham, bQUIN (100), cQUIN (200), dQUIN (300), eMK 801 (0.02), fMK 801 (0.04) in the respective region of the brain. (ANOVA followed by Tukey test). NS Not significant.

0.04 and 0.08 mg/kg) per se has no effect on the significantly reversed the histological alterations in mitochondrial enzyme complexes levels (Fig. 2 a, b, c). the QUIN treated animals (Fig. 4). MK-801 per se, MTT assay — There was no difference in the did not produce any significant histological alterations number of viable cell of naïve, sham and QUIN 100 as compared to QUIN (300 nM). nm group. Intra-striatal administration of QUIN 200 and 300 nM leads to a significant decrease in no. of Discussion viable cells, in rat brain striatum and cortex as QUIN, neuroactive metabolite of tryptophan is one compared to the sham group. Per se MK-801 did not of the endogenous excitotoxin which has been produce any significant effect on no. of viable cells, at implicated in the pathogenesis of several any of the dose. While MK-801 at doses of 0.04 and neurodegenerative and neuroinflammatory 0.08 significantly restored the no. of viable cells in the diseases8,35,36. Accordingly, QUIN administration in QUIN (300 nM) treated animals (Fig. 3). rat striatum produces similar as observed in Histological Changes — Sections of the naïve and HD patients37,38,39,40. The mechanism underlying the sham treated rats showed optimally sized, QUIN induced involves over undamaged, pyramidal shaped neuronal cells with a stimulation of NMDAr36 sub type which in turn cause clearly observable cell nucleus and continuous cell excitotoxicity12,41due to alterations in intracellular ion membrane. QUIN 100 and 200 nM produced concentrations of sodium, chloride, as well as significant histological alterations as compared to calcium42,43. Further, it has been hypothesized that naïve and sham treated animals. However, QUIN agents that block QUIN induced neurotoxicity could 300 nM showed significant swelling, appeared be used as efficacious agents in prevent neuronal scattered and dense nuclei (shown with help of degeneration associated pathologies44,45. Based on this arrows). MK-801 (0.04 and 0.08 mg/kg) treatment background, first the dose of the QUIN which mimics 886 INDIAN J EXP BIOL, NOVEMBER 2009

Table 4 — Effect of MK-801 on the total glutathione, reduced glutathione, oxidized glutathione and redox ratio in brain cortex and striatum [Values (percentage of sham) are mean ± SE from 12 animals in each group]

Group Total glutathione Reduced glutathione Oxidized glutathione Redox ratio Cortex 100.54±2.34 102.64±2.43 98.00±1.82 103.00±2.37 Naive Striatum 101±1.82 102.54±3.33 97.50±2.34 104.00±2.31 Cortex 100.00±1.56 100±9.09 100±1.92 100±1.56 Sham Striatum 100±1.92 100±15.00 100±1.56 100±1.51 Cortex 99.94±0.17NS 90.29±2.00NS 106.36±0.87NS 60.42±5.60a QUIN 100 Striatum 99.68±0.76NS 74.84±0.86a 125.44±1.70a 54.47±4.65a Cortex 99.72±0.17NS 81.14±2.22a 112.07±1.32a 51.22±3.16a QUIN 200 Striatum 99.31±0.38NS 55.53±2.97a,b 142.48±0.62a 37.82±3.06a,b Cortex 99.45±1.79NS 71,57±3.40a 117.97±2.02a 74.51±1.79a QUIN 300 Striatum 97.03±2.02 NS 38.50±1.98a,b 154.76±1.79a 24.87±1.72a,b,c Cortex 99.56±2.34 98.47±2.86 96.00±2.01 95.00±2.36 MK 801 (0.02) Striatum 96.12±2.01 100.89±2.73 96.60±2.34 96.10±2.30 Cortex 99.79±2.22 99.86±2.53 97.32±2.34 96.24±2.22 MK 801 (0.04) Striatum 97.67±2.34 101.91±2.25 97.40±2.22 97.12±2.29 Cortex 99.81±2.03 101.66±2.67 98.20±1.89 98.10±2.03 MK 801 (0.08) Striatum 98.42±1.89 103.81±2.12 98.68±2.03 98.22±2.03 Cortex 99.72±1.98 NS 78.92±2.87 NS 113.55±2.56 NS 69.31±1.98NS QUIN 300 + MK 801 (0.02) Striatum 98.30±2.56 NS 44.31±3.34 NS 148.72±1.92 NS 34.22±2.05NS Cortex 99.94±1.62 NS 88.63±3.67d,e 107.47±1.76d,e 82.22±1.62d,e QUIN 300 + MK 801 (0.04) Striatum 99.68±1.76 NS 68.77±2.98d,e 131.30±1.62d,e 49.95±1.59d,e Cortex 100.83±1.80 NS 93.62±2.34 d,e,f 105.62±1.88d,e,f 88.31±1.85d,e,f QUIN 300 + MK 801 (0.08) Striatum 101.51±1.88 NS 99.37±2.18 d,e,f 111.26±1.80d,e,f 89.70±1.81d,e,f

P<0.05 as compared to asham, bQUIN (100), cQUIN (200),dQUIN (300), eMK 801 (0.02), fMK 801 (0.04) in the respective region of the brain. (ANOVA followed by Tukey test). NS Not significant.

HD like symptoms in the rodents was standardized. region in the rodents46,47. The initial hyperactivity48-50 Three doses of QUIN (100, 200 and 300 nM) were could be due to the initial increased levels of the tried. QUIN treatment on higher doses caused excitatory . However, hypoactivity significant alterations in the locomotor activity (initial could be due to striatal neuronal degeneration and hyper activity followed by the hypoactivity), impaired energy impairment51. QUIN causes the degeneration motor function (grip strength), oxidative damage, of the striatal neurons expressing dopamine receptors, mitochondrial enzyme dysfunction and thereby attenuate dopamine signaling and causing histopathological alterations as seen in HD patients. motor symptoms like altered locomotor, motor in However, QUIN in lower doses did not produce any coordination and rotational behavior in the significant alterations in behavioral as well as rodents51,52. MK-801 being NMDA receptor biochemical and mitochondrial enzyme alterations. antagonist, significantly reversed the behavioral Therefore, dose of QUIN 300 nM was selected to alterations in QUIN treated animals, indicating its role explore the protective action of the MK-801 in the against QUIN mediated toxicity. QUIN treatment pathogenesis of HD. further caused significant reduction in the body In the present study, intrastriatal administration of weight which could be due to impaired energy the QUIN 300 nM produces the HD like alterations at metabolism and oxidative damage. There are also the behavioral level (hyperactivity followed by hypo studies reporting the impairment of the grip strength53 activity) and marked loss of neurons in the striatal in the animal model of HD. MK-801 treatment KALONIA et al.: MK-801 AGAINST QUINOLINIC ACID INDUCED ALTERATIONS IN RATS 887

Fig. 2 — Effect of MK-801 on QUIN induced altered levels of (a) mitochondrial complex-I (b) mitochondrial complex-II (c) mitochondrial complex-IV levels of brain cortex and striatum [Values are mean±SE (% age of sham). P<0.05 as compared to asham, bQUIN (100), cQUIN (200), dQUIN (300), eMK 801 (0.02), fMK 801 (0.04) in the respective region of the brain. (ANOVA followed by Tukey test). NS Not significant] reversed the QUIN induced reduction in body weight mediated antagonist14. Several studies reported the and altered rotarod performance (grip strength and protective action of the MK-801 in the various motor in coordination) in animals, suggesting their neurodegenerative disorders such as depression, therapeutic potential against QUIN mediated toxicity. epilepsy, ischemia, Alzheimer’s16-19,46,47 and MK-801 is a noncompetitive NMDA receptor- neuroprotective properties48. 888 INDIAN J EXP BIOL, NOVEMBER 2009

Fig. 3 — Effect of MK-801 on QUIN induced altered levels of MTT activity (no. of viable cells) of brain cortex and striatum [Values are mean±SE (% age of sham). P<0.05 as compared to asham, bQUIN (100), cQUIN (200),dQUIN (300), eMK 801 (0.02), fMK 801 (0.04) in the respective region of the brain. (ANOVA followed by Tukey test). NS Not significant]

This was demonstrated to induce the parameters can be considered as a tool to reproduce formation of hydroxyl radicals under in vivo similar pathological consequences of HD in the conditions in rat corpus striatum in a mechanism experimental model. In addition, QUIN-induced lipid which seems to be independent of NMDA peroxidation is blocked by a variety of receptors1,12,54. antioxidants54,55,58 giving further support for NMDA Other recent reports emphasize the involvement of receptor activation and ROS overproduction on its nitric oxide (NO•) and superoxide anion (O2-•) in the toxicity. neuronal damage induced by QUIN, demonstrating its In the present study, intrastriatal injection of the potential ability to produce the highly toxic RNS QUIN 300 nM lead to oxidative stress as indicated by peroxynitrite (ONOO-) 1,12,54. The origin of ONOO- in increase in the MDA, nitrite levels in the cortex and this model has been tracked toward the capability of striatum regions. There was a significant depletion of QUIN to induce both the expression and activity of the endogenous antioxidant defense systems such as inducible NO synthase (iNOS), thereby increasing the reduced glutathione, SOD, catalase levels in the levels of NO• while decreasing the activity of the striatum and cortex. Besides, intrastriatal QUIN enzyme responsible of detoxifying O2-•, superoxide administration significantly decreased redox ratio, dismutase (SOD). Such an altered cellular indicating the imbalance between oxidants and environment is favorable for the further formation of antioxidatant defense mechanism. MK-801 treatment more ROS and RNS11,12,55,56. The principle defense significantly attenuated QUIN mediated oxidative system against oxygen free radicals are SOD, GSH, damage, suggesting the role of oxidative damage in glutathione peroxidase, glutathione reductase, the HD progression and free radical scavenging action catalase. Antioxidant nutrients and imbalance in the of MK-801. expression of the GSH and associated enzymes has Mitochondrial dysfunction cause abatement in ATP been implicated in a variety of pathological production, oxidative damage and induction of conditions57-59. QUIN induces lipid peroxidation12 and involved in the pathogenesis of neuronal damage that can be retarded by neurodegenerative disorders62. Dysfunction of one or antioxidants60 and NMDAr antagonists61. Oxidative more respiratory chain complexes enhanced the stress and excitotoxicity are commonly involved in production of free radicals, associated mitochondrial several neurodegenerative diseases including striatal disturbance, oxidative damage to macromolecules and neuronal loss in the HD. The similarity between altering mitochondrial permeability transition pores QUIN mediated cell damage and selective neuronal inducing apoptosis. These high-conductance pores destruction observed in HD suggests that these offer a pathway which opens in response to certain KALONIA et al.: MK-801 AGAINST QUINOLINIC ACID INDUCED ALTERATIONS IN RATS 889

Fig. 4 — Effect of MK-801 on QUIN induced histopathological changes in rat brain striatum. [Histological sections of rat striatum stained with H & E: (a): Naive group, (b): Sham, (c): QUIN 100, (d): QUIN 200] stimuli, leading to apoptosis progress63. QUIN impaired energy metabolism due to the ATP injection significantly inhibits mitochondrial exhaustion. In the present study, QUIN treated animal complexes in striatum64. Biochemical analysis of the showed the significant alterations in the mitochondrial postmortem brain tissue sample analysis of HD functions and MK-801 showed the protective action patients showed the impaired energy metabolism with against the QUIN induced mitochondrial dysfunctions consistent decrease in the mitochondrial complex-I linked with the exitotoxicity that could be possibly by (NADH dehydrogenase activity), mitochondrial blocking NMDA receptors. As a result of NMDAr complex-II (succinate dehydrogenase (SDH) activity), blockade, there is decreased levels of the Ca2+ ions MTT activity levels and mitochondrial complex-IV which was the principle culprit for the mitochondrial (cytochrome oxidase) levels. Mitochondrial complex dysfunction and secondly, if the impairment of one or inhibitors preferentially damage more respiratory chain complexes lead to enhanced dopamine neurons in rat brain slices62. These findings production of free radicals further worsens the suggest the reason for various behavioral symptoms mitochondrial function by causing oxidative damage such as rotarod performance and locomotor activity. to macromolecules. Therefore, MK-801 being In the present study, QUIN 300 nM treatment NMDAr antagonist has neuroprotective action via significantly decreased in the activity of the all the NMDAr blockade and which down regulate the three mitochondrial complex enzymes indicating the further consequence of QUIN65. 890 INDIAN J EXP BIOL, NOVEMBER 2009

Fig. 4 — (contd.) Effect of MK-801 on QUIN induced histopathological changes in rat brain striatum. [Histological sections of rat striatum stained with H & E: (e): QUIN 300, (f): QUIN 300+MK-801(0.02), (g): QUIN 300+MK-801(0.04) and (h): QUIN 300+MK-801(0.08)]

Continuous neuronal depolarization leads to a damage due to apoptosis and MK-801 block the Ca2+ depletion of neuronal energy stores and inability to intake and so on the further cascade. It was therefore maintain neuronal ionic gradients of sodium and proposed that an endogenous “excitotoxin” may play chloride44,45. Consistent with this hypothesis, an early a role in the neuronal degeneration and hence in the morphologic finding accompanying excitotoxic pathogenesis of HD. damage is dendritic swelling4. There are several Taken together, above data suggest that MK-801 studies reporting the histopathological changes. In the shows the neuroprotective action against intra-striatal present study, histological evaluation verified that quinolinic acid induced behavioral, biochemical lesions present in QUIN treated animals as compared via multiple 49,66 and cellular alterations in rats to sham group, brain sections and MK-801 pathways. In conclusion, MK-801, has potential to significantly reversed the histological alterations such protect the QUIN induced HD like symptoms and as the neuronal damage observed by the damaged alterations in rat. body of the neurons in the QUIN treated animals as QUIN lead to the influx of Ca2+ which disrupt the Acknowledgement mitochondrial functions which in turn activate Authors acknowledge the financial support from caspase-9 and caspase-3 and lead to the neuronal the University Grant Commission, New Delhi. KALONIA et al.: MK-801 AGAINST QUINOLINIC ACID INDUCED ALTERATIONS IN RATS 891

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