Behavioural Brain Research 261 (2014) 345–355

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Mitochondrial cofactors in experimental Huntington’s disease: Behavioral, biochemical and histological evaluation Arpit Mehrotra, Rajat Sandhir ∗ Department of Biochemistry, Basic Medical Science Building, Panjab University, Sector-14, Chandigarh 160014, India

h i g h l i g h t s • • • •

Therapeutic role of mitochondrial cofactors (ALA and ALCAR) in HD was investigated. Combined supplementation with ALA and ALCAR ameliorated motor and cognitive deficits. Oxidative stress was attenuated by combined supplementation with ALA and ALCAR. ALA and ALCAR supplementation improved histological changes observed in HD.

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Article history: Received 7 September 2013 Received in revised form 21 December 2013 Accepted 26 December 2013 Available online 3 January 2014 Keywords: Alpha-lipoic acid Acetyl-l-carnitine Behavior Huntington’s disease Mitochondria

a b s t r a c t The present study was carried out to evaluate the beneficial effect of mitochondrial cofactors; alpha-lipoic acid (ALA) and acetyl-l-carnitine (ALCAR) in 3-nitropropionic acid (3-NP) induced experimental model of Huntington’s disease (HD). HD was developed by administering sub-chronic doses of 3-NP, intraperitoneally, twice daily for 17 days. The animals were assessed for their behavioral performance in terms of motor (spontaneous locomotor activity, narrow beam walk test, footprint analysis and rotarod test) and cognitive (elevated plus maze and T-maze tests) functions. 3-NP treated animals showed impairment in motor coordination such as decreased stride length, increased distance between inner toes, and increased gait angle. Increased transfer latency on elevated plus maze and T-maze tasks revealed cognition deficits in 3-NP treated animals. Increased lipid peroxidation and concomitant decrease in thiol levels were also observed. 3-NP administration also induced histopathological changes in terms of enhanced striatal lesion volume, presence of pyknotic nuclei and astrogliosis. However, combined supplementation with ALA + ALCAR to 3-NP administered animals for 21 days was able to efficiently improve behavioral deficits, attenuate oxidative stress and histological changes, suggesting a putative role of these two supplements if given together in ameliorating 3-NP induced impairments and thus could be engaged in managing HD. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Huntington’s disease (HD) is a neurodegenerative, hereditary disorder characterized by a variety of clinical symptoms. Among the most frequent symptoms are motor deficits involving chorea, emotional and behavioral disturbances manifested as depression and irritability, cognitive impairment and functional disability [1]. The neuropathological changes include progressive neuronal degeneration and atrophy affecting the striatum and other areas of the brain such as cortex, cerebellum, thalamus and sub-thalamic nucleus.

Abbreviations: ALA, alpha-lipoic acid; ALCAR, acetyl-l-carnitine; DAB, 3,3 -diaminobenzidine; 3-NP, 3-nitropropionic acid; GFAP, glial fibrillary acidic protein; GSH, glutathione; HD, Huntington’s disease; MDA, malondialdehyde; TTC, 2,3,5triphenyltetrazolium chloride. ∗ Corresponding author. Tel.: +91 0172 2534131/34. E-mail address: [email protected] (R. Sandhir). 0166-4328/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2013.12.035

This autosomal dominant disease is caused by CAG trinucleotide expansion within the first exon of huntingtin gene (htt), located near the telomere of the short arm of chromosome 4, locus 4p16.3 [2]. HD is found throughout the world with an estimated global prevalence of 4–5 per 100,000 in all ethnic groups, which makes it one of the most prevalent neurological diseases [3]. 3-Nitropropionic acid (3-NP) induced experimental model of HD has been universally accepted and aides in phenotypic replication of chronic neurodegenerative processes involved in HD pathology [4]. This animal model have shown to mimic characteristic features such as neurobehavioral impairments involving alteration in impaired locomotor activity, gait disturbances and cognitive decline followed by striatal degeneration concomitant with astrogliosis as those observed in HD [5]. Mitochondria are considered as a major source of reactive oxygen species (ROS) generation and mitochondrial dysfunctions are believed to be involved in aging and a number of neurological disorders including HD [6]. Therefore, improving mitochondrial

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function has now become a prime focus to combat neurodegeneration in HD. Mitochondrial cofactors, particularly acetyl-l-carnitine (ALCAR) and alpha-lipoic acid (ALA) have shown to be effective in reducing age-related mitochondrial dysfunction and their combination may decrease oxidative damage to neurons and improve locomotor and cognitive deficits [7]. ALCAR, an acetyl derivative of l-carnitine, is actively transported across the blood brain barrier and is required for the transport of long-chain fatty acids into the mitochondria for ␤-oxidation, ATP production and removal of excess short- and medium-chained fatty acids, thus helps to maintain efficient mitochondrial function [8]. Additionally, ALCAR participates in cellular energy production and in maintenance and repair of damaged neurons [9]. ALA also readily crosses the blood–brain barrier where it is reduced to dihydrolipoic acid, which is a powerful mitochondrial antioxidant that recycles cellular antioxidants, including coenzyme Q, vitamin C and vitamin E, glutathione (GSH) and also chelates transition metals like iron and copper [10]. Combined supplementation with ALA and ALCAR had been reported to be more effective than using either alone, in improving acquisition or memory performance in aged rats [11]. In a recent study, supplementation with a combination of both ALA and ALCAR were found to be effective in improving cognitive and motor performance [12]. The mechanism involved in protective effect offered by ALA and ALCAR appears to involve increased mitochondrial biogenesis [13]. Therefore, the purpose of the present study was to provide a more comprehensive behavioral assessment in 3-NP induced rat model of HD and to evaluate if the combination of mitochondrial cofactors possesses the propensity to improve behavioral, biochemical and histological changes. To this end, various locomotor and cognitive tasks were performed on rats to assess gait deformities, hind-limb impairment and muscular coordination. 3-NP induced oxidative stress was evaluated in terms of lipid peroxidation products and GSH levels. Lesion volume was measured using TTC staining (a dye, which is used as a marker for the presence of active dehydrogenases) and histopathological changes using hematoxylin–eosin and nissl staining were also conducted to evaluate the extent of neuro-anatomical damage produced by 3-NP administration. Here we report for the first time that, even though individual supplementation with ALA and ALCAR produced beneficial effect on various motor and cognitive performance tasks along with biochemical and histopathological changes, nevertheless combined supplementation (ALA + ALCAR) was more effective in reversing 3-NP induced changes. 2. Experimental procedures 2.1. Chemicals All the chemicals used in the present study were of analytical grade and were purchased from Sigma Chemical Co. (St. Louis, MO, USA), Merck (Mumbai, India) and Sisco Research Laboratories Pvt. Ltd. (Mumbai, India). ALA and ALCAR were received as a gift for research purposes from Sami Labs limited (Bangalore, India), GFAP polyclonal antibody was procured from Abcam Plc (Cambridge, UK). Secondary anti-rabbit antibody was obtained from Sigma Chemical Co. (St. Louis, MO, USA). 2.2. Animals and treatment schedule Female wistar rats aged 9–10 weeks, weighing between 200 and 250 g were procured from the Central Animal House facility of Panjab University, Chandigarh, India. The animals were allowed to acclimatize to the local vivarium for 7 days. All the experiments were carried out between 09:00 and 15:00 h.

The protocols followed were approved by the Institutional Animal Ethics Committee of the University and were in accordance with the guidelines for humane use and care of laboratory animals. The body weights of the animals were recorded daily and were randomly segregated into following eight groups with each group having five animals: Control (vehicle): Animals received vehicle alone. ALA treated: Animals were administered ALA at a dose of 50 mg/kg (i.p) for 21 days. ALCAR treated: Animals were administered ALCAR at a dose of 100 mg/kg (i.p) for 21 days. ALA + ALCAR treated: Animals were administered with combination of ALA + ALCAR at doses mentioned above. 3-NP treated: Animals were administered 3-NP at a sub-chronic dose twice a day intraperitoneally for 17 days. 7.5 mg/kg for the first 2 days, followed by 3.75 mg/kg for next 7 days, finally a dosage of 2 mg/kg for the last 8 days. The dose of 3-NP used in the study is based on the doses reported in literature and were standardized in our laboratory [14]. 3-NP + ALA treated: 3-NP treated animals were supplemented with ALA (50 mg/kg, i.p), once daily for 21 days. 3-NP + ALCAR treated: 3-NP treated animals were supplemented with ALCAR (100 mg/kg, i.p) once daily for 21 days. 3-NP + ALA + ALCAR treated: 3-NP treated animals were supplemented with combination of ALA (50 mg/kg, i.p) + ALCAR (100 mg/kg, i.p) once daily for 21 days. 2.3. Behavioral studies After the completion of respective dosages for each group, animals were assessed for locomotor and cognitive impairments using various neurobehavioral tasks. 2.3.1. Locomotor activity tests 2.3.1.1. Actophotometer test. The locomotor activity was measured using actophotometer [15]. The interruption of a beam of light falling on a photocell following the movement of the animal was recorded. Each rat was placed individually in the actophotometer for 3 min and the counts were recorded. 2.3.1.2. Narrow beam walk test. This behavioral test was used to evaluate motor performance in the animals, by progressively increasing the difficulty in the execution of the task as described by Masoud et al. [16]. The animals were trained in crossing a 150 cm long wooden beam, divided into three 50 cm segments, from a platform at one end to the animal’s home cage at the other end, placed horizontally 60 cm above the floor. The number of paw slips onto an under-hanging ledge and the time taken to traverse the beam was recorded. The maximum time allowed for the task was 2 min. Occurrence of bradykinesia was quantified by calculating the average velocity of walking for treated and control animals. 2.3.1.3. Rotarod test. Rotarod treadmill test was performed to assess muscular strength of the animals in all groups [17]. The rotarod apparatus (IMCORP Instruments, Ambala, India) consisted of a rotating rod, 75 mm diameter, on which rats were allowed to hold. After twice daily training for 2 successive days (speed 8 rpm on the first day and 10 rpm on second day) the rotational speed of the rod was increased to 15 rpm on the third day in a test session. The time for each rat to remain on the rotating rod was recorded. The maximum time was 120 s per trial. The apparatus automatically records the time of fall. The animals were trained on the rod, so that they could stay on it at least for the cut-off time. Data were presented as retention time on the rotating rod.

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2.3.1.4. Footprint analysis. This test was used to assess gait abnormalities in 3-NP treated animals [14]. After coating the hind paws with a non-toxic green dye and forepaws with red dye, rats were allowed to walk on a beam (100 cm length, 12 cm breadth and 10 cm high walls with an inclination of 30◦ ). The gangway was lined with white paper for recording the feet impressions. Animals in all the groups were tested for footprint length, footprint breadth, and footprint stride length for both left and right paws. Specifically, footprint stride length was quantified as the distance between two subsequent paws. Paws overlap analysis was carried out by measuring the center distance between the anterior paw and rear paw footprints. Additionally, distance between spreading inner toes 2–4 and gait angle were also recorded. 2.3.2. Cognition tests 2.3.2.1. Elevated plus maze test. Elevated plus maze test was used to evaluate spatial long-term memory according to the method of Itoh et al. [18]. The apparatus consisted of two open and two closed arms. The arms extended from a central platform, and the maze was elevated to a height of 50 cm from the floor. On the first day, each animal was placed at the end of an open arm. Transfer latency (TL) was recorded as the time taken by the rat to move into one of the closed arm. If the animal did not enter an closed arm within 90 s it was gently pushed into one of the closed arm and the TL latency was assigned as 90 s. The rats were allowed to explore the maze for 20 s and then returned to the home cage. 2.3.2.2. T-maze test. Left-right discrimination test of learning in animals was assessed by T-maze test [19]. The stem (T arm) and closed arms (left and right arms) of T-maze were each 45 cms long and 8 cms wide. The closed arms were covered by 20 cms high walls leaving the stem open on either side. During the training period, the food pellets were alternatively placed at the end of the each closed arms of maze so as to train the animals for their left or right closed arm choices. On day-21, the right arm was baited with food pellet and the animals were placed at the bottom of T arm. The latency to the first entry in the right arm and number of entries to the right arm for duration of 120 s were recorded using video tracking software (ANY-mazeTM , Stoelting, Wood Dale, IL, USA). 2.4. Biochemical analysis 2.4.1. Tissue homogenization On day 21, after the neurobehavioral studies, animals were sacrificed by decapitation under mild ether anesthesia. The striatum was separated from the brain, weighed and minced separately. A 10% (w/v) homogenate was prepared in ice-cold 50 mM phosphate buffer saline, pH-7.4, by using a Potter–Elvehjem type glass homogenizer. The biochemical assays for examining lipid peroxidation and GSH levels were immediately performed in the homogenates [20]. 2.4.2. Lipid peroxidation Malondialdehyde (MDA), a measure of lipid peroxidation was quantified by reaction with thiobarbituric acid at 532 nm as described by Dhanda et al. [21]. The values were expressed as nmol MDA/mg protein, using molar extinction coefficient of chromophore (1.56 × 105 M−1 cm−1 ). 2.4.3. GSH levels The GSH levels were estimated by the method of Roberts and Francetic [22], using DTNB (5,5 -dithiobis-(2-nitrobenzoic acid)) and results were expressed as nmol GSH/mg protein.

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2.4.4. Estimation of protein The protein content was estimated according to the method of Lowry et al. [23]. 2.5. Histological examination 2.5.1. TTC staining On day 21, after the completion of neurobehavioral tasks in all the experimental groups, the rats were sacrificed by decapitation and the brains were rapidly removed and frozen to later obtain the coronal sections of 2-mm thickness. Slices were stained in 2% of 2,3,5-triphenyltetrazolium chloride (TTC) solution at room temperature in the dark and fixed in phosphate-buffered 4% paraformaldehyde solution. The lesioned areas appeared as a pale staining, which were measured on the posterior surface of each section with an Image J software (USA), and the volume (in mm3 ) of each lesion was calculated by summing the results of multiplying each lesion area by 2 mm [24]. 2.5.2. Hematoxylin–eosin (H–E) and cresyl violet staining Animals in all the groups were initially perfused transcardially with cold saline followed by phosphate buffered 4% paraformaldehyde. The brains were then post-fixed overnight in 4% paraformaldehyde and later brain sections were processed for routine H–E [25] and cresyl violet staining [26]. 2.5.3. GFAP immunohistochemistry Animals were transcardially perfused with 4% paraformaldehyde on the last of the study. The brains were then post-fixed overnight in 4% paraformaldehyde and later brain sections were processed for GFAP staining [27]. Paraffin sections of the brain were initially deparaffinized via routine procedure, rinsed in cold PBS (0.1 M and pH 7.4) three times for 5 min each. These were then permeabilized with 0.4% Triton-X 100 for 30 min, blocked with 6% BSA and 0.1% Triton-X 100, and incubated overnight with primary antibody (1:80) at 4 ◦ C. After washing, HRP-conjugated secondary antibody (1:2000) was added and incubated for 1–2 h in the dark chamber at room temperature. Following PBS washings, brain sections were developed with DAB and H2 O2 , and were photographed at higher magnification (100×). 2.6. Statistical analysis All values are expressed as mean ± standard error of mean (SEM) of five animals per group. Data was analyzed using one way analysis of variance (ANOVA) followed by Newman–Keuls test for multiple pair-wise comparisons between the various treated groups using SPSS 14 software. Values with p < 0.05 were considered as statistically significant. 3. Results 3.1. Effect of ALA and ALCAR on neurobehavioral deficits 3.1.1. Locomotor activity The locomotor ability of all the animals in eight groups were evaluated in terms of number of photo beam counts recorded for a maximum time span of 180 s using actophotometer (Fig. 1A). On day 0, animals in all the eight groups had an average count of 245.5, which is indicative of normal locomotor activity. At the end of the study (day 21), the numbers of counts for 3-NP treated animals were reduced to an average of 114.6, suggesting a significant impairment in locomotor activity. However, individual supplementation with ALA and/or ALCAR to 3-NP treated animals for 21 days increased the average number of counts to 138.1 and 165.3, respectively. However, combined supplementation with ALA + ALCAR to

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Fig. 1. Effect of ALA and/or ALCAR supplementation on locomotor activity (A), narrow beam walk test in terms of total time (B), average velocity (C) and number of paw slips (D), rota-rod test for muscular strength (E) of 3-NP treated animals. Values are expressed as mean ± SEM; n = 5/group. a Significantly different from control group (p < 0.05), b,c,d Significantly different from 3-NP treated group (p < 0.05).

3-NP treated animals for 21 days, locomotor activity was found to be significantly higher than 3-NP treated animals (average of 192.7 counts). 3.1.2. Narrow beam walk test Narrow beam walk test was used to evaluate hind-limb impairment, wherein the time taken by the animals, to walk across a narrow beam with progressively decreasing width, was recorded. The maximum time allowed for each animal to complete the task was 120 s (Fig. 1B). On day 0, average time taken by animals in all the eight groups was 5.4 s. At the end of the study (day 21), 3-NP treated animals recorded a average time of 105.7 s, showing a significant impairment in hind-limb function. On the other hand, supplementation with ALA or ALCAR individually to 3-NP administered animals showed an average time of 14.5 s and 22.8 s,

respectively. Animals co-supplemented with ALA + ALCAR had significantly better hind-limb function and traversed the beam in 16 s. The average velocity (time taken/distance traveled) to cross the narrow beam and number of paw slips for animals in each group were also calculated (Fig. 1C and D). On day 0, average velocity of the animals in all the eight groups was 29.6 cm/s, whereas the average number of paw slips was 0. On day 21, 3-NP treated animals recorded an average velocity of 1.4 cm/s and the average numbers of paw slips were 2.8. However, individual supplementation with ALA or ALCAR to 3-NP treated animals for 21 days increased the average velocity to 10.9 cm/s and 7.6 cm/s, whereas the average numbers of paw slips were 0.7 and 0.5, respectively. Co-supplementation of ALA and ALCAR to 3-NP treated animals showed an average velocity of 11.1 cm/s and the average number of paw slips were 0.2.

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Fig. 2. Effect of ALA and/or ALCAR supplementation on footprint test for gait analysis by 3-NP treated animals. The footprint impressions obtained using non-toxic red and green color dyes indicates fore paws and hind paws of the animals, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.1.3. Rotarod test The purpose of this experiment was to detect muscular strength and motor coordination deficits following 3-NP induced HD (Fig. 1E). The results demonstrated that the rotarod task was a sensitive index of measuring 3-NP induced motor impairments. The muscular strength of all the animals in eight groups was evaluated in terms of maximum time spent by the animal on rotating rod. The maximum time allotted for each animal in performing task was 120 s. On day 0, average time taken by animals in all the eight groups was 116.3 s. On day 21, following 3-NP treatment, average time taken by 3-NP treated animals was lowered to 45 s, showing a significant impairment in muscular coordination. However, 3-NP treated animals supplemented with ALA or ALCAR for 21 days were able to hold the rod for longer duration, 78.5 s and 89.6 s, respectively. On the other hand, 3-NP treated animals supplemented with both ALA + ALCAR for 21 days were able to significantly improved muscular strength on rotating rod, taking an average time of 105.67 s. 3.1.4. Footprint analysis Increase in number of paw slips on narrow beam walk revealed motor impairments of the animals and is interpreted as a symptom of abnormal gait. So, to confirm gait abnormalities in 3-NP treated rats, footprint analysis tests was performed on day 0 and 21, wherein the hind paws and fore paws of the animals were stained with non-toxic green and red dyes, respectively (Fig. 2). For both left and right paws of animals, they were tested for footprint length, footprint breadth and footprint stride length. On the last day of the study, all of six parameters analyzed were found to be significantly affected in the animals that received 3-NP treatment. On day 0, average footprint length for both fore paws and hind paws of animals in all eight groups were 1.9 cm and 2.5 cm respectively, whereas, average footprint breadth for both fore paws and hind paws were 1.8 cm and 2.0 cm, respectively. In addition, footprint stride length for both fore paws and hind paws of animals in eight groups were 13.1 cm and 12.7 cm. On day 21, footprint length,

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footprint breadth and footprint stride length for fore paws of 3-NP treated animals were 1.3 cm, 1.2 cm and 5.9 cm, whereas the same observations for hind paws were found to be 2.8 cm, 1.6 cm and 8.2 cm, respectively. However, the individual or combined supplementation with ALA and/or ALCAR to 3-NP treated animals for 21 days showed that, footprint length, footprint breadth and footprint stride length for both left and right paws of animals were less affected. Footprint length for fore paws of 3-NP treated animals that received individual or combined supplementation with ALA and/or ALCAR for 21 days was found to be 1.7 cm, 1.7 cm and 1.6 cm, whereas for hind paws the length of footprint were found to be 2.6 cm, 2.5 cm and 2.6 cm, respectively. Also, footprint breath for fore paws of 3-NP treated animals that received individual or combined supplementation with ALA and/or ALCAR for 21 days are 1.5 cm, 1.5 cm and 1.8 cm, whereas for hind paws the breath of footprint were found to be 1.6 cm, 1.7 cm and 2.0 cm, respectively. In addition, footprint stride length, which is the distance between two consecutive paws on Y-axis was also calculated. The footprint stride length for fore paws in individually or combined supplementation with ALA and/or ALCAR to 3-NP treated animals for 21 days was found to be 10.2 cm, 12.5 cm and 13.3 cm, in comparison to footprint stride length for hind paws, which were found to be 10.8 cm, 12.1 cm and 13.9 cm, respectively. The spreading of inner toes (distance between toe 2 and 4) and gait angle was also found to be significantly affected on 3NP administration. On day 0, average distance between toe 2 and 4 and gait angle for all the animals in eight groups was 1.2 cm and 0.5◦ , respectively. On day 21, average distance between toe was significantly reduced to 0.8 cm whereas angle was increased to 3.2◦ for 3-NP treated animals. Individual or combined supplementation with ALA and/or ALCAR to 3-NP treated animals for 21 days, the distance between toe was found to be 1.2 cm, 1.1 cm and 1.1 cm, respectively. Gait angle on individual or combined supplementation was however reduced to 1.7◦ , 1.4◦ and 1.2◦ , respectively. An additional measurement of walking pattern in four-legged animal is their tendency to overlap both left and right paws while moving, which was calculated as the minimum distance (in cms) between left and right paws overlap. The basis of this measurement depends on the reality that rodents while walking usually have a propensity to step their hind paw in the same place formerly occupied by their fore paw, thus minimum is the distance, and the better is locomotor ability. Analyzing this aspect of the impaired motor coordination could then be considered as a measure to assess locomotor ability in such neurological disorder. On day 0, minimum distance between left paws and right paws of animals in all groups were 0.2 cm and 0.27 cm, respectively. On day 21, minimum distance recorded between left paws and right paws of 3-NP treated animals was significantly increased to 0.9 cm and 1.0 cm, showing difficulty in swift movement during task and reflects distinguished symptoms of gait deformities. However, minimum distance analyzed between left paws in 3-NP treated animals supplemented individually or in combination with both ALA and/or ALCAR for 21 days was significantly reduced to 0.4 cm, 0.3 cm and 0.2 cm, in comparison to right paws which were 0.5 cm, 0.3 cm and 0.1 cm. Thus, overall results of footprint analysis suggest that, combined supplementation with ALA and ALCAR to 3-NP treated animals is more effective than either of them using alone. 3.1.5. Elevated plus maze test Spatial memory was assessed in terms of transfer latency (average time taken to move into one of the closed arms of the maze) on elevated plus maze apparatus (Fig. 3). The average time taken by the animals to locate the closed arm of the maze in all the eight groups on day 0 was between 12 s. However on day 21, the average time taken by 3-NP treated animals was significantly increased to 67.5 s, showing impaired cognition. In 3-NP treated

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peroxidation is malondialdehyde (MDA). A significant increase of 222% in MDA levels was observed in 3-NP treated animals. MDA levels were decreased by 22% and 35% on ALA or ALCAR supplementation to 3-NP treated animals. Combined supplementation with ALA + ALCAR to 3-NP treated animals, further decreased the levels of MDA levels by 46%. One of the most important antioxidant present in brain tissue is GSH and its levels were found to be compromised in the striatum of 3-NP treated animals. The levels of GSH in 3-NP treated animals were lowered by 54%. GSH levels in 3-NP treated animals that received individual supplementation with ALA or ALCAR were increased by 13% and 9% fold, respectively. However, GSH levels were increased by 62% in 3-NP treated animals supplemented with combination of ALA and ALCAR. Fig. 3. Effect of ALA and/or ALCAR supplementation on memory performance assessed using elevated plus maze test of 3-NP treated animals. Values are expressed as mean ± SEM; n = 5/group. a Significantly different from control group (p < 0.05), b,c,d Significantly different from 3-NP treated group (p < 0.05).

animals supplemented with either ALA or ALCAR, the average time recorded on day 21 was significantly lowered to 28.8 s and 26.5 s, respectively. The combined supplementation of ALA + ALCAR to 3-NP treated group for 21 days, significantly improved cognitive abilities amongst animals that took a lesser time (19.8 s) in completing the task, suggesting that combined supplementation is much effective in improving cognitive functions. 3.1.6. T-maze test T-maze test was used as a measure to determine left-right discrimination of learning in 3-NP treated animals, wherein track path followed by the animals reflects its ability to locate the left or right arms of the maze (Fig. 4A). The left-right discrimination task of learning for all the animals was evaluated in terms of transfer latency (average time taken) to enter the right arm of the maze (Fig. 4B) and number of entries to the right arm (Fig. 4C). On day 21, the transfer latency and number of entries in the right arm of the maze for 3-NP treated animals were 69.6 s and 1.4 respectively. Transfer latency in the right arm of the maze by 3-NP treated animals which received individual or combined supplementation with ALA and/or ALCAR for 21 days were 12.54 s, 11.16 s and 8.11 s, whereas number of entries in the right arm of the maze recorded were 2, 1.8 and 2.6, respectively. 3.2. Effect of ALA and ALCAR supplementation on biochemical changes Lipid peroxidation and GSH levels were analyzed in striatum tissue (Table 1). One of the most abundant products of lipid Table 1 Effect of ALA and/or ALCAR supplementation on lipid peroxidation and glutathione levels in the striatum of 3-NP treated rats. Lipid peroxidation (nmol MDA/mg protein) Control ALA ALCAR ALA + ALCAR 3-NP 3-NP + ALA 3-NP + ALCAR 3-NP + ALA + ALCAR

1.81 1.16 1.60 1.86 5.84 4.54 3.76 3.13

± ± ± ± ± ± ± ±

0.09 0.05 0.11 0.12 0.56a 0.59b 0.11c 0.09d

All values are expressed as mean ± SEM. a Significantly different from control group (p < 0.05), from 3-NP treated group (p < 0.05).

GSH (nmol GSH/mg protein) 30.04 35.92 28.16 29.96 13.60 15.44 14.64 22.12 b,c,d

± ± ± ± ± ± ± ±

2.4 1.8 3.6 2.8 1.6a 2.1 0.8 3.0d

Significantly different

3.3. Effect of ALA and ALCAR supplementation on histological changes 3.3.1. Striatal lesion volume To evaluate the protective effect of combined ALA and ALCAR supplementation in 3-NP induced neuronal damage, we examined the degree of striatal tissue damage using TTC stain (Fig. 5). The appearance of red color in the tissue section, reflects the activity of various dehydrogenases present in the functional tissue and pale reflects the damaged area of tissue. 3-NP administration induced extensive neuronal damage in the striatum, since histological examination identified a paleness in the center of striatal region. However, the lesion volume in 3-NP administered animals supplemented with ALA, ALCAR and/or ALA + ALCAR was significantly lower as compared to 3-NP treated group. However, combined supplementation was more effective in reducing the 3NP-induced lesion. 3.3.2. H–E staining Histopathological changes in striatum of control and 3-NP treated animals were further assessed using routine H–E staining. Histological assessment through H–E staining (Fig. 6A) confirmed that control animals showed normal tissue morphology. On the other hand, 3-NP treated rats showed marked neuronal degeneration, wherein neurons appeared more pyknotic, condensed and damaged, confirming a significant degeneration of the striatal tissue. However, tissue section from animals supplemented with individual or combined therapy of ALA or ALCAR for 21 days, showed increased number of neurons with improved morphology in comparison to 3-NP treated group. 3.3.3. Cresyl violet staining In addition, cresyl staining showed analogous morphological changes (Fig. 6B). Striatal tissue sections from control rats, showed a higher number of nissl stained neurons, whereas, decreased number of nissl stained neurons were observed in 3-NP treated rats. Individual or combined supplementation with ALA and ALCAR to 3-NP treated animals for 21 days significantly increased the number of nissl stained neurons with improved morphological appearance. Both the staining procedures proved that combined supplementation with ALA and ALCAR is much more effective in improving neuronal morphology in striatum than either of them alone. 3.3.4. GFAP immunohistochemistry Glial fibrillary acidic protein (GFAP) is a marker for astrocytes, and GFAP immunohistochemistry showed homogeneous distribution of the astrocytes in the control striatal sections (Fig. 7A). 3-NP treated brain striatal sections showed increased size of astrocytes. The astrocytes appeared scattered in the lesion core, suggesting occurrence of reactive astrogliosis (Fig. 7B). In contrast, combined supplementation with ALA + ALCAR to 3-NP administered animals

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Fig. 4. Effect of ALA and/or ALCAR supplementation on cognitive function assessed by T-maze test for left-right discrimination, representative track plots (A), transfer latency to the right arm (B) and number of right arm entries (C) of 3-NP treated animals. Values are expressed as mean ± SEM; n = 5/group. a Significantly different from control group (p < 0.05), b,c,d Significantly different from 3-NP treated group (p < 0.05).

showed less number of reactive astrocytes, indicating their protective role (Fig. 7C).

4. Discussion The present study examines if supplementing mitochondrial cofactors ALA and ALCAR could ameliorate behavioral alterations associated with striatal degeneration in experimental HD. As striatum is involved in control of locomotor movements, increased time taken to cross the narrow beam and increased number of paw slips resulting in decreased average velocity were observed in 3NP treated animals. In addition, muscular strength of the animals following 3-NP exposure was found to be significantly impaired as assessed by rotarod test. These results were further strengthened by evaluating footprint analysis confirming gait abnormalities in 3NP treated animals which showed reduced stride length, reduced stride breadth, alterations of spreading inner toes and significant

decrease in stride length for both left and right paws in 3-NP treated animals. Among the various parameters of gait abnormalities investigated, stride length measurements for both left and right paws has been explored as a dependable marker of basal ganglia dysfunction following 3-NP exposure, leading to locomotor abnormalities [28,29]. In addition, the locomotor analysis of animals on narrow beam test further confirmed, that a progressive difficulty in accomplishing a task of increasing complexity could be due to 3-NP induced progression of gait disturbances. Further, overlapping distance for both left and right paws and gait angle was also found to be increased following 3-NP administration. These findings are in agreement with an earlier report, where a variety of motor deficits in rats following chronic administration with 3-NP were observed [30]. The main function of basal ganglia in brain is synchronization of movement patterns [31], and significant impairments in motor functions observed in the present study could be justified by the toxicity of 3-NP in degenerating this brain structure leading to impaired movement patterns. Increased oxidative stress and

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Fig. 5. Effect of ALA and/or ALCAR supplementation on striatal lesion volume in 3-NP treated animals. The lesion area observed in 3-NP treated frozen brain section is marked with white dotted circle. Values for striatal volume are expressed as mean ± SEM; n = 5/group. a Significantly different from control group (p < 0.05), b,c,d Significantly different from 3-NP treated group (p < 0.05).

Fig. 6. Effect of ALA and/or ALCAR supplementation on histological changes observed using routine H–E (A) and cresyl violet staining (B) in the tissue sections obtained from the brain of 3-NP treated rats. Magnification 40× (scale bar-100 ␮m).

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Fig. 7. Effect of ALA and ALCAR supplementation on 3-NP induced astrogliosis analyzed by immunostaining with anti-GFAP antibodies. A homogeneous distribution of the astrocytes in the control striatal section (A). 3-NP treated brain striatal sections showing hypertrophied astrocytes (B). Combined supplementation with ALA + ALCAR to 3-NP administered animals showed less number of reactive astrocytes (C). Magnification 100× (scale bar-20 ␮m).

mitochondrial dysfunctions following 3-NP administration can result in poor performance with respect to cognitive and motor functions [32]. The symptoms developed by chronic administration of 3-NP to animals are analogous to juvenile onset and late hypokinetic stages of HD [33]. However, individual or combined supplementation with ALA and/or ALCAR to 3-NP administered animals for 21 days significantly improved motor functions and gait abnormalities in animals, which shows the propensity of both the supplements if given in combination to prevent 3-NP-induced behavioral deficits. The combination therapy of ALA and ALCAR is reported to significantly improve behavioral performance with improved mitochondrial biogenesis in exhaustively exercised rats [34]. Cognitive impairment is one of the major symptoms observed in HD. Memory of HD patients often declines with degeneration of neurons in brain [35]. Cognitive dysfunctions may also be due to disruption of striatal–frontal circuits in HD patients. It is reported that, memory recall is generally affected more than memory storage [36]. In the present study, cognitive deficits were assessed using elevated plus maze and T-maze. Cognitive deficits were found to be more pronounced in 3-NP administered animals in terms of increased transfer latency in both the tests and decreased number of right arm entries in T-maze test. Sub-chronic administration with 3-NP has shown to produce lesion in hippocampal CA-1 and CA-3 pyramidal neurons, areas of the brain associated with cognitive performance [37]. These finding are in agreement with our earlier studies, where 3-NP administration was shown to significantly impair cognitive functions [28,38]. Combination of ALA + ALCAR to 3-NP treated animals was able to significantly improve their cognitive performance. How ALA and ALCAR affect short-term memory is not well understood, but may be caused by a number of factors, including increased neurotransmitter production, improved mitochondrial function and/or calcium handling by the neurons [39,40]. Since, low levels of acetylcholine in certain brain regions are coupled with age-related cognitive decline including in HD, the cognition enhancing effect of ALCAR supplementation may be caused in part by the donation of an acetyl group for the synthesis of the neurotransmitter acetylcholine through choline acetyltransferase thereby increasing the amount of free acetylcholine in the brain for improved neuronal cross-talk and thus reliving memory impairment [41]. ALA component on other hand improves neuro-cognitive function by potentially lowering iron and copperinduced oxidative stress observed in many age related disorders [42]. However, combination therapy provided a much improved memory performance due to the additive effect of both ALA and ALCAR. Such combined antioxidant diet is reported to attenuate age-dependent cognitive decline by reducing the consequences of oxidative stress than either of the two supplemented alone [43]. Oral supplementation with combination of ALA and ALCAR has shown to improve mitochondrial decay, lower RNA/DNA oxidation by decreasing oxidative damage to neurons and improved

cognitive function [11]. Thus, these results signify that combined supplementation with mitochondrial cofactors ALA and ALCAR offers benefit in attenuating cognitive deficits observed in HD. Oxidative stress is defined as an imbalance between the production and detoxification of reactive oxygen species (ROS), which is thought to play a significant role in the neurodegeneration as evident in HD. Oxidative stress is considered as a major deleterious event observed in clinical and experimental HD [44]. Increased oxidative stress is attributed to alterations in antioxidant defense system which includes lipid peroxidation and antioxidant enzymes. ROS generation is the key component of the secondary neuronal damage in neurodegenerative conditions [45]. The increased ROS formed during such condition attach to membrane polyunsaturated fatty acids, thereby inflicting lipid peroxidation and increasing membrane permeability [46]. The values of tissue MDA levels in 3-NP exposed animals were found to be significantly higher with concomitant decrease in GSH levels. A significant rise in MDA levels in the striatum, cortex and cerebellum of rats challenged with 3-NP has been suggestive of elevated oxidative stress [47]. It was observed that in 3-NP treated rats supplemented with ALA and/or ALCAR alone showed a significant decline in MDA levels. The decrease in the lipid peroxide levels seen on treatment with ALCAR could be due to its chelating property involved in sequestering of iron and preventing lipid-peroxidation [48]. ALA on the other hand has shown to directly reduce reactive free radicals, thus inhibiting the rate of MDA production promoted by iron mediated peroxidation [49,50]. In brain, neurons rely on the release and subsequent usage of GSH by astrocytes in order to maintain optimal intracellular GSH levels, which are required to protect against harmful radicals released from activated microglia and astrocytes [51]. Therapeutic strategies enabling astrocytes to provide neurons with sufficient substrates for GSH synthesis is of particular interest as reduction in GSH levels may contribute to neuronal cell death in a pro-oxidative, pro-inflammatory environment [52]. A significant reduction in GSH levels was observed in 3-NP induced HD animals. 3-NP administration had earlier been reported to augment the production of free radicals in rat striatum, which may lead to the utilization of detoxifying endogenous antioxidants such as GSH [53]. ALA and ALCAR are potent mitochondrial cofactors and their beneficial effects are well established in several diseases related with oxidative stress like diabetes mellitus, AIDS and various types of cancer [54,55]. ALA besides its potential to induce a substantial increase in cellular GSH, is also a scavenger of hydroxyl radicals, singlet oxygen and hypochlorous acid and functions as an essential cofactor in metabolic reactions involving energy utilization in mitochondria [56]. ALCAR being a mitochondrial metabolite improves the mitochondrial function and increases general metabolic activity [55]. Combined supplementation with ALA + ALCAR resulted in potentiating the protective effect of ALCAR, which agrees with the

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assumption that ALA shows beneficial effects in oxidative stress conditions, owning to its synergistic action in presence of other antioxidants such as ALCAR [39,56]. Hence, co-supplementation with ALA + ALCAR showed to decrease the levels of MDA, suggesting their propensity to reduce peroxidation of lipids and in restoring the antioxidant status, thereby ameliorating oxidative stress in 3NP induced HD animals. It has been widely reported that systemic administration of 3-NP can produce selective histopathological changes involving striatal lesions that closely replicate the histological, biochemical and clinical features of HD [57]. Neuronal death in HD evolves gradually with no intense tissue reaction and cellular response. As such, the onset of disease is subtle followed by insidious progression of pathological features [4]. 3-NP administered animals showed increased striatal lesion volume, indicating a significant loss of active dehydrogenases present in this brain region. This can be explained by irreversible inhibition of dehydrogenases by 3-NP. The lesions occur by a mechanism involving secondary excitotoxicity, which is linked to free radical generation [58]. Combined supplementation with ALA + ALCAR to 3-NP treated was able to lower the lesion volume and this could be due to improved activity of mitochondrial dehydrogenases as a result of additive effect imparted by these two mitochondrial cofactors. Routine histological staining of tissue sections obtained from 3-NP treated animals was also performed. 3-NP-induced histopathological findings have been well characterized in a number of previous reports [30]. In the present study, striatal sections from the control animals had normal neuronal morphology. 3-NP administered animals showed neurons with large, round pyknotic nuclei in striatum with conspicuous nucleoli and a difficult to define cytoplasm. Other neurons exhibited a more shrunken and hyperchromatic nucleus which may or may not be accompanied by a hyper-eosinophilic cytoplasm. Such changes mimic the histological alterations similar to that observed in human HD [4]. However, tissue sections obtained from 3-NP treated animals co-supplemented with combination of ALA + ALCAR showed normal neuronal morphology with mild degenerative changes compared to 3-NP treated animals. Cresyl staining on the other hand, showed increased number of lightly stained normal intact neurons in control striatum in comparison to 3-NP treated striatum, where increased number of darkly stained non-viable neurons and pyknotic nuclei were observed. Combined supplementation with ALA + ALCAR to 3-NP treated animals showed lesser spotted violet appearance of neurons with improved morphological features, suggesting the protective efficacy of mitochondrial cofactors. It is well known that astrogliosis frequently occurs together with the loss of neurons and fibers in HD [59]. Immunohistochemistry showed profound gliosis in the striatum of 3-NP-treated rats, with perceptible increase in the number and size of astrocytes present in the striatal lesion core, which is in accordance with the earlier studies [60,61]. 3-NP treatment has also shown to cause an increase in GFAP mediated inflammatory response leading to increased oxidative stress, mitochondrial dysfunctions and histopathological changes [62,63]. Under such conditions, inflammatory reactions mediated by cytokine release and increased oxidative stress evoked by 3-NP toxicity have shown to substantially contribute to brain damage [64]. Such a battery of sequences in the striatum may further aggravate the condition of 3-NP treated rats. However, tissue sections from 3-NP treated animals which received a combination of ALA + ALCAR showed normal distribution and appearance of astrocytes. Thus, in all the histological staining procedures, combined supplementation was much effective than either of them given alone and this could be due to diverse protective mechanisms adopted by these cofactors in preventing neuronal degeneration. The histological changes observed in the study are in accordance with the recently published reports, where combined

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