International Journal of Neuroscience, 2014; 124(9): 673–684 Copyright © 2014 Informa Healthcare USA, Inc. ISSN: 0020-7454 print / 1543-5245 online DOI: 10.3109/00207454.2013.872642
RESEARCH ARTICLE
Neuroprotective activity of L-theanine on 3-nitropropionic acid-induced neurotoxicity in rat striatum Sumathi Thangarajan, Asha Deivasigamani, Suganya Sarumani Natarajan, Prasanna Krishnan, and Sandhya Koombankallil Mohanan
Int J Neurosci Downloaded from informahealthcare.com by University of Sydney on 01/03/15 For personal use only.
Department of Medical Biochemistry, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai, Tamil Nadu, India The present study has been designed to investigate the protective effect of L-theanine against 3-nitropropionic acid (3-NP)-induced Huntington’s disease (HD)-like symptoms in rats. The present experimental protocol design includes systemic 3-NP acid (10 mg/kg intraperitonially) treatment for 14 d. L-theanine (100 and 200 mg/kg) was given orally, once a day, 1 h before 3-NP acid treatment for 14 d. Body weight and behavioral parameters (Morris water maze, open field test (OFT), forced swim test (FST) and rotarod activity) were assessed on 1st, 5th, 10th and 15th day post-3-NP acid administration. Malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) levels and mitochondrial enzyme complex. Succinate dehydrogenase (SDH) were measured on the 15th day in the striatum. Systemic 3-NP acid treatment significantly reduced body weight, locomotor activity and oxidative defense. The mitochondrial enzyme activity was also significantly impaired in the striatum region in 3-NP acid-treated animals. L-theanine (100 and 200 mg/kg b.wt.) treatment significantly attenuated the impairment in behavioral, biochemical and mitochondrial enzyme activities as compared to the 3-NP acid-treated group. The results of the present study suggest that pretreatment with L-theanine significantly attenuated 3-NP induced oxidative stress and restored the decreased SOD, GSH, CAT and SDH activity. It also decreased the neuronal damage as evidenced by histopathological analysis of striatum. Based on the above study, it has been proved that L-theanine has neuroprotective activity against 3-NP induced neurotoxicity. KEYWORDS: Huntington’s disease, oxidative stress, mitochondrial complex II, gliosis
Introduction Huntington’s disease (HD) is an incurable neuropsychiatric disease characterized by degeneration of basal ganglia, motor abnormalities, impaired cognitive function and emotional disturbance [1]. 3-NP acid (3-NP) is a mitochondrial toxin that causes striatal neuropathy similar to that seen in clinical HD. It has been demonstrated that systemic administration of 3-NP produced selective striatal lesions (preferentially in medium-sized spiny neurons) that begin in the dorsolateral quadrant of the striatum and later spread to the entire lateral striatum and behavioral, biochemical, histological and neu-
Received 29 October 2013; revised 3 December 2013; accepted 3 December 2013 Correspondence: Dr Sumathi Thangarajan, Department of Medical Biochemistry, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai 600113, Tamil Nadu, India. Tel: +91-44-24547086, Fax: +91-44-24540709. E-mail: [email protected]
rochemical features of HD [2,3]. 3-NP intoxication inhibits SDH, a key enzyme in the respiratory chain. Thus it leads to depletion of ATP, energy failure results altered calcium homeostasis, oxidative stress, excitotoxic events and neuronal death [4–6]. A major advantage of 3-NP model over other model of HD is that the lesions produced are bilateral, striatal specific and develop spontaneously after systemic administration of 3-NP. L-Theanine, γ -glutamylethylamide, is one of the major amino acid components in green tea and is synthesized from ethylamine and glutamate in green tea leaves [7]. It has been reported that theanine has an impact on brain function [8–11]. Studies have also documented the beneficial effect of antioxidants possibly by free radical scavenging activity and increased endogenous antioxidant defense against 3-NP induced neurotoxicity in rats [12–14]. L-Theanine can counteract excitotoxicity and/or mitochondrial radical formation [10,11]. Ltheanine is involved in the formation of the inhibitory neurotransmitter, gamma amino butyric acid (GABA). GABA influences the levels of 673
674
S. Thangarajan et al.
Int J Neurosci Downloaded from informahealthcare.com by University of Sydney on 01/03/15 For personal use only.
two other neurotransmitters, dopamine and serotonin, producing the key relaxation effect [15,16]. The administration of theanine into the lateral ventricle prevented ischemia induced neuronal death in the hippocampal CA1 region in a dose-dependent manner. Moreover, some reports suggested that the neuroprotective effect of theanine occurs via glutamate receptors, with theanine acting as a glutamate receptor antagonist [17,18]. Protective effects of many synthetic compounds and plant extracts have been reported on 3-NP induced toxicity. We have reported for the first time the neuroprotective activity of L-theanine against 3-NP induced neurotoxicity in rat striatum.
Materials and methods Animals Male Wistar rats weighing between 200 and 250 g bred in Central Animal House, Dr. ALMPGIBMS, University of Madras, Taramani campus, Chennai 113, Tamil Nadu, India were used. The animals were housed under standard laboratory conditions and maintained on natural light and dark cycle and had free access to food and water. Animals were acclimatized to laboratory conditions before the experiment. The experimental protocols were approved by the Institutional Animal Ethics Committee (IAEC) Dr. ALMPGIBMS, University of Madras, Taramani campus, Chennai 113, Tamil Nadu, India.
Drugs and treatment schedule The following drugs were used in the present study. 3NP (sigma chemicals) was dissolved in saline (adjusted to pH 7.4) and administered intraperitonially in a volume of 0.5 mL/100 g animal body weight. L-theanine (SRL chemicals) was dissolved in distilled water and administered orally. Rats were divided into six groups of six animals each. Group I: control; Group II: 3-NP (10 mg/kg, b.wt.) intraperitonially for 14 d; Group III: L-theanine (100 mg/kg b.wt.) orally prior to the administration of 3-NP for 14 d; Group IV: L-theanine (200 mg/kg b.wt.) orally prior to the administration of 3-NP for 14 d; Group V: L-theanine (100 mg/kg b.wt.) alone orally for 14 d: group VI: L-theanine (200 mg/kg b.wt.) alone orally for 14 d.
Measurement on body weight Body weight was recorded on the first and last day of the experiment. Percent change in body weight was calculated as body weight (1st day − 15th day) × 100. body weight (1st day)
[1]
Behavioral assessments Morris water maze test The acquisition and retention of a spatial navigation task was examined using a Morris Water Maze [19]. Animals were trained to swim to a platform in a circular pool (180 cm diameter × 60 cm) located in a test room. The pool is filled with water (28 ± 2◦ C) to a depth of 40 cm. A movable circular platform 9 cm in diameter, mounted on a column, was placed in the pool 1 cm above the water level for the test. On the 4th day prior to 3-NP administration, animals received a training session consisting of 4 trials. In all 4 trials, the starting positions were different. The latency to find the escape platform was recorded up to a maximum of 2 min. The platform was fixed in the center of one of the 4 quadrants and remained in that location for the duration of experiment. The time taken by a rat to reach the platform was recorded as initial acquisition latency. Following 24 h (day 5) after noting the initial acquisition latency, rats were randomly released individually at any one of the edges (North, South, East, or West) facing the wall of the pool and tested for the retention of the response. The time taken to reach the hidden platform on days 5 following initiation of 3-NP treatment were recorded and termed as retention latency. Percentage retention of memory was calculated from initial acquisition latency and retention latency.
Rota rod activity Motor coordination and grip strength was assessed by using rotarod apparatus. Animals were exposed to prior training session to acclimate them on rotarod before starts the actual assessing of drug treatments. Animals were placed on the rotating rod with a diameter of 7 cm (speed 25 rpm). The cut off time was 180 s. Three separate trials after 5 min gap were given to each rat. The average fall of time was recorded and expressed as count per 5 min [20].
Open field test (OFT) All rats were subjected to an OFT. Each rat was placed individually into the center of the open field apparatus. The open field apparatus was a circle made of wood, 90 cm in diameter. The test was performed between 09:00 and 12:00 h. A 60W light bulb (estimate in approximately 750 lumens and approximately 375.38 lux; lux = lumen/m2 ) was positioned 90–100 cm above the center, and provided the only source of illumination in the testing room. Each animal was placed in the center of the open field and the numbers of squares crossed were measured, through direct visual observation, for 5 min [21]. The floor was cleaned with a wet sponge and a dry paper towel between rats. International Journal of Neuroscience
Neurotoxicity
Forced swim test (FST) All rats were subjected to a FST. This study was carried out in according to the methods describe by Porsolt et al. [22]. After the OFT, the rats were placed individually in Plexiglas cylinders (height: 40 cm, diameter: 18 cm) containing 25 cm water, maintained at 23–25◦ C. Animals were removed from the water cylinder after 15 min and dried before they returned to their home cages. On the next day, they were replaced in the cylinders for 5 min, and the total duration of immobility was measured. A rat was judged to be immobile when it remained floating passively in the water.
Int J Neurosci Downloaded from informahealthcare.com by University of Sydney on 01/03/15 For personal use only.
Dissection and homogenization On day 15, animals were used for biochemical and mitochondrial complex enzymes estimations. The animals were sacrificed and the brain was removed by decapitation. Striatum was separated from each isolated brain. A 10% (W/V) tissue homogenate were prepared in 0.1 M phosphate buffer (pH 7.4). The homogenate were centrifuged at 10 000 × g at 4◦ C for 15 min. aliquots of supernatant were separated and used for biochemical estimations.
Isolation of rat brain mitochondria The brain regions were homogenized in isolated buffer. Homogenates were centrifuged at 13 000 × g for 5 min
at 4◦ C. Pellets were resuspended in isolation buffer with ethylene glycol tetra acetic acid (EGTA) and spun again at 13 000 × g at 4◦ C for 5 min. The resulting supernatants were transferred to new tubes and topped off with isolation buffer with EGTA and again spun at 13 000 × g at 4◦ C for 10 min. Pellets containing pure mitochondria were resuspended in isolation buffer without EGTA.
Biochemical analysis The protein content was measured by the method of Lowry et al. [23]. Lipid peroxidation products were estimated by the method of Okhawa et al. [24]. Reduced GSH was determined by the method of Moron et al. [25]. The superoxide dismutase (SOD) was assayed according to the method of Marklund and Marklund [26]. Catalase (CAT) activity was measured by Sinha et al. [27]. The mitochondrial enzyme complex II, SDH was determined by the method of Slaters and Bonners (1952) [28].
Histopathological studies On day 15, the animals were sacrificed by cervical decapitation after behavioral assessments and the striatum was removed and fixed in 10% formalin saline for 24 h. Washing was done in tap water and serial dilutions of (alcohol, ethyl, and absolute ethyl) were used for
Figure 1. Effect of L-theanine on body weight in 3 NP induced rats. Data presented are the mean ± SD (n = 6). a p
RESEARCH ARTICLE
Neuroprotective activity of L-theanine on 3-nitropropionic acid-induced neurotoxicity in rat striatum Sumathi Thangarajan, Asha Deivasigamani, Suganya Sarumani Natarajan, Prasanna Krishnan, and Sandhya Koombankallil Mohanan
Int J Neurosci Downloaded from informahealthcare.com by University of Sydney on 01/03/15 For personal use only.
Department of Medical Biochemistry, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai, Tamil Nadu, India The present study has been designed to investigate the protective effect of L-theanine against 3-nitropropionic acid (3-NP)-induced Huntington’s disease (HD)-like symptoms in rats. The present experimental protocol design includes systemic 3-NP acid (10 mg/kg intraperitonially) treatment for 14 d. L-theanine (100 and 200 mg/kg) was given orally, once a day, 1 h before 3-NP acid treatment for 14 d. Body weight and behavioral parameters (Morris water maze, open field test (OFT), forced swim test (FST) and rotarod activity) were assessed on 1st, 5th, 10th and 15th day post-3-NP acid administration. Malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) levels and mitochondrial enzyme complex. Succinate dehydrogenase (SDH) were measured on the 15th day in the striatum. Systemic 3-NP acid treatment significantly reduced body weight, locomotor activity and oxidative defense. The mitochondrial enzyme activity was also significantly impaired in the striatum region in 3-NP acid-treated animals. L-theanine (100 and 200 mg/kg b.wt.) treatment significantly attenuated the impairment in behavioral, biochemical and mitochondrial enzyme activities as compared to the 3-NP acid-treated group. The results of the present study suggest that pretreatment with L-theanine significantly attenuated 3-NP induced oxidative stress and restored the decreased SOD, GSH, CAT and SDH activity. It also decreased the neuronal damage as evidenced by histopathological analysis of striatum. Based on the above study, it has been proved that L-theanine has neuroprotective activity against 3-NP induced neurotoxicity. KEYWORDS: Huntington’s disease, oxidative stress, mitochondrial complex II, gliosis
Introduction Huntington’s disease (HD) is an incurable neuropsychiatric disease characterized by degeneration of basal ganglia, motor abnormalities, impaired cognitive function and emotional disturbance [1]. 3-NP acid (3-NP) is a mitochondrial toxin that causes striatal neuropathy similar to that seen in clinical HD. It has been demonstrated that systemic administration of 3-NP produced selective striatal lesions (preferentially in medium-sized spiny neurons) that begin in the dorsolateral quadrant of the striatum and later spread to the entire lateral striatum and behavioral, biochemical, histological and neu-
Received 29 October 2013; revised 3 December 2013; accepted 3 December 2013 Correspondence: Dr Sumathi Thangarajan, Department of Medical Biochemistry, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai 600113, Tamil Nadu, India. Tel: +91-44-24547086, Fax: +91-44-24540709. E-mail: [email protected]
rochemical features of HD [2,3]. 3-NP intoxication inhibits SDH, a key enzyme in the respiratory chain. Thus it leads to depletion of ATP, energy failure results altered calcium homeostasis, oxidative stress, excitotoxic events and neuronal death [4–6]. A major advantage of 3-NP model over other model of HD is that the lesions produced are bilateral, striatal specific and develop spontaneously after systemic administration of 3-NP. L-Theanine, γ -glutamylethylamide, is one of the major amino acid components in green tea and is synthesized from ethylamine and glutamate in green tea leaves [7]. It has been reported that theanine has an impact on brain function [8–11]. Studies have also documented the beneficial effect of antioxidants possibly by free radical scavenging activity and increased endogenous antioxidant defense against 3-NP induced neurotoxicity in rats [12–14]. L-Theanine can counteract excitotoxicity and/or mitochondrial radical formation [10,11]. Ltheanine is involved in the formation of the inhibitory neurotransmitter, gamma amino butyric acid (GABA). GABA influences the levels of 673
674
S. Thangarajan et al.
Int J Neurosci Downloaded from informahealthcare.com by University of Sydney on 01/03/15 For personal use only.
two other neurotransmitters, dopamine and serotonin, producing the key relaxation effect [15,16]. The administration of theanine into the lateral ventricle prevented ischemia induced neuronal death in the hippocampal CA1 region in a dose-dependent manner. Moreover, some reports suggested that the neuroprotective effect of theanine occurs via glutamate receptors, with theanine acting as a glutamate receptor antagonist [17,18]. Protective effects of many synthetic compounds and plant extracts have been reported on 3-NP induced toxicity. We have reported for the first time the neuroprotective activity of L-theanine against 3-NP induced neurotoxicity in rat striatum.
Materials and methods Animals Male Wistar rats weighing between 200 and 250 g bred in Central Animal House, Dr. ALMPGIBMS, University of Madras, Taramani campus, Chennai 113, Tamil Nadu, India were used. The animals were housed under standard laboratory conditions and maintained on natural light and dark cycle and had free access to food and water. Animals were acclimatized to laboratory conditions before the experiment. The experimental protocols were approved by the Institutional Animal Ethics Committee (IAEC) Dr. ALMPGIBMS, University of Madras, Taramani campus, Chennai 113, Tamil Nadu, India.
Drugs and treatment schedule The following drugs were used in the present study. 3NP (sigma chemicals) was dissolved in saline (adjusted to pH 7.4) and administered intraperitonially in a volume of 0.5 mL/100 g animal body weight. L-theanine (SRL chemicals) was dissolved in distilled water and administered orally. Rats were divided into six groups of six animals each. Group I: control; Group II: 3-NP (10 mg/kg, b.wt.) intraperitonially for 14 d; Group III: L-theanine (100 mg/kg b.wt.) orally prior to the administration of 3-NP for 14 d; Group IV: L-theanine (200 mg/kg b.wt.) orally prior to the administration of 3-NP for 14 d; Group V: L-theanine (100 mg/kg b.wt.) alone orally for 14 d: group VI: L-theanine (200 mg/kg b.wt.) alone orally for 14 d.
Measurement on body weight Body weight was recorded on the first and last day of the experiment. Percent change in body weight was calculated as body weight (1st day − 15th day) × 100. body weight (1st day)
[1]
Behavioral assessments Morris water maze test The acquisition and retention of a spatial navigation task was examined using a Morris Water Maze [19]. Animals were trained to swim to a platform in a circular pool (180 cm diameter × 60 cm) located in a test room. The pool is filled with water (28 ± 2◦ C) to a depth of 40 cm. A movable circular platform 9 cm in diameter, mounted on a column, was placed in the pool 1 cm above the water level for the test. On the 4th day prior to 3-NP administration, animals received a training session consisting of 4 trials. In all 4 trials, the starting positions were different. The latency to find the escape platform was recorded up to a maximum of 2 min. The platform was fixed in the center of one of the 4 quadrants and remained in that location for the duration of experiment. The time taken by a rat to reach the platform was recorded as initial acquisition latency. Following 24 h (day 5) after noting the initial acquisition latency, rats were randomly released individually at any one of the edges (North, South, East, or West) facing the wall of the pool and tested for the retention of the response. The time taken to reach the hidden platform on days 5 following initiation of 3-NP treatment were recorded and termed as retention latency. Percentage retention of memory was calculated from initial acquisition latency and retention latency.
Rota rod activity Motor coordination and grip strength was assessed by using rotarod apparatus. Animals were exposed to prior training session to acclimate them on rotarod before starts the actual assessing of drug treatments. Animals were placed on the rotating rod with a diameter of 7 cm (speed 25 rpm). The cut off time was 180 s. Three separate trials after 5 min gap were given to each rat. The average fall of time was recorded and expressed as count per 5 min [20].
Open field test (OFT) All rats were subjected to an OFT. Each rat was placed individually into the center of the open field apparatus. The open field apparatus was a circle made of wood, 90 cm in diameter. The test was performed between 09:00 and 12:00 h. A 60W light bulb (estimate in approximately 750 lumens and approximately 375.38 lux; lux = lumen/m2 ) was positioned 90–100 cm above the center, and provided the only source of illumination in the testing room. Each animal was placed in the center of the open field and the numbers of squares crossed were measured, through direct visual observation, for 5 min [21]. The floor was cleaned with a wet sponge and a dry paper towel between rats. International Journal of Neuroscience
Neurotoxicity
Forced swim test (FST) All rats were subjected to a FST. This study was carried out in according to the methods describe by Porsolt et al. [22]. After the OFT, the rats were placed individually in Plexiglas cylinders (height: 40 cm, diameter: 18 cm) containing 25 cm water, maintained at 23–25◦ C. Animals were removed from the water cylinder after 15 min and dried before they returned to their home cages. On the next day, they were replaced in the cylinders for 5 min, and the total duration of immobility was measured. A rat was judged to be immobile when it remained floating passively in the water.
Int J Neurosci Downloaded from informahealthcare.com by University of Sydney on 01/03/15 For personal use only.
Dissection and homogenization On day 15, animals were used for biochemical and mitochondrial complex enzymes estimations. The animals were sacrificed and the brain was removed by decapitation. Striatum was separated from each isolated brain. A 10% (W/V) tissue homogenate were prepared in 0.1 M phosphate buffer (pH 7.4). The homogenate were centrifuged at 10 000 × g at 4◦ C for 15 min. aliquots of supernatant were separated and used for biochemical estimations.
Isolation of rat brain mitochondria The brain regions were homogenized in isolated buffer. Homogenates were centrifuged at 13 000 × g for 5 min
at 4◦ C. Pellets were resuspended in isolation buffer with ethylene glycol tetra acetic acid (EGTA) and spun again at 13 000 × g at 4◦ C for 5 min. The resulting supernatants were transferred to new tubes and topped off with isolation buffer with EGTA and again spun at 13 000 × g at 4◦ C for 10 min. Pellets containing pure mitochondria were resuspended in isolation buffer without EGTA.
Biochemical analysis The protein content was measured by the method of Lowry et al. [23]. Lipid peroxidation products were estimated by the method of Okhawa et al. [24]. Reduced GSH was determined by the method of Moron et al. [25]. The superoxide dismutase (SOD) was assayed according to the method of Marklund and Marklund [26]. Catalase (CAT) activity was measured by Sinha et al. [27]. The mitochondrial enzyme complex II, SDH was determined by the method of Slaters and Bonners (1952) [28].
Histopathological studies On day 15, the animals were sacrificed by cervical decapitation after behavioral assessments and the striatum was removed and fixed in 10% formalin saline for 24 h. Washing was done in tap water and serial dilutions of (alcohol, ethyl, and absolute ethyl) were used for
Figure 1. Effect of L-theanine on body weight in 3 NP induced rats. Data presented are the mean ± SD (n = 6). a p
