J Basic Clin Physiol Pharmacol 2015; 26(1): 13–23
Atish Prakash*, Jaspreet Kaur Kalra and Anil Kumar
Neuroprotective effect of N-acetyl cysteine against streptozotocin-induced memory dysfunction and oxidative damage in rats Abstract Background: Growing evidences indicate that endogenous oxidants and antioxidant defense interact in a vicious cycle, which plays a critical role in the pathogenesis of cognitive dysfunction. In this study, we examined the effect of N-acetyl cysteine (NAC) against the intra cerebroventricular infusion of streptozotocin (ICV STZ)induced cognitive impairment and mitochondrial oxidative damage in rats. Methods: Male adult Wistar rats were injected with STZ (3 mg/kg) bilaterally through ICV. NAC (50 and 100 mg/ kg) was administered for 3 weeks post-surgery. The rats were sacrificed on the 21st day following the last behavioral test, and cytoplasmic fractions of the hippocampus and cortex were prepared for the quantification of acetylcholinesterase, oxidative stress parameter, mitochondrial enzymes, inflammatory mediators and caspase-3 activity. Results: ICV STZ resulted in poor retention of memory in Morris water maze. It also increased the mito-oxidative damage and tumor necrosis factor-α, interleukin 6 and caspase-3 levels in the hippocampus and cortex compared to sham animals. NAC significantly improved memory retention and attenuated oxidative damage parameters, inflammatory markers in STZ-treated rats. Conclusions: The results of the present study strongly indicate the effectiveness of NAC in preventing cognitive impairment as well as mito-oxidative stress and may be considered as a potential agent in the management of cognitive-related disorders.
*Corresponding author: Dr. Atish Prakash, PhD, Associate Professor, Pharmacology Division, ISF College of Pharmacy, Moga-142001, Punjab, India, Phone: +919815381443, E-mail: [email protected] Jaspreet Kaur Kalra: Pharmacology Division, ISF College of Pharmacy, Moga, Punjab, India Anil Kumar: Pharmacology Division, University Institute of Pharmaceutical Sciences, UGC Center of Advanced Study, Panjab University, Chandigarh, India
Keywords: memory dysfunction; mitochondria; N-acetyl cysteine; oxidative stress; streptozotocin. DOI 10.1515/jbcpp-2013-0150 Received October 19, 2013; accepted February 27, 2014; previously published online April 21, 2014
Introduction Cognitive dysfunction is known to significantly contribute to the pathology of different neurodegenerative disorders like Alzheimer’s disease (AD). Despite considerable progress in elucidating the molecular components of the brain lesions in numerous in vivo studies, the mechanism of neuronal degeneration of these disorders has been unclear. However, recent advances in molecular techniques have focused the attention on several pathogenic mechanisms. Oxidative damage is considered as one of the hallmarks of the memory deterioration process and is thought to be a primary event in this pathology [1]. Evidence of increased oxidative stress in experimental animal models has come from studies showing increased lipid peroxidation, increased carbonyl modification of proteins and increased oxidation of mitochondrial DNA [2–4]. Perhaps the most intriguing link between oxidative stress and mitochondrial dysfunction has been playing a pivotal role in neurodegenerative disorders. Recently, it has been suggested that oxidative stress and mitochondrial dysfunction are the major causative factors of cognitive impairment in the aging brain [5]. Many studies have shown that adenosine triphosphate (ATP) generations are significantly affected due to impairment of multiple mitochondrial enzymes in neurodegenerative disorders, suggesting impaired energy metabolism [6]. Supporting this hypothesis, apoptotic cell death has also been demonstrated in the pathology of this disease. The possible mechanism for initiating apoptosis could be the generation of free radicals leading to oxidative damage. The compound STZ is known to inhibit insulin receptor function, causing persistent oxidative stress, impairment in energy metabolism and activation of proapoptotic signaling pathways [7]. Numerous studies
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14 Prakash et al.: Effect of N-acetyl cysteine against streptozotocin-induced neurotoxicity have identified both an inflammatory response and oxidative stress as possible factors in the pathogenesis of cognitive-related disorders. Yet, the relative contribution of each process is not well understood. Intracerebroventricular streptozotocin (ICV STZ) in subdiabetogenic dose causes prolonged impairment of brain glucose and energy metabolism and progressive cognitive impairment in rat by inhibiting the synthesis of ATP and acetyl coenzyme A [8]. This ultimately results into cholinergic deficiency supported by reduced choline acetyltransferase activity in the hippocampus and an increased acetylcholinesterase (AChE) activity in the brain of ICV STZ rats [4]. It has also been demonstrated that ICV STZ activated the astrocytes, which further induced microglia activation and specific damage to myelinated tracts in the fornix through generation of oxidative stress [9]. Therefore, it may be concluded that antioxidant molecules should be beneficial for modulating reactive oxygen species (ROS) and may have a potential role in the management of different neurodegenerative disorders. N-Acetyl cysteine (NAC) is a precursor of glutathione that plays an essential role in oxidative damage. In particular, NAC is known to increase the intracellular stores of glutathione, thereby enhancing the endogenous antioxidant level. The pharmacological applications of NAC are mainly due to the chemical properties of the cysteinyl thiol group of its molecule and the ability of reduced thiol groups to scavenge ROS and reactive nitrogen species, leading to cellular protection against oxidative damage in vivo and in vitro [10]. NAC is a low-molecular-weight compound and is freely filterable with a free access to the blood-brain barrier and intracellular compartments [11]. Therefore, a large number of evidences have accumulated to link free radical generation with neuronal degeneration, which highlights the importance of anti-oxidants in the treatment of neurodegenerative disorders like AD. Despite the knowledge that NAC is a well-reported endogenous antioxidant, the exact molecular mechanism is still unclear. We wanted to investigate the downstream signaling pathways of NAC against ICV STZ-induced cognitive dysfunction and mito-oxidative damage in rats.
Materials and methods Animals Male Wistar rats (Central Animal House, Panjab University, Chandigarh) weighing 180–200 g at the start of the study were used. Animals were acclimatized to laboratory conditions at room temperature prior to experimentation. Following surgery, animals were kept under
standard conditions of a 12-h light/dark cycle with food and water ad libitum, in groups of two in plastic cages with soft bedding. All the experiments were carried out between 09:00 and 17:00 h. The protocol was approved by the Institutional Animal Ethics Committee and carried out in accordance with the Indian National Science Academy guidelines for the use and care of animals.
Surgery and intracerebroventricular administration of STZ Surgery was performed according to the previously described protocol [4]. All animals were anesthetized with thiopental sodium (45 mg/kg, intraperitoneally) and positioned in a stereotaxic apparatus. The head was positioned in a frame and a midline sagittal incision was made in the scalp. Two holes were drilled in the skull for the placement of the injection cannula on both sides over the lateral cerebral ventricle using the following coordinates as described by Paxinos and Watson: 0.8 mm posterior to bregma, 1.5 mm lateral to the sagittal suture, 3.6 mm beneath the cortical surface of the brain. The scalp was then closed with a suture and dental cement. Animals were given bilateral ICV injection of STZ (3 mg/kg) in two divided doses (1 day and 3 days) with the help of a Hamilton microsyringe through the cannula. STZ was prepared in artificial cerebrospinal fluid (ACSF) and a solution of 25 mg/mL was made. This solution was made freshly and just before injection. The injection of STZ (3 mg/kg) was repeated on day 3. ACSF (in mmol/L: 147 NaCl, 2.9 KCl, 1.6 MgCl2, 1.7 CaCl2 and 2.2 dextrose) was injected (10 μL) on the same days as in the STZ group. To promote diffusion, the microsyringe was left in place for 5 min following injection. Special care of the animals was taken during the postoperative period.
Drugs and treatment schedule STZ (Sigma Chemical Co., St. Louis, MO, USA) and NAC (Zydus Medica, Ahemdabad, India) solutions were made freshly at the beginning of each experiment. NAC was dissolved in 0.5% carboxymethyl cellulose (CMC) and administered in a dose of 0.5 mL/100 g body weight. STZ (3 mg/kg) was prepared in ACSF and delivered in two divided doses of a 10-μL injection volume administered intra cerebroventricularly (i.c.v.) on the first and third day. Animals were divided randomly based on their body weights into seven groups of eight animals each. Groups Group I Group II Group III Group IV Group V Group VI Group VII
Treatment Sham control+0.5% CMC (vehicle for NAC) ACSF (10 μL i.c.v.)+0.5% CMC (vehicle for NAC) STZ (3 mg/kg, 10 μL i.c.v.)+0.5% CMC (vehicle for NAC) NAC (50 mg/kg) per se NAC (100 mg/kg) per se STZ (3 mg/kg, 10 μL i.c.v.)+NAC (50 mg/kg) STZ (3 mg/kg, 10 μL i.c.v.)+NAC (100 mg/kg)
The doses of STZ and NAC were selected based on previous studies in our laboratory [4, 12] (Figure 1).
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Prakash et al.: Effect of N-acetyl cysteine against streptozotocin-induced neurotoxicity 15
Measurement of lipid peroxidation
NAC (50 mg/kg and 100 mg/kg) ICV STZ
Day 1 ICV STZ
Day 3 ICV STZ
AChE
Day 3 IAL
Day 14 1st IRL
Catpace -3
TNF-α, IL-6
Day 21 2nd Sacrifice IRL
Mitochondrial complex
Oxidative stress
Figure 1 Treatment schedule.
The extent of lipid peroxidation in the brain was determined quantitatively by performing the method as described by Wills [13]. The values were calculated using the molar extinction coefficient of chromophore (1.56 × 105 M–1 cm–1).
Estimation of nitrite
Assessment of cognitive performance Spatial navigation task: The acquisition and retention of a spatial navigation task was evaluated by using the Morris water maze [4]. Animals were trained to swim to a visible platform in a circular pool (180 cm in diameter and 60 cm in height) located in a test room. In principle, rats can escape from swimming by climbing onto the platform, and over time the rats apparently learn the spatial location of the platform from any starting position at the circumference of the pool. The pool was filled with water (28 ± 2°C) to a height of 40 cm. A movable circular platform (9-cm diameter) mounted on a column was placed in the pool 2 cm above the water level during the acquisition phase. A similar platform was placed in the pool 2 cm below the water level for the maze retention phase. During both the phases, the platform was placed in the center of one of the quadrants. Four equally spaced locations around the edge of the pool (N, S, E and W) were used as starting points and this divided the pool into four equal quadrants. 1. Maze acquisition phase (training). Animals received a training session consisting of four trials on day 13. In all four trials, the starting position was different. A trial began by releasing the animal into the maze facing toward the wall of the pool. The latency to find the escape platform was recorded to a maximum of 90 s. If the rat did not escape onto the platform within this time, it was guided to the platform and was allowed to remain there for 20 s. The time taken by the rat to reach the platform was taken as the initial acquisition latency (IAL). At the end of the trial, the rats were returned to their home cages and a 5-min gap was given between the subsequent trials. 2. Maze retention phase (testing for retention of the learned task). Following 24 h (day 14) and 8 days (day 21) after IAL, the rat was released randomly at one of the edges facing the wall of the pool and tested for retention of response. The time taken to find the hidden platform on day 14 and day 21 following central administration of STZ was recorded and termed as first retention latency (1st RL) and second retention latency (2nd RL), respectively.
The accumulation of nitrite in the supernatant, an indicator of the production of nitric oxide, was determined by a colorimetric assay with Griess reagent according to Green et al. [14]. The concentration of nitrite in the supernatant was determined from sodium nitrite standard curve.
Superoxide dismutase (SOD) activity SOD activity was assayed by the method of Kono [15].
Estimation of glutathione levels Reduced glutathione (GSH) in the hippocampus and cortex was estimated according to the method described by Ellman [16]. Results were calculated using the molar extinction coefficient of chromophore (1.36 × 104 M–1 cm–1) and expressed as percentage of control. Total glutathione analysis was done by the method of Zahler and Cleland [17]. Glutathione S-transferase (GST) was assayed by the method of Habig et al. [18]. GST catalyzes the formation of the glutathione conjugates of 1-chloro-2,4-dinitrobenzene (CDNB), which have maximum absorption at 340 nm and have an extinction coefficient of 9.6 mM-1 cm-1. Redox ratio (GSH/oxidized glutathione) was determined for all the groups by taking the ratio of GSH to oxidized glutathione.
Acetylcholinesterase (AChE) activity The AChE activity was assessed by Ellman method [19]. Results were expressed as micromoles of acetylthiocholine iodide hydrolyzed per minute per milligram protein.
Protein estimation The protein content was estimated by biuret method [20] using bovine serum albumin as a standard.
Biochemical tests
Mitochondrial complex estimation
Biochemical tests were conducted 24 h after the last behavioral test. The animals were sacrificed by decapitation. The brains were removed and rinsed with ice-cold isotonic saline and then kept on ice and the hippocampus and cortex areas were separated and weighed. Tissue homogenates [10% (w/v)] were prepared in ice-cold 0.1 mmol/L phosphate buffer (pH 7.4). The homogenates were centrifuged at 10,000 × g for 15 min, and the aliquots of the supernatant so formed were separated and used for the biochemical estimations.
Isolation of rat brain mitochondria: The whole brain (excluding the cerebellum) was used for mitochondria isolation by the method of Berman and Hastings [21].
NADH dehydrogenase activity (complex I) Complex I was measured spectrophotometrically by the method of King and Howard [22].
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16 Prakash et al.: Effect of N-acetyl cysteine against streptozotocin-induced neurotoxicity
Succinate dehydrogenase (SDH) activity (complex II) SDH was measured spectrophotometrically according to the method of King [23].
MTT (mitochondrial redox activity) assay (complex III) The method employed in the present study is based on in vitro studies to evaluate mitochondrial redox activity through the conversion of MTT tetrazolium salt to formazan crystals by mitochondrial respiratory chain reactions in isolated mitochondria by the method of Liu et al. [24].
Cytochrome oxidase activity (complex IV) Cytochrome oxidase activity was assayed in brain mitochondria according to the method of Sottocasa et al. [25].
Estimations of tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) The quantification of TNF-α and IL-6 was done with the help and instructions provided by R&D Systems (MN, USA) Quantikine rat TNF-α, IL-6 immunoassay kits.
Estimation of caspase-3 colorimetric assay The enzymatic reaction for caspase activity was carried out using an Imgenex (San Diego, CA, USA) caspase-3 colorimetric kit.
on day 13. STZ-treated rats significantly delayed acquisition latency to reach the visual platform compared to the sham group, indicating memory deficits. However, NAC (50 and 100 mg/kg) treatment did not produce any significant alteration in the IAL to reach the platform in the Morris water maze compared to STZ-injected rats on day 13 (Table 1). In retrieval trial, the mean retention latencies of STZ-injected rats were not significantly changed in the water maze on days 14 and 21 compared to the IAL on day 13. The results suggest that STZ caused significant cognitive impairment. However, chronic administration of NAC (50 and 100 mg/kg) significantly attenuated the delayed latencies in the 1st and 2nd RL compared to STZinjected rats on days 14 and 21, respectively, and improved the retention performance of the spatial navigation task (p
Atish Prakash*, Jaspreet Kaur Kalra and Anil Kumar
Neuroprotective effect of N-acetyl cysteine against streptozotocin-induced memory dysfunction and oxidative damage in rats Abstract Background: Growing evidences indicate that endogenous oxidants and antioxidant defense interact in a vicious cycle, which plays a critical role in the pathogenesis of cognitive dysfunction. In this study, we examined the effect of N-acetyl cysteine (NAC) against the intra cerebroventricular infusion of streptozotocin (ICV STZ)induced cognitive impairment and mitochondrial oxidative damage in rats. Methods: Male adult Wistar rats were injected with STZ (3 mg/kg) bilaterally through ICV. NAC (50 and 100 mg/ kg) was administered for 3 weeks post-surgery. The rats were sacrificed on the 21st day following the last behavioral test, and cytoplasmic fractions of the hippocampus and cortex were prepared for the quantification of acetylcholinesterase, oxidative stress parameter, mitochondrial enzymes, inflammatory mediators and caspase-3 activity. Results: ICV STZ resulted in poor retention of memory in Morris water maze. It also increased the mito-oxidative damage and tumor necrosis factor-α, interleukin 6 and caspase-3 levels in the hippocampus and cortex compared to sham animals. NAC significantly improved memory retention and attenuated oxidative damage parameters, inflammatory markers in STZ-treated rats. Conclusions: The results of the present study strongly indicate the effectiveness of NAC in preventing cognitive impairment as well as mito-oxidative stress and may be considered as a potential agent in the management of cognitive-related disorders.
*Corresponding author: Dr. Atish Prakash, PhD, Associate Professor, Pharmacology Division, ISF College of Pharmacy, Moga-142001, Punjab, India, Phone: +919815381443, E-mail: [email protected] Jaspreet Kaur Kalra: Pharmacology Division, ISF College of Pharmacy, Moga, Punjab, India Anil Kumar: Pharmacology Division, University Institute of Pharmaceutical Sciences, UGC Center of Advanced Study, Panjab University, Chandigarh, India
Keywords: memory dysfunction; mitochondria; N-acetyl cysteine; oxidative stress; streptozotocin. DOI 10.1515/jbcpp-2013-0150 Received October 19, 2013; accepted February 27, 2014; previously published online April 21, 2014
Introduction Cognitive dysfunction is known to significantly contribute to the pathology of different neurodegenerative disorders like Alzheimer’s disease (AD). Despite considerable progress in elucidating the molecular components of the brain lesions in numerous in vivo studies, the mechanism of neuronal degeneration of these disorders has been unclear. However, recent advances in molecular techniques have focused the attention on several pathogenic mechanisms. Oxidative damage is considered as one of the hallmarks of the memory deterioration process and is thought to be a primary event in this pathology [1]. Evidence of increased oxidative stress in experimental animal models has come from studies showing increased lipid peroxidation, increased carbonyl modification of proteins and increased oxidation of mitochondrial DNA [2–4]. Perhaps the most intriguing link between oxidative stress and mitochondrial dysfunction has been playing a pivotal role in neurodegenerative disorders. Recently, it has been suggested that oxidative stress and mitochondrial dysfunction are the major causative factors of cognitive impairment in the aging brain [5]. Many studies have shown that adenosine triphosphate (ATP) generations are significantly affected due to impairment of multiple mitochondrial enzymes in neurodegenerative disorders, suggesting impaired energy metabolism [6]. Supporting this hypothesis, apoptotic cell death has also been demonstrated in the pathology of this disease. The possible mechanism for initiating apoptosis could be the generation of free radicals leading to oxidative damage. The compound STZ is known to inhibit insulin receptor function, causing persistent oxidative stress, impairment in energy metabolism and activation of proapoptotic signaling pathways [7]. Numerous studies
Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 5/21/15 3:33 AM
14 Prakash et al.: Effect of N-acetyl cysteine against streptozotocin-induced neurotoxicity have identified both an inflammatory response and oxidative stress as possible factors in the pathogenesis of cognitive-related disorders. Yet, the relative contribution of each process is not well understood. Intracerebroventricular streptozotocin (ICV STZ) in subdiabetogenic dose causes prolonged impairment of brain glucose and energy metabolism and progressive cognitive impairment in rat by inhibiting the synthesis of ATP and acetyl coenzyme A [8]. This ultimately results into cholinergic deficiency supported by reduced choline acetyltransferase activity in the hippocampus and an increased acetylcholinesterase (AChE) activity in the brain of ICV STZ rats [4]. It has also been demonstrated that ICV STZ activated the astrocytes, which further induced microglia activation and specific damage to myelinated tracts in the fornix through generation of oxidative stress [9]. Therefore, it may be concluded that antioxidant molecules should be beneficial for modulating reactive oxygen species (ROS) and may have a potential role in the management of different neurodegenerative disorders. N-Acetyl cysteine (NAC) is a precursor of glutathione that plays an essential role in oxidative damage. In particular, NAC is known to increase the intracellular stores of glutathione, thereby enhancing the endogenous antioxidant level. The pharmacological applications of NAC are mainly due to the chemical properties of the cysteinyl thiol group of its molecule and the ability of reduced thiol groups to scavenge ROS and reactive nitrogen species, leading to cellular protection against oxidative damage in vivo and in vitro [10]. NAC is a low-molecular-weight compound and is freely filterable with a free access to the blood-brain barrier and intracellular compartments [11]. Therefore, a large number of evidences have accumulated to link free radical generation with neuronal degeneration, which highlights the importance of anti-oxidants in the treatment of neurodegenerative disorders like AD. Despite the knowledge that NAC is a well-reported endogenous antioxidant, the exact molecular mechanism is still unclear. We wanted to investigate the downstream signaling pathways of NAC against ICV STZ-induced cognitive dysfunction and mito-oxidative damage in rats.
Materials and methods Animals Male Wistar rats (Central Animal House, Panjab University, Chandigarh) weighing 180–200 g at the start of the study were used. Animals were acclimatized to laboratory conditions at room temperature prior to experimentation. Following surgery, animals were kept under
standard conditions of a 12-h light/dark cycle with food and water ad libitum, in groups of two in plastic cages with soft bedding. All the experiments were carried out between 09:00 and 17:00 h. The protocol was approved by the Institutional Animal Ethics Committee and carried out in accordance with the Indian National Science Academy guidelines for the use and care of animals.
Surgery and intracerebroventricular administration of STZ Surgery was performed according to the previously described protocol [4]. All animals were anesthetized with thiopental sodium (45 mg/kg, intraperitoneally) and positioned in a stereotaxic apparatus. The head was positioned in a frame and a midline sagittal incision was made in the scalp. Two holes were drilled in the skull for the placement of the injection cannula on both sides over the lateral cerebral ventricle using the following coordinates as described by Paxinos and Watson: 0.8 mm posterior to bregma, 1.5 mm lateral to the sagittal suture, 3.6 mm beneath the cortical surface of the brain. The scalp was then closed with a suture and dental cement. Animals were given bilateral ICV injection of STZ (3 mg/kg) in two divided doses (1 day and 3 days) with the help of a Hamilton microsyringe through the cannula. STZ was prepared in artificial cerebrospinal fluid (ACSF) and a solution of 25 mg/mL was made. This solution was made freshly and just before injection. The injection of STZ (3 mg/kg) was repeated on day 3. ACSF (in mmol/L: 147 NaCl, 2.9 KCl, 1.6 MgCl2, 1.7 CaCl2 and 2.2 dextrose) was injected (10 μL) on the same days as in the STZ group. To promote diffusion, the microsyringe was left in place for 5 min following injection. Special care of the animals was taken during the postoperative period.
Drugs and treatment schedule STZ (Sigma Chemical Co., St. Louis, MO, USA) and NAC (Zydus Medica, Ahemdabad, India) solutions were made freshly at the beginning of each experiment. NAC was dissolved in 0.5% carboxymethyl cellulose (CMC) and administered in a dose of 0.5 mL/100 g body weight. STZ (3 mg/kg) was prepared in ACSF and delivered in two divided doses of a 10-μL injection volume administered intra cerebroventricularly (i.c.v.) on the first and third day. Animals were divided randomly based on their body weights into seven groups of eight animals each. Groups Group I Group II Group III Group IV Group V Group VI Group VII
Treatment Sham control+0.5% CMC (vehicle for NAC) ACSF (10 μL i.c.v.)+0.5% CMC (vehicle for NAC) STZ (3 mg/kg, 10 μL i.c.v.)+0.5% CMC (vehicle for NAC) NAC (50 mg/kg) per se NAC (100 mg/kg) per se STZ (3 mg/kg, 10 μL i.c.v.)+NAC (50 mg/kg) STZ (3 mg/kg, 10 μL i.c.v.)+NAC (100 mg/kg)
The doses of STZ and NAC were selected based on previous studies in our laboratory [4, 12] (Figure 1).
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Prakash et al.: Effect of N-acetyl cysteine against streptozotocin-induced neurotoxicity 15
Measurement of lipid peroxidation
NAC (50 mg/kg and 100 mg/kg) ICV STZ
Day 1 ICV STZ
Day 3 ICV STZ
AChE
Day 3 IAL
Day 14 1st IRL
Catpace -3
TNF-α, IL-6
Day 21 2nd Sacrifice IRL
Mitochondrial complex
Oxidative stress
Figure 1 Treatment schedule.
The extent of lipid peroxidation in the brain was determined quantitatively by performing the method as described by Wills [13]. The values were calculated using the molar extinction coefficient of chromophore (1.56 × 105 M–1 cm–1).
Estimation of nitrite
Assessment of cognitive performance Spatial navigation task: The acquisition and retention of a spatial navigation task was evaluated by using the Morris water maze [4]. Animals were trained to swim to a visible platform in a circular pool (180 cm in diameter and 60 cm in height) located in a test room. In principle, rats can escape from swimming by climbing onto the platform, and over time the rats apparently learn the spatial location of the platform from any starting position at the circumference of the pool. The pool was filled with water (28 ± 2°C) to a height of 40 cm. A movable circular platform (9-cm diameter) mounted on a column was placed in the pool 2 cm above the water level during the acquisition phase. A similar platform was placed in the pool 2 cm below the water level for the maze retention phase. During both the phases, the platform was placed in the center of one of the quadrants. Four equally spaced locations around the edge of the pool (N, S, E and W) were used as starting points and this divided the pool into four equal quadrants. 1. Maze acquisition phase (training). Animals received a training session consisting of four trials on day 13. In all four trials, the starting position was different. A trial began by releasing the animal into the maze facing toward the wall of the pool. The latency to find the escape platform was recorded to a maximum of 90 s. If the rat did not escape onto the platform within this time, it was guided to the platform and was allowed to remain there for 20 s. The time taken by the rat to reach the platform was taken as the initial acquisition latency (IAL). At the end of the trial, the rats were returned to their home cages and a 5-min gap was given between the subsequent trials. 2. Maze retention phase (testing for retention of the learned task). Following 24 h (day 14) and 8 days (day 21) after IAL, the rat was released randomly at one of the edges facing the wall of the pool and tested for retention of response. The time taken to find the hidden platform on day 14 and day 21 following central administration of STZ was recorded and termed as first retention latency (1st RL) and second retention latency (2nd RL), respectively.
The accumulation of nitrite in the supernatant, an indicator of the production of nitric oxide, was determined by a colorimetric assay with Griess reagent according to Green et al. [14]. The concentration of nitrite in the supernatant was determined from sodium nitrite standard curve.
Superoxide dismutase (SOD) activity SOD activity was assayed by the method of Kono [15].
Estimation of glutathione levels Reduced glutathione (GSH) in the hippocampus and cortex was estimated according to the method described by Ellman [16]. Results were calculated using the molar extinction coefficient of chromophore (1.36 × 104 M–1 cm–1) and expressed as percentage of control. Total glutathione analysis was done by the method of Zahler and Cleland [17]. Glutathione S-transferase (GST) was assayed by the method of Habig et al. [18]. GST catalyzes the formation of the glutathione conjugates of 1-chloro-2,4-dinitrobenzene (CDNB), which have maximum absorption at 340 nm and have an extinction coefficient of 9.6 mM-1 cm-1. Redox ratio (GSH/oxidized glutathione) was determined for all the groups by taking the ratio of GSH to oxidized glutathione.
Acetylcholinesterase (AChE) activity The AChE activity was assessed by Ellman method [19]. Results were expressed as micromoles of acetylthiocholine iodide hydrolyzed per minute per milligram protein.
Protein estimation The protein content was estimated by biuret method [20] using bovine serum albumin as a standard.
Biochemical tests
Mitochondrial complex estimation
Biochemical tests were conducted 24 h after the last behavioral test. The animals were sacrificed by decapitation. The brains were removed and rinsed with ice-cold isotonic saline and then kept on ice and the hippocampus and cortex areas were separated and weighed. Tissue homogenates [10% (w/v)] were prepared in ice-cold 0.1 mmol/L phosphate buffer (pH 7.4). The homogenates were centrifuged at 10,000 × g for 15 min, and the aliquots of the supernatant so formed were separated and used for the biochemical estimations.
Isolation of rat brain mitochondria: The whole brain (excluding the cerebellum) was used for mitochondria isolation by the method of Berman and Hastings [21].
NADH dehydrogenase activity (complex I) Complex I was measured spectrophotometrically by the method of King and Howard [22].
Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 5/21/15 3:33 AM
16 Prakash et al.: Effect of N-acetyl cysteine against streptozotocin-induced neurotoxicity
Succinate dehydrogenase (SDH) activity (complex II) SDH was measured spectrophotometrically according to the method of King [23].
MTT (mitochondrial redox activity) assay (complex III) The method employed in the present study is based on in vitro studies to evaluate mitochondrial redox activity through the conversion of MTT tetrazolium salt to formazan crystals by mitochondrial respiratory chain reactions in isolated mitochondria by the method of Liu et al. [24].
Cytochrome oxidase activity (complex IV) Cytochrome oxidase activity was assayed in brain mitochondria according to the method of Sottocasa et al. [25].
Estimations of tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) The quantification of TNF-α and IL-6 was done with the help and instructions provided by R&D Systems (MN, USA) Quantikine rat TNF-α, IL-6 immunoassay kits.
Estimation of caspase-3 colorimetric assay The enzymatic reaction for caspase activity was carried out using an Imgenex (San Diego, CA, USA) caspase-3 colorimetric kit.
on day 13. STZ-treated rats significantly delayed acquisition latency to reach the visual platform compared to the sham group, indicating memory deficits. However, NAC (50 and 100 mg/kg) treatment did not produce any significant alteration in the IAL to reach the platform in the Morris water maze compared to STZ-injected rats on day 13 (Table 1). In retrieval trial, the mean retention latencies of STZ-injected rats were not significantly changed in the water maze on days 14 and 21 compared to the IAL on day 13. The results suggest that STZ caused significant cognitive impairment. However, chronic administration of NAC (50 and 100 mg/kg) significantly attenuated the delayed latencies in the 1st and 2nd RL compared to STZinjected rats on days 14 and 21, respectively, and improved the retention performance of the spatial navigation task (p
