Biol Trace Elem Res DOI 10.1007/s12011-015-0229-3
Protective Effect of Selenium Against Aluminum Chloride-Induced Alzheimer’s Disease: Behavioral and Biochemical Alterations in Rats B. V. S. Lakshmi & M. Sudhakar & K. Surya Prakash
Received: 23 November 2014 / Accepted: 5 January 2015 # Springer Science+Business Media New York 2015
Abstract In present study, selenium was selected for evaluating effect of selenium on aluminum chloride (AlCl3)-induced Alzheimer’s disease in rats. Thirty Wistar rats were divided into five groups of six in each. Group I (control) received distilled water, group II—AlCl3 (100 mg/kg, p.o.), group III—selenium (1 mg/kg, p.o.), group IV—AlCl3 +vitamin E (100 mg/kg, p.o.+100 mg/kg, p.o.), and group V— AlCl3 +selenium (100 mg/kg, p.o.+1 mg/kg, p.o.) for 21 days. At end of experiment, various behavioral, biochemical, and histopathological assessments were carried out. The animals showed increase in time to reach platform in Morris water maze and decreased step-down latencies in passive avoidance test indicating learning and memory impairment in aluminum chloride-treated group, but administration of selenium decreased time to reach platform in Morris water maze, increased step-down latencies, and strengthened its memory action in drug-treated animals. There was decrease in muscle strength measured by rotarod test indicating motor incoordination and decrease in locomotor activity assessed by actophotometer test in AlCl3 control group, whereas in selenium–AlCl3 group, there was improvement in muscle strength and locomotion. Biochemical analysis of the brain revealed that chronic administration of AlCl3 significantly increased lipid peroxidation and decreased levels of acetyl cholinesterase, catalase, reduced glutathione and glutathione reductase, an index of oxidative stress process. Administration of selenium attenuated lipid peroxidation and ameliorated the biochemical changes. There were marked changes at subcellular level observed by histopathology studies in AlCl3 group, and better improvement in these changes was observed in B. V. S. Lakshmi (*) : M. Sudhakar : K. S. Prakash Department of Pharmacology, Malla Reddy College of Pharmacy, Dhulapally (via Hakimpet), Maisammaguda, Secunderabad, Andhra Pradesh 500014, India e-mail: [email protected]
selenium+AlCl3group. Therefore, this study strengthens the hypothesis that selenium helps to combat oxidative stress produced by accumulation of AlCl3 in the brain and helps in prophylaxis of Alzheimer’s diseases. Keywords Alzheimer’s disease . Acetyl cholinesterase . Morris water maze . Locomotor activity . Passive avoidance test Abbreviations kg kilogram mg Milligram ml Milliliter p.o Per oral AD Alzheimer’s disease SDL Step-down latency PMSF Phenyl methyl sulfonylfluoride AChE Acetyl cholinesterase EDTA Ethylene diamine-tetra-acetic acid Alcl3 Aluminum chloride DTNB Dithiobis nitrobenzoic acid Se Selenium
Introduction Alzheimer’s disease (AD), which represents one of the most financially draining diseases to society, is a neurodegenerative disorder characterized by progressive degeneration of the hippocampal and cortical neurons that leads to impairment of memory and cognitive ability. Impairment of short-term memory is usually the first clinical feature, whereas retrieval of distant memories is preserved relatively well into the course
Lakshmi et al.
of the disease. When the condition progresses, additional cognitive abilities are impaired, such as the ability to calculate and use common objects and tools. The pathological hallmarks of AD are senile plaques, which are spherical impairment of short-term memory is usually the first clinical feature, whereas retrieval of distant memories is preserved relatively well into the course of the disease. When the condition progresses, additional cognitive abilities are impaired, such as the ability to calculate and use common objects and tools. The pathological hallmarks of AD are senile plaques, which are spherical accumulations of b-amyloid protein accompanied by degenerating neuronal processes as well as neurofibrillary tangles composed of paired helical filaments and other proteins. This corresponds to the clinical features of marked impairment of memory and abstract reasoning, with preservation of vision and movement. Moreover, other hypotheses state that inflammation plays a key role in the pathogenesis of AD. In addition, excessive reactive oxygen species levels are implicated in the etiology of AD [1–3]. Aluminum, a well-established neurotoxicant, is reported to be involved in the etiology of Alzheimer’s disease (AD) due to its easy admittance and accumulation in central nervous system [4]. The aluminum that accumulates in aged neurons probably derives mainly from the diet, considering that ingestion of aluminum additives is the major route of aluminum exposure by the general public [5]. Aluminum compounds are added to many commercially prepared products destined for ingestion. Aluminum sulphate (alum) is also added to some water for bottling as drinking water and many urban drinking water supplies to clarify them [6]. Up to 1 % of ingested aluminum is absorbed into the bloodstream. From there, small amounts are available for uptake by various organs, including the brain. Several accelerator mass spectrometry studies have shown that measurable amounts of radioactive aluminum can enter the brains of rats given the equivalent amount of aluminum contained in a single glass of alum-treated drinking water [7]. Micronutrients are dietary minerals required by the human body in a very small quantity. They probably interact with xenobiotics at several sites like during absorption and excretion, transport of metals in the body, binding to target proteins, metabolism and sequestration of toxic metals, and oxidative stress [8]. Besides this, they may also serve as required prosthetic groups in active sites or as co-enzymes for indispensable metalloenzymes. Several studies showed that antioxidant nutrients protect cells against deleterious effects of environmental agents [9]. Selenium (SE) has received considerable attention as an essential micronutrient for both animal and human beings. It has been detected that Se functions in the active site of glutathione peroxidase. It is important in many biochemical and physiological processes including the biosynthesis of coenzyme Q, regulation of ion fluxes across membranes, maintenance of the integrity of keratins, and stimulation of
antibody synthesis [8]. The protective effects of Se seem to be primarily associated with its presence in the seleno-enzymes, which are known to protect DNA and other cellular components from oxidative damage [10]. As there was no study which has investigated the biochemical and molecular role of selenium against neurotoxicity induced by aluminuim chloride (AlCl3), this study will do so by exploring the behavioral, biochemical, and molecular changes induced by AlCl3 in the brain of rats. In addition, the antioxidant effects of selenium on neurotoxicity induced by AlCl3 will be investigated.
Materials and Methods Chemicals Aluminum chloride and selenium were procured from standard reagents, Hyderabad. All the chemicals used were of high analytical grade purchased from Sigma–Aldrich company (USA). Animals Thirty male Wistar albino rats weighing 180–250 g were obtained from Sigma labs, Bachupally, Hyderabad. The rats were housed in polypropylene cages and maintained under standard conditions (12-h light and dark cycles, at 25±3 °C and 35–60 % humidity). Standard pelletized feed and tap water were provided ad libitum. All the pharmacological experimental protocols were approved by the Institutional Animal Ethics Committee (Reg no: MRCP/CPCSEA/IAEC/2013–14/ MPCOL/12).
Experimental Design Thirty Wistar albino male rats of weight 180–250 g were selected for this study. Animals were divided into five groups of six animals each. Group 1 Group 2 Group 3 Group 4
Control group (received distilled water 1 ml) Aluminum chloride (100 mg/kg, p.o.) Selenium (1 mg/kg, p.o.) Aluminum chloride+vitamin E (100 mg/kg, p.o.+ 100 mg/kg, p.o.) Group 5 Aluminum chloride+selenium (100 mg/kg, p.o.+ 1 mg/kg, p.o.). All the groups were treated once daily for a period of 21 days [11]. The animals were weighed, and behavioral observations were recorded before and at the end of the
Anti-Alzheimer’s Activity of Selenium in AlCl3 Induced Oxidative Stress
experiment. After the administration of last dose after 24 h, they were sacrificed under light ether anesthesia. The organs were removed, cleaned, washed with phosphate buffer saline (pH 7.4), and used for various studies.
15 s. During the second test, animals were removed from shock free zone if they did not step down for a period of 60 s. Retention was tested after 24 h in a similar manner, except that the electric shocks were not applied to the grid floor observing an upper cutoff time of 300 s [13, 14].
Evaluation of Behavioral Parameters
Rotarod Test
Morris Water Maze
The effect of aluminum choride as well as selenium on muscle performance was evaluated using rotarod (Techno) test. All the rats were given two initial training trials of 300 s, approximately 10 min apart, to maintain posture on the rotarod (3 cm in diameter and rotating at a constant 20rev/min). After the initial training trials, a baseline trial of 120 s was conducted. The time each animal remained on the rotarod was recorded. The animals that did not fall off the rotarod were given a maximum score of 120 s [13].
The acquisition and retention of memory was evaluated using the Morris water maze. The Morris water maze consisted of a large circular pool (150 cm in diameter, 45 cm in height, filled to a depth of 30 cm with water at 28±1 °C) divided into four equal quadrants with the help of two threads fixed at right angles to each other. The pool was placed in an illuminated room with several colored light clues. These external clues are remained unchanged throughout the experimental period and used as the reference memory. A circular platform (4.5 cm diameter) was placed in one quadrant of the pool, 1 cm above the water level during the acquisition phase. The same platform was placed 1 cm below the water level for the retention phase. The position of the platform was not changed in any quadrant during assessment of both phases. Each animal was subjected to four consecutive trials with a gap of 5 min. The animal was gently placed in the water of the pool between quadrants facing the wall of the pool, with the drop location changed for each trial. The animal was then allowed 120 s to locate the platform. Next, the animal was allowed to stay on the platform for 20 s. If the animal failed to reach the platform within 120 s, it was guided to the platform and allowed to remain there for 20 s [12].
Locomotor Activity The spontaneous locomotor activity of each rat was recorded individually for 10 min using actophotometer. The locomotor activity (horizontal activity) can be easily measured using an actophotometer which operates on photoelectric cells which are connected in circuit with a counter. When the beam of light falling on the photo cell is cut off by the animal, a count is recorded [13].
Estimation of Biochemical Parameters
Passive Avoidance Test
Tissue Sample Preparation
The apparatus consisted of a box (27 cm×27 cm×27 cm) having three walls of wood and one wall of Plexiglass, featuring a grid floor (made up of 3 mm stainless steel rods set 8 mm apart), with a wooden platform (10 cm×7 cm×1.7 cm) in the center of the grid floor. The box was illuminated with a 15-W bulb during the experimental period. Electric shock (20 V, A.C.) was delivered to the grid floor. Training was carried out in two similar sessions. Each mouse was gently placed on the wooden platform set in the center of the grid floor. When the mouse stepped down placing all its paws on the grid floor, shocks were delivered for 15 s and the step-down latency (SDL) was recorded. SDL was defined as the time taken by the rat to step down from the wooden platform to grid floor with all its paws on the grid floor. Animals showing SDL in the range of 2–15 s during the first test were used for the second session and the retention test. The second session was carried out 90 min after the first test. When the animals stepped down before 60 s, electric shocks were delivered for
Animals were sacrificed with light ether anesthesia, and the brain of each rat was removed and washed with ice-cold saline to remove blood and stored at −80 °C. Later, the brain was taken and minced into small pieces, and a total of 10 % homogenate was prepared using phosphate buffer (0.1 M, pH 7.4) containing 1 mmol ethylene diamine-tetra-acetic acid (EDTA), 0.25 M sucrose, 10 mM potassium chloride (KCL), and 1 mM phenyl methyl sulfonylfluoride (PMSF) with a homogenizer (REMI) fitted with a Teflon plunger, which was centrifuged at 800 rpm for 30 min at 4 °C to yield the supernatant. Later, the supernatant was used for the estimation of acetyl cholinesterase (AChE) and antioxidant parameters (MDA, GSH, CAT, and glutathione reductase). LPO was estimated colorimetrically by measuring malondialdehyde (MDA) formation as described by Nwanjo and Ojiako, 2005 [15]. Catalase (CAT) activity was estimated following the method of Aebi, 1993 [16]. Reduced glutathione (GSH) was determined by the method of Ellman, 1959 [17]. The Se-
Lakshmi et al. Table 1
Effect of selenium on Morris water maze in AlCl3-induced Alzheimer’s disease in rats
Groups
Quadrant 1 (latency in s)
Quadrant 2 (latency in s)
Quadrant 3 (latency in s)
Quadrant 4 (latency in s)
Control AlCl3 Selenium Vit E Se+AlCl3
17.00±1.817 44.00±5.762# 19.40±1.778 23.00±2.449* 25.00±4.000**
24.40±3.124 38.60±4.366# 20.60±2.462 22.80±2.853* 19.00±1.414**
23.40±2.227 35.80±5.669# 20.40±1.720 20.00±3.464** 23.00±2.950**
25.60±3.919 38.20±6.045# 19.60±4.118 20.80±1.855* 24.60±5.662**
Values are expressed as mean±SEM, n=6. The intergroup variation between various groups was conducted by Prism 6.0 software using one-way ANOVA followed by Dunnett’s t test #
P
Protective Effect of Selenium Against Aluminum Chloride-Induced Alzheimer’s Disease: Behavioral and Biochemical Alterations in Rats B. V. S. Lakshmi & M. Sudhakar & K. Surya Prakash
Received: 23 November 2014 / Accepted: 5 January 2015 # Springer Science+Business Media New York 2015
Abstract In present study, selenium was selected for evaluating effect of selenium on aluminum chloride (AlCl3)-induced Alzheimer’s disease in rats. Thirty Wistar rats were divided into five groups of six in each. Group I (control) received distilled water, group II—AlCl3 (100 mg/kg, p.o.), group III—selenium (1 mg/kg, p.o.), group IV—AlCl3 +vitamin E (100 mg/kg, p.o.+100 mg/kg, p.o.), and group V— AlCl3 +selenium (100 mg/kg, p.o.+1 mg/kg, p.o.) for 21 days. At end of experiment, various behavioral, biochemical, and histopathological assessments were carried out. The animals showed increase in time to reach platform in Morris water maze and decreased step-down latencies in passive avoidance test indicating learning and memory impairment in aluminum chloride-treated group, but administration of selenium decreased time to reach platform in Morris water maze, increased step-down latencies, and strengthened its memory action in drug-treated animals. There was decrease in muscle strength measured by rotarod test indicating motor incoordination and decrease in locomotor activity assessed by actophotometer test in AlCl3 control group, whereas in selenium–AlCl3 group, there was improvement in muscle strength and locomotion. Biochemical analysis of the brain revealed that chronic administration of AlCl3 significantly increased lipid peroxidation and decreased levels of acetyl cholinesterase, catalase, reduced glutathione and glutathione reductase, an index of oxidative stress process. Administration of selenium attenuated lipid peroxidation and ameliorated the biochemical changes. There were marked changes at subcellular level observed by histopathology studies in AlCl3 group, and better improvement in these changes was observed in B. V. S. Lakshmi (*) : M. Sudhakar : K. S. Prakash Department of Pharmacology, Malla Reddy College of Pharmacy, Dhulapally (via Hakimpet), Maisammaguda, Secunderabad, Andhra Pradesh 500014, India e-mail: [email protected]
selenium+AlCl3group. Therefore, this study strengthens the hypothesis that selenium helps to combat oxidative stress produced by accumulation of AlCl3 in the brain and helps in prophylaxis of Alzheimer’s diseases. Keywords Alzheimer’s disease . Acetyl cholinesterase . Morris water maze . Locomotor activity . Passive avoidance test Abbreviations kg kilogram mg Milligram ml Milliliter p.o Per oral AD Alzheimer’s disease SDL Step-down latency PMSF Phenyl methyl sulfonylfluoride AChE Acetyl cholinesterase EDTA Ethylene diamine-tetra-acetic acid Alcl3 Aluminum chloride DTNB Dithiobis nitrobenzoic acid Se Selenium
Introduction Alzheimer’s disease (AD), which represents one of the most financially draining diseases to society, is a neurodegenerative disorder characterized by progressive degeneration of the hippocampal and cortical neurons that leads to impairment of memory and cognitive ability. Impairment of short-term memory is usually the first clinical feature, whereas retrieval of distant memories is preserved relatively well into the course
Lakshmi et al.
of the disease. When the condition progresses, additional cognitive abilities are impaired, such as the ability to calculate and use common objects and tools. The pathological hallmarks of AD are senile plaques, which are spherical impairment of short-term memory is usually the first clinical feature, whereas retrieval of distant memories is preserved relatively well into the course of the disease. When the condition progresses, additional cognitive abilities are impaired, such as the ability to calculate and use common objects and tools. The pathological hallmarks of AD are senile plaques, which are spherical accumulations of b-amyloid protein accompanied by degenerating neuronal processes as well as neurofibrillary tangles composed of paired helical filaments and other proteins. This corresponds to the clinical features of marked impairment of memory and abstract reasoning, with preservation of vision and movement. Moreover, other hypotheses state that inflammation plays a key role in the pathogenesis of AD. In addition, excessive reactive oxygen species levels are implicated in the etiology of AD [1–3]. Aluminum, a well-established neurotoxicant, is reported to be involved in the etiology of Alzheimer’s disease (AD) due to its easy admittance and accumulation in central nervous system [4]. The aluminum that accumulates in aged neurons probably derives mainly from the diet, considering that ingestion of aluminum additives is the major route of aluminum exposure by the general public [5]. Aluminum compounds are added to many commercially prepared products destined for ingestion. Aluminum sulphate (alum) is also added to some water for bottling as drinking water and many urban drinking water supplies to clarify them [6]. Up to 1 % of ingested aluminum is absorbed into the bloodstream. From there, small amounts are available for uptake by various organs, including the brain. Several accelerator mass spectrometry studies have shown that measurable amounts of radioactive aluminum can enter the brains of rats given the equivalent amount of aluminum contained in a single glass of alum-treated drinking water [7]. Micronutrients are dietary minerals required by the human body in a very small quantity. They probably interact with xenobiotics at several sites like during absorption and excretion, transport of metals in the body, binding to target proteins, metabolism and sequestration of toxic metals, and oxidative stress [8]. Besides this, they may also serve as required prosthetic groups in active sites or as co-enzymes for indispensable metalloenzymes. Several studies showed that antioxidant nutrients protect cells against deleterious effects of environmental agents [9]. Selenium (SE) has received considerable attention as an essential micronutrient for both animal and human beings. It has been detected that Se functions in the active site of glutathione peroxidase. It is important in many biochemical and physiological processes including the biosynthesis of coenzyme Q, regulation of ion fluxes across membranes, maintenance of the integrity of keratins, and stimulation of
antibody synthesis [8]. The protective effects of Se seem to be primarily associated with its presence in the seleno-enzymes, which are known to protect DNA and other cellular components from oxidative damage [10]. As there was no study which has investigated the biochemical and molecular role of selenium against neurotoxicity induced by aluminuim chloride (AlCl3), this study will do so by exploring the behavioral, biochemical, and molecular changes induced by AlCl3 in the brain of rats. In addition, the antioxidant effects of selenium on neurotoxicity induced by AlCl3 will be investigated.
Materials and Methods Chemicals Aluminum chloride and selenium were procured from standard reagents, Hyderabad. All the chemicals used were of high analytical grade purchased from Sigma–Aldrich company (USA). Animals Thirty male Wistar albino rats weighing 180–250 g were obtained from Sigma labs, Bachupally, Hyderabad. The rats were housed in polypropylene cages and maintained under standard conditions (12-h light and dark cycles, at 25±3 °C and 35–60 % humidity). Standard pelletized feed and tap water were provided ad libitum. All the pharmacological experimental protocols were approved by the Institutional Animal Ethics Committee (Reg no: MRCP/CPCSEA/IAEC/2013–14/ MPCOL/12).
Experimental Design Thirty Wistar albino male rats of weight 180–250 g were selected for this study. Animals were divided into five groups of six animals each. Group 1 Group 2 Group 3 Group 4
Control group (received distilled water 1 ml) Aluminum chloride (100 mg/kg, p.o.) Selenium (1 mg/kg, p.o.) Aluminum chloride+vitamin E (100 mg/kg, p.o.+ 100 mg/kg, p.o.) Group 5 Aluminum chloride+selenium (100 mg/kg, p.o.+ 1 mg/kg, p.o.). All the groups were treated once daily for a period of 21 days [11]. The animals were weighed, and behavioral observations were recorded before and at the end of the
Anti-Alzheimer’s Activity of Selenium in AlCl3 Induced Oxidative Stress
experiment. After the administration of last dose after 24 h, they were sacrificed under light ether anesthesia. The organs were removed, cleaned, washed with phosphate buffer saline (pH 7.4), and used for various studies.
15 s. During the second test, animals were removed from shock free zone if they did not step down for a period of 60 s. Retention was tested after 24 h in a similar manner, except that the electric shocks were not applied to the grid floor observing an upper cutoff time of 300 s [13, 14].
Evaluation of Behavioral Parameters
Rotarod Test
Morris Water Maze
The effect of aluminum choride as well as selenium on muscle performance was evaluated using rotarod (Techno) test. All the rats were given two initial training trials of 300 s, approximately 10 min apart, to maintain posture on the rotarod (3 cm in diameter and rotating at a constant 20rev/min). After the initial training trials, a baseline trial of 120 s was conducted. The time each animal remained on the rotarod was recorded. The animals that did not fall off the rotarod were given a maximum score of 120 s [13].
The acquisition and retention of memory was evaluated using the Morris water maze. The Morris water maze consisted of a large circular pool (150 cm in diameter, 45 cm in height, filled to a depth of 30 cm with water at 28±1 °C) divided into four equal quadrants with the help of two threads fixed at right angles to each other. The pool was placed in an illuminated room with several colored light clues. These external clues are remained unchanged throughout the experimental period and used as the reference memory. A circular platform (4.5 cm diameter) was placed in one quadrant of the pool, 1 cm above the water level during the acquisition phase. The same platform was placed 1 cm below the water level for the retention phase. The position of the platform was not changed in any quadrant during assessment of both phases. Each animal was subjected to four consecutive trials with a gap of 5 min. The animal was gently placed in the water of the pool between quadrants facing the wall of the pool, with the drop location changed for each trial. The animal was then allowed 120 s to locate the platform. Next, the animal was allowed to stay on the platform for 20 s. If the animal failed to reach the platform within 120 s, it was guided to the platform and allowed to remain there for 20 s [12].
Locomotor Activity The spontaneous locomotor activity of each rat was recorded individually for 10 min using actophotometer. The locomotor activity (horizontal activity) can be easily measured using an actophotometer which operates on photoelectric cells which are connected in circuit with a counter. When the beam of light falling on the photo cell is cut off by the animal, a count is recorded [13].
Estimation of Biochemical Parameters
Passive Avoidance Test
Tissue Sample Preparation
The apparatus consisted of a box (27 cm×27 cm×27 cm) having three walls of wood and one wall of Plexiglass, featuring a grid floor (made up of 3 mm stainless steel rods set 8 mm apart), with a wooden platform (10 cm×7 cm×1.7 cm) in the center of the grid floor. The box was illuminated with a 15-W bulb during the experimental period. Electric shock (20 V, A.C.) was delivered to the grid floor. Training was carried out in two similar sessions. Each mouse was gently placed on the wooden platform set in the center of the grid floor. When the mouse stepped down placing all its paws on the grid floor, shocks were delivered for 15 s and the step-down latency (SDL) was recorded. SDL was defined as the time taken by the rat to step down from the wooden platform to grid floor with all its paws on the grid floor. Animals showing SDL in the range of 2–15 s during the first test were used for the second session and the retention test. The second session was carried out 90 min after the first test. When the animals stepped down before 60 s, electric shocks were delivered for
Animals were sacrificed with light ether anesthesia, and the brain of each rat was removed and washed with ice-cold saline to remove blood and stored at −80 °C. Later, the brain was taken and minced into small pieces, and a total of 10 % homogenate was prepared using phosphate buffer (0.1 M, pH 7.4) containing 1 mmol ethylene diamine-tetra-acetic acid (EDTA), 0.25 M sucrose, 10 mM potassium chloride (KCL), and 1 mM phenyl methyl sulfonylfluoride (PMSF) with a homogenizer (REMI) fitted with a Teflon plunger, which was centrifuged at 800 rpm for 30 min at 4 °C to yield the supernatant. Later, the supernatant was used for the estimation of acetyl cholinesterase (AChE) and antioxidant parameters (MDA, GSH, CAT, and glutathione reductase). LPO was estimated colorimetrically by measuring malondialdehyde (MDA) formation as described by Nwanjo and Ojiako, 2005 [15]. Catalase (CAT) activity was estimated following the method of Aebi, 1993 [16]. Reduced glutathione (GSH) was determined by the method of Ellman, 1959 [17]. The Se-
Lakshmi et al. Table 1
Effect of selenium on Morris water maze in AlCl3-induced Alzheimer’s disease in rats
Groups
Quadrant 1 (latency in s)
Quadrant 2 (latency in s)
Quadrant 3 (latency in s)
Quadrant 4 (latency in s)
Control AlCl3 Selenium Vit E Se+AlCl3
17.00±1.817 44.00±5.762# 19.40±1.778 23.00±2.449* 25.00±4.000**
24.40±3.124 38.60±4.366# 20.60±2.462 22.80±2.853* 19.00±1.414**
23.40±2.227 35.80±5.669# 20.40±1.720 20.00±3.464** 23.00±2.950**
25.60±3.919 38.20±6.045# 19.60±4.118 20.80±1.855* 24.60±5.662**
Values are expressed as mean±SEM, n=6. The intergroup variation between various groups was conducted by Prism 6.0 software using one-way ANOVA followed by Dunnett’s t test #
P
