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Abstract

The aim of this study was to explore the effects of subacute exposure to 1,2-dichloroethane (1,2-DCE) on mouse behavior and the related mechanisms focusing on alteration of oxidative stress and amino acid neurotransmitters in the brain. Mouse behavior was examined by open field test. Levels of nitric oxide (NO), malondialdehyde (MDA) and nonprotein sulfhydryl (NPSH) and activity of inducible nitric oxide synthase (iNOS) and superoxide dismutase (SOD) were determined by colorimetric method. Contents of glutamate (Glu), aspartate (Asp) and gamma-aminobutyric acid (GABA) were evaluated by high-performance liquid chromatography. Reduced locomotor and exploratory activities and increased anxiety were found in 0.45 and 0.9 g/m³ 1,2-DCE-treated mice. In contrast, increased excitability was found in 0.225 g/m³ 1,2-DCE-treated mice. Compensatory antioxidant status and increased NOS activity and NO level in the brain were found in 1,2-DCE-treated mice. Moreover, Glu contents in 1,2-DCE-treated mice and GABA contents in 0.9 g/m³ 1,2-DCE-treated mice increased, whereas GABA contents in 0.225 g/m³ 1,2-DCE-treated mice decreased significantly compared with control. Taken together, our results suggested that mouse behavior could be disturbed by subacute exposure to 1,2-DCE, and the changes of amino acid neurotransmitter in the brain might be related to the behavioral effects.

Keywords

1,2-Dichloroethane poisoning behavioral effects amino acid neurotransmitter mice

Introduction

1,2-Dichloroethane (1,2-DCE) is a high production volume halogenated aliphatic hydrocarbon that is used mainly in the manufacture of vinyl chloride.¹ In China, it is also commonly utilized as an organic solvent, especially as the thinner of adhesives. Therefore, it is mainly inhaled through respiratory route in the workplace. It has been reported that 1,2-DCE is rapidly absorbed through the lungs. Following absorption, it can cross the blood–brain barrier easily. Thus, brain is the main target organ for 1,2-DCE in the body. It is clear that subacute exposure to 1,2-DCE can cause severe toxic encephalopathy.^2,3 Based on the case reports published in the past 20 years, 34 occupational accidents induced by subacute exposure to 1,2-DCE occurred in China, and 219 workers injured, among them 19 workers died. The main clinical manifestations were the symptoms of nervous system, such as dizziness, headache, vomiting and convulsions. The postmortem study of human brain showed typical brain hemorrhage and cerebral edema.^4,5 Therefore, subacute exposure to 1,2-DCE has become one of the serious occupational problems in China. However, until now few studies in relation to the mechanisms underlying 1,2-DCE-induced neurotoxicity were reported.⁶

The available animal data provide evidence that biotransformation of 1,2-DCE is mediated through an oxidative pathway, presumably via cytochrome P450-2E1, which results in chemically reactive 2-chloroacetaldehyde, 2-chloroethanol and 2-chloroacetic acid. 2-Chloroacetic acid and 2-chloroacetaldehyde can undergo glutathione (GSH) conjugation to yield S-carboxymethyl-GSH.^7

–10 The direct conjugation of 1,2-DCE with GSH is catalyzed by glutathione-S-transferase to yield S-(2-chloroethyl)-GSH.¹¹ It has been suggested that the CYP450-mediated oxidation of 1,2-DCE exhibits high affinity but low capacity, conversely the GSH metabolic pathway displays low affinity but high capacity.^12,13 Therefore, as levels of 1,2-DCE increase, oxidative metabolism likely becomes saturated, and GSH conjugation becomes the predominant metabolic pathway. Consequently, exposure to high doses of 1,2-DCE might deplete intracellular GSH and reduce the protective efficacies against oxidants.¹⁴ When intracellular antioxidant defense system is unable to counteract oxidants, uncontrolled oxidation might impair cell structures and functions. Therefore, oxidative damage might be involved in the pathological mechanisms of 1,2-DCE-induced toxic effects in the brain.

Nitric oxide (NO), an important messenger molecule in the brain, can modulate a wide range of nervous activities, such as synaptic plasticity, locomotor activity and learning and memory.^15
–17 NO is a small, highly diffusible and reactive molecule with a short lifetime. It is generated from arginine by the nitric oxide synthase (NOS). There are three subtypes of NOS, namely endothelial, inducible and neuronal NOS (eNOS, iNOS and nNOS) in the brain. Activation of eNOS and nNOS commonly generates nanomolar levels of NO, whereas activation of iNOS can produce micromolar levels of NO.¹⁸ Excessive production of NO was thought to underlie the neurotoxic mechanisms through causing nitrosative stress. The primary reaction is to form peroxynitrite from NO and superoxide anion. The term of nitrosative stress describes the ability of the reactive nitrogen species to damage proteins and DNA.^19,20

Amino acids are the most abundant neurotransmitters in the brain, which are responsible for almost all the fast signal transduction among neurons, leaving predominantly modulatory roles for the other transmitters.^21,22 Amino acid neurotransmitters can be divided into inhibitory amino acids (IAAs) and excitatory amino acids (EAAs). Glutamate (Glu) and aspartate (Asp) are the major EAAs and are distributed in all regions of the brain. They are often found together at axon terminals and activate the postsynaptic cells.²³ Gamma-aminobutyric acid (GABA) is the one of major IAAs in the brain and depresses the activity of postsynaptic cells.²⁴ Changes in the ratio between the contents of EAAs and IAAs in the brain may seriously affect brain functions.²⁵ Findings from an animal experiment showed that contents of Asp and Glu in the brain of 1,2-DCE-intoxicated rats increased obviously; but contents of GABA in the brain did not show any changes.²⁶ Therefore, disturbed amino acid neurotransmitters appeared to be related with the development of 1,2-DCE-induced neurotoxic effects.

It is well known that behavior is the early and sensitive indicator when the central nervous system is affected by exogenous chemicals. Therefore, the aim of this study was to explore the effects of subacute exposure to 1,2-DCE on mouse behavior and the related mechanisms by focusing on the alteration of amino acid neurotransmitters and oxidative stress in the brain.

Materials and methods

Animals

Thirty-two female albino mice, weighing 25 ± 2 g, obtained from the animal laboratory of China Medical University were used in this study. Animal room was kept at a temperature of 20 ± 2°C with a 12-h light/dark cycle and a relative humidity of 50–60%. During the study, food and water were available to the animals, except during the inhalation exposure. Mice were housed five per cage in the sterilized plastic cages with wood shaving bedding. This study protocol has been approved by the Scientific Research Committee of China Medical University on Ethics in the Care and Use of Laboratory Animals and was carried out in accordance with the National Institutes of Health guidelines in a manner that minimized animal suffering and animal numbers.

Experimental procedures

After 1-week adaptation, the mice were divided randomly into four groups, comprising the control group and three 1,2-DCE-exposed groups. Mice were placed in the static exposure chamber with a capacity of 100 L, eight mice in each chamber. The exposure chamber was operated at a temperature of 22 ± 2°C. 1,2-DCE solution with the purity of more than 99% was weighed according to administered concentrations. It was placed on the filter paper in the plate suspended in the chamber and then evaporated by the fan in the chamber after sealing up. The administered concentrations of 1,2-DCE in the air were 0.225, 0.45 and 0.9 g/m³. They were calculated by the weight of 1,2-DCE divided by the volume of chamber, respectively. Mice in the exposed and control groups were kept in the chamber for 3.5 h/day for 10 days. Two hours after the last exposure, the open field tests were performed. Following the behavioral examination, the mice were killed by decapitation and the brains were removed immediately. They were kept in −40°C freezer until analysis.

Reagents and laboratory wares

All glasses and plastic wares were washed with detergent and nitric acid and rinsed with redistilled water. Doubly distilled water was used in this study. All reagents used are of analytical grade and were purchased from the Shanghai Chemical Co. (Shanghai, China). The standard solutions of Glu and Asp were the products of Bio Basic Inc., Markham, Ontario, Canada. The standard solutions of GABA were the products of Sigma-Aldrich Co., St Louis, Missouri, USA. Assay kits for detecting the activities of NOS and superoxide dismutase (SOD) and levels of malondialdehyde (MDA), nonprotein sulfhydryl (NPSH) and NO were purchased from Nanjing Jiancheng Bioengineering Co. Ltd (Nanjing, China).

Analysis process

Open field test

The open field arena generally consists of an empty and bright circular arena surrounded by walls to prevent animal from escaping. The apparatus used is a 170 cm diameter black-painted circular platform with 25 cm high surrounding walls and illuminated with a 40-W white light. The arena is divided into 45 equal area zones, among them, 24 zones along the side wall are named as peripheral zones, and the others are named as central zones. Mouse behavior was recorded and analyzed by ANY-maze video tracking system (Stoelting Co., Wood Dale, Illinois, USA). The test was started by placing a mouse in the center of the arena. Mouse behavior, including the number of line crossings, vertical activity, time in central zones and number of feces and urine, was recorded over 5 min.

MDA level

Lipid peroxides in the brain were determined by measuring the thiobarbiturate-reactive substances (TBA-RS) according to the method reported by Satoh.²⁷ Briefly, 10% brain homogenate prepared by ice-cold saline solution was mixed with 20% trichloroacetic acid and 0.67% thiobarbituric acid and then incubated at 100°C for 30 min. After cooling with tap water, the reaction product was extracted with butanol by vigorous shaking and centrifugation (3000 r/min, 10 min). The absorption of supernatants was monitored at 535 nm. 1,1,3,3-Tetraethoxypropane was used as the external standard, and the concentrations of TBA-RS were expressed as nanomoles of MDA per milligram of protein. The concentrations of total protein were determined using the Coomassie blue method with bovine serum albumin as standard.

Level of NPSH

Method was used as described by Ellman.²⁸ Briefly, 10% brain homogenate was mixed with 10% trichloroacetic acid to precipitate the proteins, thereafter, the mixtures were centrifuged at 15,000 r/min for 5 min, and the supernatant was collected and mixed with 0.04% 5,5′-dithiobis-2-nitrobenzoic acid. The absorbance of color formed was read at 412 nm. Results were expressed as milligram per gram of protein.

SOD activity

Method was followed as described by Elstner and Heupel.²⁹ Briefly, 1% brain homogenate was mixed with the solution of hydroxylamine, xanthine oxidase and hypoxanthine and incubated at 37°C, pH 8.2 for 30 min. Lastly, diazo dye-forming reagent was added, and the absorption was measured at 550 nm. One unit of SOD was defined as the amount of enzyme required to cause 50% inhibition of nitrite production per milliliter of assay solution. Results of enzyme activity were expressed as nitrite unit per gram of protein.

Activity of iNOS

The assay was based on the following chemical reactions. NOS catalyzed the formation of NO and l-citrulline from l-arginine and molecular oxygen and then NO reacted with a nucleophile to generate color compounds. Briefly, 10% brain homogenate was mixed with substrate buffer, reaction accelerator and color development reagent and then incubated at 37°C for 15 min. Lastly, they were mixed with the clearing reagent and stop solution. The absorbance of color formed was measured at 530 nm. NOS activity was calculated and expressed as units per milligram of protein. One unit of NOS activity was defined as the production of 1 nmol NO/s/mg of tissue protein. For measuring iNOS activity, an inhibitor was added before incubation according to the manufacturer's instructions.

NO level

As NO is unstable in vivo, it was quantified by determining its stable metabolites, that is nitrite and nitrate in the brain. Briefly, nitrate was first converted into nitrite by the nitrate reductase, and then nitrite was quantified using Griess reagent. The absorbance of color formed was read at 550 nm, and the results expressed as micromoles of nitrites per gram of protein.

Content of Glu, Asp and GABA

10% brain homogenate was centrifuged at 3500g for 20 min at 4°C and then the supernatant was centrifuged again at 10,000g for 20 min at 4°C. Lastly, the supernatant was collected for the following analysis by a high-performance liquid chromatography (HPLC) system, consisting of a solvent-delivery pump, an autosampler and a fluorescence detector (Waters Corporation, Milford, Massachusetts, USA).

Briefly, precolumn derivatization with o-phthaladehyde was used. Elution was carried out at room temperature with a C18 analytical column (Dalian Elite Analytical Instruments Co., Dalian, China) and a mobile phase of 0.1 M potassium acetate (pH 5.89) methanol at a flow rate of 1 mL/min. The initial methanol concentration was 20% (v/v) and then increased gradually to 47% at 2 min, to 53% at 9 min, to 100% at 12 min and kept at 100% for 5 min. Lastly, it was decreased gradually to 20% at 18 min and kept for 5 min. Fluorescence detector conditions were excitation 250 nm with detection at emission 410 nm.

Standard solutions of 0.3, 2.0, 3.0, 4.0, 5.0, 6.0, 8.0, 12.0 and 20.0 μmol/L of Glu, Asp and GABA were prepared from 1 mmol/L stock solution.

Statistical analysis

Data were expressed as mean ± SD or mean ± SEM and analyzed using SPSS for Windows, version 11.5 (SPSS Inc., Chicago, Illinois, USA). Significant difference was evaluated by one-way analysis of variance. Post hoc tests were analyzed using Student–Newman–Keuls test. Statistical significance was defined as p < 0.05.

Results

Data in Figure 1 showed the changes in mouse behavior induced by 1,2-DCE exposure. Compared with that in control, number of line crossings of mice in the exposed groups decreased in a dose-dependent manner and that in 0.45 and 0.9 g/m³ exposed groups decreased significantly. Vertical activity of mice in the exposed groups was more than that in control, however, only the difference between 0.225 g/m³-exposed group and control was significant. Time in central zones of mice in the exposed groups increased doses dependently, however, the difference between exposed groups and control failed to show any significance. The volume of feces and urine increased in mice of the exposed groups when compared with that in control, and that in the group exposed to 0.9 g/m³ the volume was significantly more than that in control.

Figure 1.

Comparison of line crossings (A), vertical activity (B), time in the central zones (C) and number of feces and urine (D) in mice among different groups. Notes: Data are expressed as mean ± SEM, n=8. * P < 0.05, ** P < 0.01, vs control.

Data in Table 1 shows the status of oxidative stress in the brain of mice exposed to 1,2-DCE. The MDA levels in the brain of mice exposed to 0.225 g/m³ and SOD activities in the brain of mice exposed to 0.9 g/m³ were significantly higher than those in control. Differences in NPSH levels in the brain of mice among all groups failed to show any significance.

Table 1.

Comparison of MDA and NPSH levels, and SOD activities in the brain among groups.

Groups	N	SOD (U/mg protein)	MDA (nmol/mg protein)	NPSH (μg/100 mg)
Control	8	48.02 ± 8.50	22.41 ± 5.70	15.68 ± 3.22
0.225 g/m³ 1,2-DCE group	8	55.92 ± 8.35	31.34 ± 7.14^a	17.01 ± 3.95
0.45 g/m³ 1,2-DCE group	8	51.44 ± 8.60	27.94 ± 4.77	17.44 ± 1.48
0.9 g/m³ 1,2-DCE group	8	59.01 ± 6.76^b	27.19 ± 5.46	17.05 ± 2.20

MDA: malondialdehyde; NPSH: nonprotein sulfhydryl; SOD: superoxide dismutase; 1,2-DCE: 1,2-dichloroethane.

^a p < 0.01 versus control.

^b p < 0.05 versus control.

Changes in NO levels and iNOS activities in the brain of mice exposed to 1,2-DCE are shown in Table 2. Activities of iNOS in the brain of mice from all exposed groups and the NO levels in the brain of mice exposed to 0.45 and 0.9 g/m³ were significantly higher than those in the control.

Table 2.

Comparison of NO levels and iNOS activities in the brain among different groups.

Groups	N	NO (μmol/g protein)	iNOS (U/mg protein)
Control	8	14.58 ± 2.71	0.65 ± 0.14
0.225 g/m³ 1,2-DCE group	8	17.65 ± 4.99	0.87 ± 0.20^a
0.45 g/m³ 1,2-DCE group	8	20.19 ± 4.30^a	0.88 ± 0.25^a
0.9 g/m³ 1,2-DCE group	8	19.78 ± 4.84^a	0.87 ± 0.14^a

NO: nitric oxide; iNOS: inducible nitric oxide synthase; 1,2-DCE: 1,2-dichloroethane.

^a p < 0.05 versus control.

Data in Table 3 describe the changes in amino acid neurotransmitters in the brain of mice exposed to 1,2-DCE. Glu contents in the brain of mice from all exposed groups were significantly higher than those in the control group. However, GABA contents in the brain of mice exposed to 0.225 g/m³ decreased, whereas in those exposed to 0.9 g/m³ increased significantly when compared with those in the control group. Although the Asp contents in the brain of mice from all exposed groups were higher than those in control, the mean differences between exposed groups and control were not significant.

Table 3.

Comparison of Asp, Glu and GABA contents in the brain among different groups.

Groups	N	Asp (μmol/g protein)	Glu (μmol/g protein)	GABA (μmol/g protein)
Control	8	23.85 ± 3.95	50.78 ± 5.15	12.83 ± 3.36
0.225 g/m³ 1,2-DCE group	8	25.01 ± 6.90	67.69 ± 9.89^a	8.08 ± 2.73^b
0.45 g/m³ 1,2-DCE group	8	28.59 ± 6.44	67.99 ± 6.23^a	16.40 ± 4.97
0.9 g/m³ 1,2-DCE group	8	26.15 ± 8.39	71.16 ± 5.96^a	19.87 ± 5.30^a

Glu: glutamate; ASP: aspartate; GABA: gamma-aminobutyric acid; 1,2-DCE: 1,2-dichloroethane.

^a p < 0.01 versus control.

^b p < 0.05 versus control.

Discussion

Open field test is designed to measure the behavioral responses such as locomotor activity, hyperactivity, anxiety-like and exploratory behaviors in rats or mice.^30

–33 It approaches the conflict between the innate fear that rodents have of the central area of a novel or brightly light open field versus their desire to explore new environments. The natural tendency of rodents is to prefer staying closed to the walls (thigmotaxis).³⁴ Number of line crossings and vertical activity can be used to reflect the exploratory behavior and excitability of animals.^35,36 Under normal circumstances, when mice are in a new environment, they will instinctively flee and look for an export. Therefore, the increased number of line crossings and vertical activity reflected an increased exploratory and excitability of mice. Time in central zones reflects the stress of animals in a new environment.^37,38 Mice in an open field will quickly leave the central zones and walk along the peripheral zones to avoid staying in open areas. The volume of feces and urine reflects the tension strength of animals.³⁹ Findings from this study indicated that exposure to 0.225 g/m³ 1,2-DCE could excite the central nervous system by enhancement of the exploratory behavior and general motor activities of mice, whereas exposure to 0.9 g/m³ 1,2-DCE could inhibit the central nervous system by depression of the exploratory behavior and general motor activities of mice. In addition, exposure to 0.9 g/m³ 1,2-DCE might increase tension strengths of mice. Study reported by Niu et al. suggested that when acutely exposed to 1,2-DCE, rats exposed to 2.5 g/m³ showed an increased motor activity; rats exposed to5.0 g/m³ showed irritability, grasping the nose and mouth and body shaking slightly; whereas rats exposed to10.0 g/m³ showed reduced motor activity, apathetic, shortness of breath and lateral position.⁴⁰ Therefore, our results were basically consistent with the findings from their study, suggesting that exposure to low dose of 1,2-DCE might cause behavioral excitement and exposure to high dose of 1,2-DCE might result in behavioral inhibition.

Glu, Asp and GABA are the most common and important neurotransmitters in the brain. As they play the key roles in synaptic transmission, abnormal changes in these neurotransmitters can seriously affect brain functions. Moreover, as Glu has the strong excitatory effect on neurons, it has been reported that in the case of traumatic brain injury, poisoning, ischemia and hypoxia, the excessive Glu might overstimulate the postsynaptic receptors and cause damage to the neuron, which is called as ‘excitotoxicity’.^41,42 Findings from this study disclosed that exposure to 1,2-DCE could consistently increase Glu contents in the brain, however, the changes in GABA contents were different according to the doses of 1,2-DCE. Exposure to low dose of 1,2-DCE might reduce GABA contents in the brain, whereas exposure to high dose of 1,2-DCE might increase GABA contents in the brain, leading to increased ratio of Glu to GABA in the brain of mice exposed to low dose of 1,2-DCE. Results reported by Guo et al. showed that when acutely exposed to 1,2-DCE, contents of Asp and Glu consistently increased in a time and dose-dependent manner, however contents of GABA did not show any evident changes.²⁶ Up to now, results of these studies suggested that acute and subacute exposure to 1,2-DCE might increase Glu contents in the brain, but the changes in GABA contents in the brain might be different due to the acute or subacute exposure and doses of 1,2-DCE. Although the mechanism underlying decreased GABA contents in the brain of mice exposed to low dose of 1,2-DCE is unclear, as GABA is produced by glutamic acid decarboxylase from Glu, it was reasonable to speculate that increased GABA contents in the brain of mice exposed to high-dose 1,2-DCE might be due to the continuing increase in Glu contents in the brain.^43,44 Since the changes in GABA contents in the brain were consistent with 1,2-DCE-induced changes in mouse behavior, it was suggested that disturbed contents of amino acid neurotransmitter in the brain might be one of the mechanisms of 1,2-DCE-induced neurotoxic effects.

MDA levels represented the total levels of lipid peroxides, reflecting oxidative situation of lipids in the brain attacked by oxidants.^45,46 High MDA levels might be the results of either overproduction of oxidants or accumulation of oxidants due to dysfunction of antioxidases or depletion of antioxidants. SOD is an important antioxidase catalyzing dismutation of superoxide anion into hydrogen peroxide and oxygen. It is thought to be essential for protecting cells against oxidative damage.⁴⁷ GSH, the most abundant NPSH in most cells, is the main antioxidants in the cells and acts as the nucleophilic scavenger of oxidants through enzymatic and nonenzymatic mechanisms.^48,49 Findings from this study disclosed that clear oxidative damage in the brain could be caused by exposure to 0.225 g/m³ 1,2-DCE, but it failed to show the evidence of oxidative damage in the brain of mice exposed to 0.9 g/m³ 1,2-DCE. On the other hand, SOD activity was upregulated in the brain of mice exposed to 0.9 g/m³ 1,2-DCE. Taken together, it was speculated that the improved oxidative status in the brain of mice exposed to 0.9 g/m³ 1,2-DCE was probably due to upregulation of SOD activity, since it could protect the brain against oxidative damage through potentiation of the intracellular antioxidant defense system. Findings from this study also suggested that although metabolism of 1,2-DCE in vivo could deplete intracellular GSH, it was in a dynamic equilibrium between depletion and compensatory in the brain of mice exposed to 1,2-DCE. In the study reported by Huang et al., levels of MDA and GSH and activities of SOD in both serum and brain of rats were determined following inhalation of 2.5, 5.0 and 10.0 g/m³ 1,2-DCE for 12 h. Although the MDA levels in both serum and brain increased dose dependently, the SOD activities in serum appeared to increase along with doses of 1,2-DCE, varying from 2.5 to 5.0 g/m³, and the GSH levels in the brain increased significantly when treated with 5.0 g/m³ of 1,2-DCE.⁵⁰ These results disclosed that exposure to 1,2-DCE within a range of doses could send brain tissue into compensatory antioxidant status.

NO is a neurotransmitter with the characteristics of free radicals. As a neurotransmitter, it can participate in many physiological and pathological processes; on the other hand, it can attack protein and DNA molecules through peroxynitrite resulted from reaction with superoxide anion.⁵¹ Until now, few study was reported to focus on the changes in NO metabolism in the brain of animals exposed to 1,2-DCE. Findings from this study disclosed that exposure to 1,2-DCE could result in overproduction of NO through activating iNOS in the brain, which might lead to brain dysfunction by nitrosative stress or disturbed signal transduction. In addition, since NOS being closely coupled with N-methyl-d-aspartate receptor (NMDAR), increased Glu content in the brain may stimulate NMDAR and activate subsequently NOS to produce excessive NO in the brain. Thus, increased NOS activity in the brain might be partly due to the abnormal changes in amino acid neurotransmitters.

Taken together, subacute exposure to high-dose 1,2-DCE could lead to compensatory antioxidant status; subacute exposure to 1,2-DCE could lead to increased NO metabolism and abnormal changes in EAAs and IAAs in the brain, which might result in a series of toxic effects on neuronal functions, possibly are the mechanisms underlying the changes in mouse behavior.

Footnotes

Funding

This work was supported by a grant of National Natural Science Foundation of China (NSFC 81172644).

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Effects of subacute exposure to 1,2-dichloroethane on mouse behavior and the related mechanisms

Abstract

Keywords

Introduction

Materials and methods

Animals

Experimental procedures

Reagents and laboratory wares

Analysis process

Open field test

MDA level

Level of NPSH

SOD activity

Activity of iNOS

NO level

Content of Glu, Asp and GABA

Statistical analysis

Results

Discussion

Footnotes

Funding

References