The Role Of D-serine In Nerve Injury Of Rats Induced By Lead Exposure

Objectives To investigate through the N-methyl-D-aspartic acid(NMDA) receptor expression, Ca2+ concentration, amino acid neurotransmitters and so on in hippocampus from lead exposed rats, and to appraise effect of D-Ser on learning and memory function of hippocampus.Methods 1 The experimental animal and grouping: A total of 60 male SPF grade SD rats aged 21 days were randomly divided into 4 groups after a week adaptive feeding. Rats in control group were given 500mg/kg sodium acetate in drinking water and rats in lead exposure group, D-Ser low dose group and D-Ser high dose group were given 500mg/kg lead acetate in drinking water. Meanwhile, rats in D-Ser low dose group and D-Ser high dose group also treated with D-Ser at dose of 30mg/kg and 60mg/kg by ip every other day, and rats in control and lead exposure group were treated with same amount saline. The period of treatment was 8 weeks. 2 Analysis neuroethology by using Morris water maze test. 3 Analysis of the content of lead in hippocampus in rats by using inductively coupled plasma mass spectrometry(ICP-MS). 4 Analysis of the apoptosis resulted, concentration of Ca2+ and Reactive oxygen species(ROS) in hippocampus cells of rats by using flow cytometry. 5 HE staining and Nissl staining were applied to observe the pathological changes of rats’ brain tissue. 6 Transmission electron microscopes were used to observe the changes of the structure and morphology of hippocampus neurons. 7 The immunohisto-chemical method was applied to observe NR2 A and NR2 B protein location. 8 Quantitative PCR was used to determine the NR2 A, NR2 B and Srr m RNA expression in the hippocampus tissue. 9 High performance liquid chromatography(HPLC) was applied to determine the changes of neurotransmitters in hippocampus. 10 Statistical analyses: The experimental data were showed with sx ±. The statistic methods included One Way ANOVA and LSD text; P<0.05 was determined as significant difference.Results 1 All experimental rats were feeding and drinking as abnormal and no significant difference was seen. The body weight grew normally of rats and no difference was showed among 4 groups. 2 Morris water maze test results showed that escape latency of lead exposure group rats was longer and the times crossing the platform were shorter than the control group. Compared with the lead exposure group, escape latency of D-Ser low dose group and D-Ser high dose group rats were shorter. The times crossing the platform of D-Ser low dose group and D-Ser high dose group rats were increase than that of the lead exposure group. 3 Compared with the control group, the lead content in hippocampus of rats in lead exposure group increased 2.49 folds. No significant difference between D-Ser low dose group and D-Ser high dose group was showed regarding of lead content. The same trends were seen regarding of lead content in blood and cortex. 4 The early and late apoptosis rate of hippocampus neural cells in lead exposed rats were 25.40±4.44% and 16.30±4.39%, higher than the control group of 16.70±5.79% and 12.43±3.40%(P<0.05). The early and late apoptosis rate in D-Ser lowdose group were 19.03±5.01 and 11.02±2.55%, significant lower than that of lead exposure group(P<0.05). The early and late apoptosis rate of D-Ser high dose group were 18.93±3.83% and 12.93±2.28%, have significant difference with lead exposure group. 5 Compared with the control group, the ROS in lead exposure group increased, and the difference was significant. There was no statistical difference between D-Ser low dose group and D-Ser high dose group and lead induced group. 6 Compared with the control group, the Ca2+ concentration of lead exposure group increased which was 1.94 times of control group(P<0.05). The Ca2+ concentration of D-Ser low dose group and DSer high dose group were decreased of lead exposed group, and there was a significant difference. 7 Under the optical microscope, hippocampus cells of control group rats were integrity and arranged well. Hippocampus cells of lead exposure group were arranged in disorder, and a large number of cells were dead as karyopyknosis and rupture. Hippocampus cells of rats in D-Ser low dose group and D-Ser high dose group arranged in a slightly disordered and the number of cells decreased. The Nissl staining showed that the neuronal cells of hippocampus in control group arranged closely and neatly, the cellular structure integrity, the nucleolus are clear. The rats’ neuron cells of lead exposure group arranged in loose and disorder, parts of the cell structure is not complete. Neuron cells in the D-Ser low dose group and D-Ser high dose group arranged a little in disorder and a few of the cell structure was not complete. Transmission electron microscopy results showed that hippocampus nerve cell in control group was nucleus full, smooth nuclear membrane, evenly distributed chromatin, and the thickness of the myelin sheath. But in the lead exposure group, the nuclei of hippocampus nerve cell had shrinkage and chromatin margination. In D-Ser low dose group and D-Ser high dose group, the nuclei of hippocampus nerve cell had slightly shrinkage and chromatin margination. In lead exposure group and D-Ser low dose group and D-Ser high dose group, the number of organelles reduced, mitochondrial swelled, the myelin sheathed thickness and the size changed small. 8 Immunohistochemistry results showed that, the NR2 A staining density in hippocampus of lead exposure group rats were less than that of control group. Compared with the lead exposure group, NR2 A staining density of D-Ser high dose group significant increased(P<0.05). There was no significant difference between leadinduced and normol rats in terms of NR2 B expression. Compared with control group, the m RNA expression of NR2 A and Srr in the lead exposure group were lower than that in the control group(P<0.05). After D-Ser treatment, NR2 A and Srr m RNA expression in D-Ser high dose group were higher than those in lead exposure group. No significant difference was seen between lead exposure group and control group in terms of NR2 B m RNA expression. 9 Compared with the control group, the content of Glu in hippocampus of lead exposure group increased 1.26 folds(P<0.05). No significant difference was seen between lead exposure group and D-Ser low dose group and D-Ser high dose group in terms of Glu content in hippocampus. Meanwhile, the Ser, Gly and GABA levels of hippocampus in lead-induced rats were as same as those of rats in control group.Conclusions Our findings support that D-ser might be an effective protestants to alleviatethe lead induced neurotoxicity by decreasing Ca2+ levels and apoptosis of hippocampus, as NR2 A expression increased.