| Objective: Methamphetamine, MA, a kind of new synthetic drugs which has obvious psychostimulant effects, otherwise called Metamfetaminum, commonly known as Amphetamin Chloride, belongs to amphetamines central stimulant. Amphetamine-type stimulants can cause strong psychological dependence, and its direct effect on the central nervous system will make people intensely excited or inhibit the central nervous system. The long-term repeated abuse can cause the body to produce abnormal compensatory neuronal adaptation, and can make changes in Central nervous system pathology, Neurochemistry and behavioral science and other central nervous system, which will make learning, memory, movement and other functions impaired and dependent on the MA. The present study shows that the long-term human use of MA can cause serious damage of the nervous system, the decline in cognitive function, attention and memory loss, as well as the executive dysfunction and so on. The neurotoxicity of MA is quite obvious, therefore, actively explore the protection of nerve damage caused by MA and abuse of drugs is very urgent and necessary.Cholecystokinin octapeptide, CCK-8, as a neurotransmitter and(or) neuromodulator involved in the regulation of the body in a variety of physiological functions, such as the feeding, anxiety, cognitive, and the adjustment of the learning and memory, etc. Previous studies in our laboratory found that exogenous CCK-8 can relieve physical and mental dependency of morphine. Lateral ventricle to CCK-8(0.01,0.1,1 μg) can significantly improve the spatial reference memory of morphine dependent mice. The lateral ventricle injection of exogenous CCK 8 can inhibit the formation and expression of behavioral sensitization induced by MA, and can also obviously alleviate the MA-induced dopaminergic neurotoxicity. However, whether CCK-8 will be able to effect methamphetamine-induced learning and memory impairment as well as mice hippocampal neuronal injury, has not been reported.In this study, by the methods of Morris water maze, HE staining, thionine staining and immunohistochemical staining, the effects of exogenous CCK-8 on the spatial reference memory impairment induced by methamphetamine and the hippocampal neuronal damage in mice were respectively observed. Thus provide experimental basis for the application of CCK-8 in terms of new drug treatment.Methods:1 In this study, sixty adult male CD-1 mice, weighing 20-25 g, were randomly divided into normal saline control groups, MA 1 mg·kg-1 group, MA 2 mg·kg-1 group, CCK 0.1 μg + MA 1 mg·kg-1 group, CCK 0.1 μg + MA 2 mg·kg-1 group. MA group of mice were treated with different doses of MA intraperitoneal injection(1 mg·kg-1, 2 mg·kg-1 ip) every day(8:00 am). Injected saline, the amount equivalent to CCK-8, into the lateral ventricle 15 min before the injection of MA. Every day(8:00 am),CCK + MA groups of mice were injected CCK-8(0.1 μg, icv) into the lateral ventricle 15 min before the intraperitoneal injection of MA. Saline control group were treated each day(8:00 am) by intraperitoneal injection of the equivalent MA amount of saline. As the above, injected saline, the amount equivalent to CCK-8, into the lateral ventricle 15 min before the injection of MA.In the Morris water maze test, Day 1 groups of mice were not administered but only did the adaptive training before the test, the purpose is to observe swimming speed difference between groups of mice, eliminate mice that do not conform to the experimental conditions. Days 2-6 were the training phase, according to the method described above, after three hours of administration was the water line maze training. Day 7 was the test phase, according to the same method of administration, three hours after the administration, do the water maze test, that is, space exploration experiments. Through the water maze test, to observe the impact of MA alone spatial reference memory in mice as well as the effect of exogenous CCK-8 to MA induced spatial reference memory impairment.2 After the Morris water maze test, continue to inject drugs to each group of mice as described above, which need to last for 2 weeks. After modeling, according to stereotaxic brain atlas standard parts cut by fixed routine, embedding, sectioning, and through the H-E staining, immunohistochemical staining(Neu N staining) observe the changes of hippocampal neurons in mice. Through counting the positive cells by statistical analysis, observe the effect of MA on mice hippocampal neurons as well as the effect of exogenous CCK-8 on MA-induced hippocampal neuronal damage.The results are expressed as mean ± standard deviation(mean ± SD). The data were subjected to one-way analysis of variance(ANOVA) followed by Bonferroni post hoc test for multiple comparisons with SPSS 16.0 statistical program, respectively. Values of P<0.05 were considered to be statistically significant.Results: 1 In this study, a total of 46 CD-1 mice were used in the final statistical analysis. 2 Effects of exogenous CCK-8 on spatial reference memory impairment induced by MA in mice 2.1 MA(1 mg·kg-1, 2 mg·kg-1) significantly impaired spatial reference memory in mice, that is, when tested, the percentage of time and distance in the target quadrant(the original platform quadrant) significantly reduced. 2.2 CCK-8(0.1 μg) significantly improved the impairment of spatial reference memory in mice induced by MA(1 mg·kg-1, 2 mg·kg-1) that is, compared with MA group of mice, the performance of mice in the target quadrant(the former platform quadrant) as well as the percentage of time and distance is significantly increased after being administered CCK-8. 3 Effects of Exogenous CCK-8 on MA-induced hippocampal neuronal injury in mice 3.1 H-E staining results showed that district hippocampus neurons of normal saline control group mice are round, oval or conical. Nuclear plasma ratio is large. The nucleus is circular or elliptic, evenly colored in light shallow blue purple. Besides, nucleoli is prominent. Compared with the saline control group, MA 1 mg·kg-1 mice hippocampal CA3 region structure shows no abnormal cells, cell arrangement is a bit disordered, some cells pyknosis, cytoplasm nucleus boundary is not clear. MA 2 mg·kg-1 mice hippocampal CA3 region structure shows no abnormal cells, cell arrangement is in disorder, a large number of neurons in the cytoplasm of the red dye, and pyknotic nucleus, cytoplasm and nucleus boundary is not clear. CCK 0.1 μg + MA 1 mg·kg-1 set of hippocampal CA3 area cells in mice compared with MA mg·kg-1 group shows no obvious abnormalities in the organizational structure, and occasionally red cytoplasmic staining, cytoplasmic and nuclear boundaries a little clearer. Compared with MA 2 mg·kg-1, the organization structure of CCK 0.1 μg + MA 2 mg·kg-1 set of hippocampal CA3 area cells in mice shows no abnormal, cells are arranged a little neater, a small amount of cell pyknosis, cytoplasmic and nuclear boundaries are a little clearer. 3.2 The results of Thionine staining show that saline control group were arranged in neat rows and Nissl bodies are clearly visible. Compared with saline control group, MA 1 mg·kg-1 group of mice hippocampal CA3 region of neurons arranged slightly disordered, neuron Aizen, some cell pyknosis, Nissl bodies disappear. MA 2 mg·kg-1 mice neurons in hippocampal CA3 region are in disorder, neuronal Aizen, large amounts of cell pyknosis, Nissl bodies disappear. As for hippocampal CA3 area cells in mice, compared with MA group 1 mg·kg-1, the cell arrangement of CCK 0.1 μg + MA 1 mg·kg-1 is relatively neat, only a small number of cells pyknosis, Nissl bodies are clearly visible. Compared with MA 2 mg·kg-1, cell arrangement of CCK 0.1 μg + MA 2 mg·kg-1 set of hippocampal CA3 area cells in mice is neat, only a small number of cells pyknosis, Nissl bodies are clearly visible. 3.3 Immunohistochemical staining(Neu N staining) shows that compared with normal saline control group, the number of positive cells of MA group(1 mg·kg-1, 2 mg·kg-1) in mice hippocampal CA3 area reduce, and with the increase of the concentration of MA, the degree of reduction in the number of positive cells becomes more obvious. Compared with the MA group, the number of positive cells of CCK + MA hippocampal CA3 area cells in mice increased significantly.Conclusion:Methamphetamine significantly undermines the spatial reference memory in mice, which impair hippocampal neurons associated with learning and memory. Exogenous CCK-8 can alleviate the spatial reference memory impairment induced by methamphetamine, and have a protective effect on hippocampal neurons. |
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