Changes in the proliferative activity of hippocampal neural stem cells from manganismus mice

Guohe Tan; Boning Yang; Guofu Tan; Bo Liang; Jiangu Gong; Xiaodong Ge; Songchao Guo

摘要：BACKGROUND: Manganese neurotoxicity presents in the form of not only extracorticospinal tract injury of central nervous system (CNS), but also learning and memory ability damage. So, the mechanism of manganese neurotoxicity will be further studied from the angle ofhippocampus.OBJECTIVE: To observe the effects of manganism on learning and memory ability and the proliferation of neural stem cells (NSCs) in hippocampus of mouse brains, and analyze whether this effect has dose-dependence.DESIGN: Randomized controlled experiment.SETTING: Department of Human Anatomy, and Department of Industrial Hygiene and Occupational Diseases, Guangxi Medical University.MATERIALS: Twenty-eight male Kunming mice, aged 2 weeks, were involved in this experiment. The involved mice were randomized into 4 groups, with 7 in each: control group, low-dose manganism group,middle-dose manganism group and high-dose manganism group. Manganese chloride was purchased from Shantou Chemicals Factory.METHODS: This experiment was carried out in the Experimental Center for Preclinical Medicine, Guangxi Medical University from November 2005 to August 2006. Mice in the low-, middle- and high-dose manganism groups were intraperitoneally injected with 5, 20 and 50 mg/kg per day manganese chloride, once a day, for 2 weeks successively. Mice in the control group were injected with the same amount of stroke-physiological saline solution. Neurobehavioral detection of all the animals was performed in Morris water maze constantly from the 7th day after the first injection of manganese chloride solution. Learning ability was detected in the place navigation test. Mice were trained for 5 consecutive days with four trials per day. The time to find the platform was latency. Memory ability was detected in spatial probe test. Platform was withdrawn on the following day of place navigation. The mice were placed in the water from a random start in the edge of the pool. The number of times they traversed the plateform's region was recorded as the performance of spatial memory. At the final two days of the water maze tests, all the animals were daily intraperitoneally injected with 50 mg/kg BrdU three times successively, once every 4 hours. At 24 hours after the final BrdU injection, all the animals were sacrificed and perfused, and their brains were harvested, fixed and successively sliced at coronary plane on a freezing microtome. Distribution and number of BrdU-positive cells in the subgranular zone ofhippocampus of brains of experimental animas were detected respectively by immunohistochemistry for reflecting the proliferation of NSCs. Single-factor analysis of variance was used for comparing the difference of measurement data. Linear correlation analysis was used among the performance record in Morris water maze test, the number of BrdU-immunopositive cells and the dose of manganism.MAIN OUTCOME MEASURES: Learning and memory ability and the number of hippocampal NSCs of mice in each group.RESULTS: ①Performance of mice in Morris water maze: In the place navigation test, there was a significant retarded learning in mice of high-dose manganism group from the 3rd day as compared with control group (P ＜ 0.01). Till the 5th day, escape latency of mice in each manganism group was prolonged,and learning performance was significantly decreased (P ＜ 0.05), while swimming speed did not affect above results. In the spatial probe test, the average frequency of middle- and high-dose manganism groups was 1.17±1.60 and 0.80±1.10, respectively, and decreased remarkably than that of control group which was 4.86 ±1.35 (P ＜ 0.01), indicating memory ability was decreased; while the average frequency of low-dose manganism group did not differ obviously from that of control group (P =0.066) although it was 2.67 ± 3.27.The difference of swimming speed in each group was still of no statistic significance (P ＞ 0.05). ②Effect of manganism on the number of NSCs: After counting, the average number of BrdU- immunopositive cells of one side in the control group, low-, middle- and high-dose manganism groups was 69.20±4.48, 36.63 ±4.50,31.00 ±6.87, 26.76 ±4.83, individually (P ＜ 0.01). ③Results of linear correlation analysis: The ability of spatial memory was significantly in positive correlation with the number of BrdU-labeled cells ( r =0.734,P ＜ 0.01), and in negative correlation with the intensity of manganese poisoning (r =- 0.598, P ＜0.01).Meanwhile, there was a significant negative correlation between the number of BrdU-labeled cells and the intensity of manganese poisoning(r = - 0.666, P ＜ 0.01).ONCLUSION: Manganese exposure in mice can affect the ability of learning and memory, which is probably caused by the inhibition of manganese to the neurogenesis of NSCs in hippocampus in dose-dependent manner.