| Objective To investigate of the function of chronic unpredictable wild stress-induced learning and memory impairment and autophagy in the hippocampal CA1region;To observe the effect of learning and memory of chronic unpredictable mil d stress rats caused by change of autophagy..Methods 48 male rats were ranndomly divided into four groups: Control group, chronic unpredictable mild stress+Vehicle(CUMS group), CQ + chronic unpredictable mild stress(CQ group), rapamycin + chronic unpredictable mild stress(Rapa group), In addition to the Control group, the rats in each group could be the treatment of chronic unpredictable mild stress from the first day, CUMS group was injected intracerebroventricularly of sterile saline(2ul), CQ group received intracerebroventricular injection of CQ(0.61mg/kg), Rapa group l intracerebroventricular injection of rapamycin(2ul/ 35 pmol/l).Sucrose intake test be made weekly for each groups, Four groups of rats were killed after Morris water maze experiment In the sixth week. Apoptosis was observed in hippocampal CA1 region by TUNEL, The number of autophagosomeof hippocampal CA1 region was observed by Transmission electron microscope(TEM), Pathomorphology of hippocampal CA1 region was abserved by Hematoxylin-eosin staining;the expression of Beclin1、LC3-I/LC3-IIwere measured by Western-Blot respectively.;the expression of P62 were measured by Immunofluorescence.Results 1. Sucrose preference: In addition to the Control group, sugar-preferred values decreased slightly in each groups at 0,1,2 weeks, but there was no difference between the each groups(P > 0.05),from the third week, CUMS significantly lower than the Control,it was statistically significant(P <0.05); CQ group is lower than CUMS group; Rapa is slightly higher than CUMS groups,,it was statistically significant(P <0.05). 2. Results of Western-Blot: The expression of Beclin-1 and LC3-II / LC3-I ratio in CUMS group was higher than Control group, the difference was significant(P <0.05),; Compared to CUMS group, LC3-II / LC3-I ratio in CQ group was increased, the difference was significant(P <0.05).In Rapa group, LC3-II / LC3-I ratio was higher than CUMS group, the difference was significant(P <0.05). In CUMS,CQ and Rapa groups,Beclin-1 expression has no significant difference. 3.Immunofluorescence: The fluorescence intensity of p62 in hippocampal CA1 region of CUMS was increased compare with Control group, The fluorescence intensity of p62 in CQ group was increased more obvious, Rapa group was decreased compared with CUMS. 4. HE staining, transmission electron microscopy: In Control groups, hippocampal CA1 pyramidal cells arranged irregularly, about 3 to 5 layers, Cellular level clear, uniform red cytoplasmic staining, the nucleus is clearly visible; Compared with control group, hippocampal CA1 cell numbers in CUMS was decreased,significantly sparse arrangement, about 1-3 layers, hierarchies confusion, the outline of neurons was disappear, the cytoplasm and the nucleus of the boundaries blur, the volume has narrowed partly stained cytoplasm, showing a little condensation nuclei; Compared with Control group,the number of neurons in Rapa group was decreased slightly,sparse arrangement, level somewhat confusing, cytoplasmic substantially uniform, no significant karyopycnosis; CQ group part hippocampus neurons in CA1 neurons in a hierarchy obvious confusion, cytoplasm stained edge set, showing obvious karyopyknosis. Electron microscopy showed: CUMS group compared with Control group showed nuclear membrane shrinkage has changed, nuclear chromatin margination, increased autophagic bodies, CQ group autophagic bodies, vacuolization of mitochondria significantly more than CUMS group, Rapa group mitochondrial damaged and autophagosome decreased compared CUMS group. 5. TUNEL staining: Control group had a small amount of nucleus were stained brown;In CUMS,Apoptotic cells was increased compared with Control;The total number of apoptosis in CQ was obviously increased compared with CUMS. In Rapa,apoptosis was decreased compared with CUMS. 6.Behavioral tests Morris water maze test: CUMS escape latency is longer than Control group, the numbers of enter the target quadrant and the time in target quadrant were decreased, the difference was significant(P <0.05). escape latency in CQ group is longer than CUMS group, the numbers of enter the target quadrant and the time in target quadrant is shorted, the difference was statistically significant(P <0.05). escape latency in Rapa group less than CUMS group, the numbers of enter the target quadrant and the time in target quadrant was decreased compare with CUMSConclusion: 1. Chronic stress results in learning and memory impairment by damaging autophagy stream in rat hippocampal neurons. 2. Inducing autophagy improve learning and memory in chronic unpredictable mild stress rats.Inhibit autophagy aggravated learning and memory impairment in rats,... |
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