| Objectives To investigate the effect of intermittent hypoxia on spatial memory and the hippocampus nerve cell autophagy in rat after ischemia.Methods 120 healthy male wister rats were randomly divided into: normal control group(UC group)(n=20), merely ischemia group(IR group)(n=20), intermittent hypoxia for 7 days ischemia group(IH7+IR group)(n=20), intermittent hypoxia for 14 days ischemia group(IH14+IR group)(n=20), intermittent hypoxia for 21 days ischemia group(IH21+IR group)(n=20), intermittent hypoxia for 21 days ischemic+m TOR inhibitors group(inhibitor group)(n=20). Intermittent hypoxia animals were laid in the hypoxic cabin, oxygen concentration control in the hypoxic cabin is cycled from 2% to 98% to form the intermittent hypoxia conditions, the exposure time was 7h/d and the duration time was 7 days, 14 days and 21 days respectively. Intermittent hypoxia-ischemia animals were prepared cerebral ischemia-reperfusion model after hypoxia for 7 days, 14 days, 21 days respectively. Ischemia animals were were prepared cerebral ischemia-reperfusion model by improved pulsinelli four vessels block(4-VO), rats were sacrificed individually at the time point 6h and 24 h after reperfusion after ischemia 15 minutes. The visual-spatial memory of rats was tested with water maze after ischemia-reperfusion for 24 hours. Method with HE(hematoxylin-eosin staining) to observe the morphological changes of hippocampus nerve cells of rat brain were detected with HE(hematoxylin-eosin staining); The expression of PI3 K, m TOR, beclin1 protein in hippocampus of rat brain was detected with immunohistochemistry and RT-PCR; Repeated application SPSS 17.0 statistical analysis software obtained experimental data to design one-way ANOVA(P<0.05), a statistically significant difference.Results 1 The water maze test result Compared with UC group, rats in IR group showed longer escape latency time and lower number of passing the platform in the water maze test, the difference had significance of statistics(P<0.05); Compared with IR group, intermittent hypoxia-ischemia rats in each group showed longer escape latency time and lower number of passing the platform, the difference had significance of statistics(P<0.05); Compared with IH7+IR group, rats in IH14+IR group and IH21+IR group showed longer escape latency time and lower number of passing the platform, the difference had significance of statistics(P<0.05); Compared with IH14+IR group, rats in IH21+IR group showed longer escape latency time and lower number of passing the platform, the difference had significance of statistics(P<0.05); Compared with IH21+IR group, rats in inhibitor group showed longer escape latency time and lower number of passing the platform, the difference had significance of statistics(P<0.05); 2 The result of change of cell morphological(HE staining). The never cells of rat hippocampus in UCgroup appeared neat arrangement, cellularity was normal, cytoplasmic was stained lightly, nucleolus was obvious; Compared with UC group, the never cells morphology of rat hippocampus in IR group changed after just 6 hours, which mainly manifested as cells lost normal structure and part of cells showed abnormal morphology; Swollen neurons could be observed after 24 hours in hippocampus, which manifested include interstitial edema, loose structure, neurons and glial cells appeared nuclear pyknosis and deeply stained, part of neuronal nucleus disappeared completely and formed vacuole structure; Nerve cells of rat brain in each intermittent hypoxia-ischemia group showed severe damage, major degeneration and necrosis nerve cells could be observed, swollen neurons could be observed in hippocampus, which manifested include interstitial edema, loose structure, neurons and glial cells appeared nuclear pyknosis and deeply stained, part of neuronal nucleus disappeared completely and formed vacuole structure; Compared with IR group, the survival rate of neurons of rats in each intermittent hypoxia-ischemia group decreased, the difference had significance of statistics(P<0.05); Compared with IH7+IR group, the survival rate of neurons of rats in IH14+IR group and IH21+IR group decreased, the difference had significance of statistics(P<0.05); Compared with IH14+IR group, the survival rate of neurons of rats in IH21+IR group decreased, the difference had significance of statistics(P<0.05); Compared with IH21+IR group, the survival rate of neurons of rats in inhibitor group increased, the difference had significance of statistics(P<0.05); 3 PI3 K immunohistochemistry and RT-PCR analysis result The immunohistochemistry result: PI3 K immunohistochemistry staining presented brownyellow and located in cytoplasm and mainly expressed in neurons; Immunoreactive cells were seen occasionally in UC group, which were stained lightly and appeared neat arrangement, cellularity was normal, nucleolus was obvious; Compared with UC group, the staining of rats in IR group increased at the time point 6h and 24 h, the number of immunoreactive cell also increased, the difference had significance of statistics(P<0.05); Compared with IR group, the staining of rats in each intermittent hypoxia-ischemia group increased at the time point 6h and 24 h, the number of immunoreactive cell also increased, the difference had significance of statistics(P<0.05); Compared with IH7+IR group,the staining of rats in IH14+IR group and IH21+IR group increased at the time point 6h and 24 h, the number of immunoreactive cell also increased, the difference had significance of statistics(P<0.05); Compared with IH14+IR group,the staining of rats in IH21+IR group decreased at the time point 6h and 24 h, the number of immunoreactive cell also decreased, the difference had significance of statistics(P<0.05); Compared with IH21+IR group, the staining of rats in inhibitor group increased at the time point 6h and 24 h, the number of immunoreactive cell also increased, the difference had significance of statistics(P<0.05). RT-PCR analysis result: Compared with UC group, the expressions of PI3 K protein of rats in IR group increased significantly at the time point 6h and 24 h, the difference had significance of statistics(P<0.05); Compared with IR group, theexpressions of PI3 K protein of rats in each intermittent hypoxia-ischemia group increased significantly at the time point 6h and 24 h, the difference had significance of statistics(P<0.05); Compared with IH7+IR group, the expressions of PI3 K protein of rats in IH14+IR group and IH21+IR group increased significantly at the time point 6h and 24 h, the difference had significance of statistics(P<0.05); Compared with IH14+IR group, the expressions of PI3 K protein of rats in IH21+IR group decreased at the time point 6h and 24 h, the difference had significance of statistics(P<0.05); Compared with IH21+IR group, the expressions of PI3 K protein of rats in inhibitor group increased at the time point 6h and 24 h, the difference had significance of statistics(P<0.05). 4 The m TOR immunohistochemistry and RT-PCR analysis result The immunohistochemistry result: m TOR immunohistochemistry staining presented brown-yellow and located in cytoplasm and mainly expressed in neurons; Immunoreactive cells were seen occasionally in UC group, which were stained lightly and appeared neat arrangement, cellularity was normal, nucleolus was obvious; Compared with UC group, the staining of rats in IR group increased at the time point 6h and 24 h, the number of immunoreactive cell also increased, the difference had significance of statistics(P<0.05); Compared with IR group, the staining of rats in each intermittent hypoxia-ischemia group increased at the time point 6h and 24 h, the number of immunoreactive cell also increased, the difference had significance of statistics(P<0.05); Compared with IH7+IR group, the staining of rats in IH14+IR group and IH21+IR group increased at the time point 6h and 24 h, the number of immunoreactive cell also increased, the difference had significance of statistics(P<0.05); Compared with IH14+IR group, the staining of rats in IH21+IR group increased at the time point 6h and 24 h, the number of immunoreactive cell also increased, the difference had significance of statistics(P<0.05); Compared with IH21+IR group, the staining of rats in inhibitor group decreased at the time point 6h and 24 h, the number of immunoreactive cell also decreased, the difference had significance of statistics(P<0.05). RT-PCR analysis result: Compared with UC group, the expressions of m TOR protein of rats in IR group increased significantly at the time point 6h and 24 h, the difference had significance of statistics(P<0.05); Compared with IR group, the expressions of m TOR protein of rats in each intermittent hypoxia-ischemia group increased significantly at the time point 6h and 24 h, the difference had significance of statistics(P<0.05); Compared with IH7+IR group, the expressions of m TOR protein of rats in IH14+IR group and IH21+IR group increased significantly at the time point 6h and 24 h, the difference had significance of statistics(P<0.05); Compared with IH14+IR group, the expressions of m TOR protein of rats in IH21+IR group increased at the time point 6h and 24 h, the difference had significance of statistics(P<0.05); Compared with IH21+IR group, the expressions of m TOR protein of rats in inhibitor group decreased at the time point 6h and 24 h, the difference had significance of statistics(P<0.05). 5 Beclin1immunohistochemistry and RT-PCR analysis result The immunohistochemistry result: Beclin1 immunohistochemistry staining presented brown-yellow and located in cytoplasm and mainly expressed in neurons; Immunoreactive cells were seen occasionally in UC group, which were stained lightly and appeared neat arrangement, cellularity was normal, nucleolus was obvious; Compared with UC group, the staining of rats in IR group increased at the time point 6h and 24 h, the number of immunoreactive cell also increased, the difference had significance of statistics(P<0.05); Compared with IR group, the staining of rats in each intermittent hypoxia-ischemia group increased at the time point 6h and 24 h, the number of immunoreactive cell also increased, the difference had significance of statistics(P<0.05); Compared with IH7+IR group, the staining of rats in IH14+IR group and IH21+IR group increased at the time point 6h and 24 h, the number of immunoreactive cell also increased, the difference had significance of statistics(P<0.05); Compared with IH14+IR group, the staining of rats in IH21+IR group decreased at the time point 6h and 24 h, the number of immunoreactive cell also decreased, the difference had significance of statistics(P<0.05); Compared with IH21+IR group, the staining of rats in inhibitor group increased at the time point 6h and 24 h, the number of immunoreactive cell also increased, the difference had significance of statistics(P<0.05). RT-PCR analysis result: Compared with UC group, the expressions of beclin1 protein of rats in IR group increased significantly at the time point 6h and 24 h, the difference had significance of statistics(P<0.05); Compared with IR group, the expressions of beclin1 protein of rats in each intermittent hypoxia-ischemia group increased significantly at the time point 6h and 24 h, the difference had significance of statistics(P<0.05); Compared with IH7+IR group, the expressions of beclin1 protein of rats in IH14+IR group and IH21+IR group increased significantly at the time point 6h and 24 h, the difference had significance of statistics(P<0.05); Compared with IH14+IR group, the expressions of beclin1 protein of rats in IH21+IR group decreased at the time point 6h and 24 h, the difference had significance of statistics(P<0.05); Compared with IH21+IR group, the expressions of beclin1 protein of rats in Inhibitor group increased at the time point 6h and 24 h, the difference had significance of statistics(P<0.05).Conclusions 1 Intermittent hypoxia can aggravate the damage on spatial memory of cerebral ischemia-reperfusion rats. 2 Intermittent hypoxia can affect the autophagy dgree of nerve cells in hippocampus and aggravate the damage on nerve cells after ischemia, associated with the activation of PI3K/m TOR. |
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