| Objective: Obstructive sleep apnea(OSA)can cause complications involving multiple systems.For the central nervous system(CNS)in adults and children OSA may cause cognitive impairment,including sleepiness,decreased attention,memory,and expression of mood disorders.Intermittent hypoxia(IH)is one of the important pathophysiological characteristics of OSA,which mainly causes cognitive dysfunction through apoptosis of hippocampal CA1 neurons.CNS IH can lead to excessive generation of reactive oxygen species,resulting in a large number of inflammatory factors resulting in neuronal apoptosis and necrosis and aggravate OSA related cognitive dysfunction.Therefore,through the study of primary cultured hippocampal neurons of 5-7d and then were divided into two groups.The expression level of different groups of inflammatory factors were measured after hypoxia exposure in order to explore the neurocognitive impairment caused by neuroinflammation.Materials and methords: The hippocampal neurons was isolated from fetal SD rats at 16-18 days of gestation and primarily cultured in vitro for 5-7 days.The hippocampal neurons was observed by inverted phase contrast microscope at different times to obtain morphology of cells.Immunofluorescent staining of microtubule-associated protein-2(MAP-2)was administered to analyse the purity of neurons: the neurons labeled by MAP-2 antibody,DAPI nuclear staining,observed and acquired images under a fluorescence microscope.The primarily cultured neurons was randomly divided into continuous normoxia group and intermittent hypoxia group.The experiment used Tianjin Medical University General Hospital Respiratory using simulation system 1.0 setting hypoxia / re-oxygenation cycle mode,hypoxic mixture containing 1.5% O2,93.5% N2,5% CO2,normal-oxygen mixture containing 21% O2,74% N2,5% CO2.The IH group was exposed in the hypoxia equipment for 4 hours and the frequency is 4 times / h.The IH groups were divided into 2h group,4h group,6h group,8h group according to the time of culture in standard incubator after IH exposure.The total m RNA of hippocampal neurons cells in the control group and IH groups was extracted after the intermittent hypoxia exposure.The relative expression level of HMGB1(High mobility group protein box 1)m RNA was measured by Real Time PCR.The expression of HMGB1 and NMDA(N-methyl-D-aspartate)in the control group and each culture group were detected by Real Time PCR and Western blotting after the normoxia culture.The data were analysed through SPSS and Graph Pad Prism.Results:(1)The primary hippocampal neurons culture were adhered obviously to the dishes,a few cells grow 1-2 tiny bumps 2 hours later.The cell refraction were stronger and no obvious nuclei were found.Most of hippocampal neuron cells had been adherent after inoculated 24 hours and the cells were round or oval and surrounded by halo,a single uniform distribution,showing three protrusions or more protruding.After 3 days of culture,the neuron protrusions increased and became longer,the cell bodies became larger and the cells gathered into clusters and the protrusions were seen as sparse network structures.After 5 days,neurons were triangular,rounded or polygonal and protrusions form a dense network.The neurons grew well which bodies were clear,and the protrusions were thickened to woven into dense neural networks.The hippocampal neurons were cultured with different excitation wave lengths in inverted fluorescence microscopy using neuron-specific markers Map-2 and nucleus DAP I.The green neuron protrusions and cell bodies and blue fluorescence nuclei were observed.The purity of neurons was up to 90 % when calculated by immunofluorescent stainin.(2)The relative expression of HMGB1 m RNA in IH group was 3.203 times higher than that in control group,and the expression level of HMGB1 m RNA in IH group was significantly higher than that in control group(P <0.05).The relative expression level of HMGB1 m RNA was significantly different between each group(P <0.05),and the relative expression level of 4h group m RNA was the highest.(P <0.05).The expression of HMGB1 was about 2.836 times(P <0.05)in the control group and 0.669 times in the control group at 6h group,and the expression of HMGB1 was lower than that of the control group(P <0.01)).And the expression level was 0.338 times higher than that of the control group(P <0.01).Compared with 2h group,the relative expression level of 6h group was higher than that of 2h group(P <0.05).Compared with 2h group,the relative expression level of 8H was higher than that of 2h group(P < 0.05).The relative expression level of 6h group was significantly higher than that of 4h group(P <0.05).The relative expression level of 8h group was higher than that of 4h group(P <0.05).(3).The relative expression level of NMDA m RNA in each group was significantly higher than that in normal group(P <0.05).The level of NMDA m RNA expression in each group was significantly higher than that in normal group(P <0.05)).(P <0.01).Compared with the control group,the relative expression level of the control group was significantly higher than that of the control group(P <0.001),and the expression level was significantly increased at 6 h compared with the control group Increased expression(P <0.001).(4).Compared with the control group,the expression level of HMGB1 protein in the hippocampal neurons was significantly higher than that in the control group(P <0.05),and the expression of 2h protein was higher than that of the control group at 6h Protein expression level was significantly lower than the control group,8h protein expression level compared with the control group was significantly reduced The expression of NMDA protein in the hippocampal neurons was significantly different(P <0.05).The expression of NMDA protein in the hippocampal neurons increased significantly with the prolonged incubation time.The highest f NMDA protein is in 8 h group in hippocampal neurons.Conclusion: Neuroinflammation plays an important role in OSA IH-induced neurocognitive dysfunction,and IH can cause neuroinflammatory cytokine HMGB1 expression.It is indicated that the neuroinflammation and damaged neurons form a vicious circle which could cause a "second strike" to the hippocampal neurons.Even if IH exposure is removed and neuronal damage continues to exist.The sustained release of HMGB1 from dying neurons is important for the formation and maintenant of this vicious cycle.Even if IH stops,inflammatory damage can continue.IH can cause inflammation,but IH is not a necessary condition for maintaining inflammation.With the prolonged duration of inflammation,HMGB1 secretion decreased,but NMDA increased significantly,indicating that HMGB1 and NMDA may play a synergistic role in neuronal apoptosis and dysfunction. |
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