Abstract：Objective To investigate the effects of intermittent hypoxia on the cognitive function of rats and the molecular mechanisms.Methods Sixty-four male Wistar rats (weight 180±10g) were selected and divided into normal control group (UC group) and 5% intermittent hypoxia group (5%IH group) by random number table method. Thirty-two rats in each group were randomly divided into four time subgroups (7 d, 14 d, 21 d and 28 d) according to different sampling time, with 8 rats in each subgroup. In the experiment chamber, the rats in the UC group were continuously injected with air with oxygen concentration of 21%, and the rats in the IH group were continuously injected with nitrogen and air while the oxygen concentration varied from 5% and 21%. Each cycle cost 120 s and the experiments conducted for 8 hours every day. Because of the four time subgroups of the two groups, the experiment time was different (7 d, 14 d, 21 d and 28 d). After the first experiment, the Morris water maze was performed to detect the learning and memory function of the rats in each group on day 7, day 14, day 21 and day 28. After the Morris water maze, hippocampal tissues of rats were taken out and made into section preparation. The transmission electron microscopy was adopted to observe the ultrastructural changes in the hippocampus CA1 pyramidal cells. Immunohistochemistry was used to detect the expression of apoptotic protease-activating factor(Apaf-1) and second mitochondria-derived activator of caspase(Smac) proteins in hippocampal CA1 region. The apoptosis of hippocampal CA1 neurons was detected by TUNEL method and the apoptosis index of neurons was calculated. Pearson test was used to analyze the correlation between Apaf-1 and Smac protein expression and apoptosis index of nerve cells at different time points.Results (1) Morris water maze: the escape latencies of intermittent hypoxia of rats in UC group respectively were (20.83±3.25)s,(22.17±2.79)s,(20.50±4.51)s and (21.17±4.17)s on day 7, day 14, day 21 and day 28; the cross target quadrant time respectively were(52.17±4.17)s,(52.50±2.74)s,(51.50±2.43)s and(52.00±4.78)s, and the difference was not statistically significant (all P values＞0.05). The escape latencies of intermittent hypoxia of rats in the 5% IH group respectively were (33.17±2.14)s, (44.33±3.45)s,(52.17±3.87)s and(64.33±2.73)s on day 7, day 14, day 21 and day 28; the cross target quadrant time respectively was (44.00±3.03)s,(34.50±3.94)s,(27.83±3.01)s and (20.83±1.94)s; with the increase of intermittent hypoxia time, the escape latent period was prolonged, and the time to cross the target quadrant was significantly shortened. The differences were statistically significant (F=106.335, 61.772, all P values＜0.05). Comparing the two groups: the escape latent of 5% IH group were longer than that of UC group at each time point, and the cross target quadrant time of 5% IH group were shorter than that of UC group at each time point; the differences were statistically significant (all P values＜0.01). (2) The transmission electron microscopy was adopted to observe the ultrastructural changes of nerve cells. The nucleus of neurons in hippocampal CA1 area of the rats in UC group was large; the nuclear membrane structure was complete, of which the shape was oval or round; the chromatin distribution was uniform in the shape of fine particles; the cell organelles were abundant, and the morphological structure was normal. In the 5%IH group, neurons started to appear edema, nuclear deformation, blurring of nuclear membrane, gradual disappearance of nucleoli, swelling and dissolution of organelles from Group 7d. With the prolongation of time, the level of change was more serious. (3) The relative expressions of Apaf-1 and Smac proteins: there was no statistically significant difference in the relative expression levels of Apaf-1 and Smac proteins in nerve cells in the CAl region of the hippocampus at each time point in the UC group (all P values were>0.05). The relative expressions of Apaf-1 and Smac proteins in the 5% IH group at all time points increased with the extension of intermittent hypoxia time, and the differences were statistically significant (F=25.328, 42.923, P＜0.01). Comparison between the two groups showed the relative expressions of Apaf-1 and Smac proteins of the 5%IH group were significantly higher than that of UC group at each time point and the differences were statistically significant (all P values＜0. 05). (4) The apoptosis of nerve cells: there were less apoptosis of nerve cells in the hippocampus CAl region of the UC group at each time point and AI differences had no statistical significance (P＞0.05). Apoptosis of nerve cells in 5% IH group increased with the extension of intermittent hypoxia time and the AI differences were statistically significant (F=25.799, P＜0.01). Comparison between the two groups showed the apoptosis index of nerve cells in the hippocampus CAl region of the 5%IH group were obviously higher than that in the UC group, and AI differences were statistically significant (all P values＜0.05). (5) Pearson tests revealed: the expressions of Apaf-1, Smac protein in the hippocampal CA1 region of the rats in the 5%IH group at all time points were positively correlated with the apoptosis index (rApaf-1=0.735, 0.736, 0.685 and 0.747, rSmac=0.735, 0.734, 0.679 and 0.751, all P values﹤0.05).Conclusions Intermittent hypoxia can lead to cognitive dysfunction in rats, which may be related to the ultrastructural changes of nerve cells in the hippocampal CA1 region of rats and the induction of the expressions of apoptotic proteins Apaf-1 and Smac, resulting to the apoptosis of neurons.
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2020, Vol. 25 
