| ObjectiveThrough the establishment of the different degrees of intermittent hypoxia animal models to simulate the hypoxia status of different degrees of sleep apnea hypopnea syndrome (S AHS) patients, we learned about the significance of the different degrees of intermittent hypoxia on cerebral apoptosis. After21%oxygen intervention on an animal model of intermittent hypoxia, we observed the impact of pathological changes in rat brain to deepen the understanding of SAHS, in order to provide the scientific basis for early intervention in patients with SAHS development. We observed the changes of HIF-la and iNOS expression in the rat brain after21%oxygen intervention, in order to explore their role in neuronal apoptosis and clarify the significance on assessing the value of treatment of ischemic cerebral vascular diseases.Methods42healthy SD rats were adaptively fed for one week. Experimental start-up phase:6rats were randomly selected from42as the reference value of0weeks (A group). The remaining36rats were randomly divided into three groups:control group (n=12), mild intermittent hypoxia group (n=12, equivalent to mild OSAS AHI=15), severe intermittent hypoxia group (n=12, equivalent to severe OSAS AHI=40). The rats fed for60days with ordinary feed, drinking. Rats in each group are placed in the experimental box daily from9:00to16:00, a total of7h. After16:00, they lived in the atmosphere. Each group was randomly selected6rats (BN, BQ, BZ group) to be taken the brain, in situ apoptosis detection (TUNEL), electron microscopy, the brain HIF-la, iNOS by immunohistochemistry, Western blot for the detection of protein levels. The remaining rats in each group (CN, CQ, CZ group) were reared under normal oxygen environment. And after30days they underwent the same examination. Statistical analysis was performed by SPSS17.0software.Results1. Morphological changes of nerve cells:1.1The nucleus was blue-black, and the cytoplasm pale was red by HE staining under a light microscope. In the control group (A, BN group), the morphology of the nerve cell in the hippocampus of rats was very regular and neat. There are different degrees of change in the hypoxia group (BQ, BZ group) from the morphological observation, which can determine the occurrence of apoptosis. After21%oxygen intervention for30days, the results showed that:neuronal apoptosis situation in the intervention group (CQ, CZ group) had improved. However, parts of the nerve cells were still irregular, morphological changes, and failed to recover to the initial level.1.2Perspective electron microscope examination revealed:After30days of intermittent hypoxia, there were significant changes in brain tissue pathology between intermittent hypoxia group and normal oxygen group, and severe intermittent hypoxia group changed more noticeably. We observed that the nerve nucleus double membrane structure of brain tissue damaged, part of the mitochondrial appeared edema expansion, and there were vacuoles in the cytoplasm. After21%oxygen intervention for30days, neuronal apoptosis situation in the intervention group (CQ, CZ group) had improved. However, parts of the nerve cells still changed in cell morphology, and failed to recover to the initial level.1.3TUNEL assay showed:In control group, the morphology of nerve cell in the hippocampus of rats was very regular and neat. In hypoxia group (BQ, BZ group), the number of apoptosis cells that were stained brown or tan was high. Severe intermittent hypoxia group increased most obviously (14.83±2.48VS18.67±2.66VS32.67±4.18VS49.17±4.36).The number of neuronal apoptosis in intermittent hypoxia group (BQ, BZ group) was significantly higher than that in the control group (A, BN group), and the difference was statistically significant (P<0.01). The difference between mild intermittent hypoxia and severe intermittent hypoxia group was statistically significant (P<0.01). After21%oxygen intervention for30days, the test results showed that:the situation of neuronal apoptosis in intermittent hypoxia intervention group (CQ, CZ group) had improved,(24.00±3.90VS31.67±2.50), but did not return to the initial level. The number of neuronal apoptosis in intermittent hypoxia intervention group (CQ, CZ group) was higher than that in the control group (A, BN, CN group), the difference was statistically significant (P<0.01). The difference between the intermittent hypoxia intervention group (CQ, CZ group) and the hypoxia group (BQ, BZ group) was statistically significant (P<0.01). Apoptosis of nerve cells was mainly concentrated in the hippocampus and cortex.2. Comparison of the expression of iNOS:Normal rat brain tissue of oxygen in the control group scattered in the small number of iNOS positive cells (23.46±3.5501,24,17±1.4452). In the experimental group, whether mild intermittent hypoxia group or severe intermittent hypoxia group showed more coloring cells (37.01±4.0473,53.34±8.0801). The number of iNOS positive cells in intermittent hypoxia group (BQ, BZ group) was significantly higher than those in the control group (A, BN group), and the difference was statistically significant (P<0.01). The difference between mild intermittent hypoxia and severe intermittent hypoxia group was statistically significant (P<0.01). After21%oxygen intervention for30days, iNOS positive staining cells expression in the intervention group (CQ, CZ group) returned to the initial level (24.6±8.1662,24.77±6.6268). The difference between the intervention group (CQ, CZ group) and intermittent hypoxia group (BQ, BZ group) was statistically significant, and the control group (A, BN, CN group) showed no significant difference with the intervention group (P>0.05). The iNOS positive staining cells were mainly concentrated in the cerebral cortex and hippocampus. They were not only found in neurons but also glial cells.WesternBlot test results showed:After hypoxia, the iNOS expression in experimental group (BQ, BZ group) increased compared with the control group (A, BN group), and severe intermittent hypoxia group increased most noticeably(140.01±1.7147VS151.52±19.9948VS376.52±38.9234VS585.17±35.1067). The iNOS expression in intermittent hypoxia group (BQ, BZ group) was significantly higher than that in the control group (A, BN group), and the difference was statistically significant (P<0.01). The difference between mild intermittent hypoxia and severe intermittent hypoxia group was statistically significant (P<0.01). After21%oxygen intervention for30days, the difference of iNOS expression between intervention group (CQ, CZ group:170.59±42.5984,172.45±30.2081)) and intermittent hypoxia group (BQ, BZ group) was statistically significant, and the control group (A, BN, CN group) showed no significant difference with intervention group (P>0.05). The expression of iNOS protein in each group returned to the initial level.3. Comparison of the expression of HIF-1α:Normal rat brain tissue of oxygen in the control group (A, BN group) showed weak HIF-la expression. The stained cells in the intermittent hypoxia group (BQ, BZ group), especially in severe intermittent hypoxia group were significantly increased (12.35±4.7915VS13.55±5.7289VS27.61±4.0600VS36.81±2.4887). The HIF-la expression in intermittent hypoxia group (BQ, BZ group) was higher than that in the control group (A, BN group), and the difference was statistically significant (P <0.01). The difference between mild intermittent hypoxia and severe intermittent hypoxia group was statistically significant (P<0.01). After21%oxygen intervention for30days, the HIF-la expression in the intervention group (CQ, CZ group) returned to the initial level (13.70±5.2587,14.22±6.5862). The difference between the intervention group (CQ, CZ group) and intermittent hypoxia group (BQ, BZ group) was statistically significant, and the control group (A, BN, CN group) showed no significant difference with the intervention group (P>0.05). The HIF-la positive staining cells were mainly concentrated in the cerebral cortex and hippocampus.WesternBlot test results showed:After hypoxia, the HIF-1α expression in experimental group (BQ, BZ group) increased compared with the control group (A, BN group), and severe intermittent hypoxia group increased most obviously C112.60d±11.2176VS136.26±19.994VS338.25±81.6125VS488.63α86.4614). The HIF-1α expression in intermittent hypoxia group (BQ, BZ group) was significantly higher than that in the control group (A, BN group), and the difference was statistically significant (P<0.01). The difference between mild intermittent hypoxia and severe intermittent hypoxia group was statistically significant (P<0.01). After21%oxygen intervention for30days, the difference of HIF-la expression between intervention group (CQ, CZ group:158.44±10.631,167.79±34.505) and intermittent hypoxia group (BQ, BZ group) was statistically significant, and the control group (A, BN, CN group) showed no significant difference with intervention group (P>0.05). The expression of HIF-la protein in each group returned to the initial level.Conclusions1. Intermittent hypoxia could undoubtedly induce nerve cells apoptosis in the specific area of the brain, mainly for the increase in neuronal apoptosis in the cortex and hippocampus, which might be the main reason for OSAHS lead to cognitive dysfunction.2. Different degrees of hypoxia could cause different levels of apoptosis of neurons, which illustrates the neuronal apoptosis was not only caused by hypoxia itself, but also the way of hypoxia.3. Intermittent hypoxia could effectively stimulate the hippocampus protein with high levels of expression of iNOS and HIF-la. Both of them participated in the nervous system hypoxia response. Different levels of hypoxia could cause atypical apoptosis of neuronal cells, and HIF-la and iNOS protein expression in the brain were also different.4. After hypoxia intervention, brain tissue injury had a certain degree of improvement, which was possibly related to the balance of metabolism of oxygen free radicals in the body. Oxidative stress intensity varied with the hypoxia extent. Correcting hypoxia and improving oxidative stress early could promote nerve cell injury and repair. |
PDF Full Text Request