| ObjectivesPrevious studies have shown that normobaric oxygen(NBO) can improve the neural function in acute cerebral ischemic rats. So this study was to focus on exploring the roles of AQP4 and MMPs in normobaric oxygen reducing ischemic brain edema, so that providing an crucial experimental evidence and guidance for implement oxygen inhalation intervention for the acute ischemic stroke patients. Methods1 Subjects and groups: We bought 75 healthy male sprague dawley rats at specified pathogen free level at animal center in Henan province. After the identification of qualified, they were fed for one week. According to weight, they were randomly divided into three groups, including the sham group(do corresponding anatomy, separation, hanging line and sitch, then inhaled air), the model group(after the ischemia/reperfusion stroke model inhaled air) and oxygen-inspiration group(after the ischemia/reperfusion stroke model inhaled oxygen)(n=25 per group). And then each group was randomly divided into five subgroups which were reperfusion for 0.5h, 3h, 6h, 12 h and 24h(n=5 per subgroup).2 Modeling: The rats of oxygen-inspiration group and the model group which would fast for 12 h before surgery used the improved middle cerebral artery ischemic reperfusion model referring to Zea Longa suture method. Use the Zea Longa 5 level and 4 point standard to make sure the success of models 2h later, excluding the rats of 1~3 point. After scoring, the rats were anesthetized with ether, and then reperfusion was initiated by withdrawal of the occluding filament. Whenever necessary, the samples were supplemented to ensure adequate quantity of rats. The rats in the sham group were subjected to the identical surgical procedure without suture occlusion.3 Intervening: The rats of oxygen-inspiration group were placed in the home-made enclosed oxygen chamber(50cm × 30 cm × 30cm), which has two holes(1cm × 1cm) connected with oxygen and nitrogen cylinder on one side and the oxygen detector on the other side to monitor the oxygen concentration of the chamber. So that we could adjust the flow of oxygen and nitrogen to ensure the concentration of oxygen could reach the level of(61±2)%. The rats in the model group inhaled air.4 Examination: Histopathological variables, Neurological Deficits Sores(NDS) were recorded. The real-time fluorescence quantitative Polymerase Chain Reaction(Q-PCR) was used to detect the expression of AQP4 m RNA. The Western Blotting method was used to determine the AQP4 protein expression. Also, we used gelatine zymography method to evaluate the relative activities of matrix metalloproteinase2/9(MMP2/9) at the end point.5 Statistical methods: The differences of neurological deficit scores were compared by wilcoxon man-whitney test and wilcoxon matched-pairs signed rank sum test. The differences of AQP4 m RNA, AQP4 protein expression and MMPs’ activities among the three groups were compared by factorial design ANOVA. Pairwise comparison used the Student-Newman-Keuls test. The test level was 0.05. Results1 The rat model: The successful model of rats showed neurological defect. We could find out the white ischemic brain tissue with the naked eye. Also, the ischemic brain tissue was s stained light.2 The results of neurological deficit scores of rats: Comparison of before and after oxygen administration in oxygen-inspiration group showed the scores of 12h(P=0.031) and 24h(P=0.015) after reperfusion decreased. Comparing the scores of oxygen-inspiration group with the model group after oxygen inhalation, rats receiving 61%NBO showed neurological deficit scores were decreased at 0.5h(P=0.042),3h(P=0.014), 6h(P=0.042), 12h(P=0.031) and 24h(P=0.015) after reperfusion. The scores of the sham group rats were 0, showing no neurological deficit.3 The results of brain histomorphology in rats: The gross appearance of brain tissue in rats: We can see a few vascular engorgement in rats` brain tissue in oxygen-inspiration group and the model group at 0.5h after reperfusion. And the brain tissue turned white with the area increased at 3h, 6h, 12 h and 24 h after reperfusion. At 24 h after reperfusion, the lesions became soft and the brain edema was obvious. Compared with the model group, the volume of brain edema of oxygen-inspiration group was decreased. What’s more, there were no significant changes of brain tissue in the sham group. The results of HE staining: The changes in morphology and the number of nerve cells were not apparent in the sham group. In the model group, the ischemic area of brain tissue was dyed slighter with nerve cells decreased and edema was found in astrocyte foot processes at 0.5h, 3h and 6h after reperfusion. Also, we saw the gap between nerve cells and small vessels at 12 h and 24 h after reperfusion which indicated brain edema. Furthermore, HE staining revealed a remarkable increase of nerve cells and loss of brain edema volume and size of nerve cells death in oxygen-inspiration compared with the model group.4 The results of Q-PCR: We used factorial design ANOVA to analyze the AQP4 m RNA expression levels of brain tissue in three groups. The group main effect had difference(Fgroup=1340.962, Pgroup<0.001). The time main effect also had difference(Ftime=104.569, Ptime<0.001). There was interaction between group and time(Fgroup×time=27.218, Pgroup×time<0.001). The AQP4 m RNA expression levels of the ischemic brain tissue in rats were significantly increased in the model group and oxygen-inspiration group relative to the sham group at each time point(P<0.001). Also, the m RNA expression levels of AQP4 were significantly lower in oxygen-inspiration group than the model group at 3h(P=0.011), 6h(P=0.008), 12h(P=0.019) and 24h(P=0.014).5 The results of Western Blotting: We used factorial design ANOVA to analyze the AQP4 expression levels of brain tissue in three groups. The group main effect had difference(Fgroup=7802.486, Pgroup<0.001). The time main effect also had difference(Ftime=2562.095, Ptime<0.001). There was interaction between group and time(Fgroup×time=652.677, Pgroup×time<0.001). The AQP4 protein expression of rats in the sham group was the lowest among three groups. At 3h, 6h, 12 h and 24 h after reperfusion, compared with the sham group, the expression levels of AQP4 protein in rats were higher in the model group and oxygen-inspiration group at each time point(P<0.001). At 6h, 12 h and 24 h after reperfusion, the expression level of AQP4 protein in the oxygen-inspiration group was lower than the model group(P<0.001).6 The results of zymography: We used factorial design ANOVA to analyze the activity of MMP2 of brain tissue in three groups. The group main effect had difference(Fgroup=439.644, Pgroup<0.001). The time main effect also had difference(Ftime=1622.719, Ptime<0.001). There was interaction between group and time(Fgroup×time=433.366, Pgroup×time<0.001). Compared with the sham group, the activity of MMP2 in rats` brain tissues was enhanced in the model group and oxygen-inspiration group at 3h after reperfusion(P<0.001). We used factorial design ANOVA to analyze the activity of MMP9 of brain tissue in three groups. The group main effect had difference(Fgroup=1728.421, Pgroup<0.001). The time main effect also had difference(Ftime=1936.229, Ptime<0.001). There was interaction between group and time(Fgroup×time=492.343, Pgroup×time<0.001). The activity of MMP9 increased like the curve rising till to the peak from 6h to 24 h after reperfusion in the model group and oxygen-inspiration group compared with the sham group. The activity of MMP9 was reduced in the oxygen-inspiration group compared with the sham group at 6h(P=0.001), 12h(P=0.001) and 24h(P=0.006) after reperfusion. Conclusions1 NBO can improve neurological function and reduce ischemic brain edema in middle cerebral artery occlusion reperfusion model rats.2 NBO can inhibit the expression of AQP4 and the activities of MMP9 in middle cerebral artery occlusion reperfusion model rats.3 NBO can inhibit the expression of AQP4 and the activities of MMP9 which maybe one of the molecular mechanisms of NBO on the reduction of ischemic brain edema. |
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