Hyperbaric Oxygen Improves Cerebral Ischemia/Reperfusion Injury in Rats Probably via Inhibition of Autophagy Triggered by the Downregulation of Hypoxia-Inducing Factor-1 Alpha

Abstract

Ischemic stroke, accompanied with high mortality and morbidity, may produce heavy economic burden to societies and families. Therefore, it is of great significance to explore effective therapies. Hyperbaric oxygen (HBO) is a noninvasive, nondrug treatment method that has been proved able to save ischemic penumbra by improving hypoxia, microcirculation, and metabolism and applied in various ischemic diseases. Herewith, we fully evaluated the effect of HBO on ischemic stroke and investigated its potential mechanism in the rat ischemia/reperfusion(I/R) model. Sixty Sprague-Dawley male rats were randomly divided into three groups-sham group, MCAO group, and MCAO+HBO group. In the latter two groups, the middle cerebral artery occlusion was performed (MCAO) for 2 hours, and then the occlusion was removed in order to establish the ischemic/reperfusion model. Subsequently, HBO was performed immediately after I/R (2 hours per day for 3 days). 72 hours after MCAO, the brain was dissected for our experiment. Finally, the data from three groups were analyzed by one-way analysis of variance (ANOVA) and followed by a Bonferroni test. In this article, we reported that HBO effectively reduced the infarction and edema and improved neurological functions to a certain extent. As shown by western blot analysis, HBO significantly reduced autophagy by regulating autophagy-related proteins (mTOR, p-mTOR, Atg13, LC3B II and LC3B II) in the hippocampus 72 hours after I/R, which was accompanied by inhibiting the expression of hypoxia inducible factor-1α (HIF-1α) in hippocampus. The results suggest that HBO may improve cerebral I/R injury, possibly via inhibiting HIF-1α, the upstream molecule of autophagy, and therefore, subsequently inhibiting autophagy in the rat model of ischemic stroke.

Figure 1

HBO effectively reduced infarct volume and brain edema and improved neurological functions. (a) The cerebral infarct volume in the sham, MCAO, and MCAO + HBO groups 72 hours after I/R injury indicated by thionine staining. The normal healthy region was stained blue. Scale bars, 1 mm. (b) Each point in the scatter plot represents the percentage of the infarct volume to the total brain volume (n = 6/group). (c) The percentage of the water content in the brains of the rats in the three different groups 72 hours after I/R injury (n = 6/group). (d) Neurological deficit assessment of different groups 72 hours after I/R injury. Data are expressed as the means ± SD from 8 individual rats in each group. ^∗p < 0.05, compared with the sham group; #p < 0.05, compared with the MCAO group.

Figure 2

HBO inhibited the expression of autophagy-related molecules in the hippocampus following MCAO. (a, c) Western blots for mTOR, p-mTOR, Atg13, LC-3B I, and LC-3B II in the three groups. (b) Bar diagram showing the difference in the mTOR and p-mTOR expression in the ipsilateral cortex of the rats (n = 8/group). (d, e) Statistical analysis showing the expression of Atg13 and LC-3B II/LC3B-I in the ipsilateral cortex of the rats (n = 8/group). ^∗p < 0.05, compared with the sham group; #p < 0.05, compared with the MCAO group.

Figure 3

HBO suppressed the expression of HIF-1α in the hippocampus of MCAO-treated rats. (a) IF analysis showing the difference in the HIF-1α expression in the ipsilateral hippocampus of the rats. Scale bars, 50 μm. (b) Western blots for the HIF-1α expression in each group. (c) Bar graph showing the quantitative analysis of HIF-1α (n = 8/group). ^∗p < 0.05, compared with the sham group; #p < 0.05, compared with the MCAO group.