This study examined the course of mitophagy following cerebral ischemia with reperfusion and the role of the PTEN-induced kinase 1 (PINK1)/Parkin/p62 signalling pathway. The middle cerebral artery of male Sprague-Dawley rats was occluded for 90 min and was followed by different time-points of reperfusion. Cerebral infarct areas were detected by 2,3,5-triphenyl tetrazolium chloride staining, while brain damage was observed by haematoxylin and eosin staining. Levels of LC3, Beclin1 and LAMP-1 were estimated by western blots. LC3B location was observed in various cells in the neurovascular unit. In addition, PINK1 accumulation in damaged mitochondria and Parkin/p62 mitochondrial translocation were investigated by double immunofluorescence staining. Finally, the levels of PINK1, Parkin and p62 expression in mitochondrial fractions were estimated by western blots. Cerebral ischemia with different time-points of reperfusion resulted in infarct in the territory of the middle cerebral artery accompanied by overall brain damage. In addition, we found up-regulation of LC3B, Beclin1, and LAMP-1, as well as mitophagy activation after reperfusion, with peak expression of these proteins at 24 h after reperfusion. Electron microscopy and immunofluorescence indicated that LC3B was primarily located in neurons, although lower levels of expression were found in astrocytes and even less in vascular endothelial cells. Moreover, significant increases in PINK1 accumulation in the outer membrane of mitochondria and increased Parkin/p62 mitochondrial translocation were shown at 24 h after reperfusion. These findings suggest that the PINK1/Parkin/p62 signalling pathway was involved in the pathophysiological processes following ischemia and reperfusion.
Keywords: Cerebral ischemia-reperfusion injury; Mitophagy; PINK1/Parkin/p62 signalling pathway.
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