Effects Of Mild Hypothermia On Mitochondrial Fission-related Dynamin-related Protein 1 In The Forebrain Cerebral Ischemia-reperfusion Injury Of Mice

Objective: To establish forebrain ischemia-reperfusion injury,blocking bilateral common carotid arteries were performed in male C57BL6 mice.Alcohol cooling method was used to reduce body temperature of mice to 32-34°C.To investigate the associations between ischemia-reperfusion injury and cell apoptosis and the effect of mild hypothermia on mitochondrial fission-related cell apoptosis following cerebral ischemia-reperfusion(IR)injury of mice,the Dynamin related protein 1 and cytochrome C expression were detected in brain tissues of mice.Methods:1.Experimental groups Ninety-six healthy male C57BL6 mice were randomized to three groups(n=32each):sham operation group(Group S),IR injury under normothermia(Group NT)and mild hypothermia combined with IR injury group(Group HT).2.Experimental method and the implementation of mild hypothermiaForebrain IR injury was induced by 2-vessel occlusion method.Bilateral common carotid arteries were transiently occluded for 15 min.For mice in the hypothermia group,rectal temperature was decreased to 32–34°C by spraying 75% alcohol onto the mouse’s body,and was brought back to normal with a feedback-controlled heating pad.Hypothermia maintained for 4 hours after reperfusion.For mice in the sham group,2 vessels were exposed but without occlusion.3.Sample preparation3.1 HE staining At 24,72 h of reperfusion,the mice were transcardially perfused with 0.9%Na Cl,followed by 4% paraformaldehyde(PFA).Brains were quickly removed and post fixed in 10% formaldehyde and brain tissue was embedded with paraffin.HE staining was performed.Then neutral balsam was used for mounting and the section was observed by the microscope.3.2 TUNEL staining TUNEL method was performed according to the manufacturer’s protocol.TUNEL positive-cells counting were observed by the microscope.3.3 Western Blot analysis When mice were sacrificed after anesthetized,the brains were quickly removed and the hippocampus and cortex region were rapidly dissected.For western blotting,the brain tissues were immediately removed and were used or frozen in liquid nitrogen and stored at-80°C until use.Drp1 and Cyt C expression were detected.3.4 Immunohistochemistry analysis After anesthetized,the mice were transcardially perfused with 0.9% Na Cl,followed by 4% paraformaldehyde(PFA).Immunohistochemical staining was used to detect the expressions of Cyt C and Drp1 protein.Results: 1.Compared with the sham group,the numbers of intact pyramidal cells of hippocampus CA1 region decreased in the NT group and in the hypothermia(HT)group,while the number of neurons with nuclear pyknosis and TUNEL-positive cells increased significantly in the two last groups.Furthermore,compared with the HT group,the neuronal degeneration in the NT group was more severe than the HT group.But the HT group had a lower levels of TUNEL-positive cells than the NT group.2.Compared with the sham group,the expressions of Drp1 and Cyt C were significantly higher in NT group,but the expressions of Drp1 and Cyt C protein were significantly lower at each time point in HT group(P<0.05).Conclusions: 1.Mild hypothermia preserved the neurons integrity and attenuated cerebral IR injury-induced apoptosis.2.Cerebral ischemia-reperfusion injury increased the expressions of mitochondria fission-related protein Drp1 and Cyt C proteins.3.Mild hypothermia down-regulated IR-induced elevated mitochondria fission-related protein Drp1 and Cyt C protein in brain tissues of mice,thus inhibiting excessive mitochondria fission-related apoptosis pathway and reducing cerebral IR injury damage.