Supplementary Material for: Melatonin Attenuates Cerebral Ischemia/Reperfusion Injury Through Inducing Autophagy

Introduction: The aim of this study was to investigate how melatonin administration for 3 days or 7 days following cerebral ischemia injury (CI/R) would affect autophagy, and therefore, survival in neurons of the penumbra region. Moreover, it was also aimed to determine how this melatonin treatment would affect the neurological deficit score and rotarod and adhesive removal test durations. Methods: Focal CI (90 min) was achieved in a total of 105 rats utilizing a middle cerebral artery occlusion model. After the start of reperfusion, the groups were treated with melatonin (10 mg/kg/day) for 3-days or 7-days. On all groups, neurological deficit scoring, rotarod and adhesive removal test were executed during reperfusion. Infarct areas were determined by TTC (2,3,5-triphenyltetrazolium chloride) staining at the end of the 3rd and 7th days of reperfusion. Beclin-1, LC3, p62 and caspase-3 protein levels were assessed using Western blot and immunofluorescence methods in the brain tissues. Moreover, penumbra areas were evaluated by transmission electron microscopy (TEM). Results: Following CI, it was observed that melatonin treatment improved the rotarod and adhesive removal test durations from day 5 and reduced the infarct area after CI. It also induced autophagic proteins Beclin-1, LC3 and p62 and suppressed the apoptotic protein cleaved caspase-3. According to TEM findings, melatonin treatment partially reduced the damage in neurons after CI. Conclusion: Melatonin treatment following CI reduced the infarct area and induced the autophagic proteins Beclin-1, LC3 and p62 via inhibiting the apoptotic caspase-3 protein. The functional reflection of melatonin treatment on neurological tests scores was became significant from the 5th day onward.

研究背景：本研究旨在探讨脑缺血再灌注损伤（cerebral ischemia injury, CI/R）后给予3天或7天褪黑素干预对自噬（autophagy）及半暗带区域（penumbra region）神经元存活的影响，同时明确该褪黑素治疗方案对神经功能缺损评分（neurological deficit score）、转棒疲劳试验（rotarod）与黏贴移除试验（adhesive removal test）时长的作用效果。
研究方法：本研究共纳入105只大鼠，采用大脑中动脉闭塞模型（middle cerebral artery occlusion model）实施90分钟局灶性脑缺血造模。再灌注（reperfusion）开始后，分组给予褪黑素（melatonin）10 mg/kg/天，分别干预3天或7天。所有组均在再灌注期间实施神经功能缺损评分、转棒疲劳试验及黏贴移除试验。于再灌注第3天及第7天结束时，通过氯化三苯基四氮唑（TTC, 2,3,5-triphenyltetrazolium chloride）染色检测脑梗死面积。采用蛋白质印迹法（Western blot）与免疫荧光法（immunofluorescence）检测脑组织中Beclin-1、LC3、p62及半胱氨酸天冬氨酸蛋白酶-3（caspase-3）的蛋白表达水平。此外，通过透射电子显微镜（TEM）评估半暗带区域的组织形态。
研究结果：脑缺血再灌注损伤后，研究观察到褪黑素治疗自第5天起可改善转棒疲劳试验与黏贴移除试验的时长表现，并缩小脑梗死面积；同时上调自噬相关蛋白Beclin-1、LC3及p62的表达，抑制凋亡蛋白剪切型半胱氨酸天冬氨酸蛋白酶-3（cleaved caspase-3）的活化。透射电子显微镜结果显示，褪黑素治疗可部分减轻脑缺血再灌注损伤后神经元的受损程度。
研究结论：脑缺血再灌注损伤后给予褪黑素治疗，可通过抑制凋亡相关蛋白caspase-3的表达，缩小脑梗死体积并上调自噬相关蛋白Beclin-1、LC3及p62的表达。褪黑素治疗对神经功能测试评分的改善作用自第5天起具备统计学显著性。