13Mitochondrial immoderate fission induces neuronal apoptosis following focal cerebral 14 ischemia-reperfusion (I/R) injury. With fewer complications, selective brain 15 hypothermia (SBH) is considered an effective treatment against neuronal injury after 16 stroke. However, the specific mechanism by which SBH influences mitochondrial 17 fission remains unknown. Mitochondrial fission 1 protein (Fis1), a key factor of the 18 mitochondrial fission system, regulates mitochondrial dynamics. This study aimed to 19 investigate whether SBH regulates Fis1 expression in focal cerebral I/R injury. In this 20 study, rat middle cerebral artery occlusion (MCAO) models were established. After 2 21 h of occlusion, cold saline or normal saline was pumped into rats in different groups 22 through the carotid artery, followed by restoration of blood perfusion. Cortical and 23 rectal temperatures showed that the cold saline treatment achieved SBH. Cerebral I/R 24 injury increased neurological deficit scores(NDS); neuronal apoptosis; Fis1 protein 25 and mRNA expression; cytosolic cytochrome c (cyto-Cyto c) protein expression at 6, 26 24 and 48 h postreperfusion; and cerebral infarct volumes at 24 h postreperfusion. 27 Interestingly, SBH inhibited Fis1 protein and mRNA expression, blocked cyto-Cyto c 28 protein expression, preserved neuronal cell integrity, and reduced neuronal apoptosis. 29 However, normal saline treatment rarely resulted in positive outcomes. Based on 30 these results, SBH inhibited Fis1 expression, thus ameliorating focal cerebral I/R 31 injury in rats.32 3 33 Introduction 34 Stroke is one of the leading causes of death and disability in the world [1-3]. Ischemic 35 stroke caused by cerebral thrombosis or endovascular embolization accounts for a 36 major portion of strokes. Early restoration of blood perfusion is the most effective 37 treatment for stroke; this treatment provides nutrients and oxygen and removes toxic 38 metabolites [4,5]. However, vessel recanalization often exacerbates the existing tissue 39 damage [6,7]; thus, this condition is called cerebral ischemia-reperfusion (I/R) injury. 40 Recent studies have suggested that morphological changes in mitochondria might be 41 relevant to I/R injury [8,9]. Within the cell, mitochondria exist in an ever-changing 42 dynamic state-constant fission and fusion-to form mitochondrial networks and 43 maintain cellular physiological function and survival [10]. Notably, growing evidence 44 has indicated that I/R injury can break this balance to induce neuronal apoptosis 45 [11-13]. Mitochondrial fission protein 1 (Fis1), a 16-kDa protein anchored to the outer 46 membrane of the mitochondria, mediates mitochondrial fission by recruiting 47 cytoplasmic dynamin-related protein1 (Drp1) into the mitochondrial outer membrane 48 [14]. Overexpression of Fis1 increases the frequency of mitochondrial fission, 49 subsequently increasing the release of Cyto c and disrupting mitochondrial membrane 50 potential, thus inducing neuronal apoptosis [15]. In addition, in cells with th...