Selective brain hypothermia-induced neuroprotection against focal cerebral ischemia/reperfusion injury is associated with Fis1 inhibition.

Selective brain hypothermia is considered an effective treatment for neuronal injury after stroke, and avoids the complications of general hypothermia. However, the mechanisms by which selective brain hypothermia affects mitochondrial fission remain unknown. In this study, we investigated the effect of selective brain hypothermia on the expression of fission 1 (Fis1) protein, a key factor in the mitochondrial fission system, during focal cerebral ischemia/reperfusion injury. Sprague-Dawley rats were divided into four groups. In the sham group, the carotid arteries were exposed only. In the other three groups, middle cerebral artery occlusion was performed using the intraluminal filament technique. After 2 hours of occlusion, the filament was slowly removed to allow blood reperfusion in the ischemia/reperfusion group. Saline, at 4°C and 37°C, were perfused through the carotid artery in the hypothermia and normothermia groups, respectively, followed by restoration of blood flow. Neurological function was assessed with the Zea Longa 5-point scoring method. Cerebral infarct volume was assessed by 2,3,5-triphenyltetrazolium chloride staining, and apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. Fis1 and cytosolic cytochrome c levels were assessed by western blot assay. Fis1 mRNA expression was assessed by quantitative reverse transcription-polymerase chain reaction. Mitochondrial ultrastructure was evaluated by transmission electron microscopy. Compared with the sham group, apoptosis, Fis1 protein and mRNA expression and cytosolic cytochrome c levels in the cortical ischemic penumbra and cerebral infarct volume were increased after reperfusion in the other three groups. These changes caused by cerebral ischemia/reperfusion were inhibited in the hypothermia group compared with the normothermia group. These findings show that selective brain hypothermia inhibits Fis1 expression and reduces apoptosis, thereby ameliorating focal cerebral ischemia/reperfusion injury in rats. Experiments were authorized by the Ethics Committee of Qingdao Municipal Hospital of China (approval No. 2019008).

Electroacupuncture preconditioning protects against focal cerebral ischemia/reperfusion injury via suppression of dynamin-related protein 1.

Electroacupuncture preconditioning at acupoint Baihui (GV20) can reduce focal cerebral ischemia/reperfusion injury. However, the precise protective mechanism remains unknown. Mitochondrial fission mediated by dynamin-related protein 1 (Drp1) can trigger neuronal apoptosis following cerebral ischemia/reperfusion injury. Herein, we examined the hypothesis that electroacupuncture pretreatment can regulate Drp1, and thus inhibit mitochondrial fission to provide cerebral protection. Rat models of focal cerebral ischemia/reperfusion injury were established by middle cerebral artery occlusion at 24 hours after 5 consecutive days of preconditioning with electroacupuncture at GV20 (depth 2 mm, intensity 1 mA, frequency 2/15 Hz, for 30 minutes, once a day). Neurological function was assessed using the Longa neurological deficit score. Pathological changes in the ischemic penumbra on the injury side were assessed by hematoxylin-eosin staining. Cellular apoptosis in the ischemic penumbra on the injury side was assessed by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling staining. Mitochondrial ultrastructure in the ischemic penumbra on the injury side was assessed by transmission electron microscopy. Drp1 and cytochrome c expression in the ischemic penumbra on the injury side were assessed by western blot assay. Results showed that electroacupuncture preconditioning decreased expression of total and mitochondrial Drp1, decreased expression of total and cytosolic cytochrome c, maintained mitochondrial morphology and reduced the proportion of apoptotic cells in the ischemic penumbra on the injury side, with associated improvements in neurological function. These data suggest that electroacupuncture preconditioning-induced neuronal protection involves inhibition of the expression and translocation of Drp1.

Electroacupuncture preconditioning attenuates ischemic brain injury by activation of the adenosine monophosphate-activated protein kinase signaling pathway.

Electroacupuncture has therapeutic effects on ischemic brain injury, but its mechanism is still poorly understood. In this study, mice were stimulated by electroacupuncture at the Baihui (GV20) acupoint for 30 minutes at 1 mA and 2/15 Hz for 5 consecutive days. A cerebral ischemia model was established by ligating the bilateral common carotid artery for 15 minutes. At 72 hours after injury, neuronal injury in the mouse hippocampus had lessened, and the number of terminal deoxynucleotide transferase-mediated dUTP nick-end labeling-positive cells reduced after electroacupuncture treatment. Moreover, expression of adenosine monophosphate-activated protein kinase α (AMPKα) and phosphorylated AMPKα was up-regulated. Intraperitoneal injection of the AMPK antagonist, compound C, suppressed this phenomenon. Our findings suggest that electroacupuncture preconditioning alleviates ischemic brain injury via AMPK activation.

[Effect of electroacupuncture pretreatment on expression of glucose-regulated protein 78 in hippocampus of rats with cerebral ischemia/reperfusion].

OBJECTIVE: To explore the effects and action mechanism of electroacupuncture (EA) pretreatment on rats with transient cerebral ischemia/reperfusion. METHODS: A total of 144 healthy SD male rats were randomly divided into a sham operation group (group S), an ischemia/reperfusion group (group I/R) and an EA pretreatment group (group EA), 48 rats in each one. The model of cerebral ischemia/reperfusion was established by using 4-vessel occlusion method in the group I/R; after 5 min of cerebral ischemia, the reperfusion was performed. The group EA was treated with EA at "Dazhui" (GV 14) and "Baihui" (GV 20) 5 days before model establishment, 30 min per time, once a day. In group S, bilateral foramen alares were exposed without burning on the vertebral arteries, and bilateral common carotid arteries were unfolded and not occluded. The rats in the group I/R and group EA were sacrificed 6 h, 12 h, 24 h and 48 h after reperfusion and those in the group S were sacrificed at corresponding time to collect hippocampus example. The Western-blot method was used to measure the expression of glucose-regulated protein 78 (GRP 78), and HE staining method was used to count the number of surviving neurons, and TUNEL method was used to measure the number of apoptotic neurons. RESULTS: Compare with the group S, the number of surviving neurons in hippocampus was reduced at each reperfusion time point and the number of apoptotic neurons was increased (all P<0.05) in the group I/R and the group EA; the expression of GRP 78 at each reperfusion time point in group I/R and group EA was increased (P<0.05). Compared with the group I/R, the number of surviving neurons in hippocampus was increased at each reperfusion time point and the number of apoptotic neurons was reduced in the group EA (P<0.05); the expression of GRP 78 at each reperfusion time point was further increased (P<0.05). CONCLUSION: The electroacupuncture pretreatment has obvious cerebral protection on rats with ischemia/reperfusion, which is related with further increasing the expression of GRP 78 in ischemia area, leading to relieved endoplasmic reticulum stress.

[Effect of electroacupuncture pretreatment on apoptotic neurons and expression of GRP 78 and GADD 153 in the hippocampus in rats with global cerebral ischemia/reperfusion injury].

OBJECTIVE: To observe the effect of electroacupuncture (EA) pretreatment on the number of survival neurons and the expression of glucose regulated protein 78 (GRP 78) and growth arrest and DNA damage-inducible gene 153 (GADD 153) in the hippocampus in rats with global cerebral ischemia /reperfusion injury (CI/R), so as to study its underlying mechanism in neuroprotective action. METHODS: SD male rats were randomly divided into 3 groups (n =48 each):sham operation,Cl/R model and EA pretreatment group. Global CI/R model was induced by 4-vessel occlusion (bilateral vertebral artery cauterization and bilateral carotid artery ligation for 5 min, followed by reperfusion). Before modeling, EA pretreatment of "Baihui" (GV 20) and "Dazhui" (GV 14, 2 Hz/15 Hz, 1 mA) was given to rats of the EA pretreatment group for 30 min, once daily for 5 days. At 6, 12, 24 and 48 h after CI/R, the hippocampus tissues of rats in different subgroups were separately sampled to be stained with H. E. method for detecting the number of the survived neurons, stained with TUNEL method for assaying the apoptotic neurons in the CA 1 region, and processed with Western blot (WB) for assaying the expression of GRP 78 and GADD 153 proteins. RESULTS: Compared with the sham group, the number of hippocampal survival neurons was significantly decreased at the time-points of 12 h, 24 h and 48 h after CI/R in the model group (P<0. 05) and was apparently increased by EA pretreatment at 24 h and 48 h (P<0. 05). The number of the apoptotic neurons in the hippocampal CA 1 region at the time-points of 6 h, 12 h, 24 h and 48 h after CI/R was significantly bigger in the model group than in the sham group (P<0. 05), and was obviously decreased at 12 h, 24 h, 48 h after CI/R in the EA pretreatment group (P<0.05). WB detection showed that the expression levels of hippocampal GRP 78 and GADD 153 proteins at the four time-points after CI/R were significantly higher in the model group than in the sham group (P<0. 05). Compared with the model group, hippocampal GRP 78 protein expression levels at the 4 time-points were further markedly up-regulated (P<0.05), while GADD 153 protein expression levels at the 4 time-points were significantly suppressed in the EA pretreatment group (P<0.05). CONCLUSION: EA pretreatment can effectively suppress the number of hippocampal apoptotic neurons and increase survival rate of neurons in CI/R rats, which may be closely associated with its effects in up-regulating the expression of GRP 78 protein and down-regulating the expression of GADD 153 protein in the hippocampus.