Metformin Pretreatment Protects Against Acute Ischemic Brain Injury In Mice

Aim:Long-term metformin treatment reduces the risk of ischemic stroke in diabetic patients. However, whether metformin protects against acute cerebral ischemia is unclear. We aimed to identify the effects and features of metformin on acute ischemic brain injury in mice and further explore the mechanisms.Methods:Male C57BL/6 mice were subjected to permanent or transient middle cerebral artery occlusion (pMCAO or tMCAO). Metformin of 3,10 and 30 mg/kg/d was intraperitoneally injected 1,3 or 7 days prior to MCAO, or at the onset, or 1,3 or 6 hours after reperfusion, respectively. At 12 h after occlusion or reperfusion, right brain cortex tissues were collected for determining cell apoptosis, both total and phosphorylated AMPK expressions. And at 24 h after occlusion or reperfusion, infarct volumes and neurological deficit scores were determined.Results:Prolonged pretreatment of 7 days of metformin (10 mg/kg/d) significantly ameliorated brain infarct size and neurological deficit scores at 24 h after pMCAO. Metformin also remarkably inhibited ischemia-induced cell apoptosis as revealed by the increase of Bcl-2/Bax and the decline of cleaved Caspase-3 expression. However, other dosages of metformin,3 mg/kg/d and 30 mg/kg/d, showed no neuroprotection. Shorter pretreatment duration with 3 days of metformin 10 mg/kg/d reduced this neuroprotection. Whereas 1 day or without pretreatment of metformin had no beneficial effects. In tMCAO mice, metformin cannot rescue ischemia-reperfusion-induced brain injury even with pretreatment. Furthermore, delayed metformin treatment at 3 h after reperfusion even aggregated the ischemic injury. The expressions of total and phosphorylated AMPK were sharply decreased only with effective metformin pretreatments in ischemic brains. Compound C, an AMPK inhibitor, showed additional neuroprotective effects to metformin against pMCAO model in mice.Conclusions:Our data indicated that pretreatment is required for the neuroprotection of metformin, a 7-days pretreatment duration of 10 mg/kg/d metformin protects against acute ischemic brain injury. Metformin is not beneficial in the cases with blood reperfusion. AMPK down-regulation may be involved in the neuroprotection of metformin pretreatment.