Neuroprotective Effects Of Limb Ischemic Postconditioning On Focal Cerebral Ischemia/reperfusion Injury In Mice And Its Mechanism

Objective: Cerebral ischemia/reperfusion(I/R) injury is the bottleneck of therapy in cerebral ischemia. Limb ischemic postconditioning(LIPost C) is a promising therapeutic intervention, which has been discovered to reduce ischemia/reperfusion(I/R) injury in heart, kidney and brain experimentally. However, its potential protective mechanisms have not been well elucidated. To observe the protective effects of limb ischemic postconditioning against I/R injury, and to investigate whether the Nrf2/ARE pathway mediated anti-oxidative effect is involved in this protection and to detect whether the regulation to KLF2、occludin is involved in the protective effect on blood brain barrier(BBB) damage in the acute phase of cerebral I/R in mice.Methods: Adult male CD1 mice were subjected to transient middle cerebral artery occlusion(t MCAO) surgical. 90 mice were divided into three groups randomly:Sham-operated group, I/R group, LIPost C group. Sham group: the sham-operated group, in which mice were underwent the same surgical procedure without inserting a filament; the I/R group: in which mice were subjected to focal cerebral ischemia; the LIPost C group: in which mice were subjected to focal cerebral ischemia and reversible hind limb ischemia was induced in bilateral hind limbs by occluding and releasing the femoral artery for three cycles(occlusion/release 5 min/5 min)immediately after stroke onset. Neurological deficit scores, brain water content,infarct volume were measured at 24 h after reperfusion. Immunohistochemistry staining, Western blot and RT-q PCR were used to analyze the expressions of Nrf2,NQO-1, HO-1 in ischemic brain cortex. SOD and MDA were measured by spectrophotometer. The loss of BBB integrity was assessed by leakage of Evans blue,Western blot was used to analyze the expressions of KLF2, occludin in ischemic brain cortex.Result: Sham-operated mice had a neurological score of zero. Whereas mice in the I/R group had the highest neurological deficit score compared with the Sham group.Compared with I/R group, there was a significant improvement in neurologicalfunction scores in the LIPost C group(P < 0.05). In I/R and LIPost C group, the brain water content was considerably increased than sham group. Following LIPost C treatment, as compared with I/R group, there was a significant reduction in brain water content in the LIPost C group(P < 0.05). No infarction was observed in Sham group. Extensive lesion was found in both striatum and cortex in I/R group. The infarct volume was significantly reduced in the LIPost C group compare with I/R group(P < 0.05).The number of positive cells in the ischemic cortex of I/R mice was significantly increased compared with Sham group( P < 0.05). In LIPost C group, the number of positive cells of Nrf2, HO-1 and NQO-1 was significantly increased than the I/R group( P < 0.05). Results from Western blot analysis and RT-q PCR analysis show that at protein levels and the m RNA level of Nrf2, NQO-1, HO-1were significantly increased than the I/R group( P < 0.05). Compared with the I/R group, we also found that LIPost C can significantly increased the activities of SOD and decreased the production of MDA( P < 0.05). Compared with I/R group, Evans blue leaked of brain tissues was significantly decreased in LIPost C groups(P < 0.05), Western blot analysis shows that at protein levels KLF2 and occludin were significantly increased than the I/R group( P < 0.05).Conclusions: LIPost C significantly improved neurological deficit, brain edema,infarct volume and Evans blue extravasation. The results indicated that LIPost C protected the brain from I/R injury after focal cerebral ischemia, and it may be through the activation of the Nrf2-ARE pathway and ameliorating BBB permeability by up-regulating the expression of KLF2 and occludin.