Sevoflurane Preconditioning Induced Neuroprotection Associated With Restoring Akt Activity Via CTMP Inhibition In Rats

It has been confirmed that dysregulation of the pro-survival signal Akt contributes toneuronal damage after ischemic stroke. Neuroprotective strategies such as ischemicpreconditioning and sevoflurane postconditioning promote neuronal survival by activatingAkt[5-7]. A robust increase of phosphorylation level of Akt occurred after ischemiaparalleling its sustained poor function in phosphorylating the downstream pro-apoptoticsubstrates[8,9]. Such data indicate that the phosphorylation level of Akt is not alwaysparallel with its activity. The endogenous inhibitor of Akt, carboxy-terminal modulatorprotein (CTMP)[11], increased robustly after ischemia, it probably participates in thedamage to neurons in the ischemic stroke and be regulated by sevofluranepreconditioning.The current study was designed to explore the role and mechanism of CTMP inischemic tolerance induced sevoflurane preconditioning, improving our understanding ofsevoflurane preconditioning and providing evidence of new research direction for the neuroprotection of brain ischemia in patients.Experiment1Single sevoflurane preconditioning induced neuroprotectionagainst focal cerebral ischemia and reperfusion injuryMethod：To determine the neuroprotection of single sevoflurane preconditioning，40SD rats were random assigned to5groups: a sham operation group (Sham), anischemia/reperfusion (I/R) group observed24h, a sevoflurane preconditioning (PC+I/R)group observed24h, an I/R group observed7d and a PC+I/R group observed7d (Sham=4;others, n=9). Preconditioned animals were exposed to2.7%sevoflurane for45mins at1hbefore ischemia. The neurological deficits and infarct volumes of the rats were evaluated24h or7d after reperfusion by a researcher blinded to the animal groups.Result：Sevoflurane preconditioning significantly reduced the infarct volume of therats suffering focal cerebral ischemia at24h and7d after reperfusion, and amelioratedneurological dysfunction at24h after reperfusion.Experiment2Akt pathway was involved in the neuroprotection of sevofluranepreconditioningMethod：To investigate the role of Akt in the induction of ischemic tolerance bysevoflurane preconditioning, we randomly assigned85rats to9groups: a Sham group(n=5), an I/R group, a PC+I/R group, a sevoflurane preconditioning with wortmannin(WT+PC+I/R) group, a sevoflurane preconditioning with LY294002(LY+PC+I/R) group,an ischemia with wortmannin (WT+I/R) group, an ischemia with LY294002(LY+I/R)group and two vehicle (VE-WT+PC+I/R and VE-LY+PC+I/R) groups. Preconditionedanimals were exposed to2.7%sevoflurane for45mins at1h before ischemia. Theneurological deficits and infarct volumes of the rats were evaluated24h after reperfusionby a researcher blinded to the animal groups. Wortmannin and LY294002wereadministered to the rats10mins before preconditioning. An equal volume of vehicle wasadministered to the rats in the same manner in the VE-WT+PC+I/R and theVE-LY+PC+I/R group.Result： Wortmannin and LY294002injection lessened the neuroprotection of sevoflurane preconditioning.Experiment3Sevoflurane preconditioning suppressed the CTMPover-expression induced by ischemia injury and restored Akt activityMethod： To determine the effect of sevoflurane preconditioning on thephosphorylation levels of Akt and its downstream target GSK3β, as well as changes inCTMP levels and the activity of Akt kinase after ischemia,64rats were random assignedto Sham, I/R, PC or PC+I/R group. Preconditioned animals not subjected to ischemia(noted as0h) were sacrificed24h after preconditioning. Rats in the I/R and PC+I/Rgroups were sacrificed1,3,12and24h after reperfusion (n=5). Akt activity wasdetermined3h after reperfusion with another16SD male rats (n=4).Result：Ischemia induced a transient increase in the phosphorylation of Akt at Ser473(pSer473-Akt), whereas sevoflurane preconditioning suppressed the phosphorylation ofAkt elicited by ischemia3h after reperfusion. Phosphorylation level of GSK3β(pSer9-GSK3β), a downstream target of Akt, was higher in the PC+I/R group compared tothe I/R group at1h and3h after reperfusion. An Akt kinase activity assay showed thatsevoflurane preconditioning increased Akt activity3h after reperfusion, which wasconsistent with the increased pSer9-GSK3β expression in vivo. Expression of CTMP inthe rats of the I/R group began to increase3h after ischemia/reperfusion and remainedhigher than that of the sham group at24h. Sevoflurane preconditioning suppressed theincrease in CTMP expression induced by I/R injury.Experiment4CTMP over-expression by LV-CTMP markedly suppressed thebeneficial effects of sevoflurane preconditioningMethod：To examine the effect of CTMP in sevoflurane preconditioning andischemia/reperfusion, we used lentiviral transduction (LV) to increase the expression ofCTMP before preconditioning. Rats received an intracerebroventricular injection of eitherLV-CTMP or LV-C (scrambled control vector)3d before preconditioning. After24h ofreperfusion, rats were subjected to evaluation of neurologic scores and sacrificed to determine infarct volume. Expression of CTMP and the activity of Akt kinase in theLV-CTMP, LV-C and sham operation groups was examined by Western Blot analysis.Result：Injection of LV-CTMP led to an approximately3-fold increase of CTMPprotein expression and a suppression of Akt kinase activity in the targeted brain regions.CTMP over-expression markedly lessened the beneficial effects of sevofluranepreconditioning.