The Role Of Notch Signaling Pathway In The Neuroprotection Induced By Sevoflurane

Sevoflurane, as a popular anesthesia, can induce the ischemic tolerance in animals. However, the machenism of neuroprotection induced by sevoflurane is poorly understood.The Notch signaling pathway is a highly conserved cell signaling system present in most multicellular organisms. The notch signaling pathway is important for cell-cell communication, which involves cell differentiation, proliferation and apoptosis during embryonic and adult life. Recent studies indicated that Notch was involved in apoptosis induced by brain ischemia-reperfusion injury, which suggested that Notch pathway might be critical to ischemic tolerance conferred by sevoflurane preconditioning. This potential machenism is promising to offer a new target to the prevention of brain ischemia-reperfusion injury.Therefore, constructing the transcriptor RBP-J knockout mice, this research is to study the role of the canonical Notch signaling pathway in the neuroprotection induced by sevoflurane in MCAO, and to verify the crosstalk Part 1 The role of Notch signaling pathway in focal cerebral ischemic injuryObjectives1. To study the influences of brain ischemia reperfusion on the expression of Notch signaling pathway.2. To study the role of Notch pathway in the brain ischemia-reperfusion injury.Methods1. The expression changes of Notch signaling after MCAO in mice Male C57BL/6 mice were devided into 2 groups:Sham (n=5) and MCAO. MCAO group received 1-h brain ischemia, ischemic penumbra tissue was distracted 2,24,72 hours (n=5) after reperfusion. NICD protein level was deteced by Western blot, the transcriptional levels of target genes Hesland Hes5 were determined by Real-time PCR.2. The role of Notch pathway inhibitor in MCAO-induced injury 36 male C57BL/6 mice were devided into 3 groups:Sham (n=12), MCAO and DAPT. MCAO group received 1-h brain ischemia, DAPT group received Notch inhibitor DAPT (100mg/kg i.p.) 2 hours before MCAO. The neurobehaviors of all animals were evaluated by Garcia and Longa methods. The brain infarct volumes were assessed 72 hours after reperfusion.3. The effets of knocking out RBP-J in MCAO-induced injury Male wild-type C57BL/6 mice, RBP-J knockout mice (RBP-J-/-) and heterozygous mice (RBP-J+/-) were used (n=12). All animals were given 1-hour MCAO. Neurobehaviors were evaluated by Garcia and Longa methods at 2h,24h, 72h after reperfusion. At 24h after reperfuion,5 mice per group were randomly perfused to stain Caspase-3 and TUNEL. The left were usd to evaluated the brain infarct volume at 72 hours after reperfusion.Results1. NICD protein level was increased by brain ischemia-reperfusion insults.Results of Western blot showed:NICD levels in ischemic penumbra were higher than those of Sham and MCAO (P<0.05); at 24h after reperfusion, NICD (2.62±0.32) was higher than that of 2h group(P<0.05), and lower than that of 72h group (3.28±0.19, P<0.05).2. Transcriptional levels of Hes1 and Hes5 in penumbra tissues were increased by brain ischemia reperfusionReal-time PCR results showed, Hesl mRNA was increased 24h after reperfusion (P<0.05 vs. Sham),72 hours after MCAO, Hes1 mRNA was higher than that of 24h group (P<0.05)。Hes5 mRNA was elevated 24h and 72h after reperfusion (P<0.05 vs. Sham). There was no difference between 24h and 72h groups.3. DAPT decreased MCAO-induced injuries in miceNeurobehaviors assessed by Garcia method in DAPT group at 24,48,72h after reperfusion were higher than those of MCAO (P<0.05). Neurologic deficit scores evaluated by Longa method at 72h after reperfusion were decreasd (P<0.05 vs. MCAO). The infarct volume of DAPT group was smaller than that of MCAO group (P<0.05).4. Construction of RBP-J knockout miceBy mating RBP-J-floxed mice with CamKIIa-Cre transgenic mice, we obtained bi-transgenic mice, which were then mated with each other to generate homozygous RBP-J knockout mice. The genotype of knockout mice was identified by PCR analysis on DNA extracted from tail biopsy. RBP-J floxed mice, CamKIIa-Cre transgenic mice and RBP-J knockout mice were viable and healthy, and showed no macroscopic abnormalities by gross observations. Inmmunohistochemistry results showed that, compared with wild-type mice, the number of NICD positive neurons in hippocampus and cortex of knockout mice were significantly lower, and the expression of Hes1 and Hes5 protein was weak.5. Koncking out RBP-J decreased the injuries induced by MCAOThe Garcia scores at 24,48 and 72h after reperfusion in RBP-J-/- group were higer than those in MCAO and RBP-J+/- gourps (P<0.05). No siginificant difference was found between MCAO and RBP-J+/- groups. Longa scores were decreased at 72 hours after reperfusion in comparison with other two groups (P<0.05)。Infarct volumes in RBP-J-/- group was smaller than those of WT and RBP-J+/- groups (P<0.05). There was no significant difference between WT and RBP-J+/- groups.Results of TUNEL and Caspase-3 Staining showed the number of TUNEL and Caspase-3 positive cells in WT group was more than those of RBP-J-/- group. Apoptotic cells in RBP-J+/- group were even less than those in RBP-J-/- group. No difference was found in WT and RBP-J+/- groups。 Part 2 The mechamisms of Notch activating the JAK-STAT signaling pathways in cerebral ischemia reperfusionObjectives1. To study the corelation between the activations of Notch and STAT3 during ischemia reperfusion.2. To study the regulatory mechanism of Notch in the STAT3 activation during ischemia reperfusion.Methods1. The activation of Notch was correlated with JAK-STAT after cerebral ischemia reperfusion20 wild-type C57BL/6 mice were devided into Naive and MCAO groups. Naive group (n=5) was decapticated without pretreatment. MCAO group suffered 1-hour ischemia.2h,24h and 72h after reperfusion (n=5), brain penumbra tissues were harvested in all animals. The expression of NICD, Hes1, Hes5, STAT3 and pSTAT were determined by Western blot.2. The influence of Notch on the activation of STAT3 after brain ischemia reperfusion16 male RBP-J knockout mice (RBP-J-/-) were grouped into Naive and MCAO. Naive group (n=4) were decapticated without pretreatment. MCAO group suffered 1-hour ischemia.2h,24h and 72h after reperfusion (n=4), brain penumbra tissues were harvested in all animals. The expression of Hesl, Hes5, STAT3 and pSTAT were determined by Western blot.3. Hes protein regulated the phosphorylation of STAT330 male wild-type C57BL/6 mice were devided into Hes1-SiRNA, Hes5-SiRNA, Vehicle, Hes1-SiRNA+MCAO, Hes5-SiRNA+MCAO, and Vehicle+MCAO. The expression of Hes1, Hes5, STAT3 and pSTAT3 24h after reperfusion were evaluated by Western blot.Results1. Brain ischemia reperfusion activated both the Notch and STAT3NICD protein expressions were increased in wild-type mice at 2h,24h,72h after MCAO. The expressions of Notch target genes Hes1 and Hes5 were higher than those of Naive and 2h groups at 24h and 72h after reperfusion. But there was no difference of Hes1 or Hes5 between 24h and 72h gorups. The level of STAT3 protein in Naive didn't have difference with MCAO. pSTAT3/STAT3 was increased at 2h after reperfusion.2. The activation of STAT3 after cerebral ischemia reperfusion was regulated by Notch signaling pathwayNICD level in RBP-J-/- group was increased 2h after reperfusion, and no significant difference was found among 2h,24h and 72h groups. Western blot showed no change of Hes1, Hes5, STAT3 and pSTAT3/STAT3 between RBP-J-/-and WT groups.3. Hes1-SiRNA and Hes5-SiRNA inhibited the expression of Hesl and Hes5 respectivelyWestern blot showed Hes1 protein in wild type mice significantly decreased 24h after Hes1-SiRNA injection, and Hes1 protein was also decreased in a mice brain.This decrease sustained 48h-72h after reperfusion. Hes5-SiRNA inhibited the expression of Hes5 24h after injection. The inihibition persisted until 48-72h after injection.4. The phosphorylation of STAT3 was regulated by Hes1The expression of Hes1 in Hesl-SiRNA was lower than that in Vehicle 24 hours after reperfusion, and Hesl in SiRNA+MCAO group was even lower than Vehicle+MCAO. There was no change of STAT3 content in 4 groups. No pSTAT3 protein was found in Hes1-SiRNA and Vehicle and only weak expression was found in SiRNA+MCAO group. But pSTAT3 expression in Vehicle+MCAO was higher than other three groups.Compared with Vehicle, Hes5 protein in Hes5-SiRNA group was lower at 24 hour after reperfusion. Hes5 in Hes5-SiRNA+MCAO was much lower than Vehicle+MCAO group. There was no change of STAT3 among 4 groups. pSTAT3 in SiRNA+MCAO and Vehicle+MCAO were higher than SiRNA and Vehicle group.Part 3 The role of Notch signaling pathway in neuroprotection induced by sevofluraneObjectives1. To study the brain ischemic tolerance induced by sevoflurane preconditioning2. To study the influence of sevoflurane on the expression of Notch signaling pathway3. To study the role of Notch signaling pathway in the neuroprotection induced by sevofluraneMethods1. The neuroprotective effects of sevoflurane preconditioning30 C57BL/6 were devided into 3 groups (n=10):Sham, MCAO and Sevo+MCAO. Sevo+MCAO group received 2.5% sevoflurane with100% oxygen preconditioning, lh/d, for consecutive 5 days. MCAO was given 24 hours after last preconditioning. The neurologic scores of all animals were assessed 24,48 and 72 hours after reperfusion; TTC staining was used to evaluate the brian infarct volume 72h after reperfusion.2. The influence of sevoflurane on the expression of Notch signaling pathway10 male C57BL/6 mice were grouped into Sham and Sevo(n=5). Sevo pretreated with 2.5% sevoflurane, 1h/d, for consecutive 5 days, while Sham group was given oxygen preconditioning at the same time. Oh,2h,24h after the last preconditioning, NICD content was detected by Western blot and Hes1 and Hes5 mRNA by Real-time PCR.3. The influence of sevoflurane preconditioning on the expression of Notch signaling pathway after brain ischemia reperfusionMale C57BL/6 mice were randomly devided into 3 groups (n=5):Sham, MCAO and Sevo+MCAO.2h,24h,72h after reperfusion, NICD content was detected by Western blot and Hes1 and Hes5 mRNA by Real-time PCR in all animals.4. The role of Notch signaling inhibitor in the MCAO-induced injuryMale C57BL/6 mice were randomly devided into 5 groups (n=10):Sham, Vehicle, DAPT, Sevo, Sevo+DAPT. Sevo+DAPT was administered DAPT 100mg/kg, i.p., DAPT and Vehicle received the DMSO (vehicle of DAPT). All animals were subject to MCAOat 24h after MCAO,24,48,72h after reperfusion, neurologic scores were evaluated, infarct volume were assessed at 72h after reperfusion.5. The effects of knocking out RBP-J on sevoflurane-induced neuroprotection24 male wild-type (WT) C57BL/6 mice,12 RBP-J knockout mice (RBP-J-/-) and 12 heterozygous mice (RBP-J+/-) were used in four groups:WT, Sevo, Sevo+RBP-J-/-, Sevo+RBP-J+/-. All animals were subjected to 1-hour MCAO,4 mice were randomly chosen to have Caspase-3 and TUNEL staining. The others were used to evaluate the neurologic function and infarct volumes.Results1. Sevoflurane preconditioning reduced the brain ischemic injuries Compared with MCAO, neurologic scores in Sevo+MCAO were significantly increased at 24,48, and 72 h after reperfusion. Neurologic scores at 72h were even higher than that at 48h in Sevo+MCAO group. The infarct volume percentages in Sevo+MCAO group were smaller than that of MCAO.2. Sevoflurane preconditioning activated Notch signaling pathway Western blot results showed that, NICD was upregulated 2h after sevoflurane preconditioning.24 hours after preconditioning, the level of NICD was higher than Sham group, but lower than that of 2h group. Real-time PCR showed Hesl mRNA were elevated at 2-24h after preconditioning. Hes5 mRNA was increased at 2h after preconditioning, but had no significant difference at 24h compared with basic level.3. Sevoflurane preconditioning reduced the activation peak of Notch signaling after MCAONICD content in Sevo+MCAO group was more than Sham at 2h and 24h after MCAO (P<0.05), but had no difference with MCAO group.72h after reperfusion, NICD in Sevo+MCAO group was higher than basic level(P<0.05), but lower than MCAO group(P<0.05).Hesl mRNA was upregulated at 24h after brain ischemia reperfusion with sevoflurane preconditioning (P<0.05 vs. Sham), but had no difference with MCAO group. In Sevo+MCAO group, Hes1 mRNA was lower than that of MCAO group at 72h after reperfusion (P<0.05). However, although Hes5 mRNA in Sevo+MCAO was elevated at 2h and 24h after reperfusion (P<0.05 vs. Sham), there was no difference between Sevo+MCAO and Sham at 72h after reperfusion. Besides, no difference of Hes5 mRNA was found at 2,24 72h after reperfusion in Sevo+MCAO and MCAO groups.4. DAPT reversed the neuroprotection of sevoflurane preconditioningDAPT or its vehicle alone had no effect on brain infarct volumes. Compared with Sevo, Sevo+DAPT group induced a larger infarct volume, which had no statistical difference with Vehicle. At 48h and 72h after MCAO, Sevo group showed a better neurologic function than Vehicle, the scores of which were even higer than those of Sevo+DAPT group. Neurologic scores in Sevo+DAPT group had no difference with Vehicle amd DAPT group.5. Koncking out RBP-J abolished the neuroprotection induced by sevoflurane preconditioningThe infarct volume in Sevo+RBP-J+/- group was smaller than that of WT group (P<0.05), but had no significance with Sevo grouop. Compared with Sevo, Sevo+RBP-J-/- conferred a larger infarct volume (P<0.05).24h,48h and 72h after reperfusion, the neurologic scores in Sevo and Sevo+RBP-J+/- groups were higher than those of WT (P<0.05), there was no difference between Sevo and Sevo+RBP-J+/-. Neurologic scores in Sevo+RBP-J-/- group were significantly lower than those of Sevo (P<0.05), and had no difference compared with WT. Less TUNEL and Caspase-3 positive cells were found in Sevo and Sevo+RBP-J+/- groups, and no difference was found between these two groups. In Sevo+RBP-J-/- group, TUNEL and Caspase-3 positive cells were more than those in Sevo group. There was no difference between Sevo+RBP-J-/- and WT groups. Conclusion1. Notch signaling pathway can be activated by cerebral ischemia reperfusion, and involved in ischemic injuries.2. Notch signaling pathway regulates the activation of STAT3 after cerebral ischemia reperfusion.3.2.5% sevoflurane preconditioning induces ischemic tolerance in mice.4. Notch signaling pathway can be activated by sevoflurane preconditioning, and involved in neuroprotection by anti-apoptosis.