| BackgroundDespite the clinical and laboratory research, which focuses on respiratory and cardiac arrest and cardiopulmonary-cerebral resuscitation, has extended over dozens of years, the survival rate of these conditions remains very low. In developed countries, 40%~50% patients who survive after emergency treatment will suffer from a long-term, severe neurological dysfunction due to transient global cerebral ischemia. However, 68% of such patients eventually die as a result of central nervous system injury, while this proportion is even higher in the developing countries. Except for cardiovascular accidents and accidental injuries in daily life, cardiac arrest caused by intraoperative massive blood loss and other factors is the major reason of global cerebral ischemia which leads to neurological dysfunction. Therefore, how to reduce the neuronal damage caused time (P<0.05), but at 72h after reperfusion the NICD expression in Pre IR group was weaker than the Con group (P<0.05); the NICD expression in the Pre group at 2h was stronger than the sham group (P<0.05), but there was no significant difference between the Pre group at 24h and Sham. 3. Real-time PCR: mRNA expression of the receptor notch-1 in the Con group increased at 2h and 72h after reperfusion, and higher than the sham group (P<0.05); notch-1 expression of the Pre IR group at 2h/24h after reperfusion were higher than the Con group at the same time point (P<0.05), but lower than the Con group at the time point of 72h after reperfusion (P<0.05). The mRNA expression levels of target gene HES-1 in the Con group at 2h/24h after reperfusion increased gradually, were higher than sham group (P<0.05); HES-1 expression of the Pre IR group at 2h/24h after reperfusion were higher than the Con group at the same time point (P<0.05), but at 72h after reperfusion, the HES-1 mRNA expression of the these two groups between Pre IR and Con groups was no significant difference compared with the sham group. The notch-1, HES-1 and NICD expression levels of Pre group at 2h were higher than the sham group (P<0.05), but there were no significant difference between the Pre group at 24h and Sham. Conclusion Isoflurane preconditioning can result in the earlier peak expression of Notch signaling pathway related molecules such as Notch-1, NICD and HES-1, indicating that notch signaling pathway is involved in the isoflurane preconditioning-induced neuroprotection.Experiment 3 The study on the influence of isoflurane preconditioning-induced neuroprotection after Notch signaling pathway was inhibitedObjective To investigate the influence of the Notch signaling pathway inhibitor on the neuroprotective effects induced by isoflurane preconditioning. Methods A total of 58 male C57BL/6 mice were randomly assigned to six groups.①D APT+Preconditioning ischemia-reperfusion group (DAPT Pre IR, n=8): mice were placed in a chamber and preconditioning with isoflurane (1.2% isoflurane, 98% O2, 1 h/day) for 5 days, 3 hours before each preconditioning injection DAPT by intraperitoneal (100mg/kg), 24h after last preconditioning, the BCCAO was performed for 20 min.②V ehicle+Preconditioning ischemia-reperfusion group (Vehicle Pre IR, n=8): mice were placed in a chamber and preconditioning with isoflurane (1.2% isoflurane, 98% O2, 1 h/day) for 5 days, 3 hours before each pretconditioning injection vehicle by intraperitoneal, 24h after last preconditioning, BCCAO was performed for 20 min.③D APT+Control group (DAPT Con, n=8): mice were placed in the same chamber but pretreated with oxygen only (98% O2, 1 h/day) for 5 days, 3 hours before each pretreated injection DAPT by intraperitoneal (100mg/kg), 24h after last pretreated, BCCAO was performed for 20 min.④Vehicle+Control group (Vehicle Con, n=8): mice were placed in the same chamber but pretreated with oxygen only (98% O2, 1 h/day) for 5 days, 3 hours before each pretreated injection vehicle by intraperitoneal, 24h after last pretreated, BCCAO was performed for 20 min.⑤Sham group (sham,n=8): mice were placed in a chamber and preconditioning with isoflurane (1.2% isoflurane, 98% O2, 1 h/day) for 5 days, but without surgery.⑥Inhibitor validation group (n=18). The total motor scores (TMS) observed in three consecutive days after reperfusion in the①~⑤groups, number of viable neurons in the CA1 region of the hippocampus and the TUNEL positive neurons were assessed by HE and TUNEL staining at 72h after reperfusion in the①~⑤groups, respectively. The animals in the inhibitor validation group were single injected of the inhibitor DAPT by intraperitoneal (100mg/kg) , and then sacrificed after 1,2,3,6,12,24 hours to observed NICD expression changes by western blot. Results 1. The total motor scores (TMS) in the Vehicle Pre IR group at 24h/48h/72h after reperfusion were better than the DAPT Pre IR group in the corresponding time points (P<0.05), but the scores of DAPT Pre IR group at 24h/48h/72h after reperfusion were significant better than the DAPT Con and Vehicle Con groups in the corresponding time points (P<0.05); the scores of DAPT Con group at 48h/72h after reperfusion were better than the Vehicle Con groups in the corresponding time points (P<0.05). 2. The numbers of viable neurons in the CA1 region were significantly increased in the Vehicle Pre IR group at 72h after reperfusion compared with those in the DAPT Pre IR group (P<0.05), but the numbers of viable neurons in the DAPT Pre IR group at 72h after reperfusion were higher than the DAPT Con and Vehicle Con groups in the corresponding time points (P<0.05), and DAPT Con group viable neurons at 72h after reperfusion were higher than Vehicle Con in the corresponding time points (P<0.05). 3. The numbers of TUNEL staining positive neurons in the CA1 region were significantly decreased in the Vehicle Pre IR group at 72h after reperfusion compared with the DAPT Pre IR group (P<0.05), the numbers of TUNEL positive neurons in the DAPT Pre IR group at 72h after reperfusion were lower than the DAPT Con and Vehicle Con groups in the corresponding time points (P<0.05), and DAPT Con group positive neurons at 72h after reperfusion were lower than Vehicle Con in the corresponding time points (P<0.05). 4. Inhibitor effect verification: DAPT injection (100mg/kg) by intraperitoneal to suppress the peak after 3 hours and 24 hours to maintain inhibition. Conclusion Notch signaling inhibitor DAPT partially reversed the effects of isoflurane preconditioning induced neuroprotection after cerebral ischemia-reperfusion, indicating that the Notch signaling pathway is activated by isoflurane preconditioning may be an important mechanism for cerebral ischemic tolerance.Conclusions1. Isoflurane preconditioning for 5 days can induce ischemic tolerance in the central nervous system and neuroprotective effects in the transient global cerebral ischemia-reperfusion in mice.2. Isoflurane preconditioning can result in the earlier peak expression of Notch signaling pathway related molecules such as Notch-1, NICD and HES-1, indicating that notch signaling pathway is involved in the isoflurane preconditioning-induced neuroprotection.3. Notch signaling inhibitor DAPT partially reversed the effects of isoflurane preconditioning induced neuroprotection after cerebral ischemia-reperfusion, indicating that the Notch signaling pathway is activated by isoflurane preconditioning may be an important mechanism for cerebral ischemic tolerance.
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