Isoflurane Damages The Developing Hippocampus Of Mice And Induces Subsequent Learning And Memory Deficits Through FASL-FAS Signaling

Part 1 Isoflurane damages the developing hippocampus of mice and induces subsequent learning and memory deficitsPurpose:To explore the damage caused by isoflurane on the hippocampus of neonatal mice.Methods:Healthy C57BL/6 newborn mice aged 7 days were randomly assigned to two groups. One was named isoflurane group, which received 1.5% isoflurane in 100% oxygen for 2 hours per day for 3 days totally. The other one was control group. Mice in this group received only 100% oxygen in the same period as the isoflurane group. Keep the temperature and humidity steady. On the 9th day, all mice in both of the groups were killed and the brain was used to test the apoptosis factor, caspase-3. And TUNEL positive cells were counted in the hippocampus. Morris water maze was used to test the subsequent learning and memory ability.Results:Compared to the control group, the expression of caspase-3 in hippocampus of mice in isoflurane group greatly increased and the number of TUNEL positive cells was more. The difference was statistically significant (p<0.05). Compared to the control group, the escape latency of isoflurane group was prolonged, and the platform crossing was decreased. The difference was statistically significant (p<0.05).Conclusion:Isoflurane induced apoptosis in the hippocampus and subsequent learning and memory deficits.Part 2 Isoflurane damages the developing hippocampus of mice and induces subsequent learning and memory deficits through FASL-FAS signalingPurpose:To discuss whether isolfurane could cause damage to the developing hippocampus of neonatal mice through FASL-FAS signalingMethods:Healthy C57BL/6 newborn mice aged 7 days were randomly assigned to two groups. One was isoflurane group, and the other one was control group. The two groups were anaesthetized in the same way as it did in part 1. Then the hippocampus was used to detect the FAS (CD95) protein and FAS ligand (FASL) protein by western blot. Additionally, we anaesthetized the 7 day-aged mice which were FAS knockout and FASL knockout in the same way as mentioned above. Another two groups mice knockout FAS or FASL were exposed to 100% oxygen only. Then the four groups of mice were killed and the brains were harvested. The expression of caspase-3 of the four groups above was detected by western blot. The brains of FAS knockout and FASL knockout groups received isoflurane were prepared for TUNEL assay. These two groups of mice were also tested in the morris water maze. In the end, the expression of caspase-3 of the four groups and the result from part 1 would be put together to draw a conclusion. And the number of TUNEL positive cells of the two gene-knockout-groups together with the result from part 1 would come to a final decision. Taking the results of these two groups in morris water maze and those two groups’in part 1 together, we could arrival at a conclusion.Results:The expression of FAS protein and FASL protein were significantly increased in isoflurane group compared with control group, and the difference was significant (p<0.05). Compared to the FAS knockout group received isoflurane and FASL knockout group received isoflurane, isoflurane clearly increased the expression of caspase-3 and the number of TUNEL positive cells in hippocampus of health mice. The difference was significant (p<0.05). However, the difference of the two indicators among the control group, FAS knockout group received isoflurane and FASL knockout group received isoflurane was not significant. And the result of two-way ANOVA analysis showed FAS or FASL knockout might attenuate the increase of caspase-3 caused by isoflurane. Compared with the two gene-knockout-groups received isolurane, the isoflurane healthy group prolonged the escape latency and reduced the platform crossing. The difference was significant (p<0.05). No significant difference appeared among the control group and the two gene-knockout-groups received isoflurane.Conclusion:Isoflurane could induce apoptosis in developing hippocampus of normal healthy neonatal mice but FAS knockout or FASL knockout mice through FASL-FAS signaling to damage the hippocampus and induce subsequent learning and memory deficits. We drew the conclusion that FASL-FAS signaling is one of the mechanisms for isoflurane affecting the developing brain.