The Role Of NMDAR-NMNAT In Sevoflurane Exposure Induced Learning Memory Deficits And The Change Of Spine In Developing Hippocampal Neurons Of Neonatal Rats

Clinically,inhalation anesthetics is the main anesthetics for infants and young children, and sevoflurane is the mostcommom inhalation anesthetics used in fetal surgery during pregnancy and neonatal surgery.Sevoflurane exposure may affect the development of structure and function of the nervous system.This subject was aimed at investigating the mechnism of sevoflurane exposure induced learning memory deficits and the change of spines in developing hippocampal neurons of neonatal rats.Objective To investigate the role of NMDAR-NMNAT in sevoflurane exposure induced learning memory deficits and the change of spines in developing hippocampal neurons of neonatal rats,and how the pathway of NMDAR-NMNAT1/2effects in the changes and if CREB/p-CREB are involved and play a role in the pathway.Methods 256 healthy male Sprague-Dawley rats, aged 7 days, weighing10~15g,were randomly divided into 4 groups(n=40): control group, in which rats inhaled oxygen of 30% for 6 h; 3% sevoflurane group(Sevo), in which rats inhaled3% sevoflurane for 6 h; 3% sevoflurane+NMDA receptor agonist D-cycloserine group(Sevo+DCS), in which D-cycloserine(6 mg/kg) was administered intraperitoneally before rats were exposed 6 h to 3% sevoflurane, and NMDA receptor antagonist AP-5( 2-amino-5-phosphonovaleric acid) group(AP-5), in which AP-5(12.5mg/kg) was administered intraperitoneally before rats inhaled oxygen of 30% for6 h. Rats in each group(n=16) were guillotined immediately and removed the hippocampus after inhaled oxygen or sevoflurane. The expressions of CREB/p-CREB and NMNAT1/2 in rat hippocampus were determined by western blot. Morris water maze was performed to detect the cognitive function when rats in 4 groups(n = 8)were raised to 2 m. Golgi staining was used to observe the morphology and number of dendritic spines in hippocampal CA1 region. Immunofluorescence technique was applied to detect the expression of NMNAT1/2 protein in hippocampal CA1 region and real-time fluorescent quantitative PCR was used to detect the contents of Nmnat1/2 gene. Immunohistochemical technique was used to detect the expression of CREB/p-CREB in hippocampal CA1 region. Results 1.Morris water maze test:Compared with control group, the escape latency and swimming distance were significantly longer in group Sevo and group AP-5(P<0.05), while the times of crossing was decreased(P<0.05); compared with group Sevo, the escape latency and swimming distance were significantly shorter in group Sevo+DCS(P<0.05), while the times of crossing was increased(P<0.05).2. Golgi staining: Compared with control group, the total number of dendritic spines in hippocampal CA1 region was decreased(P<0.05) in group Sevo,while the number of thin spines was significantly increased(P<0.05),and the number of mushroom-like spines was decreased(P<0.05);compared with group Sevo, the total number of dendritic spines in group Sevo+DCS was increased(P<0.05),while the number of thin spines was significantly decreased(P<0.05),and the number of mushroom-like spines was increased(P<0.05).3. Western blot: Compared with control group, the expression of CREB in group Sevo and group AP-5 did not change no matter rats aged at 7 days or 2 months(P>0.05),while the expression of p-CREB and NMNAT1/2 decreased(P<0.05);compared with group Sevo, the expression of p-CREB and NMNAT1/2 increased in group Sevo+DCS(P<0.05).4.Immunofluorescence staining:Compared with control group,the expression of NMNAT1 and NMNAT2 decreased(P<0.05) no matter rats were 7 days or 2months old(P>0.05) in group Sevo,while compared with group Sevo, the expression of NMNAT1 and NMNAT2 increased in group Sevo+DCS(P<0.05).5.Immunohistochemical technique:Compared with control group,the number of p-CREB positive cells in hippocampal CA1 area decreased in group Sevo(P<0.05);compared with group Sevo, the number of p-CREB positive cells increased in group Sevo+DCS(P<0.05).While there was no significant difference in the number of CREB positive cells in all groups(P>0.05).6. RT-PCR: Compared with control group,the relative content of Nmnat1/2 gene in group Sevo and group AP-5decreased(P<0.05);compared with group Sevo, the relative content of Nmnat1/2 gene in group Sevo+DCS increased(P<0.05).Conclusion Sevoflurane of high concentration exposed to neonatal rats for a long time may inhibit NMDA receptors in hippocampal neurons,decrease the levels of phosphorylation of CREB, reduce the expression of NMNAT1/2 in hippocampal neurons,and cause the changes of dendritic spines in morphology and number, and the long-term memory of newborn rat is low.