The Mechanisms Of Sevoflurane On Impairment Of Learning And Memory And Synaptic Transmission

Background:Whether general anesthetics have adverse effects on learning and memory has been an issue of concern. It has been found that some patients suffered from postoperative cognitive dysfunction (POCD). The relationship between POCD and anesthesia has been demonstrated by numerous studies. Recently, it was reported that different anesthetics or anesthesia technique may impair the function of learning and memory. Therefore, the effects of general anesthetics on learning and memory function remain further investigation.Sevoflurane is a novel inhaled anesthetic, which is widely used in clinic. In recent, some studies demonstrated that sevoflurane induced early postoperative cognitive dysfunction (POCD). For the Chinese population, sevoflurane is one of the independent risk factors for POCD in patients received non coronary artery bypass surgery.The effects of sevoflurane on learning and memory dysfunction have attracted much attention. There were a lot of literatures about sevoflurane on learning and memory in animal experiments, but the results were inconsistent. Most studies suggested that sevoflurane impaired learning and memory function both in young and older animal model. On the other hand, there were some reports that sevoflurane did not obviously damage the function of learning and memory, and even enhanced the function.Using behavioral, electrophysiological and molecular biological methods, we investigated the relationship between behavior, hippocampal synaptic plasticity and protein induced by sevoflurane in rats. The results will provide guidance for rational application of inhaled anesthetics in order to reduce the occurrence of POCD.Part One The effects of sevoflurane on learning and memory in ratsObjective:To investigate the impairment of learning and memory by different concentrations of sevoflurane in rats.Methods:32 male SD rats were randomly divided into Control group (n=8),1.5% sevoflurane group (Sevol group, n=8),2.4% sevoflurane group (Sevo2 group, n=8), and 3.0% sevoflurane group (Sevo3 group, n=8). Rats were placed in plexiglass box for anesthesia. The concentrations of sevoflurane were stable at 1.5% (Sevol group),2.4% (Sevo2 group) or 3.0%(Sevo3 group) for 2 hours, respectively. Then the Y-maze, Morris Water Maze and open field test were perfomed 24 hours after anesthesia.Results:(1) The SpO2 and heart rate were in the normal range. There were no differences in SpO2 and heart rate among 4 groups (P>0.05). (2) In the Y maze test, the number of training sessions and the training time gradually increased in all Sevo groups. Compared with the Control group, the training time in the Sevol group, Sevo2 group and in the Sevo3 group increased significantly (P<0.01), while the number of training sessions increased significantly both in the Sevo2 group (P<0.05) and in the Sevo3 group (P<0.01). (3) The Morris Water Maze test showed that the escape latency time and number of errors increased gradually with increasing concentration of sevoflurane. Both the escape latency time and the number of errors increased significantly in the Sevo2 group (P<0.01) and in the Sevo3 group (P<0.01) as compared to those in the Control group. (4) In the open field test, the dwell time in central prolonged and the number of standing declined with increasing concentration of sevoflurane. The time in centre increased significantly in the Sevo2 group (P<0.01) and in the Sevo3 group (P<0.01) as compared to that in the Control group, while the number of rearing decreased significantly in the Sevo3 group (P<0.05) as compared to that in the Control group.Conclusion:(1) Inhalation of sevoflurane for 2 hours impaired learning and memory in rats. (2) Sevoflurane gradually aggravated the impairment of learning and memory in rats with the increasing concentration. Moreover, the impairment reached the highest level at high concentration of sevoflurane.Part Two The effects of sevoflurane on synaptic plasticity in rat hippocampal slices in vitroObjective:To observe the effects of sevoflurane on synaptic plasticity in hippocampal CA1 region of rats in vitro by using electrophysiological technique.Methods:SD male rats were decapitated and the hippocampal slices were prepared. The population spikes (PS) were recorded in the CA1 pyramidal cell layer by electrical stimulation. The effects of 3% sevoflurane on PS amplitude were observed which included basic synaptic transmission, paired pulse depression (PPD), NMDA receptor-dependent long-term depression (NMDAR-LTD) and metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD). The effects of NMDAR antagonist D-2-Amino-5-phosphonovaleric-acid (APV) and GABAA receptor antagonist bicuculline methiodide (BMI) on synaptic transmission were also observed.Results:(1) The effects of sevoflurane on the basis of synaptic transmission:PS amplitude of hippocampal slices significantly decreased in the Sevo groups (P<0.01) as compared to that in the Control group. The trend of input-output (I/O) moved downward. GABAA receptor antagonist BMI partially relieved inhibition by sevoflurane on PS amplitude. PS amplitude increased significantly in the Sevo+BMI group (P<0.01) as compared to that in the Sevo group. PS amplitude decreased significantly in the Sevo+BMI group (P<0.01) as compared to that in the BMI group. (2) The effects of sevoflurane on PPD:P2/P1 ratio significantly increased in the Sevo group (P<0.01) as compared to that in the Control group. BMI partially reduced the enhancement by sevoflurane on P2/P1 ratio. The facilitatory effect on PPD was observed when the ACSF perfusate Ca2+concentrations increased, while sevoflurane reduced PPD and significantly increased P2/P1 ratio at different Ca2+concentrations. (3) The effects of sevoflurane on NMDAR-LTD:NMDAR-LTD induced by the low frequency stimulation (LFS,900 pulses stimulate, 1Hz). PS amplitude further reduced at 60 min after LFS in the Sevo group (P<0.01) as compared to that in the Control group. Moreover, NMDAR antagonist APV completely blocked the induction of NMDAR-LTD in the Sevo+APV group. Calcium chelator BAPTA inhibited the facilitatory effect on NMDAR-LTD by sevoflurane. (4) The effects of sevoflurane on mGluR-LTD:mGluR-LTD was induced by the mGluR2/3 agonist LY354740. The results showed that sevoflurane had no significant effect on PS amplitude in slices with LY354740. There was no difference on PS amplitude between in the Sevo+LY354740 group (P>0.05) and in the LY354740 group.Conclusion:(1) Sevoflurane significantly decreased the amplitude of PS in hippocampal slices, the trend of input-output (I/O) moved downward. Sevoflurane inhibited the synaptic transmission at the schaffer collateral-CAl pyramidal cells. (2) Sevoflurane increased P2/P1 ratios in hippocampal slices by presynaptic inhibition, which was related to the extracellular Ca2+ concentration. GABA receptor antagonist BMI partially eliminated the effects of sevoflurane on PPD. (3) Sevoflurane facilitated the induction of NMDA-LTD by reducing synaptic transmission efficacy, the mechanism involved in intracellular Ca2+ levels. (4) Sevoflurane had no significant effects on mGluR2/3-LTD.Part Three Molecular mechanisms of sevoflurane on learning and memory dysfunctionObjective:To investigate the relationship between presynaptic protein and learning and memory impairment induced by sevoflurane in rats.Methods:32 male SD rats were randomly divided into Control group (n=8),1.5% sevoflurane group (Sevol group, n=8),2.4% sevoflurane group (Sevo2 group, n=8), 3.0% sevoflurane group (Sevo3 group, n=8). Behavioral tests were performed 24 hours after anesthesia. All rats were instantly killed at the end of the Behavioral tests. The brains were removed and bilateral hippocampal tissues were separated. The synaptic binding protein Sytl and Syt4, mGluR2, and BDNF/TrkB signaling pathway were analyzed by using Western Blot.Results:(1)The results of behavior testing can be found in Part one. (2) With the increasing concentration of sevoflurane, Sytl protein expression decreased significantly in the Sevo2 group (P<0.01) and in the Sevo3 group (P<0.01) as compared to that in the Control group, while Syt4 protein expression increased significantly in the Sevol group (P<0.01), in the Sevo2 group (P<0.01) and in the Sevo3 group (P<0.01) as compared to that in the Control group. (3) Sevoflurane had no effect on the protein expression of mGluR2. There was no significant difference in the Sevol group (P>0.05), in the Sevo2 group (P>0.05) and in the Sevo3 group (P>0.05) as compared to that in the Control group. (4) With the increasing concentration of sevoflurane, BDNF, TrkB and p-TrkB protein expression gradually decreased. BDNF protein expression decreased significantly in the Sevol group (P<0.01), in the Sevo2 group (P<0.01) and in the Sevo3 group (P<0.01) as compared to that in the Control group, while TrkB and p-TrkB protein expressions decreased significantly in the Sevo2 group (P<0.01) and in the Sevo3 group (P<0.01).Conclusion:(1) The effects of sevoflurane on learning and memory was related to decreasing Sytl protein expression and increasing Syt4 protein expression. The effects of 2.4% and 3.0% sevoflurane were obvious. (2) Sevoflurane had no significant effect on the protein expression of mGluRa. (3) The effects of sevoflurane on learning and memory was related to suppressing BDNF/TrkB signaling pathways. Sevoflurane decreased expression of BDNF, TrkB and p-TrkB protein. The effects of 2.4% and 3% sevoflurane were obvious.Part Four Simulation study of sevoflurane binding sites in synaptotagminlObjective:To explore the specific binding sites of sevoflurane in the Sytl domain by using the bioinformatics techniques.Methods:(1) Ligand-binding sites on Sytl domain were detected using FTMap method. (2) The 3D conformation of sevoflurane was converted from its 2D structure using CORINA program. (3) The 3D molecular structure of sevoflurane was docked into Sytl ligand binding site using UCSF DOCK6 method, and then Sytl domain-sevoflurane complexes were obtained. (4) Molecular dynamics (MD) simulations of Sytl domain-sevoflurane complexes were carried out using AMBER10 force field in the AMBER 11 package.Results:Sytl C2A could be the regional structure to combine with sevoflurane. This region had six potential pockets which can be docked with sevoflurane. Among them, pocket 3 was most likely, the affinity of pocket 3 Kd (dissociation constant) =12.6μmol/L was determined, and the analysis showed that sevoflurane ligand can form two hydrogen bonds with Leu185 and Lys190 residues, respectively, and form a halogen bond with Asp188 residue in pocket 3.The chemical bonds provided a high stability on the complexes.Conclusion:There was an specific binding sites on the Sytl C2A structure, which could be bound by sevoflurane to play the pharmacological effect.