Endoplasmic Reticulum Stress Mediates Distinct Impacts Of Sevoflurane On Different Subfields Of Immature Hippocampus

Sevoflurane is a kind of typical inhaled anesthetic. It is widely used in surgeryanesthesia in recent years because of its fast onset, short recovery time, and the easily controlled depth of anesthesia that it provides. Sevoflurane is applicable for all ages such as the aged, adults and children, especially for children anesthesia induction. So,children become the main group who receive sevoflurane anesthesia.However, the mechanisms of ether intravenous anesthetics or inhaled anesthetics are not very clear so far. Despite these advantages, recent findings have raised increasing concerns about the safety of inhaled anesthetics, especially the influence on the neurodevelopment of children. Early studies showed protective effects of inhaled anesthetics in several neuronal injury models. Sevoflurane administration was reported toincrease neuronal survival in the ischemic brains of rats and in cases of reperfusion-induced injury. These were the supports of the safety of inhaled anesthetics.However, more recent studies have found that inhaled anesthetics may have neurotoxic effects and impair the cognitive of children. Anesthesia gas has been shown to induce cortical and hippocampal neuronal apoptosis and affect memory formation. More importantly, exposure to sevoflurane early in life appears to impair spatial memory in adult rats. However, the mechanism by which sevoflurane injures hippocampal neurons and impairs cognition remains unclear.Some researches indicated that the block of NMDA receptor might be involved in the neurotoxic of inhaled anesthetics. More recent study showed that the ER stress may also be one of the reasons. The endoplasmicreticulum(ER) is an important subcellular organelle that participates in many cellular processes and plays regulatory roles in cell survival, protein folding and cytoplasmic calcium homeostasis. ER dysfunction has been implicated in the pathological processes of many diseases. Many stress signals can disturb ER function and cellular calcium homeostasis, causing the accumulation of misfolded proteins in the ER and triggering dysfunction and even apoptosis of the cell. Although, ER stress may be relevant to sevoflurane induced neuronal death, do the hippocampal neurons suffer from the same process? Will the ER stress induced by sevoflurane make different impairments on the different types of hippocampal neurons? Can we propose a strategy to prevent the neurotoxic of sevoflurane according to this theory? So, our work which based on these hypothesis aims at investigating the role of ER stress on hippocampal injury under sevoflurane exposure and the protective effect of inhibiting ER stress during sevoflurane exposure.Methods:This study consists of three parts. First, we exposed HT22 hippocampal cell line to sevoflurane and detected the levels of key sensors of ER stress Bi P, PERK, p-PERK and caspase-12 by western immunoblotting. Then, we evaluated the apoptotic rates of the HT22 cells after sevoflurane exposure by flow cytometry. The second part was in vivo experiment, we exposed 3-week-old SD rats to sevoflurane and detected the levels of keysensors of ER stress Bi P, PERK, p-PERK and caspase-12 in the hippocampus by western immunoblotting. TUNEL staining was applied to observe the apoptotic cells in hippocampal slices and Double-labeling immunofluorescence was applied to confirm the type of the apoptotic cells. Then, we detected the intrinsic excitability of CA1 pyramidal neurons bywhole-cell electrophysiological recording. The Ca2+fluorescence probe was used to detect the concentration of Ca2+in primary cultured hippocampal pyramidal neurons. The third part was animal behavioral test. 4-PBA was used to inhibit ER stress during sevoflurane anesthesia and the rats were tested by morris water maze when they became adults 5 weeks later. The protective effect of inhibiting ER stress was evaluated.Results:Part one: The effect of sevoflurane exposure on HT22 cells by inducing ER stress.(1) HT22 cells were divided into three groups, control, sevo and sevo + 4-PBA and were exposed to 2 vol% sevoflurane for 5h. The results of western immunoblotting showed that the levers of Bi P and p-IRE1 in sevo group were significantly increased, and which were greatly reduced in the sevo + 4-PBA group, Р<0.01.(2) Cells of each group were harvested after sevoflurane exposure and the flow cytometry were used to investigate the apoptotic rates of each group. The results showed that the apoptotic rate of Control group was 1.52±0.60%, the Sevo group was 9.4±0.13%and the Sevo+4-PBA group was 3.93±0.42%. This confirmed that 4-PBA protects HT22 cells from sevoflurane induced apoptosis by inhibits ER stress.Part two: The effect of sevoflurane on different subfields of hippocampus.(1) Three-week-old rats were divided into control, sevo and sevo+4-PBA groups. They were exposed to 2 vol% sevoflurane in O2 for 5 h, followed by a 2-h recovery period and the brains were collected to make hippocampal lysates and brain slices. Western immunoblotting analysis of the hippocampal lysates revealed that exposure to sevoflurane the levels of Bi P and phosphorylated PERK were significantly increased and they were greatly reduced in the sevo+4-PBA group than the sevo group Р<0.01.(2) TUNEL staining of the brain slices showed obvious hippocampual neuronal deathin sevo group and it was greatly reduced by pretreatment with 4-PBA. But the of apoptotic cells were mainly distributed in DG, TUNEL-positive cells were almost not existent in the CA1-CA3 subfields.(3) Double-labeling immunofluorescence analysis of cleaved caspase-12 and Neu N showed that they were exactly merged in hippocampal slices. The fluorescence intensity of cleaved caspase-12 increased significantly in the Sevo group, which was confirmed by the result of western immunoblotting. This indicated that the neurons, rather than the glial cells, responded to severe ER stress.(4) The result of Ki-67 and TUNEL double-labeling showed that many of the TUNEL-positive cells were colocalized with Ki-67-positive cells. This result suggested that sevoflurane induced apoptosis of some NSCs in DG.(5) We prepared hippocampal brain slices of each group after sevoflurane exposure and recorded the intrinsic excitability of CA1 pyramidal neurons. Our recording analysis showed that sevoflurane exposure significantly reduced the spike number of action potentials and the first frequency compared with the control group and it’s normal in sevo+4-PBA group. This clearly suggested that sevoflurane exposure reduced the intrinsic excitability of CA1 pyramidal neurons.(6) We cultured primary hippocampal pyramidal neurons for 7 days and then exposed them to sevofluranein Ca2+-free medium. Then, we used Flou-3 Ca2+fluorescence probe to examine the Ca2+concentrations of the cells. This analysis revealed a significant increase in neuronal Ca2+concentration in the sevo group than the other groups P<0.01.Part three: The influence of early sevoflurane exposure on the cognitive ability in the adulthood.(1) We exposed 3-week-old rats to sevoflurane as described above and later subjected them to the Morris water maze test at 2 months of age. This analysis revealed that the rats in the Sevo group took significantly longer to find the hidden platform on the third and fourth training days compared with the other groups.(2) After 4 days training, we performed the spatial probe trial.The rats from the sevoflurane-treated group spent significantly less time in the target quadrant and the timesof crossing the hidden platform were less than the other groups. The perform of the rats in sevo+4-PBA group was as well as which in control group.Conclusion: Our work showed that sevoflurane could impair immature hippocampal neurons by inducing ER stress. The main innovations of this experiment lie in that we observed the effects of sevoflurane on different subfields of hippocampus were different.The administration of 4-PBA to inhibit ER stress before sevoflurane exposure could prevent the injury of sevoflurane and relieve the cognitive deficits in the adulthood. This was an important mechanism involved in the neurotoxic of sevoflurane, and will instruct the clinical effort to prevent neurotoxic of sevoflurane on children.(1)Sevoflurane induced cell ER stress in ether in vivo or in vitro experiments.(2)Sevoflurane caused hippocampal neurons death in DG by inducing ER stress, but there were no apoptotic cells in the CA1-CA3 subfields.(3)Sevoflurane exposure reduced the intrinsic excitability of CA1 pyramidal neurons,which was associated with ER stress caused ER calcium depletion and Ca2+ overload in cytoplasm.(4)Early sevoflurane exposure could impair the cognitive ability in the adulthood and the administration of 4-PBA to inhibit ER stress before sevoflurane exposure could relieve the cognitive deficits in the adulthood.