| Objective To investigate the changes of leaning and memory function after isoflurane anesthesia with Y-maze training, then to measure the alteration of proteome of brain hippocampus after isoflurane anesthesia in aged rats by the means of two dimensional gel electrophoresis and MALDI-TOF-MS in order to demonstrate the effects of isoflurane on cognition and seek the proteins related to the alteration of leaning and memory function after isoflurane anesthesia. We wish to find some kinds of special proteins related with cognitive function as far as possible.Methods The first stage of the experimentâ‘ )To randomly divided 75 22-month-old SD rats into group contrast (group C, n=35) and group isoflurane (group I, n=40) . The rats in Group C only inhaled mixed gas containing 40% oxygen for two hours. The rats in group I were induced by inhalation of 3% isoflurane combined 40% oxygen. Then the general anesthesia was maintained with 1.2% isoflurane combined 40% oxygen for two hours.â‘¡. We use Y-maze training to test the learning and memory function of aged SD rats from 24 hours after waking up( Group I) or inhalation of 40% oxygen(group C). The training time is 3 days and 20. times per day. The number of correct reaction, total reactive time, the number of initiative avoidance and the rate of acquire the task in 3 days are recorded.â‘¢. Five rats randomly selected from each group at 24h after stopping oxygen (group C24h) or isoflurane (group I24h) inhalation. And five rats randomly selected from each group at 72h after stopping oxygen (group C72h) or isoflurane (group I72h) inhalation. The left hippocampus of the aged rats mentioned above was cut down and reserved in - 80℃. The right hippocampus of the aged rats was cut down for Nissl's staining and observation of electron microscope.The second stage of the experiment: The left hippocampus was made into protein liquor by the means of homogenate, schizolysis and hydration. By the means of two dimensional gel electrophoresis, we got some pieces of gel containing protein spots. The Commassie's blue staining were used to make the gel coloration. We analyzed the images with scanning, discerning, synthesizing and matching, and we got the different protein spots among the groups. The different protein spots in the gel were removed and analyzed by the means of trypsinization, ZipTipTM desalinization and mass chromatographic analysis. So we got peptides mass fingerprints, which were analyzed by using database and software from internet to identify proteins.Resultsâ‘ The number of correct reaction in I24 is 10.97±3.48, which is less than 13.57±2.62 in C24 (P＜0.01); the number of initiative avoidance in I24 is 1.11±1.07, which is less than 3.20±2.59 in C24 (P＜0.01). The number of correct reaction in I48 is 13.53+-3.42, which is less than 16.07±2.79 in C48(P＜0.01); the number of initiative avoidance in I48 is 1.27±1.56, which is less than 6.45±4.97 in C48 (P＜0.01); the total reactive time in I48 is 81.93±45.01 sec, which is more than 59.27±26.37 sec in C48 (P＜0.05). There is no significant difference between I72 C72 in every index. The rate of acquire the task in 3 days in group C is 42.86%, which is more than 31.58% in groupâ… (P＜0.05).â‘¡There were no apomorphosis and necrosis in hippocampus with Nissl's staining. With electron microscope we found the postsynaptic density trend to thin and synaptic cleft trend to widen after isoflurane anesthesia, but there was no disorganization in the hippocampal neurone.â‘¢We got some pieces of ideal gel per group. The results showed that there were 906±46 protein spots in group C24h, 913±51 protein spots in group I24h, 936±36 protein spots in group C72h, 928±44 protein spots in group I72h, The results also showed that there were 17 (5/12) different protein spots between group I24h and group C24h, and there were 16 (12/4) different protein spots between group I72h and group C72h. Twenty two protein spots of all spots were identified by the means of MALDI-TOF-MS.Conclusions1. The results demonstrate that general anesthesia with 1.2% isoflurane for 2 hours may result in cognitive impairment in the aged rats, and this phenomenon may related to change of synaptic interface in CA3 area of hippocampus , but all of the changes almost recovery 72 hours after the anesthesia.2. The study shows that general anesthesia with 1.2% isoflurane for 2 hours may result in the alteration of proteome of hippocampus in aged rats. The changes of signal conduction and synapse correlated proteins may related to cognitive impairment in the aged rats, but the change of expression of the proteins may recovery 72h after the anesthesia.
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