Effects Of Hypothermia And Surgical Trauma On Spatial Learning And Memory In Rats

Effects of Hypothermia and Surgical Trauma on Spatial Learning and Memory in RatsIntroductionPhysiologically a highly soluble protein, tau is functionally known to bind microtubules and is critical in the processes of neuronal outgrowth and axonal integrity. In the decade of the 1980s, it was described the presence of tau, in hyperphosphorylated form, in the neurofibrillary tangles (NFTs). In the next 20 yeas, NFTs formation was thought to be a common mechanism underlying neurodegeneration. However, the accumulation of hyperphosphorylated tau that occured before NFT formation also contributed to the memory problems seen in a several studies, and in tau transgenic mice, impaired spatial memory was relevant to tauopathy. Moreover, little is known on the biochemical consequences of hypothermia on tauopathies and on the influence of learning and memory in vivo. General anesthetics markedly impair normal autonomic thermoregulatory control, and all of the anesthetics involved induced hypothermia. Several studies investigated the effect of anesthesia on tau phosphorylation metabolism in mouse brain. They found that tau phosphorylation were not a result of anesthesia per se, but a consequence of anesthesia-induced hypothermia, which led to inhibition of phosphatase activity.In recent years, much work has been devoted to the study of postoperative cognitive dysfunction. However, there is little evidence on the biochemical consequences of inflammation on tauopathies and influence of learning and memory in vivo. Several studies investigated the effect of inflammatory cytokines on the cognitive function of rodent:One found that minor surgery leads to an exaggerated neuroinflammatory response in aged mice but does not result in significantly impaired performance in the Morris water maze, the other found that surgery triggers a transient neurocognitive decline in a rat model that is temporally associated with glial activation and increase in proinflammatory cytokines in the hippocampus.GSK-3βis one of the major kinases that phosphorylates tau and precedes neurofibrillary tangle formation in AD. Also, GSK-3 promotes microglial responses to inflammation and that the utilization of GSK-3 inhibitors provides a means to limit the inflammatory actions of microglia. Interestingly, in human inclusion body myositis (IBM) muscle, suppression of GSK-3βactivity using lithium chloride significantly reduced tau phosphorylation. GSK-3βand phospho-tau were colocalized, further supporting the pathogenic role of GSK-3βin tauopathy. Therefore, we sought to determine if the immune response to surgical trauma results in increased neuroinflammation and cognitive impairment in adult rats. Furthermore, the aim of the current study was to determine whether inflammatory cytokines causes tau hyperphosphorylation through activated GSK-3 p.While hypothermia may be beneficial for critically ill patients, perioperative hypothermia following anesthesia is harmful for most patients. Hypothermia does not result in obvious neuronal damage, but may lead to cognitive dysfunction. The aim of the current study was to determine whether exposure to clinically relevant concentrations of isoflurane causes tau hyperphosphorylation and memory deficit. We also examined impairment in spatial memory in rats subjected to splenectomy with or without lithium chloride lithium chloride preconditioning. The effects of hypothermia and inflammation on cognitive performance were evaluated using a Y-maze test for working memory and reference memory.Materials and MethodsThree hundred and fifteen adult (6-month) male rats (Sprague-Dawley) weighing 350-400 g were housed at 22℃and under a 12-h light/dark cycle, with free access to food and water. Rats were anesthetized with isoflurane to allow tracheal intubation. All anesthetized rats were mechanically ventilated with isoflurane 1 MAC (minimum alveolar concentration,1.3-1.5%isoflurane) in 50%oxygen, using a rodent ventilator for a period of 2 h, with or without control of the body temperature. Electrocardiograms using three subcutaneous needle electrodes were recorded continuously. Temperature was monitored with a rectal probe.Experiment 1:Rats were then randomly assigned to three groups. Naive controls (group A; n=15) received no intervention. In the anesthesia-plus-normal temperatue group (group B; n=60), rats were anesthetized with isoflurane and stabilized rectal temperature between 36.0℃and 37.0℃by being warmed with a heating pad. The anesthesia-plus-hypothermia group (group C; n=60), rats were anesthetized with isoflurane and kept at the 22℃room temperature. Postintervention animals were recovered and then returned to their cages and housed individually. Rats in group B and C were killed immediately after 2 h of anesthesia and at day 1,3, or 7 after intervention (n=15/time point). Animals in group B and C were marked with B0, B1, B3, B7, C0, C1, C3 and C7 at each time point.Experiment 2:Rats were then randomly assigned to three groups. Naive controls (group A; n=15) received no intervention. In the surgery-plus-normal temperatue group (group B; n=45), rats were anesthetized and underwent splenectomy and stabilized rectal temperature between 36.0℃and 37.0℃by being warmed with a heating pad. The sugery-plus-hypothermia group (group C; n=45), rats were anesthetized with and underwent splenectomy and kept at the 22℃room temperature. Rats in group B and C were killed immediately after 2 h of anesthesia and at day 1,3, or 7 after intervention (n=15/time point). Animals in group B and C were marked with B1, B3, B7, C1, C3 and C7 at each time point.Experiment 3:Rats were then randomly assigned to three groups. Naive controls (group A; n=15) received no intervention. The surgery-plus-sodium chloride group (group B; n=45) underwent splenectomy during isoflurane anesthesia injected intraperitoneal sodium chloride. The surgury-plus-lithium chloride group (group C; n =45), underwent splenectomy during isoflurane anesthesia injected intraperitoneal lithium chloride. All animals in group B and C were anesthetized for 2h and stabilized rectal temperature between 36.0℃and 37.0℃by being warmed with a heating pad, and were killed at day 1,3, or 7 after intervention (n=15/time point). Animals in group B and C were marked withB1, B3, B7, C1, C3 and C7 at each time point. Postintervention animals were recovered and then returned to their cages and housed individually.Spatial learning task was evaluated using the modified Y-maze apparatus. We examined the gross anatomical pattern of tau phosphorylation, total tau and GSK-3Pusing immunohistochemistry. Relative expression levels of tau, total tau protein, GSK-3β, IL-1pand TNF-a protein were analysised by Western Blot. We performed protein phosphatase 2A (PP2A) assay using the Serine/Threonine Phosphatase Assay System. Isolation of mRNA and quantification of IL-1βand TNF-a were done by real-time RT-PCR.All values are reported as mean±SD. Parametric values in the same group were analyzed by paired t-test. Differences among the groups were determined by a one-way or a two-way analysis of variance(ANOVA) followed by the Dunnett post hoc test. Statistical significance was defined as P<0.05.ResultsExperiment1:The rectal temperature of the rats decreased very rapidly after isoflurane exposure(34℃,30min;30℃,60min 26℃,120min). The results suggest that anesthesia with lower body temperature induces a temporary impairment in working memory on test day 1. Phosphorylation-dependent antibody, phospho-Thr205, displayed an approximately twelvefold increase in phosphorylation, whereas phospho-Ser396 exhibited 3 times more phosphorylation, but total tau levels did not change. There was no significant difference in PP2A activity between control and anesthetized mice samples when incubated at 37℃, whereas nearly 45%PP2A inhibition was detected when the reaction was performed at 26℃. Experiment 2:These results suggest that the surgical trauma induces a temporary impairment in learning and memory on test day 1,3. Phosphorylation-dependent antibody, phospho-Thr205, displayed an approximately 2.7 times increase in phosphorylation, whereas phospho-Ser396 exhibited 1.1 times more phosphorylation, but total tau levels did not change. On days 1 and 3 after splenectomy group B, the level of IL-1pmRNA was increased by 1.6 times and 60%, respectively, when compared with the controls. The level of IL-1pmRNA for group C was increased by 17.3 times and 1.6 times, respectively, when compared with the controls. Protein expression of IL-1(3 for group B and C was significantly increased by 73%and 1.35 times respectively on the first postsurgical day, but not at other time points. On postsurgical day 1 of group B and C, the level of TNF-amRNA was increased by 32% and 1.0 times, respectively, when compared with the controls. On postsurgical day 3 of group B, the level of TNF-amRNA was increased by 60%.There was no change in the expression of TNF-aprotein at any time after surgery. The increase of phospho-tau epitopes correlated well with the activation of GSK-3βkinase, as measured by the significant reduction of phospho-GSK-3(3(nearly 55%,53%respectively), an inactive state, whereas total levels of GSK-3βwere unchanged in group B and C.Experiment 3:These results suggest that the surgical trauma induces a temporary impairment in learning and memory on test day 1,3 in group B.On days 1 and 3 after splenectomy group B, the level of IL-1βmRNA was increased by 1.5 times and 50%, respectively, when compared with the controls.The level of IL-1βmRNA for group C was increased by 1.4 times and 50%, respectively, when compared with the controls. Protein expression of IL-1p for group B and C was significantly increased by 90%and 67%respectively on the first postsurgical day, but not at other time points.The general anesthetic did not induce an increase of TNF-amRNA expression at any time point when compared with the naive controls. However, on postsurgical day 1 of group B and C, the level of TNF-amRNA was all increased by 23%, but no difference in transcription was noted at days 3 and 7. There was no change in the expression of TNF-aprotein at any time after surgery. Treatment with lithium chloride, group C versus group B, had no significant impact on the expression of inflammatory cytokines. Phosphorylation-dependent antibody, phospho-Thr205, displayed an approximately 2.7 times increase in phosphorylation, whereas phospho-Ser396 exhibited 1.2 times more phosphorylation, but total tau levels did not change. When we treated animals with lithium chloride (group C), and this protocol restored the activity of GSK-3βto normal and completely restored phosphorylation to control levels, total tau did not change.Conclusion1. The principal finding in the present study is that anesthesia with 1.5%isoflurane impaired acquisition of working and reference memory in hypothermal animalsⅠday after intervention. Furthermore, We found that anesthesia induced rapid and massive hyperphosphorylation of tau, prolonged hypothermia, and inhibition of PP2A. Reestablishing normothermia during anesthesia completely rescued tau hyperphosphorylation to normal levels. Our results indicate that tau phosphorylation were not a result of anesthesia per se, but a consequence of anesthesia-induced hypothermia, which led to inhibition of phosphatase activity and subsequent hyperphosphorylation of tau. The deficits of spatial learning and memory following anesthesia in hypothermal rats may be attributed to the effect of phospho-tau.2. Furthermore, Inflammation in hippocampus was induced by surgery, which activated GSK-3βand concomitantly increased phospho-tau levels. We suggest that these changes in phosphor-tau underpin hippocampal function leading to learning and memory impairment.3. we found that surgery induced rapid and massive hyperphosphorylation of tau, and activation of GSK-3β. Inhibition of GSK-3βwith lithium completely restored tau hyperphosphorylation to normal levels. Our results indicate that tau phosphorylation was a result of surgical trauma, a consequence of surgery-induced immune response, which led to activation of kinases activity and subsequent hyperphosphorylation of tau. The deficits of spatial learning and memory following splenectomy in rats may be attributed to the effect of phospho-tau.