| Post-operative cognitive dysfunction(POCD) is one of important complications following major surgery and anesthesia in elderly patients, presenting cognitive function impairment such as learning and memorizing, which can increase the mortality.According to the latest research, the pathogenesis of POCD include neuron apoptosis raise, neurogenesis decline, synaptic plasticity impairment, and neurodegeneration caused by neuroinflammation, oxidative stress, cholinergic system disorders and amyloid-?deposition in the central nervous system. MicroRNA (miRNA) are a class of small non-coding RNAs, fulfilling post-transcriptional regulatory function by binding 3'-untranslated region(3'UTR) of target mRNA. MicroRNA are rich in the brain and play an important role in the procession of neuron development and differentiation, synaptic connection and dendritic spine formulation. The recent studies suggest that aging, stress,inflammation, and ischemic-reperfusion injury and other harmful injuries can lead miRNA expression aberration, and miRNA are involved in the pathogenesis of several cognitive disorders such as Alzheimer's disease(AD), schizophrenia and depression by regulating synaptic plasticity.Based on the above findings, we hypothesized that one of pathogenesis of POCD may be the miRNA expression aberration in hippocampus leading to a decline in synaptic plasticity. Therefore, this study is designed to explore the miRNA expression aberration in hippocampus and its association with postoperative cognitive impairment in aged mice.Part I Effects of surgery and general anesthesia on postoperative cognitive capacity and hippocampus synaptic plasticity in aged miceObjective: To investigate the effects of isoflurane inhalation anesthesia and abdominal surgery on postoperative cognitive function in aged mice and synaptic plasticity in hippocampus.Methods: Experiment 1: Aged C57/BL6 mice( 18 months old,n=64) were randomly allocated into four groups: control group(con), isoflurane group(iso), surgery group(sur),isoflurane plus surgery group(iso+sur), 16 in each group. Sham surgery, 1.8% isoflurane inhalation for 1.5h, abdominal surgery under local anesthesia, abdominal surgery under local anesthesia combined 1.8% isoflurane inhalation for 1.5h were exerted respectively to mice in each group. All mice were sent to open field test before operation and Morris water maze and fear conditioning test after operation to access learning and memory function. Experiment 2: Aged C57BL/6 mice(18 months old, n=48) were randomly allocated into four groups as Experiment 1, 12 in each group, and received the same treatment as Experiment 1. All mice were sacrificed and brain samples were dissected at day3 and day7 after operation and the expression status of Synapsis-1(SYN-1) and Postsynaptic density 95(PSD95) in hippocampus were detected by Immunofluorescence and Western blot respectively.Results: Experiment 1: In the open field test, lines crossing numbers and rears times of mice in four groups showed no significant difference(P>0.05). In the Morris water maze,compared with the control group, mice in the surgery group had longer latency time(P<0.05), shorter duration in target quadrant(P<0.01) and reduced platform crossing times(P<0.05). While compared with the surgery group, the isoflurane group and isoflurane plus surgery group had shorter latency time(P<0.05), longer duration in target quadrant(P<0.05). In fear conditioning test, compared with the control group, mice in surgery group had lower percentage of freezing time in context test session on dayl,day3 and day7 after operation(P<0.05), lower percentage of freezing time in tone test session on day3 after operation(P<0.05). Compared with the surgery group, isoflurane group had increased percentage of freezing time both in context test and in tone test session on day3 after operation(P< 0.05),and isoflurane plus surgery group had increased percentage of freezing time in context test session both on day3 and day7(P<0.05).Experiment 2: The immunofluorescence test showed that the expression of SYN-1 was reduced(P<0.05),namely declined synapse density,in hippocampus of mice in the surgery group compared with the other groups on day3 after operation, and no significant difference on day7. The western blot test showed that, compared with the control group,the expression of PSD95 was decreased in hippocampus of mice in surgery group on day3 after operation(P< 0.05),and compare with the surgery group,the expression of PSD95 in isoflurane group was increased on day3. There was no difference of the expression of PSD95 in all four groups on day7 after operation.Conclusion: Abdominal surgery can induce early postoperative learning and memory impairment and hippocampal synaptic plasticity decline in aged mice, while operating abdominal surgery under isoflurane inhalation anesthesia can attenuate this impairment,which further confirmed one of pathogenesis of POCD is synaptic plasticity decline.Part ? Preliminary analysis and investigation of hippocampal microRNA expression differences in aged mice model of POCDObjective: To compare and analyze the changes in the expression of hippocampal microRNA in aged mice model of POCD and to investigate the possible pathogenesis in which it impaired cognitive function.Methods: Experiment 1: Aged C57BL/6 mice(18 months old, n=28) were randomly divided into two groups: the control group(con) and the surgery group(sur), 14 in each group. Sham surgery and abdominal surgery under local anesthesia were exerted respectively to mice in each group. After 24h, 3 mice in each group were randomly selected and sacrificed to obtain hippocampus tissues, and total RNA were extracted. The miRNA expression profile in hippocampus of mice in each group were detected by miRNA microarray gene chip experiment. Meanwhile, another 3 mice in each group were randomly selected and sacrificed, and total RNA in hippocampus were extracted and preserved in liquid nitrogen for subsequent qRT-PCR test. Another 8 mice left in each group were sent to Morris water maze behavior test to access post-operative cognitive status. Experiment 2: A subset of differentially expressed miRNA, which changed more than 1.5 folds, were selected based on data from microarray gene chip, and target genes were predicted using online data base such as miRanda, miRDB, miRWalk, and GO analysis and KEGG pathway prediction were performed. A few of differentially expressed miRNA were randomly selected and sent to qRT-PCR to verify the accuracy and consistency of microarray. Experiment 3: Aged C57BL/6 mice(18 months old, n=36)were randomly allocated into four groups: control group(con), isoflurane group(iso),surgery group(sur) isoflurane plus surgery group(iso+sur), 9 in each group, and received the same treatment as Experiment 1 in Part ?. 3 mice in each group were randomly selected and sacrificed respectively on dayl, day3 and day7 after operation, and hippocampal miRNA related to cognitive function such as miR-155-5p, miR-582-5p,miR-134-5p were sent to qRT-PCR test. The expression of target protein such as CREB and BDNF were tested by western blot.Results: Experiment 1: MiRNA microarray showed that, 29 miRNA from hippocampus of mice in surgery group were up-regulated more than 1.5 folds while 34 miRNA were down-regulated more than 1.5 folds at 24-hours after surgery compared with the control group. Morris water maze suggested that abdominal surgery could induce longer latency time(P<0.05), shorter duration in target quadrant (P<0.01) and decreased platform crossing times(P<0.05). Experiment 2: By performing target genes prediction of above miRNA,we found that 36 miRNA were associated with gene creb1, 23 miRNA were associated with bdnf. In qRT-PCR experiment, randomly selected miR-16-3p,miR-290a-5p, miR-217-5p, miR-382-3p, miR-466b-3p and miR-183-5p showed no significant difference compared with microarray experiment. By performing GO analysis,we found there were 447 GO terms in biological process(BP), 74 GO terms in molecular function(MF), and 36 GO terms in cellular component(CC), which were mostly associated with inflammation, neuron development and differentiation,synapses formation and function. By performing KEGG pathway prediction we found that several signaling pathways related to cognition were activated such as Neurotophin signaling pathway, cAMP signaling pathway, Long-term potentiation, MAPK signaling pathway.Experiment 3: 3 miRNA associated with all crebl and bdnf were selected for qRT-PCR test,namely miR-155-5p,miR-582-5p,miR-134-5p. Compared with other groups,these 5 miRNA were up-regulated in surgery group on day1 after operation, while miR-155-5p was still up-regulated on day3 and others were recovered to normal expression level.There were no significant differences in all groups on day7. In western blot test,compared with other groups, CREB activity of mice in surgery group was inhibited on dayl and day3 after operation. Compared with other groups, the expression of BDNF were decreased in surgery group on day3 and day7.Conclusion: Abdominal surgery can induce early postoperative cognition impairment which are associated with miRNA expression aberration in hippocampus. In response to trauma, neuro-inflammation, oxidative stress, ischemic-reperfusion injury caused by surgery, miRNA were up/down regulated and thus influencing many signaling pathways about neuron growth and apoptosis, synapse formation and function, gliacytes activation,which change synaptic plasticity and cognitive function. MicroRNA-CREB pathway may be involved in the pathogenesis of POCD. |
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