The Mechanism Of SIRT1-p53,cAMP/CREB Signaling On The Impairment Of Learning And Memory After Inhalation Anesthesia In Aged Rats

Background:Many elderly patients experience difficulties with learning, memory, concentration, and attention after surgery and anesthesia. The mechanism is not well-understood, and general anesthesia has been implicated as a possible cause. Recent studies have raised concerns about the neurotoxicity of inhalational anesthetics and their contribution to impairment of learning and memory. As sevoflurane and nitrous oxide are widely used in clinical anesthesia practice, the aim of the current study is to assess the contribution of general anesthesia with sevoflurane combined with N2O to the POCD in aged rats. To better understand the neural effects, we studied the behavioral and biochemical changes in aged rats that were exposed to sevoflurane and nitrous oxide for 4 hours. This study includes two parts:Part 1:The effect of general anesthesia with sevoflurane and nitrous oxide on learning and memory in aged ratsObjectives:To explore the changes of learning and memory induced by 1.3% sevoflurane combined with 50% nitrous oxide in aged ratsMethods:Eighteen month old rats were randomly assigned to receive 1.3% sevoflurane and 50% nitrous oxide/50% oxygen or 50% oxygen for 4h. Spatial learning and memory were tested with the Morris water maze 48h after exposure, including navigation task for consecutive six days (day 1-day 6).24h after navigation task, the first probe test was conducted (day 7), and on day 14 the second probe test was conducted again.Results:Repeated-measures two-way ANOVA revealed longer escape latencies in the anesthesia group than control grou (p=0.001). The significant difference was observed on day 2 (p=0.044), day 3 (p=0.003) and day 5 (p=0.033). Latencies decreased significantly across sessions during the 6 days of training (p<0.001), but no significant differences were found regarding the swimming speeds (p=0.11). The probe test conducted on day 7 indicated that exposed rats spent significantly less time as well as the distance travelled in the target quadrant (p=0.022, p=0.009, respectively), which induced a significant deficit in spatial learning acquisition and memory retention. However, in this part, we didn’t observe any difference in the performance of rats in the probe test conducted on day 14.Conclusions:Aged rats exposed to 1.3% sevoflurane plus 50% N2O showed impairment of learning and memory.Part 2:The mechanism of the SIRT1-p53 pathway involving in learning and memory impairment in aged rats after general anesthesia with sevoflurane and nitrous oxideObjectives:Silent information regulator 1 (SIRT1), a member of the sirtuin family of class III histone deacetylases, is expressed in the brain and mediates intracellular responses that promote cell survival, enhances the repair of DNA damage, and reduces cell division, all of which have important neuronal roles. The objects of this part is to explore the biochemical changes of SIRT1-p53 signaling in the hippocampus in aged rats after general anesthesia, investigate the correlation between SIRT1-p53 signaling and neurotoxicity induced by inhalation anesthesia, and explore the neuroprotection of SIRT1 activator resveratrol and effect to the cognitive function.Methods:1) At 48h after anesthesia with 1.3% Sev+N2O and immediately after probe test, rats were killed and hippocampi were dissected. The expression of SIRT1, p53 deacetylation, cleaved caspase-3, Bax and the DNA damage sensor PARP-1 was tested by Western Blot and immunostaining. Double staining with NeuN and TUNEL was used to measure the neuronal loss and cell apoptosis.2) Primary hippocampal neurons were cultured from Sprague-Dawley rat pups on postnatal day 0. Neurons were preincubated with resveratrol (100μmol/L), sirtinol (50μmol/L), salermide (50μmol/L), or an equal volume of DMSO for 24 hours and then exposed to sevoflurane plus N2O for 2 hours. Western blot and immunostaining were used to assess the neurotoxicity and expression of SIRT1, acetylated p53.3) Rats were administered 100 mg/kg resveratrol or vehicle intraperitoneally for 7 days. On the 7th day, general anesthesia was administered to all the rats.48h after general anesthesia, Morris Water Maze was used to assess the behavioral performance. At 48h and immediately after probe test, neurotoxicitiy and SIRT1 expression was measured.Results:1) at 48h after exposure and immediately after probe test 1 conducted on 7th day, we found a significant increase in levels of the apoptotic markers cleaved caspase-3, Bax by Western Blot, as well as neuronal loss and cell death by double staining with NeuN and TUNEL in the hippocampus of exposed rats compared to the control rats. Meanwhile, the results also showed an endogenous increase in SIRT1 expression and p53 deacetylation. However, after the second probe test 2 conducted on 14th day, no significant difference was observed about the expression of cleaved caspase-3 and Bax between the two groups, and the expression of SIRT1 also decreased in the exposed rats.2) In vitro, by treatment with SIRT1 inhibitor sirtinol, salermide and SIRT1 activator resveratrol to the primary hippocampal neurons, we found that resveratrol further activate the expression of endogenous SIRT1 and deacetylated p53, while the expression of cleaved caspase-3, Bax, PARP-1, yH2AX decreased. The results showed a certain protection of SIRT1 against the neuronal insults of anesthetics. Otherwise, sirtinol and salermide inhibited the expression of SIRT1 and p53 deacetylation, then increased the cleaved caspase-3, Bax, PARP-1, yH2AX levels in the neurons.3) Aged rats pretreated with resveratrol exhibited better performance during the navigation phase of the Morris Water Maze. The escape latency of rats treated with resveratrol was shorter than controls (p=0.013), and the significant difference was found on day 3 (p=0.020), day 4 (p=0.049) and day 6 (p=0.044). No significant differences were observed regarding the swimming speeds between the two groups (p=0.912). However, the time spent and distance traveled in the target quadrant was no significant difference between the two groups during the probe test. By activating the SIRT1 and enhancing its deacetylated activity, resveratrol inhibited neuronal apoptosis in hippocampus and provided some neuroprotection against neurotoxicity induced by inhalational anesthetics.Conclusions:In vivo and in vitro studies showed that exposure to sevoflurane plus N2O induced hippocampal neuronal apoptosis accompanied by endogenous increase in SIRT1 expression and p53 deacetylation. Pretreatment with SIRT1 activator resveratrol promoted neuronal survival and protected against the neurotoxicity induced by general anesthesia.Part 3:Impaired spatial learning and memory after sevoflurane-nitrous oxide anesthesia in aged rats is associated with down-regulated cAMP/CREB signalingObjectives:The cyclic AMP response element-binding protein (CREB) has been extensively implicated in learning and memory, long term potentiation (LTP), and neuroprotection. Phosphorylation/activation of CREB (pCREB) on Ser 133 by cyclic AMP-or Ca2+-dependent protein kinase is critical for long-term memory consolidation. This part is to explore the correlation between cAMP/CREB-neuronal apoptosis/neurogenesis and cognitive dysfunction induced by inhalation anesthesia.Methods:At 48h after anesthesia with 1.3% Sev+NaO and immediately after probe test 1, levels of cAMP in the hippocampus were detected by ELISA. Expression of pCREB, CREB and Bax was measured by Western Blot. The neurogenesis markers nestin and DCX was assessed by immunofluorescence. Double labeling with Annexin V-FITC and NeuN was used to mark the neuronal apoptosis. Nissl staining was used to number the functional neurons in the hippocampus.Results:In this part, experiments revealed that the cAMP and pCREB levels in the dorsal hippocampus were decreased in rats with anesthetic exposure in comparison with control rats at 48h as well as after the probe trial, but there were no significant differences in CREB expression. Besides these, quantitative analysis of Nissl-positive cells in hippocampal CAl showed a neuronal loss in exposed rats compared with their controls at 48h and after probe test. Annexin V-FITC by immunofluorescence revealed the more neurons were underwent apoptosis in exposed rats, moreover double staining of Annexin V and the neuronal marker NeuN indicated that most of the apoptotic cells in these structures were indeed neurons. At 48h after anesthesia, Bax expression significantly increased in the exposed rats compared with the control rats. However, after probe test, the Bax expression was only slightly higher in the exposed rats. Furthermore, in this part, we also found less nestin-positive neurons as well as DCX-positive neurons in the DG area of hippocampus in exposed rats compared to the control rats, which suggested neuronal progenitor proliferation and differentiation was inhibited.Conclusions:Down-regulation of cAMP/CREB signaling, neuronal apoptosis, decrease of CREB-dependent neurogenesis in the hippocampus is associated with the neurotoxicity and cognitive dysfunction induced by general anesthesia with sevoflurane and nitrous oxide.