Branched-chain Amino Acids Improve Postoperative Delirium-like Behavior And Cognitive Impairment In Mice

Objective: Postoperative delirium(POD)is an early postoperative cognitive dysfunction,which is manifested as acute,transient and reversible fluctuating disorders of attention,cognition,mood and psychomotor behavior occurring after anesthesia and surgery.Pod often leads to prolonged hospitalization,long-term cognitive dysfunction,and increased morbidity and mortality.At present,there is no perfect prevention and treatment of postoperative delirium,and the effect of branched-chain amino acid(BCAA)on cognitive impairment caused by postoperative delirium is still unknown.Based on the metabolism of BCAA in the brain and its effect on improving cognitive impairment after craniocerebral injury,BCAA is likely to play an important role in the prevention and treatment of postoperative delirium and cognitive improvement.By establishing a postoperative delirium model,this study aims to clarify the expression level of BCAA in the postoperative delirium model,verify whether a high level of BCAA diet can improve delirium-like behavior and cognitive impairment after anesthesia and surgery in mice,and analyze the potential mechanism of BCAA improving postoperative cognition,so as to provide new ideas for subsequent related research and clinical prevention and treatment of postoperative delirium.Methods: Part I: An anesthesia/postoperative delirium model was established and a sham group was set up for comparison.Then,the changes of attention,consciousness and thinking of the mice were evaluated by combinative behavior,and the Z-score was calculated to analyze the incidence of delirium in the model mice.The mitochondrial and synaptic ultrastructures of hippocampal neurons were observed by transmission electron microscopy,and serum BCAA levels were detected by enzyme-linked immunosorbent assay.High-throughput sequencing technology was used to screen important differentially expressed genes that may be involved in the occurrence of delirium after anesthesia/surgery in mice,and qPCR was used to verify.PartⅡ:the mice were fed with BCAA before the establishment of anesthesia/postoperative delirium model.The combinative behavior and Z-score were used to evaluate the changes of attention,consciousness and thinking and the incidence of delirium in each group of mice.Nissl staining was used to observe the damage of hippocampal nerve cells.The protein expression levels of synapse-associated proteins(PSD95,SYP,α-syn)and STAT5 a were detected by Western blot.Results: The first part of the experimental results: the open field experiment showed that the total movement distance(% to baseline)of mice in the A+S group was significantly decreased than that in the Sham group at 6h after surgery(P < 0.05).Y maze experiment showed that the number of mice entering the new arm 6h after operation in A+S group(% to baseline)was significantly lower than that in Sham group(P < 0.01).Food burial experiment showed that the feeding latency of mice in A+S group(% to baseline)was significantly prolonged at 6h and 9h after surgery compared with that in Sham group(P < 0.05).A positive Z score was calculated for the diagnosis of delirium in each mouse,and the overall incidence of delirium was 50% over the24-hour test period.Ultrastructural analysis of mitochondrial morphology of hippocampal neurons in CA1 area showed that acute ultrastructural changes of neuronal mitochondria were observed in mice in group A+S,and obvious mitochondrial swelling was observed under microscope,which was manifested as reduced mitochondrial matrix density,shorter and fewer crists,and increased mitochondrial volume.The serum BCAA level of mice after operation was detected by ELISA,and the serum BCAA level of mice in A+S group was significantly lower than that in Sham group(P < 0.001).The differentially expressed genes were screened by high-throughput sequencing technology,and three genes were selected for RT-q PCR verification.The results showed that compared with Sham group,the relative m RNA expression levels of Stat5 a,Cdkn1a and Wnt3 in hippocampal tissue of A+S group were significantly increased(P < 0.05),and the change levels were consistent with the sequencing results.The second part of the experimental results: The open field experiment showed that,compared with the A+S+B group,the total movement distance(%to baseline)at 6h after operation were significantly improved(P < 0.05),and there were no significant differences between the A+S+B group and the Sham group(P > 0.05).Y maze experiment showed that compared with A+S+B group,the number of mice entering the new arm at 6h after operation(% to baseline)was significantly improved(P < 0.05),and there was no significant difference between group A+S+B and Sham group(P > 0.05).Food burial experiment showed that the feeding latency of group A+S+B had no significant improvement compared with group A+S at 6h and 9h after surgery(P > 0.05),and there was no significant difference compared with Sham group(P > 0.05).A positive Z score was calculated for the diagnosis of delirium in each mouse,and the overall incidence of delirium was 10% in group A+S+B mice during the24-hour test period.The serum BCAA level of mice after operation was detected by ELISA,and the serum BCAA level of mice in group A+S+B was significantly higher than that in group A+S(P<0.001).However,there were significant differences compared with Sham group(P<0.01).Observation of Nishi staining showed that Nishi staining was heavy and clear in CA1 area of hippocampal tissue in Sham group and A+S+B group,while Nishi staining was light and fuzzy in some nerve cells in CA1 area of A+S group.Western blotting results showed that compared with Sham group,the expression level of PSD95 protein in hippocampus of mice in A+S group was significantly decreased(P <0.01).Compared with A+S group,PSD95 protein expression level in hippocampus of mice in A+S+B group was significantly increased(P < 0.05),and had no significant difference compared with Sham group(P > 0.05).There were no significant differences in the expression levels of SYP protein andα-syn protein in hippocampus among the three groups(P > 0.05).Compared with Sham group,STAT5 a protein expression level in hippocampus of mice in A+S group was significantly increased(P < 0.05).Compared with A+S group,STAT5 a protein expression in hippocampal tissue of mice in A+S+B group was significantly decreased(P < 0.01),and had no significant difference compared with Sham group(P > 0.05).Conclusion: Abdominal surgery under isoflurane anesthesia induced postoperative delirious-like behavior and cognitive impairment in mice,and serum BCAA levels were reduced in this model.Dietary supplement of BCAA through absorption can increase serum BCAA level with good effectiveness,and can significantly improve delirious-like behavior and hippocampal neuron damage in mice.BCAA reduces compensatory up-regulation of Stat5 a after stress,which is an important mechanism to improve postoperative delirium and nerve injury in mice.As a potential predictive factor or therapeutic target of postoperative delirium,BCAA is worthy of further study by researchers in order to obtain good clinical effects in the future.