| Background:Delirium is a common,serious and fatal disease,and the neuropathogenesis of delirium is still unknown.The mechanism research in this field is limited,and there are no effective drugs to prevent or treat delirium.At present,there are only a few animal models that can be used for delirium research.One of the obstacles to advance the research on the mechanism of delirium is the lack of animal models.Therefore,the establishment of an animal model of delirium to study the mechanism of delirium is a problem that needs to be solved urgently.Lancet and many other documents have confirmed that there is a significant correlation between indwelling urethral catheter implantation and delirium in hospitalized patients and nursing home populations,and the incidence of delirium in the recovery period after anesthesia surgery in patients with indwelling catheters is also significantly increased.Glucose is the most important and basic energy source for living things.At present,the research on the mechanism of delirium is mostly focused on neuro inflammation and mitochondrial dysfunction.Whether it is related to glucose metabolism,there is no relevant report.This topic uses Glucose transporter-1(GLUT1)as the key protein to deeply study the effect of urethral catheter implantation on delirium-like behavior in mice,and explore its molecular mechanism to provide new ideas and treatments for the study of delirium Program.Part I Establishment of an animal model of delirium with Indwelling Urinary Catheter,the effect of indwelling catheter on mouse breathing and delirium-like behavior Objective:To explore the respiratory and behavioral changes in mice caused by indwelling catheter.Method:1.Divide 18 16-week-old C57BL/6 female mice into three groups(Control group lsoflurane group and indwelling catheter group)randomly,with 6 mice in each group.The mice in the control group received 60% oxygen + 50 mg/kg pentobarbital intraperitoneal injection,the mice in the isoflurane group received 60% oxygen +1.4% isoflurane anesthesia,and the mice in the indwelling catheter group received60% oxygen + 1.4% isoflurane.Under anesthesia,the catheter was placed under anesthesia,and the maintenance time was controlled at 10 minutes.After the operation,blood was collected from the carotid artery for blood gas analysis to study the effect of indwelling catheter and anesthesia on the respiratory function of mice.2.Divide 24 16-week-old C57BL/6 female mice into two groups(Control group and indwelling catheter group)randomly,with 12 mice in each group.Each group of mice underwent a series of behavioral tests(foraging test,open field test and Y maze test)24 hours before operation,and the basic values ??of behavioral indicators were recorded.On the second day,the mice in the control group received 60%oxygen + 1.4% isoflurane anesthesia for 10 minutes.The indwelling catheter group also received 60% oxygen + 1.4% isoflurane anesthesia.At 3,6,9,and 24 hours after operation,each mouse was subjected to these tests again(foraging test,open field test and Y maze test)to study the changes in behavior with acute and fluctuating nature of delirium animal model mice.3.A comprehensive Z score was calculated for each mouse to represent the total change of these behaviors [standardized with the scores of the control group],used to evaluate the severity of the behavior changes of each mouse,similar to the use of CAM in humans,The scale assesses the severity of delirium.In order to verify whether the animal model of indwelling catheter can be used as a model of delirium for further research.Result:1.Indwelling catheter and isoflurane anesthesia have no significant effects on the PH value of arterial blood,arterial partial pressure of oxygen(Pa O2)and arterial partial pressure of carbon dioxide(Pa CO2).2.Catheter insertion selectively impaired certain components of mouse behavior:In the foraging experiment,compared with the control group,the incubation period of mice seeking food was significantly prolonged 6 hours after catheter insertion(P<0.05);3.In the open field experiment,compared with the control group,6 hours after the catheter was inserted,the incubation period for the mice to reach the center was prolonged(P<0.05),9 hours after the catheter was inserted,the time spent by the mice in the center was significantly reduced(P<0.05);4.In the Y maze test,compared with the control group,indwelling catheter mouse number of entry into the novel arm of mice in 6 hours was reduced(P<0.05),the number of entry into the novel arm of mice in 9 hours and the exploration time of the new arm were reduced(P<0.05)5.Compared with the control group,the composite Z scores of mice increased significantly at 3,6,and 9 hours after indwelling catheter,and there was no significant difference in the composite Z scores of mice at 24 hours after indwelling catheter.Furthermore,compared with control conditions,catheter placement may cause significant behavioral disorders in mice in a time-dependent manner.Conclusion:Catheter insertion and anesthesia did not significantly affect the results of mouse breathing and blood gas analysis.Catheter insertion impaired the natural(foraging experiment and open field experiment)and the learning behavior(Y maze test)of the mice in a time-dependent manner.Part II The mechanism of urethral catheter implantation causing abnormal glucose metabolism in the brain of mice Objective: To investigate the changes of glucose transporter 1 in brain tissue and glucose levels in extracellular interstitial fluid of mice after urethral catheter implantation.Method:1.Thirty 16-week-old C57BL/6 female mice were randomly divided into two groups(Control group and Catheter group),15 mice in each group.The mice in the Control group received60% oxygen + 1.4% isoflurane anesthesia for 10 minutes.Catheter group mice were catheterized under60% oxygen + 1.4% isoflurane anesthesia,and the operation time was controlled at 10 minutes.6 hours after the catheter was inserted,the mouse cerebral cortex was taken for Western blot analysis and ATP determination(n=6),RNA was extracted for real-time fluorescent quantitative PCR analysis(n=3),After the mice heart was perfused with 4%paraformaldehyde and PBS,brain tissues were taken for immunofluorescence analysis(n=6).2.Twelve 16-week-old C57BL/6 female mice were randomly divided into two groups(Control group and Catheter group),with 6 mice in each group.Two days before the catheter insertion,the two groups of mice were implanted with a microdialysis probe under60% oxygen + 1.4% isoflurane anesthesia.When the catheter was inserted,the mice in the Angel Control group received60% oxygen +1.4% isoflurane anesthesia for 10 minutes.Catheter group mice were catheterized under60% oxygen + 1.4% isoflurane anesthesia,and the operation time was controlled at 10 minutes.Collect the mouse brain interstitial fluid in the first 1-6hours after the catheter inserted,use the glucose determination kit to detect the extracellular interstitial fluid glucose concentration of the mouse brain tissue,and monitor the mouse blood glucose once every hour(by tail Blood sampling from marginal vein).Result:1.Urethral catheter implantation reduced the levels of Slc2a1 and GLUT1 in the cerebral cortex of mice(P<0.05);2.urethral catheter implantation reduced the glucose transport in the brain of mice,and reduced the level of glucose in the extracellular interstitial fluid of the mouse brain tissue(P<0.05);3.Insertion of urinary catheter reduced the level of ATP in the brain tissue of mice(P<0.05);4.The insertion of the urinary catheter did not affect the blood glucose concentration of the mice.Conclusion:The insertion of a urinary catheter reduces the glucose transport in the brain of mice,thereby causing the glucose concentration and ATP content of the interstitial fluid of the mouse brain tissue to decrease.Part III Recombinant VEGF protein rescues delirium-like behavior changes in urethral catheter implantation mice Objective:To explore the rescue effect and mechanism of recombinant VEGF protein on delirium-like behavior changes in mice after urethral catheter implantation.Method:1.Forty-eight 16-week-old C57BL/6 female mice were randomly divided into four groups(Control+NS group,Catheter+NS group,Control+VEGF group,and Catheter+VEGF group),with 12 mice in each group.Starting four days before the catheter placement,mice in the Control+NS group and Catheter+NS group were injected with 100 ul normal saline via the tail vein for three consecutive days,once a day.Mice in the Control+VEGF group and the Catheter+VEGF group were injected with recombinant VEGF protein(0.02 mg/kg dissolved in 100 ul normal saline)via the tail vein for three consecutive days,once a day.On the day of catheter placement,mice in the Control+NS and Control+VEGF groups received60% oxygen + 1.4%isoflurane anesthesia for 10 minutes.Catheter+NS group and Catheter+VEGF group mice were catheterized under60% oxygen + 1.4% isoflurane anesthesia,and the operation time was controlled at 10 minutes.Each group of mice underwent a series of behavioral tests(buried food test,field test and Y maze test)24 hours before operation and 6 hours after operation.We calculated a comprehensive Z score for each mouse to represent the total change of these behaviors [standardized with the control group's scores] to evaluate the severity of each mouse's behavior changes.After the behavioral studies,the mouse cerebral cortex tissue was taken for Western blot analysis and ATP determination(n=6),RNA was extracted for real-time fluorescence quantitative PCR analysis(n=3),After cardiac perfusion with 4%paraformaldehyde and PBS,the mouse brain tissue was extracted for immunofluorescence analysis(n=6).2.Twenty-four 16-week-old C57BL/6 female mice were randomly divided into four groups(Control+NS group,Catheter+NS group,Control+VEGF group,and Catheter+VEGF group),with 6 mice in each group.Starting four days before the catheter placement,mice in the Control+NS group and Catheter+NS group were injected with 100 ul normal saline via the tail vein for three consecutive days,once a day.Mice in the Control+VEGF group and the Catheter+VEGF group were injected with recombinant VEGF protein(0.02 mg/kg dissolved in 100 ul normal saline)via the tail vein for three consecutive days,once a day.Two days before the catheter insertion,the two groups of mice were implanted with a microdialysis probe under60% oxygen + 1.4% isoflurane anesthesia.On the day of urethral catheter implantation,mice in the Control+NS group and Control+VEGF group received60%oxygen + 1.4% isoflurane anesthesia for 10 minutes.Catheter+NS group and Catheter+VEGF group mice were catheterized under60% oxygen + 1.4% isoflurane anesthesia,and the operation time was controlled at 10 minutes.Collect the mouse brain interstitial fluid from the first 1-6 hours after the catheter is inserted,use the glucose determination kit to detect the glucose concentration of the mouse brain interstitial fluid,and monitor the mouse blood glucose once an hour(to collect blood from the tail vein).Result:1.Recombinant VEGF protein reduced the behavioral changes caused by catheter placement,shortened the incubation period of looking for food in the food search experiment,and shortened the incubation period to the central area in the open field experiment(P<0.05).2.Recombinant VEGF protein alleviated the decrease of Scl2a1,GLUT1,ATP content in mouse brain tissue caused by urethral catheter implantation.3.Recombinant VEGF protein alleviated the decrease of glucose level in mouse brain tissue interstitial fluid caused by catheter placement.There was no obvious effect on the blood sugar of mice.Conclusion:Recombinant VEGF protein can increase the decrease of GLUT1 content in mouse brain caused by urethral catheter implantation,increase the glucose concentration and ATP content of interstitial fluid in mouse brain tissue,and Selectively rescued the behavioral changes in mice caused by catheter placement. |
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