The Role And Mechanism Of LHb-VTA Circuit In Postoperative Cognitive Dysfunction

Background:Postoperative cognitive dysfunction(POCD)is a common neural complication after anesthesia and surgery,which is closely related to the high mortality and disability rate of patients,but the specific mechanism is still unclear.In order to better understand and intervene in POCD,understanding the underlying neural circuit mechanism of the occurrence and development of POCD will help to accurately intervene the abnormal neural circuits of POCD and improve abnormal behaviors.In recent years,with the development of optogenetic and chemogenetic techniques,we can artificially activate or inhibit the activities of neurons in different brain regions to understand the neural circuits mechanisms related to behavior.Lateral habenula(LHb)functionally interacts with the dorsal hippocampus and is the hub that connects the limbic system and midbrain monoaminergic nucleus,and plays an important role in regulating motivation,cognition and movement processes.Ventral tegmental area(VTA)is a nucleus where dopaminergic neurons are concentrated in the midbrain.It releases dopamine to the prefrontal cortex,hippocampus and many other brain areas,and plays an important role in regulating the growth of dendritic spine and cognitive function.This study intends to discuss the role and potential mechanism of the LHb-VTA circuit in POCD and provide a new target for the prevention and treatment of POCD.Part one:The relationship between limbic system-LHb-VTA network and postoperative cognitive dysfunctionObjective:To explore the neural activation of brain areas related to cognition after surgery and look for the potential neural circuits that mediate POCD.Methods:(1)Thirty CD1 male 8-week-old mice were randomly divided into 2 groups(n=15):control group and surgery group.Animal surgery model establish:mice were anesthetized with 1.8%isoflurane.a 1.5 cm midline neck incision was made after the mouse was exposed to sevoflurane for about 30 minutes.One centimeter long right common carotid artery was carefully dissected free from adjacent tissues without any damage on vagus nerve and explored gently.The operation time was about 15 minutes.The incision was sutured,and the total time of anesthesia was 2 hours.After the operation,all animals were injected with bupivacaine 3 mg/kg subcutaneously.Control group inhaled air/oxygen(1:1)for 2 hours without anesthesia and surgery.Open field(OF)test was performed on the 4th postoperative day.Novel object recognition(NOR)test was performed on the 5th postoperative day,and the Barnes maze(BM)test was performed on the 6th postoperative day.After 4 consecutive days of training,short-term and long-term memory tests of Barnes maze were performed on the 10th and 17th days after surgery.Fear conditioning(FC)test was performed on the 18th day after surgery.24 hours after the training section,context-related memory and tone-related memory tests were performed to observe the freezing time.(2)Twelve CD1 male 8-week-old mice were randomly divided into 2 groups(n=6):control group and surgery group.Mice were decapitated 3 hours after surgery and their brains were immersed in 10%neutral formalin at 4℃ for 72 hours,then paraffinembedded and sectioned.The number of c-Fos positive cells in hippocampus(HIP),prefrontal cortex(PFC),medial habenula(MHb),lateral habenula(LHb),paraventricular thalamic nucleus(PVT),mediodorsal thalamic nucleus(MD),amygdala and ventral tegmental area(VTA)were counted by immunofluorescence staining.Results:(1)There was no significant difference in spontaneous activity among the groups(P>0.05).The exploration time of new object in surgery group was significantly shorter than that of the control group(P<0.01).In the training phase of the Barnes maze experiment,the time to identify the target box in the two groups decreased with the increase of training section(P<0.05),but the reduction degree of the surgery group was significantly less than that of the control group(P<0.05).In the short-term and long-term test phases of the Barnes maze,mice in surgery group took more time to recognize the target box than control group(P<0.01,P<0.01,respectively).In fear conditioning test,the freezing time of the context-related memory and tone-related memory were significantly impaired in surgery group compared with control group(P<0.01,P<0.05,respectively).(2)The immunofluorescence staining results showed that the number of c-Fos positive cells in PFC,PVT,MD,and amygdala in surgery group was significantly higher than that in control group(P<0.01).C-Fos was almost not expressed in LHb and VTA in control group,but it increased significantly after surgery(P<0.01).Conclusion:Our results indicate that the right carotid artery exploration surgery cause postoperative cognitive dysfunction of mice.Surgery causes neural activation in multiple brain regions,and LHb and VTA neurons are significantly activated after surgery.The LHb and VTA may be involved in mediating the occurrence and development of POCD.Part two:The role of LHb-VTA circuit in postoperative cognitive dysfunctionObjective:Animal behavior tests and molecular biological techniques were used to explore the role of the LHb-VTA circuit in POCD.Methods:(1)Virus of pAAV2-hSyn-mCherry was injected into LHb to study the neural projection and type from LHb to VTA.(2)Western blot,immunofluorescence staining and chemogenetic techniques were used to study the activity of LHb-VTA circuit after surgery.(3)Chemogenetic technique was used to artificially control the activity of LHb-VTA circuit after surgery and observe the effect of the LHb-VTA circuit in postoperative cognitive function.Open field test was performed on the 4th postoperative day,Novel object recognition test was performed on the 5th postoperative day,and the Barnes maze test was performed on the 6th postoperative day.After 4 consecutive days of training,short-term and long-term memory tests of Barnes maze were performed on the 10th and 17th days after surgery.Fear conditioning test was performed on the 18th day after surgery.24 hours after the training section,context-related memory and tone-related memory tests were performed to observe the freezing time.(4)Chemical lesion technology was used to observe the role of LHb in postoperative cognitive function.Open field test was performed on the 4th postoperative day,Novel object recognition test was performed on the 5th postoperative day,and the Barnes maze test was performed on the 6th postoperative day.After 4 consecutive days of training,short-term and long-term memory tests of B arnes maze were performed on the 10th and 17th days after surgery.Fear conditioning test was performed on the 18th day after surgery.24 hours after the training section,context-related memory and tone-related memory tests were performed to observe the freezing time.Results:(1)There was a direct neural projection from LHb to VTA,and the LHb projection fiber co-stained with vGluT2 in VTA.(2)The expression of c-Fos in LHb and VTA was significantly higher than that of the control group at 3,24 and 48 hours after operation(P<0.01,P<0.01,P<0.05,respectively),and decreased to control level at 72 hours after surgery(P>0.05).Chemogenetic technique effectively inhibited the activation of the LHb-VTA circuit and the expression of c-Fos in LHb and VTA was significantly reduced after surgery(P<0.01,P<0.01,respectively).(3)Inhibiting the abnormal activation of the LHb-VTA circuit after surgery significantly increased the time exploring new object(P<0.05),improved the learning curve of mice in the Barnes maze training phase(P<0.05),reduced the time to recognize the target box in the short-term and long-term test phases of the Barnes maze experiment(P<0.05,P<0.01,respectively),and significantly increased context-related memory and tonerelated memory in fear conditioning test after surgery(P<0.01,P<0.05,respectively).(4)Chemically LHb lesion significantly reduced VTA c-Fos expression after surgery(P<0.01)and had no impact on the normal cognitive function(P>0.05).The LHb lesion significantly increased the time to explore new object(P<0.05),improved the learning curve of mice in the Barnes maze training phase(P<0.05),reduced the time to recognize the target box in the short-term and long-term memory tests of Barnes maze(P<0.01,P<0.01,respectively),and increased context-related memory and tonerelated memory in fear conditioning test after surgery(P<0.05,P<0.01,respectively).Conclusion:There is a direct glutamatergic projection from LHb to VTA.The LHbVTA circuit is over-activated after surgery.Inhibiting the activation of the LHb-VTA circuit improve the postoperative cognitive function of mice;The LHb-VTA circuit is involved in mediating the occurrence of POCD.Part three:The LHb-VTA circuit activates NMDA receptor to cause endoplasmic reticulum stress mediating the occurrence of POCDObjective:Western blot,ELISA,immunohistochemistry,TUNEL staining,Golgi staining and behavioral tests were used to explore the relationship between LHb-VTA circuit activation after surgery and VTA endoplasmic reticulum stress and to explore whether NMDA receptor is involved.Methods:(1)Using immunohistochemistry,Western blot,TUNEL staining and ELISA to explore the effect of postoperative LHb-VTA circuit activation on NMDA receptor activity,endoplasmic reticulum stress,inflammation and apoptosis in VTA.The Golgi staining was used to study the effect of LHb-VTA circuit activity on the dendritic spine density of prefrontal cortex and hippocampus.(2)Molecular biological techniques and animal behavior tests were used to explore the effect of NMDA receptor of VTA on postoperative cognitive function.At 24 hours after operation,immunohistochemistry,Western blot,TUNEL staining,and ELISA were used to explore the effect of inhibiting NMDA receptor activity of VTA on endoplasmic reticulum stress,inflammation and apoptosis.Open field test was performed on the 4th postoperative day,Novel object recognition test was performed on the 5th postoperative day,and the Barnes maze test was performed on the 6th postoperative day.After 4 consecutive days of training,short-term and long-term memory tests of B arnes maze were performed on the 10th and 17th days after surgery.Fear conditioning test was performed on the 18th day after surgery.24 hours after the training section,context-related memory and tone-related memory test were performed to observe the freezing time.(3)Molecular biological techniques and animal behavior tests were used to determine the effect of endoplasmic reticulum stress on postoperative cognition.At 24 hours after operation,immunohistochemistry,Western blot,TUNEL staining,and ELISA were used to study the effect of inhibiting endoplasmic reticulum stress on the inflammation and apoptosis of VTA.Open field test was performed on the 4th postoperative day,Novel object recognition test was performed on the 5th postoperative day,and the Barnes maze test was performed on the 6th postoperative day.After 4 consecutive days of training,short-term and longterm memory tests of Barnes maze were performed on the 10th and 17th days after surgery.Fear conditioning test was performed on the 18th day after surgery.24 hours after the training section,context-related memory and tone-related memory test were performed to observe the freezing time.Results:Compared with the control group,p-NR1 in VTA increased significantly at 24 and 48 hours after operation(P<0.01,P<0.01,respectively),and decreased to the control level at 72 hours after surgery(P>0.05),which was consistent with the activation of the LHb-VTA circuit after operation.The expression of endoplasmic reticulum stress-related proteins CHOP and XBP1s in VTA increased significantly(P<0.01,P<0.01,respectively),and the concentration of inflammatory factors IL-1βand IL-6 increased significantly(P<0.01,P<0.01,respectively)after operation.Cleaved-Caspase 3 level and cell apoptosis rate increased significantly in VTA after surgery(P<0.01,P<0.01,respectively).Dopaminergic neurons amount in VTA and dendritic spine density of prefrontal cortex and hippocampus decreased significantly after surgery(P<0.01,P<0.01,P<0.01,respectively).Inhibition of postoperative abnormal activation of the LHb-VTA circuit significantly decreased NMDA receptor activation(P<0.05),the expression of CHOP and XBP1s(P<0.01,P<0.01,respectively),the concentration of IL-1β and IL-6(P<0.01,P<0.01,respectively),cell apoptosis(P<0.01),the loss of dopaminergic neurons(P<0.05)in VTA and significantly increased dendritic spine density of prefrontal cortex and hippocampus after surgery(P<0.01,P<0.01,respectively).Inhibiting the activation of NMDA receptor and endoplasmic reticulum stress in VTA after surgery significantly reduced the expression of CHOP and XBP1s(P<0.05),the concentration of inflammatory factors IL-1β and IL-6(P<0.01),cell apoptosis(P<0.01),and the loss of dopaminergic neurons(P<0.01)in VTA.Inhibiting the activation of NMDA receptor and endoplasmic reticulum stress in VTA after surgery significantly increased the time exploring new object(P<0.05),improved the learning curve of mice in the Barnes maze training phase(P<0.05),reduced the time to recognize the target box in the short-term and long-term test phases of the Barnes maze experiment(P<0.05),and significantly increased context-related memory and tone-related memory in fear conditioning test after surgery(P<0.05).Conclusion:After the operation,the LHb-VTA circuit is over-excited,which induces VTA endoplasmic reticulum stress by activating NMDA receptor,causing inflammation,apoptosis and the loss of dopaminergic neurons in VTA.These changes lead to a decrease in the dendritic spine density of prefrontal cortex and hippocampal,which ultimately leads to postoperative cognitive dysfunction.