Regulation Of Postoperative Delirium By Endocannabinoid Signaling In Prefrontal Cortex

Every year,there are approximately 313 million surgical patients,the majority of whom will benefit from general anesthetic.Nonetheless,general anesthetics may have an effect on perioperative cerebral functions,leading to postoperative complications such postoperative psychosis（POD）.POD,which affects up to 73.5%of surgical patients,is characterized by fluctuations in consciousness,attention,learning memory function,and mood within 5 days following surgery,resulting in long-term postoperative cognitive dysfunction,dementia,and a threefold increase in mortality.However,the pathophysiology of POD,particularly its brain circuit mechanisms,is still poorly understood.The medial prefrontal cortex（mPFC）,a high-order cortex,plays a vital role in consciousness and cognitive control.Recent research also showed that increased pro-inflammatory cytokines in the mPFC induced by lipopolysaccharide（LPS）led to delirium-like behavior in mice.Furthermore,general anesthesia and surgery（AS）decreased excitatory synaptic transmission in mPFC pyramidal neurons,but did not change the firing properties of the mPFC pyramidal neurons.We previously revealed that endocannabinoid（e CBs）signal could modulate general anesthesia recovery through the presynaptic CB1R in the dorsomedial hypothalamus（DMH）,which is innervated by mPFC.Considering e CBs modulate synaptic and neuronal functions as a widely distributed neuromodulator,we proposed e CBs signaling in the mPFC may participate in both of consciousness and cognition alteration during general anesthesia and postoperative period.In the present study,we applied chemical genetics,molecular probe,in vivo electrophysiology,combined with behavioral tests and pharmacological intervention to investigate the e CB associated perioperative alteration of neurological functions.We mainly focused on the mPFC in e CB associated POD progressing,to screened the cell specificity and its functional circuit.Furthermore,the effect of plasma membrane CB1R（pm CB1R）and mitochondrial CB1R（mt CB1R）was evaluated in POD development and general anesthesia.Part I.Involvement of endogenous cannabinoid signaling of the prefrontal cortex in postoperative deliriumObjectives:After establishment of the POD model,we aimed to clarify whether the endogenous cannabinoid signaling is involved in POD development using behavioral tests,pharmacological intervention,and molecular probes.And then we will reveal the role of mPFC in e CBs associated POD regulation,including the changes of e CB signaling in the mPFC during the perioperative period.Methods:1.Establishment of POD model.C57 mice was exposed to 1.4%isoflurane anesthesia for 2h with a simple laparotomy,to simulate the anesthetic surgical（AS）procedure.Behavioral tests,including buried food experiment,open field experiment and Y-maze experiment,were performed at 24h before AS,and at 6h,9h and 24h after AS,respectively.The POD-like behavioral impairment was evaluated by composite Z score,manifesting as the sum of Z scores of six behavioral tests normalized with the SD for that sum in the Shams.2.Antagonists of CB1R,including AM281（3μg/mg body weight,antagonizes both mt CB1R and pm CB1R）and Hemopressin（1μg/mg body weight,antagonizes only pm CB1R）,was injected intraperitoneally to clarify the effect of systemic antagonism of CB1R on POD.Next,AM281（0.1μg/0.2μl/side）and Hemopressin（0.1μg/0.2μl/side）was administered intra the mPFC through bilateral cannulas to confirm the involvement of mPFC in e CB associated POD development.3.Throughout the AS and postoperative behavioral tests,fiber photometry was applied to record the e CB signaling,which was achieved by AAV-h Syn-e CB microinjection and optic fiber implantation into the mPFC.The western blot technique was applied to verify further the CB1R expression changes in the mPFC of mice before and after AS.Results:1.The POD model was successfully established.The composite Z score of mice was significantly decreased at 6h（Sham group 0.00±0.55 vs AS group 4.93±1.358,P=0.042）and 9h（Sham group 0.00±0.695 vs AS group 4.368±1.271,P=0.0167）after AS,but were not significantly different from the Sham group at 24h（Sham group 0.00±0.438 vs AS group 2.426±0.69,P=0.1153）,suggesting that the POD model was successfully constructed.In the further experiments,we will mainly investigate behavioral tests at 6h and 9h after AS.2.Systemic antagonism of CB1R could alleviate POD.Intraperitoneal injection of CB1R antagonist significantly reduced the composite Z score at 9h after AS[F（3,44）=7.263,P=0.0005],while improved the freezing time in the open field and latency to eat buried food.3.The e CB signaling in the mPFC was activated after AS.The signal strength of e CB in the mPFC decreased followed by an increase during anesthesia induction,increased significantly during surgery,and gradually leveled off after the end of anesthesia.Furthermore,the e CB signal was significantly higher during the Y-maze test at 6h after AS compared to the Sham group[F（1,6）=7.184,P=0.0365].CB1R expression in the mPFC was time-dependently increased,with statistical difference at 9h after AS（Sham group1.000±0.1364 vs AS group 1.419±0.06338,P=0.0360）.4.Antagonism of CB1R in the mPFC could alleviate POD.The administration of the CB1R antagonist AM281 in mPFC significantly reduced composite Z scores at 6h and 9h after AS[6h:F（3,36）=8.869,P<0.0001;9h:F（3,6）=6.405,P=0.0014].Conclusion:AS significantly induced POD like cognitive impariments at 6h and 9h after AS.The e CB signaling in the mPFC was activated until at 9h after AS.Antagonism of CB1R in the mPFC could significantly increase the composite Z score at 6h and 9h after AS.Part II A study of the neurological circuitry in endocannabinoids associated postoperative delirium modulationObjectives:After confirming the involvement of mPFC in e CB-regulated POD processes,this part of the study was focused on exploring the neuronal and circuitry specificity of mPFC involved in e CB associated POD development using behavioral tests,chemogenetics,transgenic animals,immunofluorescence and in vivo electrophysiology.Methods:1.Establishment of POD model.Same as part I.2.Using v Glut2-Cre and v Gat-Cre mice,the glutamatergic and GABAergic neurons of the mPFC were selectively activated by chemogenetic,respectively.Furthermore,exogenous cannabinoid,Nabilone（3μg/mg body weight）,was microinjected into the mPFC to clarify whether the the PFC neurons regulating POD was influenced by e CBs.3.In vivo electrophysiological techniques was applied to verify the electrical activity of glutamatergic neurons in the mPFC during AS and behavioral tests.4.The neuronal specificity of Mediodorsal thalamus（MD）and hippocampus（HIP）,which innervated the glutamatergic neurons in the mPFC,was validated using a combination of RV reverse tracing and immunofluorescence.5.Chemogenetics with Cre-lox P and Flp-FRT dual enzyme systems was used to activate glutamatergic projections from mediodorsal thalamus（MD）and hippocampus（HIP）to the mPFC in v Glut2-Cre mice.Application of Nabilone（0.1μg/0.2μl/side）was used to explore the influence of e CB in POD regulation effect of above circuits.6.To selectively knock out the presynaptic CB1R of MD-mPFC projection,AAV-h Syn-f DIO-Cre-m Cherry was microinjected into the MD,while AAV-retro-h Syn-FLP-EGFP was microinjected into the mPFC in CNR1-flox mice.This experiment aimed to verify the role of presynaptic CB1R of MD-mPFC projection in postoperative cognitive impairments.7.We further investigated the effect of mt CB1R in the mPFC in POD regulation using AAV-h Syn-mito-cre microinjection into the mPFC in CNR1-flox mice to selective knockout of mt CB1R.Results:1.The mPFC glutamatergic neurons mediated the regulatory effects of e CB on POD.Activation of the mPFC glutamatergic neurons significantly reduced the composite Z score at 6h and 9h after AS[6h:F（3,20）=8.079,P=0.001;9h:F（3,20）=11.50,P=0.0001],which could be reversed by Nabilone microinjection.On the other hand,activation of the mPFC GABAergic neurons also reduced the composite Z score at 9h after AS[F（3,20）=7.754,P=0.0013],however,this effect could not be reversed by Nabilone.The mPFC glutamatergic neurons exhibited a significantly lower firing frequency in the Y-maze test 6h after AS（Sham group 5.810±1.518 vs AS group 4.138±0.4466,P=0.0395）,indicating a relative inhibiting of the mPFC glutamatergic neurons may contribute to POD.2.The glutamatergic neurons of mPFC were mainly innervated by MD glutamatergic neurons.Retrograde tracing showed that glutamatergic neurons in the mPFC mainly received projections from MD glutamatergic neurons rather than GABAergic neurons（glutamatergic neurons 0.475±0.058 vs GABAergic neurons 0.285±0.067,P=0.0476）.HIP had almost no projection neurons to the mPFC region.3.MD^glu-mPFC^glumediates the modulatory effects of e CB on POD.Chemogenetic activation of MD^glu-mPFC projection significantly decreased the composite Z score at 6h and 9h after AS[6h:F（3,20）=7.606,P=0.0014;9h:F（3,20）=9.646,P=0.0004],which could be reversed by administration of Nabilone intro mPFC.However,activation of HIP^glu-mPFC projection had no influence on the composite Z score after AS.Further study revealed that activation of MD^glu-mPFC^glucircuitry could statistically reduce the composite Z score at 6h after AS[F（3,20）=8.470,P=0.0008],which could be also significantly reversed by Nabilone microinjection.4.Selective knockout of presynaptic CB1R of MD-mPFC projection significantly improved cognitive impairments.Selective knockout of CB1R in MD glutamatergic neurons,which projected to the mPFC,significantly shortened the latency to eat food at6h and 9h after AS[F（2,15）=6.076,P=0.0117],increased the number to novel arm in Y-maze at 9h[F（2,15）=6.755,P=0.0081]and increased the exploration index RI in new object recognition in mice at 9h after AS[F（2,15）=5.092,P=0.0205].5.Knock out of mt CB1R in the mPFC could attenuate POD.Selective knockout of the mt CB1R in mPFC neurons significantly decreased the composite Z score at 9h after AS[F（2,15）=6.177,P=0.0110].Conclusion:The mPFC glutamatergic neurons,but not GABAergic neurons,mediated the effects of the e CBs on POD.Furthermore,e CBs regulated POD through the MD^glu-mPFC^glubut not the Hip^glu-mPFC circuitry.Interestingly,both of the presynaptic pm CB1R of MD^glu-mPFC^glucircuitry and the mt CB1R in the mPFC may be involved in the cognitive impairments after AS.Part III Research on the consciousness alteration during general anesthesia modulated by endocannabinoids associated mitochondrial function in the mPFCObjectives:Considering the relationship between POD and alteration of neurological function during general anesthesia,we further investigated the effects of endocannabinoids signaling in the mPFC in general anesthesia associated consciousness alteration,specifically focused on the role of mt CB1R in mPFC using anesthesia behavioral tests,electroencephalogram（EEG）,fiber optic recording and molecular biology.Methods:1.Mice were subjected to 1.4%isoflurane for 30min.The time of loss of righting reflex（LORR）and recovery of righting reflex（RORR）were recorded using pharmacological intervention and anesthesia behavior,to explore the effects of CB1R in the mPFC on general anesthesia.2.Experimental method of anesthesia sensitivity.Anesthesia sensitivity during the induction period:The ISO concentration started from 0.2%and increased in the order of0.1%each time,with each concentration lasting for 15 min and the maximum concentration being 1.4%.The LORR was determined when each concentration lasted for15 min,and if LORR occurred,the ISO concentration at this time was determined as the effective concentration for induction of anesthesia.Anesthesia sensitivity in the emergency period:The ISO concentration started from 1.4%and reduced in the order of 0.1%each time,with each concentration lasting for 15 min and the minimum concentration being0.2%.The RORR was determined when each concentration lasted for 15 min,and if RORR occurred,the ISO concentration at this time was determined as the effective concentration for anesthesia emergency.3.EEG recording and analysis during general anesthesia were conducted to observe the effect of CB1R on the the power spectrum and burst suppression ratio（BSR）.4.Fiber optic and pharmacological intervention were used to record the intracellular ATP（i ATP）fluctuation in the mPFC during general anesthesia.5.Using UV spectrophotometry and pharmacological intervention,the activity of mitochondrial respiratory chain in the mPFC after 2h of isoflurane exposure.Results:1.Blockade of CB1R promoted general anesthesia induction and recovery.Intraperitoneal injection of AM281,which could blockade both the pm CB1R and mt CB1R,could significantly shorten LORR[F（2,21）=9.608,P=0.0011]and RORR[F（2,21）=4.151,P=0.0303].As well,the anesthesia-induced EC50（AM281 group 0.800 to 0.873 vs Veh group 0.957 to 0.971,P<0.001）and EC50 for anesthesia awakening（AM281 group0.550 to 0.572 vs Veh group 0.425 to 0.442,P<0.001）was left shifted.Systemic administration of Hemopressin,which is an antagonist of pm CB1R,only shortened the LORR（Hemo group 7.094±0.4354 vs Veh group 8.781±0.2773,P=0.0239）.2.Blockade of mt CB1R in the mPFC reduced LORR and RORR.Microinjection of AM281 intra the mPFC significantly shorten the LORR[F（2,26）=4.037,P=0.0297]and RORR[F（2,13）=9.892,P=0.0024]compared to vehicle,while AM281 left shifted the anesthesia-induced EC50（AM281 group 0.847 to 0.870 vs Veh group 0.994 to 0.997,P<0.001）and EC50 for anesthesia awakening（AM281 group 0.496 to 0.504 vs Veh group0.396 to 0.404,P<0.001）.Microinjection of Hemopressin into the mPFC had no effect on LORR and RORR.3.Blockade of the mt CB1R in the mPFC accelerated the EEG changes after general anesthesia.During isoflurane induction,the total power ofδ-band as significantly higher in the AM281 group than in Veh or Hemopressin groups[F（2,6）=19.57,P=0.0023],compared with reduction in theα-band power[F（2,6）=16.54,P=0.0036]andβ-band power[F（2,6）=5.711,P=0.0408]in the AM281 group.The AM281 also promoted the time of BSR appearance[F（2,10）=6.019,P=0.0192]and the time of BSR disappearance[F（2,8）=5.688,P=0.0291],resulting in a much lower BSR during 28-30 min of anesthesia compared to Veh group and Hemopressin group[F（2,6）=12.95,P=0.0067].4.Blockade of mt CB1R in the mPFC intensified the energy reduction during induction and accelerated the energy increase during recovery.In the AM281 group,the slope of i ATP reduction[ΔF/F_ana,F（2,9）=5.807,P=0.0240]during isofluran induction and elevation[ΔF/F_awake,F（2,9）=8.550,P=0.0083]during recovery from anesthesia were significantly higher compared with the other groups.5.Blockade of mt CB1R could reverse the inhibition of mitochondrial functions induced by 2h isoflurane anesthesia.Two hour exposure of isoflurane could significantly inhibit the activity of mitochondrial complexⅠ-Ⅴ,while AM281 application could reverse the inhibition of mitochondrial complexⅠ[F（3,16）=37.21,P<0.0001],mitochondrial complexⅢ[F（3,16）=37.10,P<0.0001],mitochondrial complexⅣ[F（3,16）=94.95,P<0.0001]and mitochondrial complexⅤ[F（3,12）=47.60,P<0.0001].However,Hemopressin only improved the function of mitochondrial complexⅤ.Conclusion:Not only postoperative period,endocannabinoids signaling influence the procedure of general anesthesia mainly through the mt CB1R in the mPFC.Blockade of mt CB1R in the mPFC could accelerated the energy inhibition during anesthesia induction to promote consciousness lost,and accelerate the energy elevation during stage of awakening to promote consciousness recovery.However,AS activated the endocannabinoids signaling even after AS,which may inhibited the function of MD^glu-mPFC^glucircuitry,leading to POD development.