The Role Of CX3CL1-CX3CR1 Signaling On Microglia M2 Polarization And Surgery-induced Cognitive Deficits In Aged Rats

Objective: Postoperative cognitive dysfunction(POCD)is a well-known complication following major surgery in elderly patients.Although the mechanisms underlying the pathophysiology of POCD remain elusive,age has been increasingly reported as a significant and independent risk factor for POCD.Accumulating evidence has demonstrated that the neuroinflammatory response plays a key role in the pathogenesis of POCD.Proinflammatory cytokines inhibit neuronal functions,including long-term potentiation(LTP),dendritic branching,oxidative stress activation,and increased permeability of the blood brain barrier(BBB).Microglia play a major role in the development of POCD.Microglia are primary immune cells and are the major source of pro-inflammatory cytokines in the central nervous system(CNS).The activated microglia play a dual role because of distinct microglia phenotypes,including deleterious M1 and neuroprotective M2 phenotypes.M1 represents a detrimental state of microglia,characterized by high expression of proinflammatory mediators(e.g.IL-1β,IL-6 and TNF-α).Conversely,the M2 phenotype may reverse neuron loss,accelerate the removal of cellular debris,repair neural networks and enhance the production of neurotrophic mediators.C-X3-C motif chemokine ligand 1(CX3CL1)is expressed on neurons and is thought to modulate microglial function through interactions with its receptor C-X3-C motif chemokine receptor 1(CX3CR1).CX3CL1 has been demonstrated to play a neuroprotective role by reducing neurotoxicity,inhibiting microglial activation,and promoting the release of neuroprotective soluble factors(e.g.,adenosine).The increase of immunomodulatory miRNA may lead to the dysregulation of microglia and the enhancement of inflammation.There are numerous identified miRNAs,we focused on immunomodulatory miRNAs.These miRNAs have significant regulatory effects on multiple target genes and multiple signaling pathways.MiRNA can play a role in learning and memory by targeting CX3CL1 to regulate the inflammatory process.This study investigated the potential role of age-related differences of surgery-induced cognitive deficits and neuroinflammatory responses.The potential role of microglia M2-type polarization on postoperative cognitive dysfunction was also investigated.Thisstudy investigated the potential role of CX3CL1-CX3CR1 signaling in age-related differences of surgery-induced cognitive deficits and neuroinflammatory responses.The mechanism of microRNA-29b-3p regulating the effect of CX3CL1 mRNA on microglia function was also investigated.Methods:Part I: Effect of microglia M2 polarization on surgery-induced cognitive impairment and neuroinflammation in aged rats Both adult and aged Sprague-Dawley male rats were subjected to partial hepatectomy or partial hepatectomy with a cisterna magna infusion of IL-4.On postoperative days 1,3,and 7,the rats were subjected to a reversed Morris water maze test.Hippocampal IL-1β,IL-6,IL-4,and IL-4 receptor(IL-4R)were measured at each time point.Brain derived neurotrophic factor(BDNF),Ionized calcium-binding adapter molecule 1(Iba-1),microglial M2 phenotype marker Arg1 were also examined in the hippocampus.Part II: The role of CX3CL1-CX3CR1 signaling on surgery-induced cognitive deficits and neuroinflammation in aged rats Both adult and aged Sprague-Dawley male rats were subjected to partial hepatectomy or partial hepatectomy with intracerebroventricular infusion of CX3CL1.On postoperative days 3,7,and 14,the rats were subjected to Open field test and Morris water maze test.Hippocampal IL-1β,CX3CL1,and CX3CL1 receptor(CX3CR1)were measured at each time point.Brain derived neurotrophic factor(BDNF),Ionized calcium-binding adapter molecule 1(Iba-1),and microglial M2 phenotype marker Arg1 were also examined in the hippocampus.Part III: MicroRNA-29b-3p affects surgery-induced cognitive deficits and neuroinflammation by regulating CX3CL1 mRNA The effect of miR-29b-3p on CX3CL1 was confirmed by cell experiments.Mimics and inhibitor of miR-29b-3p were added into HT22 cells to detect the expressions of miR-29b-3p and CX3CL1 by PCR.Animal experiments confirmed that mimics and inhibitor of miR-29b-3p were injected in vitro,and the expression of CX3CL1 and inflammatory cytokines in the hippocampal tissues of mice in each group were detected by real-time PCR and Western blot.Results:Part I: Effect of microglia M2 polarization on surgery-induced cognitive impairment and neuroinflammation in aged rats Age induced an exacerbated cognitive impairment and an amplified neuroinflammatory response triggered by surgical stress on postoperative days 1 and 3.A corresponding decline in the anti-inflammatory cytokine IL-4 and BDNF were also found in the aged rats at the same time point.Treatment with IL-4 downregulated the expression of proinflammatory cytokines(e.g.,IL-1β and IL-6),increased the levels of BDNF in the brain and improved the behavioral performance.An increased Arg1 expression was also observed after a cisterna magna infusion of IL-4.Part II: The role of CX3CL1-CX3CR1 signaling on surgery-induced cognitive deficits and neuroinflammation in aged rats Age induced an exacerbated cognitive impairment and an amplified neuroinflammatory response triggered by surgical stress on postoperative day 3.The surgery-related decreases in CX3CL1 and CX3CR1 proteins were accompanied by amplified microglial activation as indicated by increased Iba-1 expression in the aged rats on postoperative days 3,and 7.A corresponding decline in Arg1 and BDNF were also found in the aged rats on postoperative days 3,and 7.Treatment with CX3CL1 downregulated the expression of proinflammatory cytokines(e.g.,IL-1β),increased the levels of BDNF and Arg1 in the brain and improved the behavioral performance.After LPS treatment of microglia,the proinflammatory cytokine(IL-1β)increased.After treatment with exogenous CX3CL1 lead to microglia M2 polarization,the IL-1β level was significantly decreased,and Arg1 expression was significantly increased.Part III: MicroRNA-29b-3p affects surgery-induced cognitive deficits and neuroinflammation by regulating CX3CL1 mRNA The expression of mir-29b-3p was increased after surgery in elderly rats.In HT22 cells,overexpression of mir-29b-3p down-regulated the expression of CX3CL1,and inhibition of mir-29b-3p up-regulated the expression of CX3CL1.In vivo experiments,mir-29b-3p can regulate the expression level of CX3CL1 protein.Injection of mir-29b-3p mimics into the lateral ventricle reduced CX3CL1 and increased the proinflammatory factor il-1in the hippocampal area of elderly rats;on the contrary,injection of mir-29b-3p inhibitor into the lateral ventricle increased CX3CL1 and decreased the proinflammatory factor il-1 in the hippocampal area of elderly rats.Conclusion:1.An age-related decrease in IL-4 expression exacerbated surgery-induced cognitive deficits and exaggerated the neuroinflammatory responses.Treatment with IL-4potentially attenuated these effects by enhancing BDNF expression,inhibiting microglia activation and decreasing the associated production of proinflammatory cytokines.2.The surgery-related decreases in CX3CL1 and CX3CR1 expression exacerbated postoperative cognitive deficits and exaggerated neuroinflammatory responses in this rodent model.Treatment with CX3CL1 potentially attenuated these effects by inhibiting microglial activation,decreasing the associated production of proinflammatory cytokines and enhancing BDNF expression.3.The surgery-related increases the microRNA-29-3p expression.Overexpression of miR-29b-3p can down-regulate the expression of CX3CL1,while inhibition of miR-29b-3p can up-regulate the expression of CX3CL1.Inhibition of miR-29b-3p can regulate the up-regulation of CX3CL1 mRNA expression,promote the M2 polarization of microglia,and reduce the production of proinflammatory cytokines.