The Role And Of Central IL-6 Trans-signaling In Neuroinflammation And Perioperative Neurocognitive Disorders Caused By Surgery

BACKGROUND:Perioperative Neurocognitive Disorders(PND)is a new term proposed in 2018 by well-known scholars in the field of anesthesia neurocognitive and multidisciplinary experts after a joint discussion,and is used in the field of anesthesiology.The term of PND were published in many of well-known journal at the same time after its nomenclature,the two concepts of Postoperative Cognitive Dysfunction(POCD)and Postoperative Delirium(POD)were officially renamed PND to promote the communication between different medical specialties for this type of perioperative neurological deficit syndromes.The incidence of PND was about 17% to 43% at one week after surgery,and 17% at 3 months after surgery.With the increasing number of operations in elderly patients year by year,the incidence of PND is also rising,bringing a heavy burden to patients and society.Although domestic and international scholars have studied perioperative neurocognitive dysfunction for many years,its pathogenesis is still not fully understood.In recent years,the influence of central nervous system inflammatory response on cognitive function has received extensive attention,and the research on its mechanism is deepening.Tissue damage at the surgical site can activate the body's innate immune system,trigger the release of tissue cytokines,and lead to an inflammatory response.The blood-brain barrier(BBB)is a dynamic barrier between brain tissue and blood.The massive release of inflammatory cytokines can damage the function of BBB and endothelial cells,resulting in increased BBB permeability.Peripheral inflammatory cytokines enter the brain and cause an inflammatory response in the central nervous system,eventually leading to the occurrence of PND.Studies have shown that the level of peripheral inflammation,the amount of cytokines entering the central nervous system and the severity of central nervous system inflammation are related to circulating interleukin-6 levels.Interleukin-6(IL-6)plays a key role in the pathogenesis of inflammatory diseases and the physiological homeostasis of neural tissues.Typical neuropathies,such as multiple sclerosis,Parkinson's disease and Alzheimer's disease,are associated with elevated expression of IL-6 in the central system.IL-6 can regulate cells through two signaling pathways,one of which is anti-inflammatory.In limited cell types expressing membrane-bound IL-6 receptor(m IL-6R),IL-6 can directly bind to IL-6R and activate ?-receptor glycoprotein 130(gp130)and subsequent downstream proteins for signal transduction called IL-6 Classic-signaling,this signaling pathway is essential for the body's immune defense.The other is pro-inflammatory,called IL-6 Trans-signaling,which relies on the soluble form of IL-6 receptor(s IL-6R),which can bind to IL-6 to form A complex that can signal transduction in cells expressing gp130.Most neurons lack m IL-6R expression,but widely express gp130,so IL-6 Trans-signaling is particularly important for IL-6 signal transduction in the CNS.It is currently known that IL-6 is associated with a variety of central nervous system inflammatory diseases,especially IL-6 is a key cytokine involved in peripheral and central inflammation,but IL-6 Trans-signaling is involved in perioperative neuroinflammation is little known.It has been reported that IL-6 Trans-signaling is the key to LPS-induced neuroinflammation and cognitive behavioral changes.This provides inspiration for our further study on the role of Trans-signaling in PND.In view of the dominant role of Trans-signaling in the occurrence and promotion of inflammation,especially Trans-signaling is the main signaling pathway of IL-6 in the central inflammatory pathological state,and the previous studies on IL-6 and PND did not provide any evidence for Trans-signaling,especially how the key receptor s IL-6R in Trans-signaling participates in central inflammation and mediates behavioral changes in animals has not been fully revealed.Based on the above theories,we speculate: 1.IL-6 Trans-signaling may be the main mechanism of IL-6-mediated post-traumatic central nervous system inflammation and cognitive decline.2.Hippocampal neurons may be involved in the regulation of cognitive behavior by responding to IL-6 Trans-signaling,and the main source of s IL-6R in the center is provided by microglia.In addition,s IL-6R may also be in the periphery.it penetrates the blood-brain barrier together with other inflammatory mediators and plays a role in the central nervous system during inflammation.Based on the above assumptions,we plan to carry out two batches of experiments.The first is clinical research.We plan to observe the occurrence of postoperative delirium in elderly patients undergoing major lower limb surgery;to detect postoperative inflammatory markers IL-6 and s IL-6R.The expression of IL-6 Trans-signaling and the occurrence of POD were explored to provide clinical clues for later animal experiments.The second research is animal experiments.The IL-6 Classic-signaling and Trans-signaling in the central hippocampus of mice were distinguished by a variety of transgenic model mice combined with AAV-Cre virus injection,and IL-6 Trans-signaling was discussed.role in PND.Immunofluorescence and ELISA were used to analyze the expression of signaling proteins and molecules downstream of IL-6 Trans-signaling in cells,and to clarify the potential mechanism of IL-6 Trans-signaling in the central and hippocampus leading to cognitive function changes in peripheral trauma.We hope this project will provide a theoretical basis for clinical improvement of PND treatment measures,and provide a target for the development of PND prevention and treatment drugs,thereby improving patients' postoperative recovery and reducing social burden.METHOD:1.The clinical study adopts a prospective longitudinal observational study design,and selects patients aged ? 60 years who undergo elective major lower limb surgery under general anesthesia.Peripheral venous blood was collected from preoperative and postoperative day 1 to postoperative day 4 to measure serum interleukin-1?(IL-1?),interleukin-2(IL-2),interleukin-4(Interleukin-4,IL-4),IL-6,s IL-6R,interleukin-8(Interleukin-8,IL-8)and tumor necrosis factor-?(Tumor Necrosis Factor-?,TNF-?).Postoperative delirium was assessed using the Delirium Measurement Scale twice daily for four days.The patients were divided into two groups according to the occurrence of postoperative delirium.The generalized linear mixed model was used to analyze the differences of perioperative serum cytokines between the two groups.The perioperative trajectories of cytokines with differences were analyzed by group-based trajectory modeling(GBTM).2.In animal research,First,the C57/BL6 mice were used to establish a model of cognitive dysfunction after tibial fracture and peripheral intraperitoneal injection of IL-6,to detect the expression of IL-6 and s IL-6R in the center,and to further observe the intracerebral IL-6 on cognitive function.The second part is based on IL-6R?flox/flox and CAMKII-Cre transgenic mice,specifically knocking out IL-6R in hippocampal CA1 neurons,and exploring the role of IL-6 Classic-signaling in peripheral trauma and central IL-6 in cognitive decline.The third part is based on IL-6R?flox/flox and CX3XR1-Cre transgenic mice to specifically knock out IL-6R in microglia,and explore the role of IL-6 Trans-signaling in peripheral trauma and central IL-6 in cognitive decline.The fourth section will use IL-6R-/-mice to describe the role of IL-6 Trans-signaling in peripheral trauma-induced postoperative cognitive dysfunction.Finally,gp130flox/flox transgenic mice were injected with AAV-CAMKII-Cre virus to specifically knock out gp130 to specifically knock out IL-6 Trans-signaling in the hippocampal CA1 region,while using IL-6 Trans-signaling specific blockers recombinant Glycoprotein 130(soluble Glycoprotein 130,sgp130),based on genetic and pharmacological means to verify the role of central IL-6 Trans-signaling in peripheral trauma-induced postoperative cognitive dysfunction.In the experiment,the behavioral evaluation of the mice was carried out by Contextual Fear Conditioning,and the genes and proteins of the mice were identified by western blot,PCR and flow cytometry,and the expression of IL-6 and s IL-6R in the central were detected by ELISA,the expression of IL-6 Trans-signaling signal in the hippocampal CA1 region was detected by immunofluorescence staining of p STAT3.RESULT:1.A total of 188 patients were included in the clinical study,129 of whom were eligible and underwent surgery,126 participants had complete delirium assessment data,and the final analysis was performed.Postoperative delirium occurred in 31 of 126 patients(24.6%).Longitudinal analysis of serum cytokines using Generalized Linear Mixed Model(GLMM)found that patients with postoperative delirium had higher levels of IL-6 and s IL-6R than those without postoperative delirium.The results of the group-based trajectory model analysis showed that the two-cluster model(stable lower level and fluctuating higher level)were the most statistically suitable model for the perioperative trajectory of IL-6 and s IL-6R.Patients who experienced fluctuating higher levels of IL-6 in the perioperative period were more likely to develop delirium.Likewise,patients who experienced fluctuating higher levels of s IL-6R in the perioperative period also tended to develop delirium.2.Based on the basic research of genetic model animals,the results of behavioral and molecular biology show that: 1.After surgery and peripheral administration of IL-6,the contextual fear memory ability of mice is decreased,and the expression of IL-6 were increased in hippocampus,and the content of s IL-6R in the cerebrospinal fluid also increased;2.The direct central administration of IL-6 can also lead to the decline of the contextual fear memory ability of mice.The number of p STAT3 positive cells in the hippocampal CA1 area increased;the use of IL-6R non-specific blocker BE0047 in the center can block the decline of contextual fear memory caused by surgical trauma and IL-6;3.Specific knockout except mice IL-6R? in CA1 area neurons can block the classic-signaling of hippocampal CA1 neurons.After surgery and central administration of IL-6,it can also induce the decline of contextual fear memory in mice,and it is also accompanied by p STAT3 expression increased in CA1 neurons.4.After specific knockout of IL-6R? in mouse microglia,the central Trans-signaling block is achieved,and surgical trauma can induce the decline of contextual fear memory in mice and the increase of p STAT3 expression in neurons in the CA1,while the central administration of IL-6 could not cause the change of contextual fear memory and the expression of p STAT3 in neurons in CA1;5.For IL-6R-/-mice,the specific IL-6 Trans-signaling activator Hyper IL-6 can lead to the decline of contextual fear memory and the increase of the number of p STAT3 positive cells in the CA1,while surgery and IL-6 cannot induce this phenomenon;6.Central sgp130Fc(specific IL-6 Trans-signaling antagonist)can block the decline of contextual fear memory caused by surgical trauma and IL-6 and the increase of p STAT3 expression in neurons in CA1;7.After the specific knockout of gp130 in hippocampal CA1 region neurons to achieve the loss of IL-6 Trans-signaling in hippocampal neurons,surgical trauma,central administration of IL-6 or Hyper IL-6 could not induce the decline of contextual fear memory and the loss of contextual fear memory in mice and the increases of p STAT3-positive cells in the CA1 region.CONCLUSION:1.Compared with patients without POD,patients with POD will be accompanied by increased circulating IL-6 and s IL-6R,and the formation of IL-6/s IL-6R complex predicts the enhancement of IL-6 Trans-signaling,suggesting that the downstream effector cells of IL-6 Trans-signaling may be involved in mediating the occurrence and development of POD.In addition,the combined elevation of IL-6 and s IL-6R in the perioperative period may serve as a serum marker for the development of postoperative delirium.2.By using a variety of transgenic mice,it was confirmed that central IL-6 Trans-signaling is involved in mediating postoperative cognitive dysfunction caused by peripheral trauma,and specific activation of IL-6 Trans-signaling in the central center can also lead to Changes in cognitive behavior in mice.Furthermore,IL-6 classical-signaling in hippocampal CA1 neurons was not involved in the peripheral trauma-induced decline in contextual fear memory in mice.During peripheral inflammation,circulating s IL-6R may enter the central nervous system,providing the possibility for IL-6 to conduct trans signal transduction.