The Role Of Neuroinflammation Mediated By TLR4/MyD88 Signal Pathway In Central Pain After Thalamus Hemorrhage

BackgroundIntracerebral hemorrhage(ICH)refers to the hemorrhage caused by nontraumatic intraparenchymal vascular rupture,which accounts for 20% and 30% of all stroke,compared with high mortality and disability.In addition to mechanical trauma such as hematoma compression,secondary injury caused by neuroinflammation,brain edema and erythrocyte lysate play an important role in the pathophysiological mechanism after ICH.Neuropathic pain refers to a disease or injury of the somatosensory nervous system involving the peripheral nerve or central nervous system.CPSP is widely regarded as a severe chronic neuropathic pain.CPSP refers to the pain associated with the lesion that occurs after hemorrhage or ischemic stroke,which is persistent or discontinuous in a part of the paralyzed body,accompanied by sensory abnormalities.Any injury or disease affecting the spinothalamic tract pathway or cortical projection fibers may cause CPSP.The ventral posterolateral nucleus(VPL)of thalamus plays an important role in CPSP,which is the third level of neuron body in pain conduction pathway.CPSP is usually diffuse,difficult to locate and diverse in nature,and there are significant differences between patients.With the aging of the population,the incidence of the disease is increasing day by day.At the same time,due to the difficulties in examination,diagnosis,treatment and social and family reasons,not only the patients themselves suffer from pain,families and society also pay a great price for the torment of disease.Because the clinicians know little about CPSP and the patients are in pain for a long time,they may suffer from depression,anxiety and other emotional changes,which will aggravate the pain of patients and form a vicious circle.However,the specific pathogenesis of CPSP is unclear,it is necessary to establish a stable animal model,explore its pathogenesis,and provide an effective drug target for clinical treatment.Microglia act as locally immune effector cells in the central nervous system.Microglia and its mediated neuroinflammation play a very important role in the damage of central nervous system and the prognosis of diseases.Minocycline,a microglia inhibitor,has been confirmed in many studies that it can reduce nerve damage after cerebral hemorrhage and reduce the expression of exogenous TNF-? and IL-1?.In the meantime,it can reduce brain edema,decrease brain atrophy,reduce neurological impairment.Therefore,hematoma clearance of microglia / macrophages after intracerebral hemorrhage is considered to be the target of treatment.Martinez found that microglia/macrophages have M1/M2 polarization type.In other words,microglia are functionally differentiated.Under normal physiological conditions,M1 and M2 microglia maintain a dynamic balance,which will be broken once disease occurs.The results show that M1 polarized microglia produced inflammatory cytokines,such as TNF-?,IL-1 ?and IL-6,oxidase(inducible nitric oxide synthase),glutamate and other harmful cytokines.On the contrary,M2 polarized microglia have arginase activity and can produce neurotrophic factor and IL-10.M2 polarized microglia are related to the neuroprotective and regenerative effects after brain injury,and plays an important role in the late stage of cerebral hemorrhage.Toll-like receptor4(TLR4)is a kind of the pattern recognition receptor.It has been proved to be closely related to immunological or inflammatory diseases.It can produce and release a large number of pro-inflammatory factors through NF-?B signaling pathway,such as TNF-?,IL-1? and IL-6 after activated.It has been found that TLR4 is mainly expressed on the surface of microglia in the central nervous system,takes part in the activation of microglia and participates in the immune inflammation of the central nervous system.Recent studies have shown that TLR4 is closely associated with inflammatory pain and neuropathic pain.At the same time,inhibiting the expression of TLR4 after intracerebral hemorrhage(ICH)can decrease the volume of brain injury,reduce the content water of brain,protect the injured neurons and improve the neural function.However,its role in the pathophysiological process of CPSP is unclear.As a result,we propose such a hypothesis: after thalamic VPL hemorrhage,microglial cells were activated excessively,and the percentageof M1 polarization microglia increased.At the same time,TLR4/MyD88 signaling pathway activated and produced the inflammatory factor TNF-??IL-6?IL-1?,which are involved in the generation and maintenance of central poststroke pain.PurposesExplore the appropriate dosage of collagenaseVII-s and establish a stable CPSP model.Observe the behavioral changes and evaluate objectively the behavioral characteristics of CPSP models.Analyze the activation of microglia around intracerebral hemorrhage(ICH),the expression of inflammatory associated protein on the surface of microglia,the serum level of TNF-? and IL-6 and the surface phenotypic characteristics of microglia.The purpose of this study is to explore the role of neuroinflammation in the pathogenesis of CPSP,and to study the process of its occurrence and development at molecular level,providing a theoretical basis for the treatment of clinical CPSP patients.Methods(1)Setting different doses of collagenaseVII-s groups to explore the appropriate doseAnimals were divided into four groups: sham group and CPSP(0.1U,0.2U,0.3U)groups.we injected 0.1U,0.2U and 0.3U collagenase?-s(dissolved in 0.5ul saline)into VPL respectively.The stereotactic coordinates were 3.5 mm posterior to bregma,3.3 mm lateral to the midline,and 6.0 mm in depth from the skull surface,which is the most visible location of the VPL in the rat brain map.Sham control rats received the same treatment,including needle insertion,but without collagenase injection.In order to determine the final dose of collagenaseVII-s and establish a stable rat CPSP model,we detected the change of pain threshold on 0d,7d,14 d,21d,28 d after the operation(2)Detecting the relevant behavior after thalamus surgey with the appropriate dose of collagenaseVII-sAnimals were divided into two groups: sham group and CPSP group.we observed the changes of motor function in rats which include the Longa score,Corner Turn Test,Forelimb Placing Test and Hindlimb Placing Test.At the same time,we observed the changes of mechanical thermal and noxious cold pain stimulation threshold.And we measured depression,anxiety and other emotional changes include anxiety-related behavioral indicator(the open field test)and depression-related behavior indicators(the forced swimming test and the sucrose preference test).Evaluate objectively the behavioral characteristics of CPSP animal model.(3)Analyze glial cells activation and the changes of TLR4/MyD88 signal pathway related proteins around thalamus hemorrhageAnalyze qualitatively and quantitatively the activation of microglia and astrocytes around thalamus hemorrhage by immunofluorescence and Western blot;Validy double-labeled situation between microglia and TLR4;measure TLR4 and downstream factors expression of MyD88,NF-?B in the CPSP model rats brain.The concentration of proinflammatory factors TNF-?/IL-6 in serum were measured by Enzyme-linked immune sorbent assay(ELISA).Analyze surface phenotype of microglia were analyzed with flow cytometry to verify hypothesis.(4)Investigate the changes in behavior and expression of related molecules after intraperitoneal TAK242,an inhibitor of TLR4.The animals were divided into three groups: sham group,CPSP+vehicle group,CPSP+TAK242 group.In CPSP+TAK242 group.TAK242 was injected intraperitoneally 6h post thalamus surgey and the following four days.In CPSP+vehicle group,the same dose of saline was injected,and the other procedures were the same.The pain threshold(the mechanical,thermal and cold thresholds)were measured before and 7d,14 d,21d and 28 d post thalamic hemorrhage.At the same time,the activation of microglia and the expression of TLR4/MyD88 signal pathway relate proteins around injury were detected.Results(1)The optimal dose of collagenase?-s for the rat model of thalamus hemorrhage is 0.2U: compared with the sham group,mechanical withdraw threshold of the contralateral hindlimb in each experimental group was significantly decreasing,and lasted for 28d(***P<0.001).However,the thermal hypersensitivity of the contralateral hindlimb in 0.1U group decreased significantly at 14 d and 21 d and showed no change at 7d post surgey.However rats in 0.2U group and 0.3U group showed an obvious decrease at 7d,14 d and 21 d compared with sham group in thermal hypersensitivity.In addition,0.3U group rats showed the mortality rate within 24 h while no rats died in other groups.In addition the area of injury was beyond thalamus.To sum up,we selected collagenase?-s of 0.2U as the most optimal dose.(2)The motor function was not impaired,pain threshold was decreased and compared with the changes of emotion after thalamus hemorrhage:compared with the sham group,the Longa score of the CPSP group was 0,that means,there was no neurological defect.There was no significant impairment at 3d,5d,7d,14 d in Corner Turn Test,Forelimb Placing Test and Hindlimb Placing Test in CPSP rats at different time points(P > 0.05)compared to baseline.It showed that the motor function of CPSP group rats were not impaired,and on this basis,we could detect the difference of pain threshold more significantly.Compared to baseline pre-injury values observed at 0d,the mechanical withdraw threshold of the contralateral hindlimb in the CPSP group decreased significantly(***P<0.001).The mechanical withdraw threshold was measured to 28 d and the difference was still significant.This change persisted for at least 7 weeks compared with the sham control group.A significant hyperalgesia in response to thermal pain stimulation was observed in 7d and 14 d post thalamus hemorrhage(**P <0.01).while the threshold of noxious cold stimulation was significantly different at 7d after surgey(**P <0.01)and the difference was still significant at 28 d.Mechanical withdraw threshold and cold stimulation threshold reached a minimum level on the 21 st day,and gradually reached a stable state.Compared with the sham group,the rats in the CPSP group showed a tendency of anxiety and depression after thalamus hemorrhage.In the open field test,the time spent in the out ring and central areashowed a significant difference in CPSP group rats compared with sham group during the 5 minutes testing period.In the forced swimming test,there was a statistical difference in immobility of thalamic hemorrhage rats compared with sham control rats.In the sucrose preference test,the percentage of the sucrose preference of CPSP rats was lower than sham control rats.This phenomenon showed that CPSP rats exist an hedonia.(3)Glial cells were activated and TLR4/MyD88 signal pathway related proteins were upregulated around thalamus hemorrhage:compared with the sham group,the expression of microglia and astrocytes around hematoma in the CPSP group was significantly increased,and the expression of microglia and astrocytes were activated.The expression of TLR4 and microglia was completely co-labeled,but not with astrocyte.CPSP group rats showed that the expression of TLR4,MyD88,NF-?B was upregulated around hematoma tissue of injury,suggesting that the signal pathway of TLR4/MyD88 was activated.The expression of downstream TNF-? and IL-6 increased,which was consistent with our expected results.(4)After intraperitoneal TAK242,pain threshold was increased and neuroinflammatory related indicators were decreased:compared with sham group rats,the brain water content of CPSP + vehicle group rats were higher.However the brain water content of ipsilateral hemisphere were lower in TAK242-treated ratsthan in vehicle-treated rats(78.06±2.626 in TAK242-treated group vs 81.34±1.903 in vehicle-treated group).After intraperitoneal injection of TAK242,compared with CPSP + vehicle group,the mechanical threshold increased at 7d?14d?21d? 28 d.Meanwhile,the cold stimulation threshold also changed obviously.The thermal stimulation threshold increased on the 7d and 14 d.Western-blot showed that the expression of TLR4,MyD88,p-p65 were down-regulated.Elisa showed that the expression of TNF-?/IL-6 was also significantly down-regulated.Therefore,TAK242 may alleviate CPSP by reducing the expression of TLR4/MyD88 signaling pathway related protein and its downstream inflammatory factors.Conclusions:(1)In this study,the rat model of thalamic hemorrhage was successfully established with collagenase ?-s.The behavioral results showed that hemorrhage in the thalamic VPL can induce central poststroke pain.At the same time,rats with CPSP exhibited depression-and anxiety-like behaviors.(2)Microglia were activated after thalamic VPL hemorrhage.Neuroinflammation mediated by the TLR4/MyD88 signaling pathway might be involved in the occurrence and maintainance of central poststroke pain.