The Inflammatory Reactions Evoked By MG Participate METH-induced Striatum Neurotoxicity

BACKGROUNDBeing the pharmacological target of the addictive drugs,the damages of the CNS by these drugs has long been the primary concern.METH produces strong CNS stimulation and drug dependence,and the tendency being used becomes seriously recent years.By now extensive investigations of METH addiction both clinically and in animal models have led to the consensus that the neurotoxicity of METH is closely related to the production of some active substances such as ROS and ONOO^-.The cascade of the neurotoxic events,followed the generation of these active substances, results in the substances lesions to the sensitive encephalic region(the monoaminergic nerve endings).In the process of substances lesions,MG play a crucial role especially.Inflammation is a defensive reaction normally,once out of control,will be harmful to closed tissue.MG participate the chronic inflammatory reactions in a moment of pathological conditions,such as the lesions of neurotoxicity,chronic neuropathy(Parkinson's disease,etc.),brain ischemia and brain trauma.Identically, after toxicity dose METH administration,MG become active and aggregate in the sensitive encephalic region.These phenomenons demonstrate that being the resident immunocell of CNS,MG participate the neurotoxic lesions.But there are many questions waiting to clarify.What is the relationship between METH administration and the activation of MG? Which events are relevant with the activation of MG in the sensitive encephalic region? Is there some agent could regulate the activation of MG in the sensitive encephalic region? By now,few reports have been available to clarify those questions,especially a systemic investigation of this kind. On the base of the METH neurotoxicity studies recent years of our group,this paper will carry out studies aimed at above questions through both in vivo and in vitro experimental systems.OBJECTIVEDue to the strong selectivity of METH-induced neurotoxicity in the CNS,we chose the striatum as the target region for our observation,where the damage can be the most severe.This study aims to investigate the pathological events in the striatum, the time course,morphological changes and phenotypic conversion of activated MG, and the signal transductions involved in the inflammatory lesion mediated by the MG under OS in vitro,to thereby understand the roles of MG in METH-induced neurotoxicity.METHODS1.The inflammatory lesion evoked by MG in the METH-induced neurotoxicity in the striatum of rats1.1 To duplicate the METH poisoning model and characteristics of MG activation in the striatumAdult male Wister rats(n=48),weighting 200±20g,were divided randomly into control group and experimental group equally.The rats of experimental and control group received i.p.injections of METH and saline(8 injections,15mg/Kg,at 12h intervals).The samples were harvested post treatments at 0.5d,1d,2d,3d,4d,5d,6d and 7d.The ratio of activated MG and the activation time course were analyzed by immunohistochemistry with CD-11b antibody.The ultrastructures of the activated MG were observed using transmission electron microscopy.The percentage of the cells positive for both CD-11b and MHC-â…¡antibodies following immunofluorescence labeling was determined using flow cytometry,for the identification of MG activation.1.2 Agents caused and modulating to MG activation respectivelyOn the base of identification of ratio and time course of MG activation,the agents caused and modulating to MG activation had been studied followed.The content of DA in the striatum was measured by high-performance liquid chromatography,The concentration of ROS was detected using ROS detection kit. And the CD200 protein expressions were essayed using Western blotting.1.3 The inflammatory lesions in striatum evoked by MG and the ways of inflammatory reactions in MGAs followed,the ways of inflammatory reactions in MG post METH administrations and the degree of injury had been studied in striatum.The contents of TNF-αand IL-1βwas assayed using ELISA-kit.The level TH protein expressions were essayed using Western blotting and immunohistochemistry.The ultrastructures of the nerve fibers in striatum were observed using transmission electron microscopy.2.The course of MG activation,under OS condition,and the modulations of CD200 to activation and inflammatory reactions of MG2.1 To establish the method of MG primary culture and the isolation of cortical neurons from neonatal Wistar ratsThe experiments in vitro include listed below.MG from cortex neonatal Wistar rats were isolated,purified and cultured.The cortical neurons were isolated of the neonatal Wistar rats.The experimental model of MG activation under OS was established,followed by studying of the modulations of CD200 to activation and inflammatory reactions of MG.The neonatal rat MG were isolated and purified through lidocaine treatment(12mmol/L) and differential adhesion-time methods.The growth activity of the purified MG was analyzed using MTT assay.The purity and status of the cultured MG were evaluated with morphological observation and the immunofluorescence labeling of CD-11b and MHC-â…¡,through TEM and flow cytometry respectively.The cortical neurons were isolated,through the gradient micropore filtration and high glucose medium methods.The positivity rate for CD200 of cortical neurons was detected using flow cytometry,being aimed at the investigations of the modulations of CD200 to activation and inflammatory reactions in MG.2.2 To establish the model of MG activation under OS condition and the related regulation of CD200 in vitro Proposing to clarity the modulations of CD200 to MG activation and inflammatory reactions,MG were treated with H₂O₂(100μmol/L) and METH (200μmol/L) to test the properties of MG activation in vitro,with LPS(0.5mg/L) as positive control.Basement above,the location and proteins expression levels of p-p38 MAPK,the proteins expression levels of IκB-αin MG was analyzed by laser scanning confocal microscopy and Western blotting.The contents of TNF-αand IL-1βin the culture medium were assayed using ELISA kit also.RESULTS1.The inflammatory lesion evoked by MG in METH poisoning model in striatum1.1 Model duplication and characteristics of MG activation in the striatumAfter METH administration,rats showed typical stimulated symptoms, indicating the model was established successfully.In experimental group,the activated MG displayed larger cell body,shorter cell processes,resembling bush or amoeba-like.Under TEM,the activated MG displayed lowered electron density in the nuclei with shortened and thickened cell processes and rich cell organelles(including lysosomes) in the cytoplasm.The MG were found in close quarters with the neurons with cytomembrane contact.In the experimental group,the ratio of activated MG was increased significantly(n=9,P＜0.001),with rising continuously from 1^st to 5^thd,as compared with the control group.The cell percentage positive for both CD-11b and MHC-â…¡increased continuously also.1.2 Agents caused and modulating to MG activation respectivelyAs compared with the control group,METH treatment resulted in DA content decreasing significantly(n=3,P＜0.05),ROS content increasing significantly(n=3, P＜0.05) significantly,and CD200 protein expression decreased,in the striatum of experimental group.1.3 The inflammatory Iesions in striatum evoked by MG and the ways of inflammatory reactions in MGAs compared with the control group,METH treatment resulted in both TNF-α and IL-1βcontents decreasing significantly(n=3,P＜0.05),the protein expression level and immunoreactive intensity of TH decreasing,and dropsy and vacuolization of never fibers,ending and chondriosomes inspectely,in the striatum of experimental group.2.The course of MG activation,under OS condition,and the modulations of CD200 to activation and inflammatory reactions of MG2.1 To establish the method of MG primary culture and the isolation of cortical neurons from neonatal Wistar ratsThe survival rate of the isolated primary MG exceeded 95%,which presented intact cell structures under TEM.Of the MG obtained,97.0%were positive for CD-11b,and 2.47%were positive for MHC-â…¡.The CD200 positive ratio of the isolated cortical neurons was 87%.2.2 To establish the model of MG activation under OS condition and the related regulation of CD200 in vitroSaline,LPS,METH and H₂O₂ were used to treat the primary cultured MG. Application of LPS and H₂O₂ resulted in increased MHC-â…¡positive ratio and contents of TNF-αand IL-1β,but neither saline nor METH produced such effect. Compared with exclusive treatment of the primary cultured MG with H₂O₂,the treatment of H₂O₂+CD200 resulted in lowered the positive cell percentage of MHC-â…¡, the level of p-p38 MAPK protein expression and the continents of TNF-αand IL-1β, but higher the level of IκB-αprotein expression.CONCLUSIONS1.Continuous METH stimulation of the CNS results in MG activation,which in turn showing active inflammatory reaction.As the modulator,activated MG achieved the ability to recruit lymphocyte participating the inflammatory reactions.2.METH administration resulted in the exhaustion of DA and releasing ROS in the striatum,in turn inducing MG activation.On the one hand,activated MG released massive ROS,causing the continent of ROS rising continuously.That mains activated MG by releasing ROS to participate the lesion.As the modulator,the decreased level of CD200 protein expression in striatum regulated MG activation. 3.The increased production of TNF-αand IL-1βafter METH administration, indicate that activated MG by releasing pro-inflammatory cytokines to induce secondary lesions in the striatum.The decreased level of TH protein expression and morphological changes of never fibers and endings indicate that MG through above manner cause striatum lesion.Otherwise,the depletion of DA after METH administration initially did not result from the decreased level of TH protein.4.The primary MG and cortical neurons obtained in this study provides the basis for further study the role of MG-mediated inflammatory injuries in the neurotoxicity of METH.5.In vitro,the results about the increased of MHC-â…¡positive cell percent and TNF-αand IL-1βcontinents confirmed the conclusion that ROS triggered MG activation,but METH did not.By inhibiting the activation of p38 MAPK pathway, CD200 attenuates the activity of NF-κB in MG-mediated inflammatory response. Accordingly,decreased level of CD200 expression induced by METH further enhances the inflammatory reactions mediated by MG in vivo or in vitro.