The Inhibition Of Minocycline On Microglial Activation In Axonal Protection Of Chronic Experimental Autoimmune Encephalomyelitis

Objective:To explore the activation of microglia in the mechanism of axonal injury and the therapeutic effect of minocycline in C57BL/6 mice with chronic non-remitting experimental autoimmune encephalomyelitis.Methods:(1) In vivo:46 adult female C57BL/6 mice with MOG 35-55 peptides induced EAE were randomly divided into control group(n=10), EAE group(n=18) and EAE+mino group(n=18).The mice of each group were evaluated for clinical neurological score until they were put to death on the day of preclinical stage(～9d),onset stage(12～17d),peak stage (21～25d) and chronic stage(32d～) after the first immunization.The brain tissue and spinal cord were stained by the methods of Haematoxylin Eosin stain,Luxol Fast Blue stain and Bielschowsky stain.The expression of Iba-1 in microglia and APP,NF in brain and spinal cord were detected by immunochemical method.The mRNA expression of IFN-γ,and IL-10 were dynamically detected at every stage of clinical course by RT-PCR method.The expression of phosphorylated p38 MAPK protein was ascertained by Western Blot method.(2) In vitro:The primary cultured microglial cells were divided into control group,LPS group and LPS+mino group.The supernatant of each group were taken for detection at 3,6,12,24 and 48 hour after LPS treated.The expression of TNF-αand IL-1βwere measured by ELISA.The production of NO was measured by Griess Reagent assay.Results:(1) In vivo:1.The clinical course of experimental mice:The controlled mice had no symptoms of disease.The mice of EAE group and EAE plus mino group developed signs on 12～17 days after induction with no difference between groups.On day of 21～25,the symptoms were developed to peak stage,the EAE mice showed more severe clinical symptoms than the mino group.After 32 days,the mice of both groups were improved with mino group better,and the disease sustained for whole chronic stage with classical chronic non-remitting course.2.Neuropathological evaluation of brain tissue and spinal cord:Mice with EAE were found with obvious pathlogical changes.At peak stage, HE stain showed vasculitis throughout the CNS,the vascular dilatation and perivascular infiltration of inflammatory cells seemed sleeve-like changes.The myelin in white matter was found with discontinuity, segmentation,vacuolus change and white lamellar loss area by LFB stain. Immunochemical staining for Iba-1,which identified microglia,showed activated microglial cell swelling,proliferated and with prominence. Abnormal staining after acute axonal injury was detected by APP immunochemical staining.At chronic stage,the axons were seen with swelling,disrupted,deformed,and ovoid formation by Bielschowsky staining and NF immunochemical staining.Lesions in minocyclinetreated group were less frequent,markedly reduced destructions.3.The mRNA expression of inflammatory cytokine at different stage of clinical course in brain tissue and spinal cord:①The expression of IFN-γmRNA:There was slight expression of IFN-γmRNA detected in control group,with stable expression for all stages.The expression in EAE group had no difference to control group at preclinical stage, significantly elevated at onset stage and sustained to peak stage for 8～6 times,markedly weakened at chronic stage but still more than control.In EAE plus mino group,the expression was not different to EAE group at onset stage,but significantly lower than EAE group at peak stage,still lower at chronic stage and nearly to control level.②The expression of IL-10 mRNA:There was also a little expression of IL-10 mRNA in control group,with stable expression for all stages.In EAE group,the expression had no difference to control group at preclinical stage,began to elevate from onset to peak stage,till to 6 times at chronic stage.In EAE plus mino group,the expression was not different to EAE group at onset stage,but significantly higher at peak stage,still higher than EAE group at chronic stage.4.The expression of and phosphorylated p38 MAPK protein at peak stage in brain tissue and spinal cord:The expression of p-p38 MAPK protein was a little in control group,significantly elevated to 3 times in EAE group,markedly lower than mino group but still higer than control level.(2) In vitro:1.The primary cultured microglia:The cultured microglial cells were round with strong refraction,and verificated by FITC-IB4 to 95% purity.After activated by LPS,the cell bodies relatively increased than normal,with multiple slender prominences like macrophages.2.The expression of cytotoxic cytokines by microglia at different time points after LPS treated:The expression in control group was a little with time-dependently.In LPS group,the level of TNF-αwas elevated earliest at 3 hours after LPS treated,IL-1βwas significantly elevated at 6 hours,while NO elevated at 12 hours.In LPS plus mino group,the expression of TNF-αwas significantly decreased than LPS group at 3～48 hours after LPS treated,IL-1βwas significantly decreased at 6～48 hours, while NO decreased at 12～48 hours. Conclusion:1.The activation of microglia might be an essential mechanism for axonal injury in chronic EAE.2.The inhibition of microglial activation by minocycline may participate the protection of axonal injury.3.P38 MAPK pathway might be a mechanism for the inhibition of microglial activation by minocycline.