The Roles Of Macrophages In The Pathogenesis Of Acute Inflammatory Demyelinating Polyneuropathy In A Rabbit Model

Objective:Guillain-Barre syndrome(GBS)is an autoimmune disease that causes severe disability in the nervous system.It can involve respiratory muscles and lead to dyspnea,which is one of the most common acute and severe diseases in neurology.The pathogenesis of GBS is still unclear.Current studies suggest that the basic mechanism of GBS is that foreign pathogens induce the body to produce autoimmune antibodies through molecular mimicry mechanism,which activates complement,then forms membrane attack complex,and eventually leads to peripheral nerve axonal or myelin damage.Acute inflammatory demyelinating polyneuropathy(AIDP)is the most classic and common demyelinating type of GBS.Macrophage infiltration in the spinal nerve root has been demonstrated in autopsy specimens of patients with AIDP,but its pathological significance is uncertain.The aim of this study was to investigate the role of macrophages in the pathogenesis of AIDP.Methods:The AIDP model was established by galactocerebrosides(GalC),which was mixed with keyhole limpet hemocyanin and complete Freund’s adjuvant to make an antigen emulsion,which was injected subcutaneous in the back and immunized Japanese white rabbits at intervals of three weeks until the disease occurred(immunized 10 times at most).The auricular venous blood was collected weekly into the anticoagulant tube,and the plasma after centrifugation was used for enzyme-linked immunosorbent assay to detect the anti-GalC IgG antibodies;Electrophysiological tests were performed after 3%sodium pentobarbital anesthesia in auricular vein to observe the changes of nerve conduction in AIDP model;The spinal nerve roots were then removed after cardiac perfusion with 2%paraformaldehyde solution;Paraffin sections were used for toluidine blue staining to determine pathological changes of demyelination in AIDP models.Frozen sections were used for immunofluorescence staining.Anti-myelin basic protein antibody(myelin),anti-βⅢ-tubulin antibody(axon)and anti-rabbit IgG antibody were stained by immunofluorescence,and their relative fluorescence intensity was measured to observe whether demyelination occurred in AIDP model.The anti-voltage-gated sodium channels(nodes of Ranvier)and anti-contact-associated protein 1 antibody(paranodes)were stained by immunofluorescence and the occurrence frequency was counted to observe whether the nodes/paranodes of Ranvier was damaged.The position of complement deposition was observed by immunofluorescence staining of complement C3c.The role of macrophages in AIDP model was observed by immunofluorescence staining with anti-macrophage RAM11 antibody.Electron microscopy was used to observe the ultrastructural changes of spinal nerve roots in AIDP model and to clarify the role of macrophages.Results:Two rabbits(AIDP1 and AIDP2)developed limb weakness with a maximum clinical score of 13 for AIDP1 and 8 for AIDP2.AIDP models produced different titers of anti-GalC IgG antibodies by enzyme-linked immunosorbent assay.Electrophysiological results showed that both AIDP1 and AIDP2 had demyelination.Immunofluorescence staining showed IgG binding,complement deposition,myelin destruction,and macrophage phagocytosis of myelin fragments.Toluidine blue staining showed focal demyelination of spinal nerve roots with a large accumulation of macrophages around the lesion.Electron microscopy showed that myelin damage preceded macrophage infiltration,and macrophages were recruited to phagocytose and remove myelin debris during severe demyelination.Conclusion:In the AIDP model,macrophage infiltration occurs after complement-mediated demyelination,and macrophages may promote nerve regeneration in AIDP by removing damaged myelin debris.In the AIDP model,there is voltage-gated sodium channel breakdown in nodes of Ranvier,which can exacerbate peripheral nerve block and lead to limb weakness.