Feasibility Study On The Underlying Immune Mechanisms Of Regulatory T Cells In Experimental Autoimmune Neuritis Served As The Animal Model Of Guillain-Barré Syndrome

Background:Guillain-Barré syndrome(GBS) is an acute monophasic immune-mediated polyradiculoneuropathy, including humoral and cellular immunity. Different infectious agents(bacterial or viral infection) and genetic background(genetic susceptibility to infection) played a certain role in the pathogenesis of GBS, which definite etiology and immunological pathogenesis had not been understood clearly and had no specific treatment yet. Experimental autoimmune neuritis(EAN) is a helper T cell-mediated inflammatory demyelinating disease of the peripheral nervous system and has been widely recognized and applied as an animal model to investigate therapy and disease mechanisms of GBS around the world, which was immunized by P253-78 peptide. Regulatory T cells(CD4+CD25+regulatory T cells, Tregs) play a very important role in the maintenance of immune homeostasis and prevention of autoimmune diseases. Relevant researches confirmed that the reduction of number or functional deficiency of Tregs could lead to the occurrence of autoimmune diseases. However, there have little researches on the relationship between Tregs and GBS,the same as the role of Tregs in immune mechanisms of GBSand immunological effect of back transfusion of Tregs at home and abroad.To detect the change of the proportion of circulating Tregs of EAN rats and relationship with disease process, we make the model of EAN that mirrors many clinical and immunological features of the human GBS. Therefore, this investigation lays a good foundation for the further study of the role of Tregs in immune mechanism of GBS.Objective: To investigate the effect of Tregs in the immune mechanism in Guillain-Barré syndrome.Method: 1.All animals were divided into 2 groups(n=8), including EAN model group and normal group. The rats of model group were immunized by subcutaneous injection into both hind footpads of P253-78 peptide. All rats were weighed and inspected for disease severity daily for 30 days. To assess and compare the degree of inflammation and demyelination of peripheral nerves, twp rats from each group were sacrificed randomly under 10%chloral hydrate anesthesia respectively at the peak stage and in recovery period of EAN. Sciatic nerves were rapidly harvested.2.The spleen were removed under aseptic conditions from healthy rats of same population. Single cell suspensions of mononuclear cells(MNC) from individual rats were prepared separately. Tregs were sorted by flow cytometry. The suppressive activity and purity of CD4+CD25highTregs were detected after isolation by FACS to research the effect of Tregs to EAN rats.3.The proportions of circulating Tregs of rats were detected from the groups before the onset of disease,at the peak and in recovery period of EAN. And these data were statistically analyzed and compared each other.4. One rat from each group were sacrificed randomly and sciatic nerves were rapidly harvested at the peak stage and in recovery period of EAN respectively. Through the pathological HE staining and electron microscope analysis, we observed the improvement of inflammatory cells infiltration and demyelination and axonal injury.These data help us to further evaluate the effect of Tregs in EAN.Results: 1. We constructed the EAN with P253-78 peptide successfully. HE staining and electron microscopy showed that inflammatory cells infiltration and demyelination,even axonal dissolution were found in sciatic nerves of EAN rats.2. After the rats were immunized, we discovered weight loss, higher neurological score and lower proportion of circulating Tregs in EAN model group compared with healthy control. This difference has statistical significance(P<0.05). Nevertheless, the body weight and the proportion of circulating Tregs of EAN rats were not enhanced and the paralysis symptoms were not relieved compared with healthy control in recovery period. This difference also has statistical significance(P<0.05).3.In recovery period, there has no significant improvement of inflammatory cells infiltration and demyelination damage in sciatic nerves of EAN rats.Conclusions: The proportion of circulating Tregs in EAN model group was reduced at the peak stage of the disease compared with healthy control,which indicated that Tregs involved in the cellular immune mechanism of GBS.These data provide a new theoretical basis for further clinical applications of Tregs immunotherapy in GBS.