The CD4～+T Cell And CD4～+CD25～+T Cell In The Repair And Regeneration Of Facial Nerve

On the basis of establishing stable and reliable assessment system on immunedeficiency mouse model with facial nerve paralysis, investigation on specific T cellsubpopulation and the relationship with facial motoneuron in facial nerve repair andregeneration are the essential questions that we want tackled in the research onimmunobiological mechanism of facial nerve injury. Focusing on these issues willeventually make immunological interventions practicable for facial nerve injury in theclinic.Objective1. To investigate the T cells activation of the facial nerve axotomized mouse withmethods of flow cytometry for the latent neuroimmunologic mechanism of traumaticfacial paralysis.2. To establish stable and reliable assessment system on immune deficiency mousemodel with facial nerve paralysis by the application and assessment of fluorogoldretrotracer on the nude mouse with facial nerve transection and comparing with wildtype mouse model, so as to provide proper animal model for revealing the latentneuroimmunologic mechanism of traumatic facial paralysis.3. To investigate the specific T cell subpopulation and the relationship with facialmotoneuron in immune deficiency mouse model with facial nerve paralysis, so as to find information for new strategy of facial palsy treatment.Method1. (Part 1) Establishment of the murine model with facial nerve transection, then thelymphocytes separated from the cervical drainage lymphoid nodes (CDLN) ofBALB/c mouse at specific time were collected and estimated the expression rate ofCD69 on T cells by flow cytometry following two-color immunofluorescent staining.2. (Part 2) The nude mouse (BALB/c background) group and the wild type mouse(BALB/c background) group all were subjected to a right facial nerve axotomy. Thenapplication and assessment with fluorogold retrotracer at specific time. After collectingthe slices of brain stem two weeks post the operation, the facial motoneuron wasobserved under fluorescence microscope, then analyzed and count with the softwareImage Pro Plus5.1.2. (Part 3) To analyze the T cell infiltration and the expression of inducible nitricoxidesynthase in facial motoneuron. Then purifying the CD4~+T cell from wild type mouseand reestablishing the immune function of nude mouse by infusing the CD4~+T cell andCD4~+T cell with regulatory T cell deleted through the tail vein a week before thesurgery. Then the nude mouse (BALB/c background) group and the wild type mouse(BALB/c background) group all were subjected to a right facial nerve axotomy. Thenapplication and assessment with fluorogold retrotracer at specific time, after collectingthe slices of brain stem three days post the operation and two weeks post the operation,the facial motoneurons was observed under fluorescence microscope, then analyzed andcount with the software Image Pro Plus5.1.Results1. (Part 1) At the time of three days post the operation (DPO3) and two weeks post theoperation (DPO14), the expression rate of CD69 on the T cells from the cervicaldrainage lymphoid nodes of the groups with facial nerve transection had bothupregulated; and the expression rate of CD69 on the T cells at DPO14 had significantdifference with that of the sham-operation group (p=0.0007)。.2. (Part 2) After transection of the facial nerve, the facial motoneuron retrotraced with fluorogold is luminous and easy for counting; There was significant difference ofsurvival facial motoneuron between the wild type mouse group and the nude mousegroup (2723.00±54.07, 2419.33±84 respectively, P=0.0011).3. (Part 3) There was the T cell infiltration and the expression of inducible nitricoxidesynthase in facial motoneuron of mouse model. There was significant difference ofsurvival facial motoneurons between nude mouse infused with CD4~+T cell and PBS atthree days and 14 days post the operation (P=0.0003 and 0.0004, respectively). Therewas also significant difference of survival facial motoneurons between the mouseinfused with CD4~+T cell and CD4~+T cell with regulatory T cell deleted (P=0.0132).Conclusion1. There was the T cell activation in mouse model with facial nerve injury at early stage. Itsuggest that there is a T cell immune response following the facial nerve injury, which ishave benefits on coordinating with the immune response to maintain the homeostasis ofimmune environment.2. The assessment system of nude mice facial nerve paralysis model together withfluorogold retrotracing method is effective and feasible. Which provided proper animalmodel for revealing the latent neuroimmunologic mechanism centering on T cellbehavior of the pathogenesis and progression of facial nerve trauma.3. It is the transplantation of CD4~+ T cell that rescued the survival facial motoneuron to thelevel of wild type. It could be inferred that the CD4~+CD25~+Treg have the ability torescuing the injuring FMN indirectly by down-regulating the CD4~+ T cell activation to aproper level. It may suggest that there is a critical role of the specific T cellsubpopulation in facial nerve repair and regeneration. Which provided basic informationfor revealing the latent neuroimmunologic mechanism centering on T cell behavior ofthe pathogenesis and progression of facial nerve trauma.