| Objective:Traumatic optic neuropathy(TON)is a common cause of mechanical optic nerve injury.Early direct pathological damage caused the death of neurons and glial cells.After the initial injury,a large number of harmful factors will be produced,which will cause secondary damage to the neurons or glial cells that have not been directly injured.The secondary neurodegeneration caused by TON is mainly reflected in the continuous apoptosis of retinal ganglion cells(RGCs)in the late stage of inflammatory response.Existing research shows that mechanical damage of the central nervous system(CNS)can activate the endogenous autoimmune T cell group,which is Trauma-induced autoimmunity(TIA).At present,there is still much controversy about the role of TIA in body damage repair,but more and more people believe that TIA plays a key role in maintaining CNS homeostasis and damage repair.Some studies believe that autoreactive T cells can not only trigger and maintain pathological processes in the CNS,but also participate in the repair and regeneration of damaged nerves and play a protective role.However,it is unclear how the adaptive autoimmune response participates in the pathological process of TON,and what is the activation state of the relevant immune cells?Participate in nerve damage or repair process?This study used optic nerve transection to simulate acute axonal degeneration(AAD)caused by mechanical optic nerve injury,and explored whether autoreactive T cells were involved in the optic nerve injury process.At the same time,the C57BL/6 mice were immunized subcutaneously with the antigen molecule Interphotoreceptor Retinoid Binding Protein(IRBP)to determine whether autoreactive T cells are involved in inflammation or repair?And what kind of specific autoreactive T cell subsets are involved?What role did it play?Clarifying the mechanism of self-reactive T cell group in TON has potential clinical significance for further elucidating the pathological mechanism of TON and searching for potential treatments.Methods:C57BL/6 mice of the same sex and age were selected and divided into three groups:sham operation group,optic nerve transection group,and optic nerve transection group after immunization,the sham operation group served as the control group.After the optic nerve was transected,three time points of 7 days,14 days and 28 days were selected for observation,and 10 mice were selected for each time point in each group,5 females and 5 males.The simple optic nerve transection operation was performed on the optic nerve transection group to establish a mechanical optic nerve injury model.After the modeling was completed,the mice were sacrificed at specific time points,and the eyeballs and optic nerves of the mice were taken and fixed,frozen sections were performed and stored after sectioning.Haematoxylin-eosin(HE)staining and immunofluorescence staining were used to observe RGC and count.Immunofluorescence staining was used to observe innate immune response and whether T cells were produced in the retina and optic nerve.The spleen of the mouse was aseptically collected at a specific time point,and the lymphocytes were isolated and frozen.Flow cytometry(FCM)was used to detect changes in lymphocyte groups,that is,effector T cells(Teff)and regulatory T cells(Treg)before and after injury.At the same time,specific T cell groups were detected before and after injury by the enzyme-linked immunospot assay(ELISPOT).In addition,the mice were actively immunized with autoantigen-specific T cell epitopes,IRBP.Flow cytometric analysis of changes in lymphocyte(Teff and Treg)groups after immunization and confirmation of the peak change in each T cell group,the mice optic nerve transection model was established at the optimal time of T cell change,that is,the optic nerve transection group after immunization.After successful modeling,the mice were sacrificed and the eyeballs were taken at a specific time,and the number of RGCs transected after immunization was compared with the number of RGCs transected to determine whether autoreactive T cells reduced RGC apoptosis.The immunofluorescence staining of the anti-inflammatory factor IL-10 and the brain-derived neurotrophic factor BDNF was used to analyze whether the presence of autoreactive T cells had a protective effect on mechanical optic nerve damage.Results:After the optic nerve was transected,there was no bleeding at the surgical site,no swelling or protrusion of the eyes,no change in the size of the eyes,no discoloration of the lens,and no concurrent infection.By optimizing the surgical method,the success rate of the surgery was significantly improved.After fisher's accurate test,the bleeding rate after the optimization of the surgery was significantly reduced(P<0.05).Retina HE staining results showed that the number of RGCs in the retinal ganglion cell layer of the optic nerve transection group was significantly reduced,the size of the nucleus was different,the cell distribution was uneven,the cell arrangement was disordered,and the inner layer was thin.Immunofluorescence staining showed that RGCs were counted at different time points after injury.Compared with control eyes,there was a statistically significant difference in the reduction of RGC in the injury group(P<0.05).After the optic nerve is transected,the retina produces activated microglia and astrocytes.Fluorescently labeled T lymphocytes appeared in the optic nerve and eyes of the optic nerve transection group,but not in the optic nerve and eyes of the control group.After the optic nerve was transected,the results of flow cytometry detection of lymphocyte groups showed that Thl in Teff increased at each time point,and significantly increased at 14 and 28 days(P<0.05).However,there was no significant change in Th2 compared with the control group(P>0.05).Treg decreased somewhat,and significantly decreased at 14 and 28 days(P<0.05).Elispot results showed that the increase of specific autoreactive T cells(Th1)after optic nerve transection was not significant,and the increase was statistically significant only at the 28-day time point(P<0.05).However,there was no significant change in Th2 compared with the control group(P>0.05)The mice were stimulated by injection of specific autoantigen epitopes,and the results of flow cytometry detection of lymphocyte populations showed that Thl in Teff increased at each time point and significantly increased at each time point after antigen immunization(P<0.05),the increase was most obvious at the 7-day time point after antigen immunization(P<0.01).However,there was no significant change in Th2 compared with the control group(P>0.05).Treg decreased significantly at each time point(P<0.01).Elispot results showed that specific autoreactive T cells(Th1)increased significantly at various time points after immunization with antigen(P<0.05),and Th1 increased most significantly(P<0.01)7 days after antigen immunization.However,there was no significant change in Th2 compared with the control group(P>0.05).The results of immunofluorescence staining showed that more CD4+T cell infiltration occurred around the retina of mice 7 days after antigen immunization than that of mice with simple optic nerve transection(P<0.05).By counting the number of RGCs in mice after antigen immunization and comparing with the control group,the results showed that injection of specific antigens did not affect the number of RGCs in mice(P>0.05).Therefore.the mice selected for 7 days after immunization with specific antigens were selected for optic nerve transection.RGC counts were performed at specific time points on days 7,14,and 28.By comparing with the number of RGCs in mice with optic nerve transection alone,it was found that the autoreactivity In the presence of T cells,RGC apoptosis did not decrease after optic nerve transection(P>0.05).The immunofluorescence staining of the anti-inflammatory factor IL-10 and the brain-derived neurotrophic factor BDNF was used to analyze whether the presence of autoreactive T cells had a protective effect on mechanical optic nerve damageConclusion(s):1.An adaptive immune response occurred in the optic nerve transection model of C57BL/6 mice.The autoreactive T cells infiltrated the retina and the optic nerve,and there was an imbalance of Th1/Treg ratio,but the specific response is weak.2.Immunization with specific autoantigens enhanced the activation of autoreactive T cells(Th1 in Teff),exacerbated the Th1/Treg imbalance,and found that CD4+T cells infiltrated from around the retina into the retina.3.Activated self-reactive Th1 may trigger chronic inflammation and participate in subsequent secondary degenerative lesions.Th1/Treg imbalance and chronic inflammation caused by insufficient autoreactive Treg activation may be the main obstacles to optic nerve repair. |
PDF Full Text Request