| Background:Through long-term observation of clinical work,corneal injuries(trauma,chemical injury,surgical injury,etc.)usually cause refractory glaucomatous syndrome and irreversible optic nerve damage,which could damnify the life quality of patients and affect the recovery of visual acuity after surgery.Recent studies shown that the cytokines of tumor necrosis factor family and interleukin family may be involved in the process of corneal injury-related retinal inflammation,but the specific mechanism of retinal neural cell loss after corneal injury remains unclear so far.There is no unified standard on how to prevent corneal injury-related retinal damage.Objective:In order to explore the mechanism of immune cells infiltration in retinal inflammatory response,we established four different corneal injury models to detect the changes of related cytokines and macrophage/microglia infiltration in retinal tissue after corneal injuries.Three immunosuppressive drugs,glucocorticoid,TNF-a monoclonal antibody and IL-1? antagonist,were used to achieve the inhibitory and neuroprotective effects of different regimens on retinitis and to provide a new strategy for clinical treatment.Method:Four different models of acute corneal injuries were established.C57BL/6 mice were used for 1N NaOH corneal burn,corneal suture,penetrating keratoplasty and penetrating keratoplasty combined lens extraction in allgroups.Comeal and retinal specimens were collected at different time points for qPCR,flow cytometry,TUNEL and confocal microscopy to observe the expression of inflammatory cytokines.Bone marrow transfer chimera were prepared to verify the source of macrophage/microglia activation in retina and further identify the immune cells.Thyl-YFP mice were used to examine the survival rate of retinal nerve cells 7 days after corneal injury.Use Glucocorticoid,TNF-a monoclonal antibody and IL-1? antagonist to treat the four groups of mice respectively,then observe the inhibitory effects of different drug regimens.By using microfluidic single cell sequencing technology,we preliminarily analyzed the phenotypes of immune cells from different sources of retina and the effect of immunotherapy.Results:1.24 hours after corneal alkali bum,the result of flowcytometry showed that the proportion of CD45 CDllb inflammatory cells in cornea and retina increased significantly.Inflammatory cells can be divided into two groups:CD45hiCD11b+cells and CD45loCD11b+cells,which indicated that there might be two different sources of immune cells.Meanwhile,the upward trend of CX3CR1+CD45+CD11b+ MHC?+ cells proportion proved that macrophages/microglia were activated and participated in the process of inflammation.The expression of TNF-? and IL-1? in cornea and retina were obviously up-regulated,suggesting that both cornea and retina had inflammatory reaction after comeal alkali burn.We used CX3CRIGFP/+mice to produce bone marrow chimera model,confocal microscopy and flow cytometry were used to venfy the existence of peripheral monocytes/macrophages.The number of GFp cells increased prominently,which represented peripheral immune cells passed through blood-eye barrier into the retina after corneal alkali bum.2.24 hours after penetrating keratoplasty(PK),the proportion of CD45 CDllb+inflammatory cells in cornea and retina increased significantly.The substantially increase of CX3CR1+CD45+CDllb+MHCll+cells indicated that PK could activate retinal macrophages/microglia infiltration.TUNEL labeling confirmed the apoptosis of retinal cells clearly,while the results of qPCR showed that the expression of TNF-?,IL-1? and IL-6 in retina increased after PK,suggesting that antagonism of above-mentioned cytokines might alleviate the surgical inflammation.However,the elevation of three cytokines was not as high as that of corneal alkali burn model.The existence of peripheral monocytes/macrophages was confirmed by using bone marrow chimera model.Thyl-YFP mice model conbined with confocal microscopy was used to observe the functional status of neural cells,we observed retinal neural cells decreased significantly 7 days after PK.3.The therapeutic effect of single comeal suture group showed that corticosteroid had less inhibitory effect on TNF-a than infliximab alone or double treatment with anakinra.The results of post-PK mice showed that all four treatments were effective,but the effect of immunosuppressant was superior than corticosteroid.Compared with PK group and suture group,retinal inflamnation after PK combined lens removal surgery showed the worst because the expression of TNF-?,IL-1? and IL-6 increased most significantly,the therapeutic efifect of immunosuppressive combination was better than single drug or corticosteroid.4.Single-cell sequencing results indicated the transcriptome information of retinal microglia and macrophages in mice treated with immunosuppressive agents was similar to control group 6 weeks after corneal alkali burn.2 years after corneal alkali burn,the transcription message of retinal microglia and macrophages in untreated mice deviated significantly from naive microglia.Retinal microglia and macrophages of mice treated with immunosuppressive agents remained relatively quiet,and there was no sign of drastic changes in their genomes.Conclusions:This study showed that four models of different corneal injuries could induce inflammatory damage of retina.The activation of inflammatory reaction in retina was highly related to peripheral CD45 CDllb?macrophage/monocyte infiltration.The group of penetrating keratoplasty combined lens removal group performed the most obvious retinal damage,which indicated that the severity of corneal injury was positively correlated with the expression of inflammatory cytokines.In addition,the decrease of idflammatory cytokines in retina after immunosuppressive therapy confirmed that blocking the activation pathway of immune cells can effectively inhibit the growth of macrophages/monocytes,which not only provides a theoretical basis for studying the mechanism of glaucomatous syndrome after corneal injury,but also provides a novel strategy for clinical treatment. |
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