The Diversity Of Immune Cells In The Iris And Ciliary Body During Penetrating Keratoplasty

Purpose To study the relationship between the anterior chamber microenvironmentand allograft rejection after penetrating keratoplasty and further to reveal the mechanismof allograft rejection.MethodsExperimental groupsA, Syngeneic keratoplasty (donor: BALB/c mouse, recipient: BALB/c mouse)B, Allograft keratoplasty (donor: C57BL/6 mouse, recipient: BALB/c mouse)C, Without operationBiomicroscopic examinationThe eyes were routinely examined by slit lamp microscopy on postoperative 3days, when the sutures of eyelid were removed, until 2 months after transplantation. Thefirst two weeks of daily observation, after all mice 2-3 times a week to 2 months.Immunohistoehemistry studiesMice from 2 groups were treated with whole-body perfusion in the time of 0,5,10 and 18days postoperation before iris wholemount were prepared. Then immunohistochemicalstainings were carried out in these wholemounts using monoclonal antibodies to MHCclassâ…¡, F4/80 (the macrophage marker) and costimulatory molecules CD86/CD80, andCD4+T-lymphocyte to identify the diversity of immune cells in the iris during cornealgraft rejection. Each wholemount were observed three different field under high powermicroscope (20-fold objective lens), two mice were observed. Image-J analysis software(a total of six different vision) were used to count the share of positive cells.RT-PCR to identify cytokine in the irisAt 5,7,10 and 18 days postoperation, 2 mice from each group were executed. And CCR5.CCR7, IL-12, IL-4 in iris were dected by RT-PCR. Image-J statistical image analysissoftware was used to assess the ratio of each primer and intemal parameters. ResultsBiomicroscopic examinationCorneal grafts opacity combined with angiogenesis occurred in group B, and resulted ingrafts rejection within 12 to 20 days postoperation ultimately. But in group A, the graftsonly had a mild edema at the early stage postoperation, and resumed transparencycompletely after the sutures were moved.ImmunohistochemistryIn group B, except MHC classâ…¡+ cells, the rest, including F4/80+ cells,CD86/CD80+cells, and CD4+T-lymphocyte, increased promptly after PK, and arrivedthe climax in the time of rejection with a distinct difference from normalmouse,(P=0.000). While, MHC classâ…¡+ cells decreased promptly on day 5, andincreased obviously on day 10 and thereafter, arrived the climax in the time of rejectionwith a distinct difference from normal mouse,(P=0.000). In group A, distinct changeswere not found in MHC classâ…¡+ cells, F4/80+ cells, CD86/CD80+cells, CD4+T-lymphocyte, with P=0.271, 0.249, 0.870, 0.786, respectively.RT-PCRIn group B, CCR5 and CCR7 were detected in all time in the iris and ciliary body tissues,but increased gradually, and to exclude up to the peak during rejection. IL-12 was alsodetected in the iris and ciliary body in all time, and increased gradually, but none wasdetected in group A. IL-4 was not detected in all groups.ConclusionsThe anterior chamber immune micro-environment had changed, when corneal transplantrejection, high expression of MHC classâ…¡molecules and costimulatory molecules onantigen-presenting cells, and CD4+T lymphocytes. Anterior chamber micro-environmentis likely to be another important factor of keratoplasty rejection.