Expremental Study On Mechanism Of Chronic Corneal Allograft Dysfunction After Corneal Transplantation

PurposeTo establish a murine model of chronic corneal allograft dysfunction (CCAD) thatpermits molecular evaluation of alloantigen specific factors and non-alloantigenspecific factors in the process of chronic allograft dysfunction after cornealtransplantation.MethodsPartⅠ: Establishment of murine model of CCAD1) Penetrating keratoplasty (PK) model in mice: Semiallogeneic CB6F1 (BALB/c×C57BL/6) mice (H-b/d) were obtained from matching of female BALB/c (H-2d)mice and male C57BL/6 mice. C57BL/6, CB6F1 and BALB/c corneal grafts weretransplanted orthotopically to BALB/c recipients respectively. 2)Groups: Seventy-twoBLAB/c mice were randomly divided into four groups (18 mice per group): Group A,allogenic transplantion group, C57BL/6 corneal grafts were transplanted to BALB/crecipients; Goups B, semiallogeneic transplantation group, CB6F 1 corneal grafts weretransplanted to BALB/c recipients; Group C, syngeneic transplantation group,BALB/c corneal grafts were transplanted to BALB/c recipients. Group D, controlgroup, BALB/c mice without corneal transplantation. 3) Postoperative examination:The follow-up time was more than 100 days, and the graft was evaluated by slit-lampbiomicroscopy two times every week. Graft survival time and corneal opacity scorewere monitored, and corneal endothelium were examined by alizarin red andPI/Hoechst stain. CD4~+ and CD8~+ T lymphocytes were examined byimmunohistochemistry. Ultrastructure changes of the grafts were examined byelectromicroscopy. PartⅡ: Effect of ailoantigen specific and non-alloantigen specific factors inCCAD1) Animal model: Animals were divided into 4 groups and consult partⅠinexperiment. 2) Lab examination: F4/80, TGF-βand bFGF were examined byimmunohistochemistry, F4/80 and TGF-βwere examined by RT-PCR, and cellapoptosis was examined by TUNEL analysis at 3 weeks, 2 and 3 monthspostoperation.ResultsPartⅠ: Establishment of murine model of CCADAllograft examination: Mild corneal edema and opacity occurred in the grafts in allgroups immediately after PK, and then disappeared after removal of corneal sutures.Corneal grafts opacity combined with angiogenesis reoccurred in group A, andresulted in grafts dysfunction within 12 to 28 days postoperation ultimately. In groupB, the grafts became transparent completely at 1 to 2 months, and then mostly becameopacity gradually without corneal angiogenesis at 2 to 3 months. In group C, thegrafts remained transparent and survived over 100 days. All the corneas remainedtransparent in group D. Median graft survival times were 17d, 85.5d,＞100d and＞100d in 4 groups, respectively.Immunohistochemistry examination: A large amount of CD4~+ and CD8~+ Tlymphocyte infiltration was present in allografts in group A at 3 weeks after PK, withmore expressions at 2 months, and hardly seen at 3 months; Few CD4~+ and CD8~+ Tlymphocytes were observed in allografts in group B and C at 3 weeks after PK, andno can be seen at 2 and 3 months; CD4~+ and CD8~+ T lymphocyte infiltration was notobserved in group D.The endothelium can not be counted because the blurred image after the alizarin redcombined PI/Hoechst stain and apoptotic and necrotic cells can be seen in group A;The endothelial cell density decreased and few apoptosis can be detected in group Band C. No apoptotic and necrotic endothelial cells were found in group D. Ultrastructural characteristic changes mainly include fibrosis formation andendothelium atrophy and degeneration in failed grafts in group A, B and C by electronmicroscopy examination.PartⅡ: Effect of alloantigen specific and non-alloantigen specific factors inCCAD1) Immunohistochemistry examination: F4/80, TGF-β, bFGF were detected at 3weeks, 2 and 3 months in group A, B and C. Alpha-SMA could only be detected at thelate stage in group A, B and C. 2) RT-PCR examination: F4/80 was positiveexpression in group A, B and C, and the relative expression amount was low at earlystage and increased at the late stage; Positive expression of TGF-βwas present in allgroups, and the relative expression amount was high at early stage and decreased atthe late stage. 3) TUNEL analysis: TUNEL positive cells could be seen in thecolumnar epithelium and in the stroma at 3 weeks and 2 months in group A, B and C,but not in group D. No TUNEL positive endothelial cells could be found in all groups.Conclusions1) Semiallogeneic and syngeneic transplantation groups present the changessimilar to CCAD in clinical study, and both can be regarded as the model that permitsmolecular evaluation of CCAD. The former model is applicable for the research ofalloantigen specific and non-alloantigen specific factors, and the latter is applicablefor study of non-alloantigen specific factors.2) Early infiltration of immune and inflammatory cells and production ofnon-immune related cytokines might take part in the pathogenesis of chronicdysfunction of corneal allograft.