The preliminary study of establishing animal model of chronic allograft dysfunction after penetrating keratoplasty

Objective: To establish a murine model of chronic corneal allograft dysfunction (CCAD) after penetrating keratoplasty (PK). Methods: Experimental study. PK model in mice: Semiallogeneic CB6F1 mice were obtained from matching of female BALB/c and male C57BL/6 mice. C57BL/6 (allogeneic group), CB6F1 (semiallogeneic group) and BALB/c (syngeneic group) grafts were transplanted orthotopically to BALB/c recipients respectively, and BALB/c mice as a control group. The follow-up time was more than 100 d, and graft survival time and corneal opacity score were monitored, and corneal endothelium were examined by alizarin red and PI/Hoechst stain. CD4 ⁺ and CD8 ⁺ T lymphocytes were examined by immunohistochemistry. Ultrastructure changes of the grafts were examined by electromicroscopy. Log-rank test were used to compare survival curves. Results: (1) Graft examination; Median graft survival times were 17.0 d, 85.5 d, >100d and >100 d in allogeneic, semiallogeneic, syngeneic and control groups, respectively (F = 344.0, P<0.01). (2) Immunohistochemistry examination: There were large amount of CD4 ⁺ and CD8 ⁺ T lymphocyte infiltration in allografts in allogeneic group at 3 weeks after PK; Few CD4 ⁺ and CD8 ⁺ T lymphocytes were observed in semiallogeneic group and syngeneic goups at 3 weeks after PK; CD4 ⁺ and CD8 ⁺ T lymphocyte infiltration was not observed in the control group. (3) Endothelium examination: The endothelium can not be counted because the blurred image after the alizarin red combined PI/Hoechst stain and apoptotic and necrotic cells can be seen in allogeneic group; the endothelial cell density decreased and few apoptosis can be detected in semiallogeneic and syngeneic groups; no apoptotic and necrotic endothelial cells were found in the control group. (4) Ultrastructural characteristic changes mainly include fibrosis formation and endothelium atrophy and degeneration in failed grafts in all transplanted groups by electron microscopy examination. Inflammation cells can only be found in the allogeneic group. Conclusions: Semiallogeneic and syngeneic transplantation groups present the changes similar to CCAD in clinical study, and both can be regarded as the model that permits molecular evaluation of CCAD.