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Purpose: To investigate the effect of VEGF-C and VEGF-D blockade via soluble VEGFR-3 (sVEGFR-3) on T cell allosensitization, corneal neovascularization, and transplant survival., Methods: Corneal intrastromal suture placement and allogeneic transplantation were performed on BALB/c mice to evaluate the effect of sVEGFR-3 on corneal neovascularization. Soluble VEGFR-3 trap was injected intraperitoneally to block VEGF-C/D (every other day starting the day of surgery). Immunohistochemical staining of corneal whole mounts was performed using anti-CD31 (PECAM-1) and anti-LYVE-1 antibodies to quantify the levels of hem- and lymphangiogenesis, respectively. Mixed lymphocyte reaction (MLR) was performed to assess indirect and direct host T cell allosensitization and the frequencies of IFN-γ-producing T cells in the draining lymph nodes were assessed using flow cytometry. Graft opacity and survival was evaluated by slit-lamp biomicroscopy., Results: Treatment with sVEGFR-3 resulted in a significant blockade of lymphangiogenesis 2 weeks post-transplantation and significantly prolonged corneal allograft survival compared to the control group at 8 weeks post-transplantation (87.5 % vs. 50 %), and this was associated with significant reduction in the frequencies of allosensitized T cells and decreased frequencies of IFN-γ-producing CD4 T cells., Conclusions: Soluble VEGFR-3 suppresses corneal lymphangiogenesis and allograft rejection and may offer a viable therapeutic modality for corneal neovascularization and corneal transplantation.

We evaluated whether corneal graft survival in presensitized corneal transplantation was affected by subconjunctival ranibizumab in a rat model. The effect of ranibizumab in the presensitized corneal transplantation has not been previously reported, although anti-VEGF was attempted on a non-presensitized model in other studies. Corneas were transplanted from Brown Norway to Spraque Dawley rats. The recipient rats were randomly assigned to three groups: Group 1, skin autograft and subconjunctival injection of PBS; Group 2, skin allograft and injection of PBS; and Group 3, skin allograft and injection of ranibizumab (vascular endothelial growth factor antibody). A skin graft was performed 2 weeks before corneal transplantation. On days 3, 7, 11, and 14 after transplantation, the grafts were scored. The number of corneas with graft rejection on day 14 was significantly higher in Group 2 than in Group 1 or 3 (6/15 [40.0%] in Group 1, 13/15 [86.7%] in Group 2, and 4/15 [26.7%] in Group 3). The mean clinical scores for edema, opacity, and new vessels in Group 3 were significantly lower than those in Group 2, while the edema score in Group 1 was significantly lower than that in Group 2 on day 14. Before corneal allotransplantation, presensitization by skin grafting accelerated the graft rejection process. In a short-term presensitized rat model of keratoplasty, application of subconjunctival ranibizumab prevented graft rejection., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objective: The effect of human leukocyte antigen (HLA) matching on immune rejection after keratoplasty was studied., Methods: Immunoserologic HLA typing was performed on thirty-three eyes with corneal vascularization of 33 high-risk patients before keratoplasty, and HLA matching was made before the surgery. The donor corneas used for these cases were selected on the basis of obtaining a negative HLA cross-match test and matched ABO antigen compatibility of blood group before surgery. The mean follow-up was fifteen months., Results: The findings suggested that there should be highly statistic significance (t = 7.36, P < 0.01) between the rate of rejection of well matched group and that of the poorly matched one. There was no correlation between the number of HLA antigen matched and the rejection rate (t = 0.917, P > 0.05)., Conclusion: Lower incidence of corneal graft failure from allograft rejection occurs when well HLA matching is used for selecting donor in high-risk patients with corneal vascularization.

Immunologic rejection is the main cause of corneal graft failure, especially in vascularized corneal beds. The purpose of this study was to investigate the effect of systemic Cyclosporine-A (CsA) on the survival of corneal allografts in heavily vascularized rabbit corneal beds, following alkali burn. Heavy corneal vascularization was induced in one eye of 20 rabbits by alkali burn. Forty-five days later, penetrating keratoplasty was performed in all the heavily vascularized corneas. Twenty-five mg/kg/day of CsA was intramuscularly administered to 10 rabbits for 30 days. The other 10 rabbits were treated with the solvent without CsA and were used as a matched control group. The results show a significant difference in corneal allograft survival between the two groups. All corneal grafts in the untreated group were intensely rejected and vascularized within 3 weeks. Nine of the 10 corneal transplants, in the CsA-treated group, remained transparent without signs of immunologic rejection for > 180 days. In one corneal transplant, minor signs of rejection occurred. We suggest that CsA, when given systemically, is a potent drug in the prevention of immunologic rejection in high-risk corneal transplantations, such as allografts, in heavily vascularized corneas following alkali burn.

Using a vascularized cornea rabbit model closely resembling human high-risk keratoplasty, corneal allografts were performed on three groups of animals that were paired and tested preoperatively by mixed lymphocyte cultures (MLCs). The three groups were group A, unmodified controls with clear corneas; group B, untreated animals with vascularized corneas; and group C, animals treated with topical cyclosporine (CSA) with vascularized corneas. The MLC results were expressed as stimulation indices (SIs) and divided into low (SI < or = 20) and high (SI > 20) responders and were correlated with final outcome of grafts using survival analysis estimates. In group A, five of 13 (38.5%) grafts rejected, the chance of failure depending on the degree of MLC mismatch between donor and recipient (p = 0.02). All allografts in group B rejected regardless of the degree of mismatch. In group C, seven of 12 (58.3%) grafts rejected, indicating that topical CSA significantly improved survival (p = 0.003) compared with group B. Grafts with mild degrees of MLC mismatch (low responders) survived better (p = 0.0003) than did higher degrees of MLC mismatch (high responders), all of which rejected despite treatment. Our results indicate that both corneal vascularization and the degree of donor-recipient matching play important roles in determining corneal graft survival.

We used a rat model of orthotopic penetrating keratoplasty to study splenic cell cytotoxicity in the host. DA (RT1avl) rats received grafts of Fischer (F344, RT1lv1) rat corneas. Cytotoxic T lymphocyte (CTL) activity was measured by the cell-mediated lymphocytotoxicity assay using 4/4R.M.-4 cells as target cells. CTL activity became detectable in vitro 2 weeks after grafting and peaked at 3 weeks. This simple method of quantifying CTL activity in the orthotopic penetrating keratoplasty model should provide a reliable tool for studying the CTL response in corneal transplantation.