The Critical Role of Lymph Nodes in Corneal Alloimmunization and Graft Rejection

purpose. To elucidate the role of draining cervical lymph nodes (CLNs) in corneal alloimmunity. methods. Fully mismatched orthotopic corneal transplantation was performed in BALB/c hosts that had their CLNs excised before transplantation (CLN−). Normal hosts (CLN+), splenectomized mice (Sp−), and those without either CLNs or spleen (CLN−/Sp−) served as comparison groups. To determine the contribution of CLNs to alloimmunity more directly, CLN− mice were reconstituted by grafting LNs from other BALB/c mice to their cervical lymphatic chains, thus deriving CLN−/+ mice. Tetramethyl rhodamine isothiocyanate’s (TRITC) flow to draining CLNs was used as a measure of afferent lymph flow. Graft survival and allospecific delayed-type hypersensitivity (DTH) were used as measures of alloreactivity. results. Fifty percent of normal control and 12% of Sp− hosts accepted the allografts. In contrast, 100% of CLN− and 88% of CLN−/Sp− hosts accepted allografts indefinitely (P < 0.01). Additionally, all CLN− hosts failed to demonstrate allospecific DTH (P < 0.001). CLN−/+ mice reconstituted with LN from naïve animals showed graft survival rates and DTH responses that were indistinguishable from those of naïve CLN+ mice. Of particular interest, however, is that mice reconstituted with CLNs from hosts with rejected corneal grafts had swift rejection of subsequent corneal grafts and exhibited strong donor-specific DTH. In contrast, mice reconstituted with CLNs from hosts with accepted corneal grafts showed rejection of subsequent corneal grafts in a manner that was indistinguishable from rejection in naïve CLN+ hosts. conclusions. Draining CLNs play a critical role in allosensitization and rejection. In contrast to the spleen, draining CLNs do not appear to play a critical role in tolerance induction in corneal transplantation

Tissue engineering of corneal stroma with rabbit fibroblast precursors and gelatin hydrogels

PURPOSE: To isolate fibroblast precursors from rabbit corneal stroma using a sphere-forming assay, to engineer corneal stroma with the precursors and gelatin, and to establish the therapeutic application of precursors in a rabbit corneal stroma. METHODS: In the in vitro study, a sphere-forming assay was performed to produce precursors from rabbit corneal stroma. Corneal stroma was engineered by cultivating precursors in porous gelatin for one week. In the in vivo study, the engineered corneal stromal sheet with precursors (precursor/gelatin group) or with fibroblasts (fibroblast /gelatin group) or without cells (gelatin group) was transplanted to a pocket of rabbit corneal stroma. Gene expression and extracellular matrix production were examined immunohistochemically in each group one week and four weeks after surgery. RESULTS: In the in vitro study, cells in the spheres were BrdU-positive, and their progeny were keratocan-positive. The study also showed that the corneas transplanted with a porous gelatin sheet did not show any opacity four weeks after transplantation in any group. In the gelatin sheet of the precursor/gelatin group, a more intense expression of type I collagen was observed relative to the other two groups four weeks after the surgery. CONCLUSIONS: Our findings demonstrate that the transplantation of fibroblast precursors combined with gelatin hydrogel into the corneal stroma is a possible treatment strategy for corneal stromal regeneration