Long-term survival of allogeneic donor cell-derived corneal epithelium in limbal deficient rabbits.

PURPOSE: To investigate the capability of cultivated allogeneic epithelial stem cells to restore a functional ocular surface in a limbal deficient cornea; to verify the long term survival of epithelial allograft; and to examine the host immune response to heterologous cell transplant in a rabbit model. METHODS: Limbal deficiency was established by performing limbectomy on rabbits (n = 100). Corneal epithelial stem cells were obtained from the limbus and replicated in vitro without a supporting layer. The cell (3 x 10(5)) suspension was then transplanted via topical application as eye drops. Animals were divided into allograft, autograft, and control groups. Females were used as recipients and males as donors for the allograft. Corneas were collected at 7, 14, 21, 40 days as well as 2, 3, 7 and 8 months after cell transplantation. Experimental corneas were evaluated by histology, immunofluorescence, immunohistochemistry and Y chromosome analysis. RESULTS: A well-differentiated corneal epithelium was recognized at 14 to 40 days after cell transfer overlying an infiltrated corneal stroma. Corneal re-epitheliazation was confirmed in 31 of 36 allograft corneas. No significant immune rejection was noted. Stromal abnormality caused by previous limbal deficiency was mostly resolved three months after the regeneration of corneal epithelium. CONCLUSIONS: Transplanted corneal epithelial stem cells were able to differentiate into normal corneal epithelium in vivo without the use of membrane scaffolding. This non-autologous donor cell-derived corneal epithelium survived up to 8 months without immunosuppression and was able to reverse the stromal scarring. Thus, cultivated epithelial stem cells have great potential as an alternative to multiple-surgical procedures in the treatment of limbal deficiency states.

Wound healing patterns following perforation sustained during laser in situ keratomileusis.

BACKGROUND AND PURPOSE: Corneal perforation during laser in situ keratomileusis (LASIK) may interfere with flap adhesion and wound healing. The purpose of this study was to investigate wound healing patterns following corneal perforation sustained during LASIK in rabbits. METHODS: Forty-two pigmented rabbit eyes underwent LASIK surgery with 5.0-mm excimer laser treatment under the corneal flap. Animals were divided into two groups: group I (n = 19) underwent the regular LASIK procedure with -10.0 D treatment, without perforation; in group II (n = 23), the cornea was perforated with the excimer laser. Treatment was discontinued once perforation was observed, and the corneal flap was replaced without sutures. Slit-lamp biomicroscopy, photography, and scatterometry were performed preoperatively and at 1 and 2 days, 1 week, and weekly up to 1 month, 2 months, and 3 months postoperatively. Animals were killed at 1 day, 1 week, 1 month, and 3 months postoperatively and processed for light microscopic, electron microscopic, and immunohistochemical examinations. RESULTS: In group I, the corneas remained clear throughout the experiment. In all eyes, the interface was not readily discernable clinically or histologically. Corneal wound healing was accompanied by minimal cell infiltration. Epithelial hyperplasia at the flap edge was noted at 1 week. Myofibroblast activation was found at the epithelial wedge where there was an epithelial basement membrane break. In group II, the anterior chamber was shallow with no iris incarceration at the end of surgery. The corneas were clear (n = 6) or showed mild to moderate edema (n = 12). Corneal edema peaked at 3.6 +/- 5.0 days and subsided thereafter. Corneal wounds healed similarly to those in group I except at the perforation site. The break in Descemet's membrane and endothelium was covered with a fibrin plug on day 1, which resolved thereafter. There was no statistically significant difference in the incidence of postoperative infection (p = 1.0) or flap displacement (p = 0.69) rates between the two treatment groups. The scatterometry index peaked at 2 to 3 weeks postoperatively and was significantly higher in group II than in group I (p < 0.001). CONCLUSIONS: Although corneal perforation during LASIK surgery may interfere with immediate postoperative flap adhesion, corneal wound healing following LASIK perforation may be similar to that after an uncomplicated LASIK procedure.