The excimer laser has been undergoing rapid development for clinical use since the early 1980s. The authors report 2-year follow-up results from studies in 31 eyes (15 women and 14 men) to evaluate the excimer laser in performing photokeratectomy. Patients were divided into two groups: 27 eyes (group 1) underwent phototherapeutic keratectomy, and 4 eyes (group 2) underwent photorefractive keratectomy. Visual function improved in 21 of 27 eyes in group 1 and in 2 of 4 eyes in group 2. Complications were minimal and manageable. The authors describe a procedure to minimize induced hyperopia in phototherapeutic keratectomy patients, and, although not statistically significant, less induced hyperopia was noted in these patients. Photokeratectomy may be an alternative to penetrating or lamellar keratoplasty and more invasive refractive procedures, but the long-term effects must be carefully observed.

To determine if the corneal epithelium prevents the collagen cross-linking effect. Using immunofluorescence microscopy after CXL, we indirectly analyzed the role of the epithelium as ultraviolet-A (UVA) shield as well as a barrier to riboflavin penetration. Fifteen freshly enucleated porcine eyes were divided into 3 groups. The corneal epithelium was kept intact in all groups. Five eyes served as control (Group 1). On group 2, eyes received tetracaine anesthetic drops and topical 0.1% riboflavin solution (10 mg riboflavin-5-phosphate in 10 mL 20% dextran-T-500). On Group 3, riboflavin was injected into the anterior chamber to allow penetration of the drug through the endothelium. Groups 2 and 3 were exposed to UVA (365 nm, 3 mW/cm(2)) for 30 minutes. Ultra-thin sections (8 µm) of the corneas were stained with anti-collagen type I and DAPI (4,6-diamidino-2-fenilindole dihydrocloride) and analyzed with fluorescence microscopy. Corneas treated with UVA irradiation and intracameral injection of riboflavin (Group 3) showed greater pattern of collagen organization compared to groups 1 (Control) and 2 (riboflavin and tetracaine eye drops). A yellow stromal staining, which represents the riboflavin diffusion into the stroma, was only observed in eyes injected with riboflavin into the anterior chamber. Using immunofluorescence microscopy in porcine corneas, we demonstrated that the corneal epithelium reduces the effectiveness of CXL by preventing the penetration of the drug and not by limiting the UVA transmittance. An inadequate intrastromal concentration of riboflavin may impair CXL effect.

Purpose: To assess ultrastructural stromal modifications in porcine corneas after riboflavin and ultraviolet A (UVA) exposure using immunofluorescence confocal imaging. Methods: Twenty-five freshly enucleated porcine eyes were enrolled in the study. Five eyes served as control (group I). Twenty eyes had their epithelium removed (groups I, II, IV, and V) and five eyes had their epithelium intact (group III). Groups II and III were cross-linked with riboflavin 0.1% solution (10 mg riboflavin-5-phosphate in 10 mL 20% dextran-T-500) and exposed to UVA (365 nm, 3 mW/cm ² ) for 30 minutes. Group IV included five eyes soaked with riboflavin without posterior irradiation, and group V included five eyes irradiated, without previous exposure to riboflavin. Ultra-thin sections (8 µm) of the corneas were stained with anti-collagen I and DAPI and their fluorescence was revealed under confocal microscopy. Results: Only the cross-linked corneas (group II) showed a pronounced, highly organized anterior fluorescence zone of 182.5±22.5 µm. Using DAPI staining, an anterior and concentrated displacement of cell nuclei due to collagen compaction was observed after cross-linking (group II). No structural changes were observed in all other groups. Conclusions: The cross-linking treatment effect can be directly visualized using confocal fluorescence imaging, allowing for a quantitative analysis. Cross-linked corneas showed a pronounced and limited anterior zone of organized collagen fibers, which was not observed in the other groups. Treatment of the cornea with riboflavin and UVA without previous deepithelialization did not induce any cross-linking effect. Consequently, to facilitate diffusion of riboflavin throughout the corneal stroma, the epithelium should be removed as an important initial step in the treatment. [ J Refract Surg . 2008;24:S715–S719.]

Purpose. We characterized fluorescent bone marrow cells (YFP⁺ BMCs) in the thy1-YFP mouse and determine if they promote trigeminal ganglion (TG) cell neurite growth. Methods. Excimer laser annular keratectomy was performed in thy1-YFP mice, and corneas were imaged. BMCs were harvested from femur and tibia, and the expression of surface markers on YFP⁺ BMCs was analyzed by flow cytometry. The immunosuppressive action of BMCs (YFP⁺ and YFP⁻) was evaluated in an allogenic mixed lymphocyte reaction (MLR). Neurotrophic action of BMCs (YFP⁺ and YFP⁻) was determined in compartmental and transwell cultures of dissociated TG cells. Results. Following annular keratectomy, YFP⁺ BMCs infiltrated the cornea. YFP⁺ BMCs shared surface markers (CD11b+Gr1+Ly6C+Ly6G-F4/80^low) with monocytic myeloid-derived suppressor cells (MDSCs), had similar morphology, and suppressed T-cell proliferation in allogenic MLR in a dose-dependent manner. YFP⁺ BMCs, but not YFP⁻ BMCs, significantly increased growth of TG neurites in vitro. When cultured in a transwell with TG neurites, YFP⁺ BMCs expressed neurotrophins and secreted nerve growth factor (NGF) in conditioned medium. YFP⁺ BMCs that infiltrated the cornea maintained their phenotype and actions (neuronal and immune). Conclusions. YFP⁺ BMCs in thy1-YFP mice have immunophenotypic features of MDSCs. They secrete NGF and promote neuroregeneration. Their immunosuppressive and neurotrophic actions are preserved after corneal infiltration. These findings increase our understanding of the beneficial roles played by leukocyte trafficking in the cornea and may lead to therapeutic strategies that use NGF-secreting myeloid cells to repair diseased or injured neurons.