The role of apoptosis in the early corneal wound healing after excimer laser keratectomy in the rat.

BACKGROUND: The potential role of apoptosis in corneal wound healing after excimer laser keratectomy was investigated in a rat model. METHODS: Lewis rats underwent laser keratectomy using a 193-nm excimer laser. The central corneas were ablated in three depths: group A, epithelium; group B, superficial stroma; group C, deep stroma. Eyes were collected at 1, 12, 24, and 36 h and 1 week. Cellular markers associated with apoptosis--Fas, Fas ligand (FasL), Bcl-2, and Bax were examined by immunohistochemistry. Keratocyte depletion and endothelial changes were evaluated histologically. In situ end labeling of double-stranded DNA breaks was used to demonstrate apoptosis in corneal sections. RESULTS: Keratocyte depletion was observed in 6 (50%) of 12 rats (total from groups A, B, and C) at 12 h, 11 (73%) of 15 at 24 h, 3 (20%) of 15 at 36 h, and 2 (15%) of 13 at 1 week after laser surgery. Corneal endothelial edema was observed in the ablation zone. Expression of Fas, FasL, Bcl-2, and Bax in corneal cells showed dynamics similar to that of keratocyte depletion and endothelial changes. There was less expression of apoptotic molecules in newly generated epithelial cells and more in endothelial cells of the stromal ablation groups. CONCLUSIONS: Excimer laser keratectomy triggered apoptosis of corneal keratocytes and endothelial cells. More endothelial edema was observed in the stromal ablation than in the epithelial ablation group. The expression of apoptotic molecules coincided with the period of keratocyte depletion and regeneration and of endothelial recovery, suggesting that apoptosis is a dynamic part of corneal wound healing and remodeling after excimer laser keratectomy.

Estimation of fetal hemoglobin levels in individual red cells via fluorescence image cytometry.

A method for estimating fetal hemoglobin (Hb F) levels in individual red blood cells was developed. Cell smears were prepared using a slide maker to ensure uniform thickness and were then stained with immunofluorescence. An antifading gel was applied to preserve a stable fluorescence. The total fluorescence intensities from the same number of red cells in different slide specimens correlated with their hemolysate Hb F levels, which were determined via column chromatography (R = 0.95). Hb F level in individual cells was estimated from fluorescence intensity and cell area, which were determined via image analysis techniques and the hemolysate Hb F level. Blood from a normal subject, a subject with hereditary persistence of fetal hemoglobin, and from sickle cell patients with varying Hb F levels was analyzed. Our analyses showed a wide distribution of Hb F among cells for the normal subject and a gaussian distribution with a peak at the hemolysate Hb F level for the subject with hereditary persistence of fetal hemoglobin. The Hb F distributions were unique to the patients with sickle cell disease. Because Hb F level in individual sickle cells is crucial to the inhibition of cell sickling, the unique hb F distribution may be important in determining the clinical course of this disease.