PURPOSE. To study corneal wound healing after two cross-linking techniques using either rose bengal and green light (RGX) or the conventional treatment using riboflavin and UVA radiation (UVX). METHODS. Corneas of New Zealand rabbits were monolaterally treated with UVX (21 eyes) or RGX (25 eyes). Treatments involved corneal de-epithelialization (8-mm diameter), soaking with photosensitizer (0.1% riboflavin in 20% dextran for 30 minutes for UVX; 0.1% rose bengal for 2 minutes for RGX), and light irradiation (370 nm, 3 mW/cm 2 , 30 minutes for UVX; 532 nm, 0.25 W/cm 2 , 7 minutes for RGX). Contralateral eyes were used as controls. Clinical follow-up included fluorescein staining, haze measurement, and pachymetry. Healing events analyzed after euthanasia at 2, 30, and 60 days included cell death (TUNEL assay), cell proliferation (BrdU [bromodeoxyuridine] immunofluorescence), and differentiation to myofibroblasts (a-SMA [alpha smooth muscle actin] immunohistochemistry). RESULTS. Re-epithelialization and pachymetries were similar after RGX and UVX. The haze from day 1 to 15 was greater after UVX. Cell death was deeper after UVX, being localized in the anterior and middle stroma, and was superficial (anterior third) after RGX. Cell proliferation appeared after 2 days and was localized in the middle and posterior stroma in the UVX group but was superficial in the RGX group. After 60 days the number of stromal cells had not returned to the control number in either group. CONCLUSIONS. The deeper and longer-lasting cell damage caused by UVX compared to RGX may underlie the slower cell repopulation after UVX and other differences in healing. Shallower damage and a shorter treatment time suggest that RGX may be appropriate for stiffening thin corneas.

The extracellular matrix (ECM) confers transparency to the cornea because of the precise organization of collagen fibrils and a wide variety of proteoglycans. We monitored the corneal wound healing process after alkali burns in rabbits. We analyzed the location and expression of collagens and proteoglycans, the clinical impact, and the recovery of optical transparency. After the animals received both general and ocular topical anesthesia, the central cornea of the left eye was burned by placing an 8-mm diameter filter paper soaked in 0.5 N NaOH for 60 s. The eyes were evaluated under a surgical microscope at 1, 3, and 6 months after burning. At each time point, the clinical conditions of the burned and control corneas were observed. The arrangement of collagen fibers in the corneal stroma was visualized by Picrosirius-red staining, Gomori's silver impregnation and transmission electronic microscopy. Corneal light transmittance was also measured. Myofibroblasts presence was analyzed by immunohistochemistry. mRNA expression levels of collagen types I and III, lumican, decorin, keratocan and alpha-smooth muscle actin were determined by quantitative real-time polymerase chain reaction. One month after alkali burn, the ECM was disorganized and filled with lacunae containing different types of cells and collagen type III fibers in the wound area. Corneal opacities were present with attendant loss of light transmittance. Collagen and proteoglycan mRNA expression levels were up-regulated. After three months, wound healing progress was indicated by reduced corneal opacity, increased light transmittance, reorganization of collagen fibers and only collagen type I expression levels were at control levels. After six months, the wound area ECM morphology was similar to controls, but transmittance values remained low, denoting incomplete restoration of the stromal architecture. This multidisciplinary study of the stromal wound healing process revealed changes in corneal transmittance, collagen organization, myofibroblasts presence and ECM composition at 1, 3, and 6 months after alkali burning. Documenting wound resolution during the six-month period provided reliable information that can be used to test new therapies.

No abstract

The organization of the corneal stoma is modified due to different factors, including pathology, surgery or external damage. Here the changes in the organization of the corneal collagen fibers during natural healing after chemical burn are investigated using second harmonic generation (SHG) imaging. Moreover, the structure tensor (ST) was used as an objective tool for morphological analyses at different time points after burn (up to 6 months). Unlike control corneas that showed a regular distribution, the collagen pattern at 1 month of burn presented a non-organized arrangement. SHG signal levels noticeably decreased and individual fibers were hardly visible. Over time, the healing process led to a progressive re-organization of the fibers that could be quantified through the ST. At 6 months, the stroma distribution reached values similar to those of control eyes and a dominant direction of the fibers re-appeared. The present results show that SHG microscopy imaging combined with the ST method is able to objectively monitor the temporal regeneration of the corneal organization after chemical burn. Future implementations of this approach into clinically adapted devices would help to diagnose and quantify corneal changes, not only due to chemical damages, but also as a result of disease or surgical procedures.