Rose Bengal and Green Light Versus Riboflavin–UVA Cross-Linking: Corneal Wound Repair Response

Abstract

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.

Arginine as an Enhancer in Rose Bengal Photosensitized Corneal Crosslinking

“…17 Effects of RB CXL with arg on stromal cells was limited to the region closest to the anterior surface below the Bowman membrane, similar to that found for aerobic RB CXL, and substantially less deep than produced by riboflavin CXL. 5 As previously reported, after oxygen-dependent RB CXL, cell death occurred only to the same depth as found after de-epithelialization alone (no treatment control; see Fig. 5), which is about the same depth as RB penetration into the stroma.…”

“…5), which is about the same depth as RB penetration into the stroma. 5 After anaerobic RB CXL with arg, TUNEL-labeled cells were found at a slightly but statistically significant increased depth from the anterior surface, which correlates well with the increased penetration depth of RB in arg pretreated corneas. Further studies have to determine whether this increase has any consequences in a clinical setting, especially when compared to the results observed in riboflavin crosslinking.…”

“…[6][7][8] After crosslinking, it causes cell death in the cornea comparable to the approved and clinically widely used riboflavin and UV corneal crosslinking. 5 Yet, there is no data on arginine and RB in combination. Arginine appears to be a safe choice for potential clinical applications.…”

Medical Applications of Rose Bengal‐ and Riboflavin‐Photosensitized Protein Crosslinking

“…In animal models, RF-CXL increased corneal stiffness as determined by air puff corneal deformation imaging with finite element modeling and by optical coherence elastography (93,94), consistent with previous studies in ex vivo-treated corneas that indicated~2-fold increase in Young's modulus (95)(96)(97)(98)(99). Responses to RF-CXL in rabbit corneas included downregulation of genes for ECM components and upregulation of glycan and proteoglycan glycosylation (100) as well as stromal cell death throughout the anterior and middle stroma (65).…”

“…In more recent studies, RB 2− phototoxicity to keratocytes in the corneal stroma has been observed and studied , which was consistent with earlier studies showing that R5P 2− photosensitization causes phototoxicity in the same tissue . The reason for greater photocytotoxicity in corneal keratocytes compared with dermal fibroblasts is not clear.…”