Evaluating the Toxicity/Fixation Balance for Corneal Cross-Linking With Sodium Hydroxymethylglycinate (SMG) and Riboflavin-UVA (CXL) in an Ex Vivo Rabbit Model Using Confocal Laser Scanning Fluorescence Microscopy

• Published in: Cornea Vol. 35; no. 4; pp. 550 - 556
• Main Authors: Kim, Su-Young, Babar, Natasha, Munteanu, Emilia Laura, Takaoka, Anna, Zyablitskaya, Mariya, Nagasaki, Takayuki, Trokel, Stephen L, Paik, David C
• Format: Journal Article
• Language: English
• Published: United States Copyright Wolters Kluwer Health, Inc. All rights reserved 01-04-2016
• Subjects: Animals; Calorimetry, Differential Scanning; Collagen - metabolism; Cornea - drug effects; Cornea - metabolism; Cornea - pathology; Corneal Stroma - metabolism; Cross-Linking Reagents - toxicity; Disease Models, Animal; Endothelium, Corneal - drug effects; Endothelium, Corneal - metabolism; Endothelium, Corneal - pathology; Microscopy, Confocal; Photosensitizing Agents - toxicity; Rabbits; Riboflavin - toxicity; Sarcosine - analogs & derivatives; Sarcosine - toxicity; Tissue Fixation; Ultraviolet Rays
• ISSN: 0277-3740; 1536-4798
• DOI: 10.1097/ICO.0000000000000743

PURPOSE:To develop methods to delineate the relationship between endothelial cell toxicity and tissue fixation (toxicity/fixation) using sodium hydroxymethylglycinate (SMG), a formaldehyde releaser, and riboflavin-UVA photochemical corneal cross-linking (CXL) for therapeutic tissue cross-linking of the cornea.
METHODS:Eleven fresh cadaveric rabbit heads were used for ex vivo corneal cross-linking simulation. After epithelial debridement, the tissue was exposed to 1/4 max (9.8 mM) or 1/3 max (13 mM) SMG at pH 8.5 for 30 minutes or riboflavin-UVA (CXL). The contralateral cornea served as a paired control. Postexposure, cross-linking efficacy was determined by thermal denaturation temperature (Tm) and endothelial damage was assessed using calcein AM and ethidium homodimer staining (The Live/Dead Kit). Confocal laser scanning fluorescence microscopy was used to generate live/dead cell counts using a standardized algorithm.
RESULTS:The ΔTm after CXL, 1/3 SMG, and 1/4 SMG was 2.2 ± 0.9°C, 1.3 ± 0.5°C, and 1.1 ± 0.5°C, respectively. Endothelial cell damage was expressed as the percent of dead cells/live + dead cells counted per high-power field. The values were 3 ± 1.7% (control) and 8.9 ± 11.1% (CXL) (P = 0.390); 1 ± 0.2% (control) and 19.5 ± 32.2% (1/3 max SMG) (P = 0.426); and 2.7 ± 2.4% (control) and 2.8 ± 2.2% (1/4 max SMG) (P = 0.938). The values for endothelial toxicity were then indexed over the shift in Tm to yield a toxicity/fixation index. The values were as follows2.7 for CXL, 14 for 1/3 max, and 0.1 for 1/4 max.
CONCLUSIONS:Quarter max (1/4 max = 9.8 mM) SMG effectively cross-linked tissue and was nontoxic to endothelial cells. Thus, SMG is potentially a compound that could achieve both desired effects.