title: 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.
abstract: OBJECTIVE
  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 follows: 2.7 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.
authors.affiliation: *Department of Ophthalmology, College of Physicians and Surgeons, Columbia University, New York, NY; †Department of Ophthalmology, Uijeongbu St. Mary's Hospital College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea; and ‡Confocal and Specialized Microscopy Shared Resource, Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY.
authors.forename: Su-Young
authors.initials: SY
authors.lastname: Kim
authors.forename: Natasha
authors.initials: N
authors.lastname: Babar
authors.forename: Emilia Laura
authors.initials: EL
authors.lastname: Munteanu
authors.forename: Anna
authors.initials: A
authors.lastname: Takaoka
authors.forename: Mariya
authors.initials: M
authors.lastname: Zyablitskaya
authors.forename: Takayuki
authors.initials: T
authors.lastname: Nagasaki
authors.forename: Stephen L
authors.initials: SL
authors.lastname: Trokel
authors.forename: David C
authors.initials: DC
authors.lastname: Paik
mesh_terms: D000818:Animals; D002152:Calorimetry, Differential Scanning; D003094:Collagen / Q000378:metabolism; D003315:Cornea / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D003319:Corneal Stroma / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000633:toxicity*; D004195:Disease Models, Animal; D004728:Endothelium, Corneal / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D018613:Microscopy, Confocal; D017319:Photosensitizing Agents / Q000633:toxicity*; D011817:Rabbits; D012256:Riboflavin / Q000633:toxicity*; D012521:Sarcosine / Q000031:analogs & derivatives* / Q000633:toxicity; D016707:Tissue Fixation; D014466:Ultraviolet Rays
chemical_list: D003432:Cross-Linking Reagents; D017319:Photosensitizing Agents; C020420:monomethylolglycine; D003094:Collagen; D012256:Riboflavin; D012521:Sarcosine