title: Safety profile of accelerated corneal cross-linking versus conventional cross-linking: a comparative study on ex vivo-cultured limbal epithelial cells.
abstract: OBJECTIVE
  To compare the effects of accelerated corneal collagen cross-linking (ACXL) and corneal collagen cross-linking (CXL) on ex vivo-cultured limbal epithelial cells (LECs).
  
  
  METHODS
  Day 14 cultured LECs were either unexposed (control) or exposed to different intensities of ultraviolet-A (UV-A) irradiance for different durations (3 mW for 30 min, 9 mW for 10 min, 18 mW for 5 min and 30 mW for 3 min) in the presence and absence of riboflavin. These cells were further processed for quantitative real-time PCR, vital staining, immunofluorescence staining and fluorescence-activated cell sorting (FACS) staining to evaluate the apoptotic status. Statistical analysis was performed using a Student t test.
  
  
  RESULTS
  Vital staining showed a significantly higher (p=0.004) dead cell population with 3 mW for 30 min when compared with 30 mW for 3 min exposure (p=0.225). Quantitative PCR results revealed significantly reduced abcg2 and Δnp63 mRNA levels, while FACS analysis showed an increase in ABCG2-Annexin V positive population in cells exposed to 3 mW for 30 mins. Neither reduction of mRNA expression of abcg2 and Δnp63 nor increase in FACS-stained ABCG2-Annexin V positivity was detected in cells exposed to 30 mW for 3 min. Additionally, enhanced caspase activity was detected with fluorochrome inhibitor of caspases staining and mRNA expression of caspase 3 and 9 was upregulated in cells exposed to 3 mW for 30 min, but not at 30 mW for 3 min.
  
  
  CONCLUSIONS
  The 30 mW UV-A irradiation used in ACXL appears to be safe on cultured LECs in comparison with 3 mW used in CXL.
authors.affiliation: Department of Cornea & Refractive Surgery, Narayana Nethralaya, Bangalore, Karnataka, India.
authors.forename: Rohit
authors.initials: R
authors.lastname: Shetty
authors.affiliation: Department of Cornea & Refractive Surgery, Narayana Nethralaya, Bangalore, Karnataka, India Stem Cell Research Laboratory, Narayana Nethralaya, Bangalore, Karnataka, India.
authors.forename: Himanshu
authors.initials: H
authors.lastname: Matalia
authors.affiliation: Department of Ophthalmology, Maastricht University Medical Center, Maastricht, The Netherlands.
authors.forename: Rudy
authors.initials: R
authors.lastname: Nuijts
authors.affiliation: Stem Cell Research Laboratory, Narayana Nethralaya, Bangalore, Karnataka, India.
authors.forename: Murali
authors.initials: M
authors.lastname: Subramani
authors.affiliation: Stem Cell Research Laboratory, Narayana Nethralaya, Bangalore, Karnataka, India.
authors.forename: Kamesh
authors.initials: K
authors.lastname: Dhamodaran
authors.affiliation: GROW Laboratory, Narayana Nethralaya, Bangalore, Karnataka, India.
authors.forename: Ramanan
authors.initials: R
authors.lastname: Pandian
authors.affiliation: Department of Vitreo-Retina Services, Narayana Nethralaya, Bangalore, Karnataka, India.
authors.forename: Chaitra
authors.initials: C
authors.lastname: Jayadev
authors.affiliation: Stem Cell Research Laboratory, Narayana Nethralaya, Bangalore, Karnataka, India.
authors.forename: Debashish
authors.initials: D
authors.lastname: Das
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis; D053148:Caspase 3 / Q000235:genetics; D053453:Caspase 9 / Q000235:genetics; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism*; D003319:Corneal Stroma / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000627:therapeutic use*; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D017319:Photosensitizing Agents / Q000627:therapeutic use*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D012256:Riboflavin / Q000627:therapeutic use; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D017304:Annexin A5; D003432:Cross-Linking Reagents; D009363:Neoplasm Proteins; D017319:Photosensitizing Agents; D012333:RNA, Messenger; C115051:TP63 protein, human; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D003094:Collagen; D053148:Caspase 3; D053453:Caspase 9; D012256:Riboflavin
keywords: Apotosis; Cornea; Ocular surface; Stem Cells