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Safety profile of accelerated corneal cross-linking versus conventional cross-linking: a comparative study on ex vivo-cultured limbal epithelial cells
  1. Rohit Shetty1,
  2. Himanshu Matalia1,2,
  3. Rudy Nuijts3,
  4. Murali Subramani2,
  5. Kamesh Dhamodaran2,
  6. Ramanan Pandian4,
  7. Chaitra Jayadev5,
  8. Debashish Das2
  1. 1Department of Cornea & Refractive Surgery, Narayana Nethralaya, Bangalore, Karnataka, India
  2. 2Stem Cell Research Laboratory, Narayana Nethralaya, Bangalore, Karnataka, India
  3. 3Department of Ophthalmology, Maastricht University Medical Center, Maastricht, The Netherlands
  4. 4GROW Laboratory, Narayana Nethralaya, Bangalore, Karnataka, India
  5. 5Department of Vitreo-Retina Services, Narayana Nethralaya, Bangalore, Karnataka, India
  1. Correspondence to Dr Debashish Das, Stem Cell Research Laboratory, Narayana Nethralaya, 258/A, Bommasandra Industrial Area, Narayana Health City, Bangalore, Karnataka 560 099, India; drdebashish{at}narayananethralaya.com, dasdebashish{at}yahoo.co.uk

Abstract

Aim/background 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.

  • Apotosis
  • Cornea
  • Ocular surface
  • Stem Cells

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