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Potential apoptotic effect of ultraviolet-A irradiation during cross-linking: a study on ex vivo cultivated limbal epithelial cells
  1. Himanshu Matalia1,2,
  2. Rohit Shetty2,
  3. Kamesh Dhamodaran1,
  4. Murali Subramani1,
  5. Vincent Arokiaraj1,
  6. Debashish Das1
  1. 1Stem Cell Research Laboratory, Narayana Nethralaya, Bangalore, Karnataka, India
  2. 2Department of Cornea and Refractive Surgery, 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 study the effects of ultraviolet-A (UV-A) irradiation, in the presence or absence of riboflavin, on ex vivo cultured limbal epithelial cells (LECs).

Methods The study was carried out in a super specialty ophthalmic hospital. Ex vivo cultured LECs were grown on denuded amniotic membranes and exposed to similar levels of UV-A radiation used during corneal cross-linking (CXL), in the presence or absence of the photosensitiser, riboflavin. These cells were then used for extraction of RNA, cDNA conversion, and antibody staining. Quantitative PCR and immunofluorescence staining were performed to evaluate the apoptotic state of treated and non-treated LECs. Statistical analyses were evaluated using a Student's t test.

Results We found that bcl-2, an antiapoptotic gene, was downregulated, whereas, bax, a proapoptotic gene, was upregulated. After LECs were exposed to UV-A radiation, a significant upregulation of both caspase 3 and caspase 9 was observed in treated cells when compared with untreated LECs.

Conclusions These results indicate that exposure of LECs to UV-A dosages similar to those used in the CXL procedure promotes the expression of genes known to promote apoptosis. In the presence of riboflavin, the damage caused by UV-A treatment was marginalised, but not totally blocked.

  • Stem Cells
  • Biochemistry
  • Cornea

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