April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Phototoxic Effects on the Corneal Endothelium During (Riboflavin/UV-A)-Mediated Collagen Cross-Linking
Author Affiliations & Notes
  • C. L. Springs
    Ophthalmology, Indiana University, Indianapolis, Indiana
  • M. Balasubramanian
    School of Optometry,
    Indiana University, Bloomington, Indiana
  • S. Cheluvaraja
    Department of Physics,
    Indiana University, Bloomington, Indiana
  • R. S. Pinapati
    School of Optometry,
    Indiana University, Bloomington, Indiana
  • S. P. Srinivas
    School of Optometry,
    Indiana University, Bloomington, Indiana
  • Footnotes
    Commercial Relationships  C.L. Springs, None; M. Balasubramanian, None; S. Cheluvaraja, None; R.S. Pinapati, None; S.P. Srinivas, None.
  • Footnotes
    Support  NIH grant R21-EY019119 (SPS) and Faculty Research Grant, VP of Research, IU Bloomington, IN (SPS)
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 6422. doi:
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      C. L. Springs, M. Balasubramanian, S. Cheluvaraja, R. S. Pinapati, S. P. Srinivas; Phototoxic Effects on the Corneal Endothelium During (Riboflavin/UV-A)-Mediated Collagen Cross-Linking. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6422.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Photochemical cross-linking of stromal collagen to halt progression of corneal ectasia is a new approach to treat keratoconus. The treatment consists of irradiation of the affected cornea with UV-A (365 nm; 2 mW/cm2) concomitant with topical riboflavin (Rf; 0.1%). We have characterized the phototoxicity of the treatment on corneal endothelium using electrical-cell substrate impedance sensing (ECIS).

Methods: : Bovine cells were grown to confluence on gold electrodes for ECIS-based measurements of trans-endothelial electrical resistance (TER), which decreases upon cell death and stress-induced loss of integrity of the apical junctions. After steady state in TER in 24 hrs, cells were challenged with Rf and UV-A light (30 min, 0.5 mW/cm2). Lethal dose for 50% loss in TER was calculated from response to 1-200 µM of Rf. UV-A transmittance across the bovine cornea, with and without topical Rf, was assessed using a fiber-optic spectrometer.

Results: : UV-A alone did not induce a loss in TER except for a small transient decrease that lasted for the duration of UV-A exposure (i.e., 30 min). In the presence Rf (> 30 µM), similar exposure to UV-A led to an acute fall followed by a sustained loss for > 20 hrs. This biphasic response obtained was similar to that produced by rose bengal (40 nM)/white light (0.1 mW/cm2; 30 min) exposure.LD50 for Rf was 160, 80, and 90 µM for loss in TER at 8, 12, and 16 hrs, respectively. When the medium contained catalase (7000 units/mL), Rf/UV-A had no effect on TER. Rf/UV-A produced both apoptosis and necrosis as measured by flow cytometry. Upon topical Rf, transmittance at 375 nm reduced by 50% immediately, but recovered to ~ 50% in 30 min.

Conclusions: : At Rf levels > 30 µM, UV-A irradiation employed for CxL is phototoxic to corneal endothelial cells. Although Type-1 reaction mediated singlet oxygen is implicated in collagen cross-linking, catalase effect suggests that the toxicity involves H2O2 production (i.e., Type II reaction). The immediate loss in UV transmittance is secondary to formation of a thick film of Rf on the stromal surface upon administration of the drop.

Keywords: cornea: endothelium • apoptosis/cell death • radiation damage: light/UV 
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