April 2009
Volume 50, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2009
Effects of Riboflavin and UV upon Glycosaminoglycan Synthesis in Human Keratoconic Corneas
Author Affiliations & Notes
  • C. M. Diniz
    Ophthalmology,
    UNIFESP, São Paulo, Brazil
  • F. M. Gadelha
    Ophthalmology, UFPB, João Pessoa, Brazil
  • Y. Michelacci
    Biochemistry,
    UNIFESP, São Paulo, Brazil
  • M. Campos
    Ophthalmology,
    UNIFESP, São Paulo, Brazil
  • Footnotes
    Commercial Relationships  C.M. Diniz, None; F.M. Gadelha, None; Y. Michelacci, None; M. Campos, None.
  • Footnotes
    Support  None.
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 5461. doi:
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      C. M. Diniz, F. M. Gadelha, Y. Michelacci, M. Campos; Effects of Riboflavin and UV upon Glycosaminoglycan Synthesis in Human Keratoconic Corneas. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5461.

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Abstract

Purpose: : To investigate the effects of agents that induce collagen crosslinking - riboflavin and UVA - upon the synthesis of glycosaminoglycans (GAG) by human keratoconic corneas

Methods: : Fifteen human keratoconic corneas that were removed from patients submitted to corneal transplantation were used. All procedures were performed under sterile conditions. After epithelial debridement, the corneas were cut in two halves: one half was exposed to an ultraviolet A (UVA) light source (wavelength of 365 nm and delivering an irradiance of 3 mW/cm2, at a distance of 1cm) while applying 50µl of 0.1% riboflavin solution (10 mg riboflavin 5-phosphate in 10 ml of 20% dextran T-500) every five minutes, for 30 minutes; the other half received no treatment, and was used as matched control. The corneal explants were washed with 5 ml of a 4 mg/ml gentamycin solution in PBS and immediately placed in 12 ml of Ham F-12 nutrient mixture, as previously described1. For the metabolic labeling of glycosaminoglycans, fresh Ham F-12 culture medium (without serum) containing 100 mCi/ml of 35S-sulfate was added. The corneas were labeled with 35S-sulfate for 24 h. After 35S incorporation, the corneas were retrieved, washed twice with PBS, and the GAGs were extracted and analyzed by agarose gel electrophoresis.

Results: : The corneal treatment with UVA and riboflavin caused a marked decrease in GAG synthesis by corneal explants, as compared to the matched controls. This decrease was more evident for dermatan sulfate and keratan sulfate, than for heparan sulfate.

Conclusions: : The corneal crosslinking procedure here used caused a marked decrease in the GAG biosynthesis, possibly due to stromal cell death (by necrosis and/or apoptosis). This decrease was even more evident for dermatan sulfate and keratan sulfate, possibly due to the higher turnover rate of heparan sulfate.[1] Soriano ES, Campos MS, Michelacci YM. Clin Chim Acta. (2000) 295(1-2):41-62.

Keywords: cornea: basic science • keratoconus • proteoglycans/glycosaminoglycans 
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