Abstract
Purpose Collagen crosslinking using ultraviolet- A (UVA) -irradiation combined with the photosensitizer riboflavin is a new technique for treating progressive keratoconus. It has been shown to increase effectively the biomechanical strength of the cornea and to stop or even reverse the progression of keratoconus. As part of a safety evaluation, the present study was undertaken to investigate in vitro the possible cytotoxic effect of combined riboflavin/UVA-treatment on corneal keratocytes and to compare it to UVA-irradiation alone.
Methods Cell cultures established from porcine keratocytes were treated with 0.025% riboflavin solution and various UVA (370 nm)-irradiances ranging from 0.4 to 1.0 mW/cm2 and with UVA alone between 2 and 9 mW/cm2 for 30 min. The cell cultures were evaluated for cell death 24 h after irradiation using trypan-blue and Yopro-fluorescence staining.
Results An abrupt cytotoxic irradiance level was found at 0.5 mW/cm2 for keratocytes after UVA-irradiation combined with the photosensitizer riboflavin, which is 10-fold lower than the cytotoxic irradiance of 5 mW/cm2 after UVA-irradiation alone.
Conclusions A cytotoxic effect of combined riboflavin/UVA-treatment on keratocytes is to be expected at 0.5 mW/cm2, which is reached in the clinical setting in human corneas down to a depth of 300 μm using the standard surface UVA-irradiance of 3 mW/cm2.
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References
Wollensak G, Spoerl E, Seller T . Riboflavin/ultraviolet-A-induced collagen crosslinking for the treatment of keratoconus. Am J Ophthalmol 2003; 135: 620–627.
Wollensak G, Spoerl E, Seller T . Stress–strain measurements of human and porcine corneas after riboflavin-ultraviolet-A-induced cross-linking. J Cataract Refract Surg 2003; 29: 1780–1785.
Spoerl E, Huhle M, Seiler T . Induction of cross-links in corneal tissue. Exp Eye Res 1998; 66: 97–103.
Spoerl E, Schreiber J, Hellmund K, Seiler T, Knuschke P . Untersuchungen zur Verfestigung der Hornhaut am Kaninchen. Ophthalmologe 2000; 97: 203–206.
Spoerl E, Wollensak G, Seiler T . Increased resistance of crosslinked cornea against enzymatic digestion. Curr Eye Res 2003, accepted for publication.
Schnitzler E, Spoerl E, Seiler T . Bestrahlung der Hornhaut mit UV-Licht und Riboflavingabe als neuer Behandlungsversuch bei einschmelzenden Hornhautprozessen, erste Ergebnisse bei vier Patienten. Klin Mbl Augenheilkd 2000; 217: 190–193.
Seiler T . Iatrogenic keratectasia: academic anxiety or serious risk? (Editorial). J Cataract Refract Surg 1999; 25: 1307–1308.
Wilson SE . Keratocyte apoptosis in refractive surgery. CLAO J 1998; 24: 181–185.
Mitooka K, Ramirez M, Maguire LJ, Erie JC, Patel SV, McLaren JW et al. Keratocyte density of central human cornea after laser in situ keratomileusis. Am J Ophthalmol 2002; 133: 307–314.
Kim W-J, Helena MC, Mohan RR, Wilson SE . Changes in corneal morphology associated with chronic epithelial injury. Invest Ophthalmol Vis Sci 1999; 40: 35–42.
Campos M, Szerenyi K, Lee M, McDonnell JM, Lopez PF, McDonnell PJ . Keratocyte loss after corneal deepithelialization in primates and rabbits. Arch Ophthalmol 1994; 112: 254–260.
Gurelik BG, Bilkgihan K, Sezer C, Akyol G, Hasanreisoglu B . Effect of mechanical vs dilute ethanol epithelial removal on keratocyte apoptosis and polymorpho-nuclear leukocyte migration. Eye 2002; 16: 136–139.
Borderie VN, Lopez M, Lombet A, Carvajal-Gonzalez S, Cywiner C, Laroche L . Cryopreservation and culture of human corneal keratocytes. Invest Ophthalmol Vis Sci 1998; 40: 1511–1519.
Idziorek T, Estaquier J, De Bels F, Ameisen J-C . YOPRO-1 permits cytofluorometric analysis of programmed cell death (apoptosis) without interfering with cell viability. J Immunol Meth 1995; 185: 249–258.
Kolozsvári L, Nógrádi A, Hopp B, Bor Z . UV absorbance of the human cornea in the 240- to 400-nm range. Invest Ophthalmol Vis Sci 2002; 43: 2165–2168.
Wollensak G, Spoerl E, Wilsch M, Seiler T . Keratocyte apoptosis after corneal collagen-crosslinking using riboflavin-UVA treatment. Cornea 2003, accepted for publication.
Tsubai T, Matsuo M . Ultraviolet light-induced changes in the glucose-6-phosphate dehydrogenase activity of porcine corneas. Cornea 2002; 21: 495–500.
Ringvold A, Davanger M . Changes in the rabbit corneal stroma caused by UV-radiation. Acta Ophthalmol 1985; 63: 601–606.
Pitts DG, Cullen AP, Hacker PD . Ocular effects of ultraviolet radiation from 295-365 nm. Invest Ophthalmol Vis Sci 1977; 16: 932–939.
Cho K-S, Lee EH, Choi J-S, Joo C-K . Reactive oxygen species-induced apoptosis and necrosis on bovine corneal endothelial cells. Invest Ophthalmol Vis Sci 1999; 40: 911–919.
Dollery C Therapeutic Drugs, Vol. 2. Churchill Livingstone: Edinburgh, 1991, pp 24–25.
Podskochy A, Fagerholm P . Cellular response and reactive hyaluronan production in UV-exposed rabbit corneas. Cornea 1998; 17: 640–645.
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Wollensak, G., Spoerl, E., Reber, F. et al. Keratocyte cytotoxicity of riboflavin/UVA-treatment in vitro. Eye 18, 718–722 (2004). https://doi.org/10.1038/sj.eye.6700751
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DOI: https://doi.org/10.1038/sj.eye.6700751
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