Purpose To examine the effect of riboflavin/UVA corneal crosslinking on stromal ultrastructure and hydrodynamic behaviour. Methods One hundred and seventeen enucleated ungulate eyes (112 pig and 5 sheep) and 3 pairs of rabbit eyes, with corneal epithelium removed, were divided into four treatment groups: Group 1 (28 pig, 2 sheep and 3 rabbits) were untreated; Group 2 (24 pig) were exposed to UVA light (3.04 mW/cm2) for 30 minutes and Group 3 (29 pig) and Group 4 (31 pig, 3 sheep and 3 rabbits) had riboflavin eye drops applied to the corneal surface every 5 minutes for 35 minutes. Five minutes after the initial riboflavin instillation, the corneas in Group 4 experienced a 30 minute exposure to UVA light (3.04 mW/cm2). X-ray scattering was used to obtain measurements of collagen interfibrillar spacing, spatial order, fibril diameter, D-periodicity and intermolecular spacing throughout the whole tissue thickness and as a function of tissue depth in the treated and untreated corneas. The effect of each treatment on the hydrodynamic behaviour of the cornea (its ability to swell in saline solution) and its resistance to enzymatic digestion were assessed using in vitro laboratory techniques. Results Corneal thickness decreased significantly following riboflavin application (p<0.01) and also to a lesser extent after UVA exposure (p<0.05). With the exception of the spatial order factor, which was higher in Group 4 than Group 1 (p<0.01), all other measured collagen parameters were unaltered by cross-linking, even within the most anterior 300 microns of the cornea. The cross-linking treatment had no effect on the hydrodynamic behaviour of the cornea but did cause a significant increase in its resistance to enzymatic digestion. Conclusions It seems likely that cross-links formed during riboflavin/UVA therapy occur predominantly at the collagen fibril surface and in the protein network surrounding the collagen.
References
[1]
Hart RW, Farrell RA (1969) Light Scattering in the Cornea. Journal of the Optical Society of America 59: 766–774.
[2]
Maurice DM (1957) The structure and transparency of the cornea. Journal of Physiology 136: 263–286.
[3]
Scott JE (1992) Morphometry of cupromeronic blue-stained proteoglycan molecules in animal corneas, versus that of purified proteoglycans stained in vitro, implies that the tertiary structures contribute to corneal ultrastructure. Journal of Anatomy 180: 155–164.
[4]
Lewis P, Pinali C, Young R, Meek K (2010) Structural interactions between collagen and proteoglycans elucidated by three-dimensional electron tomography in the bovine cornea. Structure 18: 239–245.
[5]
Rahman I, Carley F, Hillarby C, Brahma A, Tullo A (2009) Penetrating keratoplasty: indications, outcomes, and complications. Eye 23: 1288–1294.
[6]
Wollensak G, Spoerl E, Seiler T (2003) Riboflavin/Ultraviolet-A-induced collagen crosslinking for the treatment of keratococnus. American Journal of Ophthalmology 135: 620–627.
[7]
Spoerl E, Wollensak G, Dittert D, Seiler T (2004) Thermomechanical behaviour of collagen crosslinked porcine cornea. Ophthalmologica 218: 136–140.
[8]
Wollensak G, Spoerl E, Seiler T (2003) Stress-strain measurements of human and porcine corneas after riboflavin-ultraviolet-A-induced cross-linking. Journal of Cataract and Refractive Surgery 29: 1780–1785.
[9]
Spoerl E, Wollensak G, Seiler T (2004) Increased resistance of crosslinked cornea against enzymatic digestion. Current Eye Research 29: 35–40.
[10]
Iseli H, Thiel M, Hafezi F, Kampmeier J, Seiler T (2008) Ultraviolet A/riboflavin corneal cross-linking for infectious keratitis associated with corneal melts. Cornea 27: 590–594.
[11]
Makdoumi K, Mortensen J, Sorkhabi O, Malmvall B, Crafoord S (2012) UVA-riboflavin photochemical therapy of bacterial keratitis: a pilot study. Graefe's Archive for Clinical and Experimental Ophthalmology 250: 95–102.
[12]
Wollensak G, Aurich H, Wirbelauer C (2007) Potential use of riboflavin/UVA cross-linking in bullous keratopathy. Ophthalmic Research 41: 114–117.
[13]
Kreuger R, Ramos-Esteban J, Kanellopoulos A (2008) Staged intrastromal delivery of riboflavin with UVA cross-linking in advanced bullous keratopathy: laboratory investigation and first clinical case. Journal of Refractive Surgery 24: S730–736.
[14]
Ghanem R, Santhiago M, Berti T, Thomaz S, Netto M (2010) Collagen crosslinking with riboflavin and ultraviolet-A in eyes with pseudophakic bullous keratopathy. Journal of Cataract and Refractive Surgery 36: 273–276.
[15]
Gharaee H, Ansari-Astaneh M, Armanfar F (2011) The effects of riboflavin/Ultraviolet-A corneal cross-linking on the signs and symptoms of Bullous keratopathy. Middle East African Journal of Ophthalmology 18: 58–60.
[16]
Bottos K, Hofling-Lima A, Barbosa M, Barbosa J, Dreyfuss J, et al. (2010) Effect of collagen cross-linking in stromal fibril organization in edematous human corneas. Cornea 29: 789–793.
[17]
Brummer G, Littlechild S, McCall S, Zhang Y, Conrad G (2011) The role of nonenzymatic glycation and carbonyls in collagen cross-linking for the treatment of keratoconus. Investigative Ophthalmology and Visual Science 52: 6363–6369.
[18]
Wollensak G, Sp？rl E, Mazzotta C, Kalinski T, Sel S (2011) Interlamellar cohesion after corneal crosslinking using riboflavin and ultraviolet A light. British Journal of Ophthalmology 95: 876–880.
[19]
Zhang Y, Conrad A, Conrad G (2011) Effects of Ultraviolet-A and riboflavin on the interaction of collagen and proteoglycans during corneal cross-linking. The Journal of Biological Chemistry 286: 13011–13022.
[20]
Wollensak G, Aurich H, Pham D, Wirbelauer C (2007) Hydration behaviour of porcine cornea crosslinked with riboflavin and ultraviolet A. Journal of Cataract and Refractive Surgery. 33: 516–521.
[21]
Meek KM, Quantock AJ (2001) The use of X-ray scattering techniques to determine corneal ultrastructure. Progress in Retinal and Eye Research 20: 95–137.
[22]
Van Horn D, Doughman D, Harris J, Miller G, Lindstrom R, et al. (1975 ) Ultrastructure of human organ-cultured cornea. II. Stroma and epithelium. Archives of Ophthalmology 93: 275–277.
[23]
Muller LJ, Pels E, Vrensen GFJM (2001) The specific architecture of the anterior stroma accounts for maintenance of corneal curvature. British Journal of Ophthalmology 85: 437–443.
[24]
Lee D, Wilson G (1981) Non-uniform swelling properties of the corneal stroma. Current Eye Research 1: 457–461.
[25]
Fullwood NJ, Meek KM (1994) An ultrastructural, time-resolved study of freezing in the corneal stroma. Journal of Molecular Biology 236: 749–758.
[26]
Meek KM, Fullwood NJ, Cooke PH, Elliott GF, Maurice DM, et al. (1991) Synchrotron x-ray-diffraction studies of the cornea, with implications for stromal hydration. Biophysical Journal 60: 467–474.
Wollensak G, Spoerl E, Wilsch M, Seiler T (2004) Keratocyte apoptosis after corneal collagen cross-linking using Riboflavin/UVA treatment. Cornea 23: 43–49.
[29]
Kikkawa Y, Hirayma K (1970) Uneven swelling properties of the corneal stroma. Investigative Ophthalmology and Visual Science 9: 735–741.
[30]
Boote C, Dennis S, Newton RH, Puri H, Meek KM (2003) Collagen fibrils appear more closely packed in the prepupilliary cornea- optical and biomechanical implications. Investigative Ophthalmology and Visual Science 44: 2941–2948.
[31]
Rawe IM, Meek KM, Leonard DW, Takahashi T, Cintron C (1994) Structure of corneal scar tissue - an x-ray-diffraction study. Biophysical Journal 67: 1743–1748.
[32]
Maroudas A, Wachtel E, Grushko G, Katz E, Weinberg P (1991) The effect of osmotic and mechanical pressures on water partitioning in articular cartilage. Biochimica et Biophysica Acta 1073: 285–294.
[33]
Worthington CR, Inouye H (1985) X-ray diffraction study of the cornea. International Journal of Biological Macromolecules 7: 2–8.
[34]
Faber C, Scherfig E, Prause J, S？rensen K (2008) Corneal Thickness in Pigs Measured by Ultrasound Pachymetry In Vivo. Scandinavian Journal of Laboratory Animal Science 35: 39–43.
[35]
Raiskup-Wolf F, Hoyer A, Spoerl E, Pillunat L (2008) Collagen crosslinking with riboflavin and ultraviolet-A light in keratoconus: long-term results. Journal of Cataract and Refractive Surgery 34: 796–801.
[36]
Wollensak G, Spoerl E, Seiler T (2003) Riboflavin/Ultraviolet-A-induced collagen crosslinking for the treatment of keratoconus. American Journal of Ophthalmology 135: 620–627.
[37]
Malik NS, Moss SJ, Ahmed N, Furth AJ, Wall RS, et al. (1992) Aging of the human corneal stroma - structural and biochemical changes. Biochimica Et Biophysica Acta 1138: 222–228.
[38]
Malik N, Meek K (1994) The inhibition of sugar-induced structural alterations in collagen by aspirin and other compounds. Biochemical and Biophysical Research Communications 199: 683–686.
[39]
Malik N, Meek K (1996) Vitamins and analgesics in the prevention of collagen ageing. Age and Ageing 25: 279–284.
[40]
Fullwood NJ, Meek KM (1993) A synchrotron X-ray study of the changes occurring in the corneal stroma during processing for electron microscopy. Journal of Microscopy 169: 53–60.
[41]
Meek KM, Hayes S, Kamma-Lorger CS, Boote C, Quantock AJ, et al. (2008) The Effect of Collagen Crosslinking Using Riboflavin/UVA on the Molecular and Fibrillar Architecture of the Corneal Stroma. ARVO Fort Lauderdale: Investigative Ophthalmology and Visual Science. 3913.
[42]
Hayes S, Boote C, Kamma-Lorger C, Rajan M, Harris J, et al. (2011) Riboflavin/UVA collagen cross-linking-induced changes in normal and keratoconus corneal stroma. PLoS One 6: e22405.
[43]
Blanco T, Merayo-Lloves J, Fabiani L, Del Olmo Aguado S, Mar-Sardana S, et al. (2008) Corneal Collagen Cross-Linking With Riboflavin and UVA Improves Transparency in Hen Corneas. ARVO Fort Lauderdale: Investigative Ophthalmology and Visual Science. 3911.
[44]
Slack J, Kangas T, Edelhauser H, Geroski D, McDermott M (1992) Comparison of corneal preservation media for corneal hydration and stromal proteoglycan loss. Cornea 11: 204–210.
[45]
Wollensak G, Aurich H, Wirbelauer C (2009) Potential use of riboflavin/UVA cross-linking in bullous keratopathy. Ophthalmic Research 41: 114–117.
[46]
Spoerl E, Terai N, Raiskup F, Pillunat L (2012) Amylase reduces the biomechanical stiffness of the cornea ARVO. Fort Lauderdale: Investigative Ophthalmology and Visual Science. In Press.
[47]
Wollensak G, Wilsch M, Spoerl E, Seiler T (2004) Collagen fiber diameter in the rabbit cornea after collagen crosslinking by riboflavin/UVA. Cornea 23: 503–507.
[48]
Fratzl P, Daxer A (1993) Structural transformation of collagen fibrils in the corneal stroma during drying: an X-ray scattering study. Biophysical Journal 64: 1210–1214.
[49]
Craig AS, Robertson JG, Parry DA (1986) Preservation of corneal fibril structure using low-temperature procedures for electron microscopy. Journal of Ultrastructure Molecular Structure Research 96: 172–175.
[50]
Craig AS, Parry DAD (1981) Collagen fibrils of the vertebrate corneal stroma. Journal of Ultrastructure Molecular Structure Research 74: 232–239.
[51]
Twersky V (1975) Transparency of pair-correlated, random distributions of small scatterers, with applications to the cornea. Journal of the Optical Society of America 65: 524–530.
[52]
Scott J (1988) Proteoglycan-fibrillar collagen interactions. Biochemical Journal 252: 313–323.
[53]
Verzijl N, DeGroot J, Thorpe S, Bank R, Shaw J, et al. (2000) Effect of collagen turnover on the accumulation of advanced glycation end products. The Journal of Biological Chemistry 275: 39027–39031.
