• Endothelial cell damage (due to UVA irradiation). Apoptosis has been detected histologically using either TUNEL (in rabbits) [1] or trypan blue/Yopro staining (in pigs) [2]. To protect the endothelial cells requires precise knowledge of how much radiation penetrates the stroma, and that in turn requires careful measurement of the absorption coefficient and the effects of riboflavin.  This parameter has been measured in human donor corneas with and without riboflavin. The riboflavin led to a 50% increase in absorbance after 30 minutes of riboflavin treatment, [3] with an absorbance coefficient of 56.36±4.80 cm-1 although other workers have found a significantly lower value [4] which may be a cause for concern. This level of absorbance has been calculated to yield a UVA irradiance at a depth of 400 µm of 0.18 mWcm-2,which is less than half the toxic level [5]. For this reason, the maximum thickness of the cornea that can be treated by the standard method was set at 400 µm.
  • Damage to the corneal limbus (location of epithelial stem cells). As an added protection it is advised that polymethacrylate rings or other forms of masking should be used to ensure absolute limbal protection, particularly in low-compliance patients who cannot maintain fixation adequately during the 30 minute CXL procedure [6].
  • Impairment of corneal sensitivity due to nerve damage (a consequence of the epithelial removal process). Immediately after CXL the subepithelial plexus and anterior/mid-stromal nerve fibres disappear. In humans and rabbits, regeneration of nerve fibres is complete after about six months [6,7] and plexus structure after one year [6] by which time sensitivity returns to normal [8].

[1] Wollensak G, Spoerl E, Seiler T. Endothelial cell damage after riboflavin-ultra-violet-A-treatment in the rabbit. Journal of Cataract and Refractive Surgery. 2003;29:1786-90.

[2] Spoerl E, Mrochen M, Sliney D, Trokel S, Seiler T. Safety of UVA-Riboflavin Cross-Linking of the Cornea. Cornea. 2007;26(4):385-9.

[3] Wollensak G, Aurich H, Wirbelauer C, Sel S. Significance of the riboflavin film in corneal collagen crosslinking. Journal of Cataract and Refractive Surgery 2010;36(1):114-20.

[4] Koppen C, Gobin L, Tassignon M. The absorption characteristics of the human cornea in ultraviolet-A crosslinking. Eye and Contact Lens. 2010;36:77-80.

[5] Spoerl E, Mrochen M, Sliney D, Trokel S, Seiler T. Safety of UVA-Riboflavin Cross-Linking of the Cornea. Cornea. 2007;26(4):385-9.

[6] Mazzotta C, Traversi C, Baiocchi S, Caporossi O, Bovone C, Sparano C, et al. Corneal healing after riboflavin ultraviolet-A collagen cross-linking determined by confocal laser scanning microscopy in vivo: Early and late modifications. American Journal of Ophthalmology. 2008;146:527-33.

[7] Xia Y, Chai X, Zhou C, Ren Q. Corneal nerve morphology and sensitivity changes after ultraviolet A/riboflavin treatment. Experimental Eye Research. 2011;93:541-7.

[8] Mazzotta C, Traversi C, Baiocchi S, Sergio P, Caporossi T, Caporossi A. Conservative treatment of keratoconus by riboflavin-uva-induced cross-linking of corneal collagen: quantitative investigation. European Journal of Ophthalmology. 2006;16:530-5.