Effects of riboflavin/UVA corneal cross-linking on keratocytes and collagen fibres in human cornea.
OBJECTIVE
To evaluate the effects of corneal cross-linking on keratocytes and collagen fibres in human corneas.


METHODS
Fifteen corneal buttons were examined. Ten were from patients with keratoconus submitted to penetrating keratoplasty and five of them were treated with cross-linking 6 months before penetrating keratoplasty. Five normal corneal buttons from healthy donors were used as controls. All samples were prepared for TUNEL assay and Western blot analysis for the detection of keratocyte apoptosis and immunohistochemical analysis for the morphological evaluation of keratocytes and collagen fibre diameter.


RESULTS
Normal corneas exhibited no TUNEL-positive keratocytes and keratoconic and cross-linked corneas showed moderate apoptotic cells mainly in the anterior part of the stroma. This apoptotic trend was confirmed by the cleavage of poly (ADP-ribose) polymerase assessed using Western blot. The Ki-67 staining showed a significant increase in the keratocyte proliferation in cross-linked corneas compared with normal and keratoconus. In cross-linked corneas CD34-positive keratocytes were regularly distributed throughout the whole corneal stroma as in the control, and keratoconus was associated with patchy loss of immunoreactivity. The immunohistochemical analysis of collagen type I showed a significant increase in fibre diameter of cross-linked corneas compared with control and keratoconus.


CONCLUSIONS
Corneal cross-linking leads to keratocyte damage; after 6 months a repopulation by proliferating cells, a distribution of CD34-positive keratocytes as in control and an increase in collagen fibre diameter were observed. These modifications are the morphological correlate of the process leading to an increase in biomechanical stability.
Mencucci
Rita
R
Department of Oto-Neuro-Ophthalmological Surgical Sciences, Eye Clinic, Florence, Italy. rita.mencucci@unifi.it <rita.mencucci@unifi.it>
Marini
Mirca
M
Paladini
Iacopo
I
Sarchielli
Erica
E
Sgambati
Eleonora
E
Menchini
Ugo
U
Vannelli
Gabriella B
GB
D000328:Adult; D018952:Antigens, CD34 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D049109:Cell Proliferation; D003094:Collagen / Q000378:metabolism*; D003319:Corneal Stroma / Q000378:metabolism* / Q000473:pathology*; D005347:Fibroblasts / Q000473:pathology; D006801:Humans; D007124:Immunoenzyme Techniques; D020287:In Situ Nick-End Labeling; D007640:Keratoconus / Q000188:drug therapy* / Q000378:metabolism / Q000601:surgery; D015948:Keratoplasty, Penetrating; D019394:Ki-67 Antigen / Q000378:metabolism; D010778:Photochemotherapy; D017319:Photosensitizing Agents / Q000627:therapeutic use*; D012256:Riboflavin / Q000627:therapeutic use*; D014466:Ultraviolet Rays
D018952:Antigens, CD34; D019394:Ki-67 Antigen; D017319:Photosensitizing Agents; D003094:Collagen; D012256:Riboflavin