Abstract
Purpose: :
To develop a method to photo-crosslink stromal collagen, thus stiffening the cornea, which is more rapid than riboflavin/UVA treatment, is non-toxic to stromal keratocytes and can be used for treatment of keratoconus.
Methods: :
Rabbit eyes, previously frozen and freshly obtained, were used. Rose Bengal dye (RB, 0.1% in PBS) was applied to the de-epithelialized cornea surface for 2 min and the cornea was then exposed to green (532 nm) light from a KTP laser for 1 to 5 min (24.4-122 J/cm2). For comparison, additional corneas were treated with 0.1% riboflavin (Rf, 20% dextran solution) for 30 min, then exposed to UVA (5.4 J/cm2; 30 min) while applying drops of Rf solution, similar to the clinical treatment. Results of mechanical testing using a tensiometer were used to calculate Young’s modulus, a measure of stromal stiffness. Depth of penetration of RB and Rf into corneal stroma was determined by measuring the dye fluorescence intensity, using confocal microscopy, every 10 µm from the anterior surface. Keratocyte nuclei were counted on H&E stained sections 24 hr after treatments to determine cell viability.
Results: :
The same increase in stromal stiffness was produced by RB and Rf photo-crosslinking, when RB-treated corneas were treated with only 5 min of green light. Values of Young’s modulus for RB-treated (34.0 ± 7.4 MPa) and Rf-treated (31.8 ± 11.2 MPa) corneas were greater than that for untreated corneas (15.4 ± 5.1 MPa) (n=6-8/group; p<0.05). RB penetrated less deeply than Rf into the stroma, as indicated by the decrease in RB fluorescence with distance from the anterior surface, which reached 10% of the initial intensity by 93 ± 6 µm. Thus RB/green light-initiated crosslinks are produced closer to the anterior surface than with Rf/UVA, since Rf penetrated throughout the cornea. RB/green light did not affect keratocyte viability whereas Rf/UVA decreased cell viability to less than 5%.
Conclusions: :
RB/green light effectively crosslinked stromal collagen in less than 7 min total treatment time and was not toxic to keratocytes suggesting that this procedure has potential as a second generation photo-crosslinking treatment for keratoconus.
Keywords: keratoconus • cornea: stroma and keratocytes