Abstract
Purpose: :
Collagen crosslinking (CXL) using riboflavin and ultraviolet-A (UVA CXL) is a promising treatment for keratoconus. Since CXL by other mechanisms results in increased collagen autofluorescence (CAF), we hypothesized that CAF might be used to monitor corneal CXL. The purpose of this study was to evaluate the effect of UVA CXL on corneal CAF intensity.
Methods: :
Rabbit eyes from an abattoir (PelFrez, Az) were used in this study. Eyes were separated into four groups (3 eyes each) including, control 1 (intacted epithelium), control 2 (epithelium removed), riboflavin control, and riboflavin with UVA treatment for 15 and 30 min. Riboflavin-5-phosphate solution (0.1%) was applied every 2 min for 30 min prior to and during UVA irradiation. CXL was achieved using 3mW/cm2, 370 nm light source. Corneal stiffness after CXL was measured by indentation after corneal removal using a 250 µm diameter probe with round tip fixed to force transducer. Corneas were fixed in 2% paraformaldehyde, washed and sectioned at 300 µm thickness using a vibratome. Tissue sections were then scanned using a Zeiss 510 Meta CLSM (Zeiss, Jena, Germany) and Chameleon femtosecond laser (Coherent Inc, Santa Clara, CA) to generate two photon excited CAF signals using 760 nm infrared light and the image was collected over the 400-450 nm spectrum. Fluorescence intensity was analyzed across cornea in 88µm width.
Results: :
The indenting force at 500 µm depth was 0.97mN (STD 0.15), 0.77mN (STD 0.15) and 1.17mN (STD 0.23) for control 1, control 2 and riboflavin alone treated corneas and no CAF was detected. Indenting force increased to 2mN (STD 0.26) and 2.5mN (STD 0.62) and CAF was detected to a depth of 308µm (STD 15) and 307µm (STD 18) in the 15 min and 30 min UVA irradiated groups. No autofluorescence was detected in the control groups. The overall intensity of autofluorescence was 7805 (STD 5421) arbitrary units (a.u) x µm in the 15 min and 11406 (STD 5693) a.u x µm in the 30 min UVA treatment groups. There was a significant relationship between CAF and change in stiffness (R2=0.81, p<0.001)
Conclusions: :
These findings indicate that CAF signal might be used to assess UVA CXL in cornea, especially, the depth and amount of CXL induction by CXL dependent on irradiation variables such as intensity and duration of irradiation.
Keywords: keratoconus • cornea: stroma and keratocytes • extracellular matrix