Papain Digestion Method for Analysis of Cross-linking in Corneal Flaps

Abstract

 

Purpose

 

To evaluate the use of papain digestion and spectrofluorometer analysis as a means to quantify the amount of cross-linking in porcine corneal flaps that have undergone various UVA-RF based corneal cross-linking protocols.

 

Methods

 

Fresh whole porcine eyes were obtained <24 hours postmortem in saline on ice from Sioux-preme (Sioux City, IA), de-epithelialized, soaked for 20 minutes with 0.1% riboflavin in distilled water, and exposed to 30mW/cm² of UVA light for 3 minutes (5.4J/cm²) or 4 minutes (7.2J/cm²). Using an Intralase femtosecond laser (Abbot Medical Optics, Santa Ana, CA) corneal flaps of 380 µm were excised after cross-linking. The thickness of the corneal flaps was checked using ultrasonic pachymetry. To prepare for digestion the corneal flaps were washed with distilled water 15X to remove remaining riboflavin, and dried in a vacuum until the weight change became less than 10%. Each flap was digested for 2.5 h at 65°C with 2.5 units/ml of papain (from Papaya latex, Sigma) in 1 ml of papain buffer [1x PBS (pH 7.4), 2 mM L-cysteine and 2 mM EDTA]. Papain digests were centrifuged for 20 seconds at 2200xG (Mini centrifuge 05-090-100, Fisher Scientific) and diluted .5 times with 1x PBS solution. Fluorescence of the solutions was measured with excitation of λex = 360nm in a QM-40 Spectrofluorometer (Photon Technology Int., London, Ontario, Canada).

 

Results

 

The spectrofluorometer recorded fluorescence of the corneal flap digested solutions from 375-650nm. After subtracting out the increase in fluorescence from 450-650nm due to remaining riboflavin, the fluorescence at 450nm is indicative of the amount of cross-linking in the corneal flap. The fluorescence readings increased as total energy increased, as expected. Statistically significant results (P<.05) are observed between the groups.

 

Conclusions

 

Papain digestion and spectrofluorometer analysis of cross-linked corneal flaps is an effective method to evaluate the amount of cross-linking in a flap. Multiple flaps can be excised from one cornea to analyze the amount of cross-linking at different depths. The method can be used to help determine the optimal protocol for corneal cross-linking.

 

 

The relative fluorescence of porcine corneas soaked for 20 minutes with 0.1% riboflavin in distilled water, exposed to 30mW/cm² UVA light for 3 minutes (5.4J/cm²) or 4 minutes (7.2J/cm²)

 

Keywords: 480 cornea: basic science • 574 keratoconus • 671 radiation therapy