Correlation of Multiphoton Microscopy, Tissue Morphological Changes and Enzymatic Resistance in Riboflavin-UVA Crosslinked Human Corneas

Abstract

 

Purpose

 

Collagen crosslinking (CXL) is a well established procedure to treat ocular disorders with cornea ectasia, and yet today there is no standardized technique to evaluate corneal CXL in clinical settings. To explore the utility of multiphoton imaging as a non-invasive tool to assess CXL efficacy, we investigated the correlation between second harmonic generation (SHG) imaging and histological/biochemical changes of corneas after riboflavin (RF)-induced UVA CXL.

 

Methods

 

De-epithelialized human corneoscleral tissues were imaged by Leica SP5 multiphoton microscope to study (1) RF tissue diffusion profile and (2) SHG signals before and after CXL. Z-stack images were acquired throughout 500 μm of corneas with a 15 μm interslice interval. The emission of the MP laser was set to a wavelength of 800 nm. RF 0.1% was installed for 5, 10 and 20 min with 20 % Dextran vehicle as controls, followed by UVA irradiance at 3 J/cm². Masson’s trichrome staining (MTS) and collagenase digestion assay were employed to assess the collagen ultrastructural changes and enzymatic resistibility.

 

Results

 

The corneal stromal absorption of RF was significantly higher in 20 min compared to 5 and 10 min drug instillation. Intensity of SHG signals was remarkably reduced post RF-UVA CXL at all RF instillation time points compared to dextran controls. However, correlation between RF and SHG profile was only observed in 10 and 20 min RF instillation (R² = 0.13 and 0.28, respectively, all p <.05), but not in 5 min group. In contrast to a rugged, heterogenous collagen texture of unirradiated controls, MTS revealed homogenous collagen fibril lamellae in crosslinked corneas in an RF dose-dependent manner. The intercalated collagen fibril texture in the anterior stroma was observed in all RF groups, while such a lattice structure in the posterior stromal segment was only noticed in 20 min RF group. Lastly, collagenase assay showed that tissue strength was significantly increased by higher doses of RF in UVA CXL. Histological and biochemical evaluations further suggest that the RF doses are related to the depth of CXL in the corneal stroma.

 

Conclusions

 

An RF-defined threshold determines the applicability of SHG imaging as a non-invasive technique in assessing the efficacy of RF-UVA CXL for the treatment of cornea ectasia.  

 

Axial riboflavin diffusion profile imaged by multiphoton microscopy