Differential impact of LIRIC and femto-LASIK on keratocyte viability in cat cornea

Abstract

Purpose : Laser refractive surgeries cause unwanted post-operative side effects. We recently developed an alternative refractive procedure, LIRIC, which changes the refractive index of cornea using a femtosecond (fs) laser. We assessed the extent to which LIRIC damages corneal cells, contrasting it with fs laser in-situ keratomileusis (LASIK).

Methods : LIRIC was performed on five, excised feline eyes using a 400nm Ti:Sapphire laser. A water-immersion objective (NA=1.0) was raster-scanned across the applanated cornea, creating a 2.5mm-wide refractive pattern. Three layers were written either 10μm or 30μm apart, inducing -1 to -1.5D cylinder. Two eyes underwent LASIK: a 9mm flap was cut in applanated corneas using a fs laser. With the flap retracted, a Technolas 217 excimer laser was used to produce a -1.5D cylinder across a 4mm ablation zone. All eyes were immersed in Optisol-GS at 4°C for 6 hours and processed for histology. Corneal sections were stained using the TUNEL assay or immunostained for γ-H2AX before counter-staining with DAPI. 109 photomicrographs of 26 sections were analyzed with a custom MATLAB cell counting program to assess the distribution of TUNEL and γ-H2AX-positive cells.

Results : In LIRIC-treated eyes, no TUNEL or γ-H2AX-positive epithelial or endothelial cells were seen. TUNEL-positive keratocytes were only within LIRIC layers, and γ-H2AX-positive cells were seen directly adjacent to these layers. Layer separation was important. Tight patterns caused 86±10% of cells in the LIRIC region to become TUNEL-positive, relative to only 53±8% if the spacing was 30µm. In contrast, in LASIK, all cells along the flap cut were TUNEL-positive, in addition to 72±14% of cells in the sub-ablation zone. No γ-H2AX reaction was seen with LASIK, but ~20% of stromal cells within ±50μm of all LIRIC patterns stained for γ-H2AX.

Conclusions : LIRIC causes stromal cell death in highly localized regions. LASIK causes cell death along the flap, within and deep beneath the ablation zone. The differential expression of γ-H2AX in LASIK and LIRIC suggests that LIRIC may induce a DNA repair response adjacent to the laser focal zone that is absent following LASIK. Finally, since structural disruption of the cornea, together with cell death usually precede a stronger wound-healing response, LIRIC, which involves no ablation and exhibits only minimal cell death, may cause fewer side effects than traditional laser vison correction.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.