Temporal evolution of the biological response to laser-induced refractive index change (LIRIC) in rabbit corneas

Laser-induced refractive index change (LIRIC) is a new, non-incisional, non-ablative, femtosecond photo-modification technique being developed for vision correction in humans. Prior, exvivo studies showed intra-tissue refractive index change to induce minimal cell death, restricted to the laser focal zone in the corneal stroma, and with no observable damage to the epithelium or endothelium. Here, we used live rabbits to ascertain longer-term consequences of LIRIC in vivo. Specifically, we assessed cell death, fibrosis, corneal nerve distribution, endothelial cell density, and corneal structure for up to 3 months after LIRIC. A +2.5 D gradient-index LIRIC Fresnel lens was inscribed inside 20 applanated corneas of Dutch Belted rabbits, over a circular region of the mid-stroma measuring 4.5 mm in diameter. Twelve additional rabbit eyes were used as applanation-only controls to differentiate the effects of laser treatment and suction applanation on biological and structural parameters. In vivo optical measurements were performed pre-operatively, then immediately, 2, 4, and 12 weeks after the procedure, to measure endothelial cell density and changes in corneal structure. Groups of four rabbits were sacrificed at 4 hours, 2, 4, and 12 weeks after LIRIC for histological determinations; the TUNEL assay was used to evaluate cell death, H&E staining was used to assess inflammatory infiltration, and immunostaining for α-smooth muscle actin (α-SMA) and βIII tubulin (Tuj-1) was performed to assess myofibroblast differentiation and corneal nerve distribution, respectively. Consistent with prior ex vivo data, only minimal cell death was observed in the laser focal zone, with TUNEL-positive cells restricted to the stromal region of refractive index change 4 h after LIRIC. No TUNEL-positive cells were evident anywhere in the cornea 2, 4, or 12 weeks after LIRIC. Applanation-only corneas were completely TUNEL-negative. Neither LIRIC-treated nor applanation-only eyes exhibited α-SMA-positive staining or altered corneal nerve distributions at any of the time points examined. In vivo confocal imaging revealed normal endothelial cell densities in all eyes (whether LIRIC-treated or applanation-only) at all time points. Optical coherence tomography showed suction applanation to cause a temporary decrease in central corneal thickness, which returned to normal within 4 h. Corneas into which LIRIC Fresnel lenses were written while applanated did not undergo major structural or shape changes beyond the temporary thinning already described for suction applanation. The present findings suggest that LIRIC patterns, which generated a clinically-relevant refractive correction in the mid-stromal region of live rabbit corneas, induced little-to-no disruption to corneal structure and biology for 3 months after the procedure. This affirms the relative safety of LIRIC and predicts that compared to traditional laser vision correction surgeries, common post-operative complications such as dry eye, haze, or patient discomfort may be entirely avoided.