Contrasting cellular damage after Blue-IRIS and Femto-LASIK in cat cornea

Abstract

Blue-intra-tissue refractive index shaping (Blue-IRIS) is a new approach to laser refractive correction of optical aberrations in the eye, which alters the refractive index of the cornea rather than changing its shape. Before it can be implemented in humans, it is critical to establish whether and to what extent, Blue-IRIS damages the cornea. Here, we contrasted the impact of -1.5 D cylinder refractive corrections inscribed using either Blue-IRIS or femtosecond laser in-situ keratomileusis (femto-LASIK) on corneal cell viability. Blue-IRIS was used to write a -1.5 D cylinder gradient index (GRIN) lens over a 2.5 mm by 2.5 mm area into the mid-stromal region of the cornea in six freshly-enucleated feline eyes. The same correction (-1.5 D cylinder) was inscribed into another four cat eyes using femto-LASIK. Six hours later, all corneas were processed for histology and stained for terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) and p-γ-H2AX to label damaged cells. In Blue-IRIS-treated corneas, no tissue was removed and TUNEL-stained cells were confined to the laser focal zone in the stroma. In femto-LASIK, photoablation removed 14 μm of anterior stroma, but in addition, TUNEL-positive cells clustered across the femto-flap, the epithelium at the flap edges and the stroma below the ablation zone. Keratocytes positive for p-γ-H2AX were seen adjacent to all Blue-IRIS focal zones, but were completely absent from femto-LASIK-treated corneas. Unlike femto-LASIK, Blue-IRIS attains refractive correction in the cornea without tissue removal and only causes minimal, localized keratocyte death within the laser focal zones. In addition, Blue-IRIS induced DNA modifications associated with phosphorylation of γ-H2AX in keratocytes adjacent to the laser focal zones. We posit that this p-γ-H2AX response is related to alterations in chromatin structure caused by localized changes in osmolarity, a possible mechanism for the induced refractive index changes.

Keywords: GRIN lens; Photoablation; Refractive correction; Refractive index modification; Stroma; TUNEL; γ-H2AX.

Copyright © 2017 Elsevier Ltd. All rights reserved.

Figures

Figure 1. Blue-IRIS apparatus

An 800 nm, 100 fs Coherent Vitesse laser beam was frequency doubled to produce 400 nm laser pulses, which were tightly focused through a 1.0 NA, water immersion objective, into the stromal layer of applanated feline corneas. A complementary metal-oxide semiconductor (CMOS) camera was used to find the interface between the applanator and cornea, thus locating the surface of the enucleated globe. The objective was then lowered such that the focal spot was located in the mid stromal region. A commercial, oscillating, vibration exciter was used in combination with a linear translation stage to raster-scan across a 2.5 mm × 2.5 mm area in the center of each cornea. ND filter: neutral density filter.

Figure 2. Blue-IRIS in excised cat cornea

A. The focal region of the objective was scanned through the cornea using 3-axis delivery system to inscribe a cylindrical GRIN lens. B. Schematic of a corneal segment containing an inscribed, mid-stromal, 3-layer GRIN pattern. A top-view, schematic representation of the refractive index change distribution induced by the femtosecond laser across a single Blue-IRIS GRIN layer is shown below the corneal segment. C. Photomicrograph of the entire stroma (epithelium to endothelium), containing a 3-layer Blue-IRIS pattern (green auto-fluorescence). The tissue was counterstained with DAPI (blue fluorescence).

Figure 3. Method for quantitative analysis of TUNEL/p-γ-H2AX staining in sectioned corneas

Magnification is the same for all photomicrographs. A and B show en-face, schematics of corneas with the approximate size and positioning of Blue-IRIS and femto-LASIK procedures respectively. The straight line through each schematic cornea illustrates approximate source of the putative cross-section, with typical staining patterns (in this case, TUNEL/DAPI) imaged in the photomicrographs below each schematic. A. In Blue-IRIS-treated cornea, the region of interest (ROI) was set to encompass the lateral edge of the GRIN lens (green auto-fluorescence). B. The ROI in the two femto-LASIK-treated corneal sections encompassed the edge of the femtosecond flap (green auto-fluorescence) and the femto-flap + sub-ablation zone, which was recognized because it possessed an additional region of TUNEL-positive cells extending into the stroma, below the flap, in the approximate center of the cornea.

Figure 4. TUNEL assay in Blue-IRIS-and femto-LASIK-treated feline corneas

In all photomicrographs (A, C, E, G), green auto-fluorescence indicates areas of Blue-IRIS or flap cut for femto-LASIK-treated corneas. Red/pink staining indicates TUNEL-positive cells. Blue fluorescence denotes DAPI-positive cell nuclei. Corneal sections are oriented with the epithelium uppermost. Magnification is identical in all photomicrographs. All graphs (B, D, F, H) plot the percentage (%) of DAPI-positive cells also positive for TUNEL, as a function of distance from the epithelial-stromal interface. The overlaid green area shows size and position of each ROI relative to the epithelium; the overlaid blue box in H indicates the size and position of the sub-ablation zone.

Figure 5. p-γ-H2AX antibody staining for Blue-IRIS-and femto-LASIK-treated feline corneas

Photomicrographs in A, C, E, show stained corneal sections with green auto-fluorescence denoting areas of Blue-IRIS or flap cut for femto-LASIK-treated corneas, red/pink indicating p-γ-H2AX-positive cells and blue denoting DAPI-positive cell nuclei. Corneal sections are oriented with the epithelium uppermost. Magnification is identical in all photomicrographs. Adjacent graphs (B, D, F) plot the percentage (%) of DAPI-labeled cells positive for p-γ-H2AX, as a function of distance from the epithelial-stromal interface. The overlaid green boxes inside each graph shows the approximate size and position of each ROI relative to the epithelium.