Axial mechanical and structural characterization of keratoconus corneas

Highlights

• The anterior is stiffer than the posterior in the keratoconus cornea cone.

• The keratoconus cornea cone is significantly less stiff than the healthy cornea.

• Collagen complexity in the anterior keratoconus cone is greater than the posterior.

• Collagen complexity in the keratoconus cone is less than in the healthy cornea.

• Collagen complexity outside of the keratoconus cone is greater than within.

Abstract

Purpose

Previous studies indicate that there is an axial gradient of collagen lamellar branching and anastomosing leading to regional differences in corneal tissue stiffness that may control corneal shape. To further test this hypothesis we have measured the axial material stiffness and quantified the collagen lamellar complexity in ectatic and mechanically weakened keratoconus corneas (KC).

Methods

Acoustic radiation force elastic microscopy (ARFEM) was used to probe the axial mechanical properties of the cone region of three donor KC buttons. 3 Dimensional second harmonic generation microscopy (3D-SHG) was used to qualitatively evaluate lamellar organization in 3 kC buttons and quantitatively measure lamellar branching point density (BPD) in a separate KC button that had been treated with epikeratophakia (Epi-KP).

Results

The mean elastic modulus for the KC corneas was 1.67 ± 0.44 kPa anteriorly and 0.970 ± 0.30 kPa posteriorly, substantially below that previously measured for normal human cornea. 3D-SHG of KC buttons showed a simplified collagen lamellar structure lacking noticeable angled lamellae in the region of the cone. BPD in the anterior, posterior, central and paracentral regions of the KC cornea were significantly lower than in the overlying Epi-KP lenticule. Additionally, BPD in the cone region was significantly lower than the adjacent paracentral region in the KC button.

Conclusions

The KC cornea exhibits an axial gradient of mechanical stiffness and a BPD that appears substantially lower in the cone region compared to normal cornea. The findings reinforce the hypothesis that collagen architecture may control corneal mechanical stiffness and hence corneal shape.

Introduction

Keratoconus (KC) affects roughly 1 in 2000 people and is characterized by progressive paracentral corneal thinning and steepening (Rabinowitz, 1998). As a result, the cornea takes on a conical shape leading to myopia, astigmatism, and markedly reduced visual acuity. The onset of keratoconus typically occurs in childhood to early adulthood and progresses before stabilizing by the fourth decade (Gordon-Shaag et al., 2015; Bykhovskaya et al., 2016). The pathogenesis of KC is commonly accepted to involve both environmental and genetic factors, with environmental factors possibly acting as triggers for the condition in genetically predisposed individuals. Among environmental factors are eye rubbing, atopy, and UV exposure, while genome wide association studies (GWAS) and genome wide linkage studies (GWLS) have identified numerous genes related to KC (Gordon-Shaag et al., 2015; Bykhovskaya et al., 2016). Regardless of the specific pathogenesis, abnormal collagen structure in the KC cornea is thought to weaken overall mechanical properties thus resulting in the formation of the hallmark cone in response to intraocular pressure (IOP).

The healthy human cornea possesses a unique collagen structure, which has been proposed to play a key role in the maintenance of corneal shape, biomechanics, and in turn visual acuity (Winkler et al., 2013; Kling and Hafezi, 2017). 3-dimensional second harmonic generation imaging (3D-SHG) has shown that lamellae in the anterior cornea branch and anastomose with one another and run at oblique angles relative to Bowman's layer, while in the posterior cornea, lamellae run parallel to the anterior and posterior limiting lamina with significantly reduced branching (Jester et al., 2010a, Jester et al., 2010b; Winkler et al., 2011a, Winkler et al., 2011b; Winkler et al., 2013). It has also been established that there are bow spring fibers which extend from Bowman's layer and intertwine with deeper fibers (Morishige et al., 2011; Winkler et al., 2011; Mercatelli et al., 2017). Recent studies have also shown that these structural differences are associated with distinctly different mechanical properties in the cornea leading to increased mechanical stiffness and elastic modulus in the anterior cornea that is two to three fold higher than the posterior cornea (Winkler et al., 2011; Petsche et al., 2012; Dias et al., 2013; Dias and Ziebarth, 2013; Mikula et al., 2016). The KC cornea stands in stark contrast to the healthy cornea, both in regard to mechanics (Andreassen et al. 1980; Edmund 1988, 1989; Scarcelli, Besner et al. 2014, 2015) as well as collagen structure (Meek et al., 2005; Morishige et al., 2007; Morishige, Shin-gyou-uchi et al., 2014). Specifically, Morishige et al. have shown that collagen structure varies axially in the KC cornea when compared to the healthy cornea (Morishige et al., 2007; Morishige, Shin-gyou-uchi et al., 2014). Given that collagen structure is inextricably tied to biomechanical properties and that KC structure from anterior to posterior varies significantly compared to healthy corneas, it is expected that biomechanical properties will also vary in the axial direction.

The primary objective of this study was to assess the axial variation in mechanical properties of keratoconic corneal buttons using acoustic radiation force elastic microscopy (ARFEM). For structural comparison to biomechanical results, collagen architecture was qualitatively evaluated in KC corneal buttons, and then quantitatively analyzed in KC button obtained from a patient that had received Epikeratophakia (Epi-KP) using 3D-SHG. The analysis of the Epi-KP button allowed for the assessment of lamellar collagen branching point density (BPD) in both the KC cornea and overlying Epi-KP lenticule, allowing for direct comparison in the same sample.