Axial Anisotropy of Corneal Collagen Organization and Biomechanics

Abstract

Purpose: : Visual acuity depends on corneal curvature, which is determined by its mechanical properties. We aim to characterize and quantify the lamellar organization of human corneas in three dimensions using non-linear optical High Resolution Macroscopy (NLO-HRMac), and to correlate these findings with mechanical data obtained by compression testing of corneal flaps, to establish a link between the supramolecular organization and corneal shape.

Methods: : Vibratome sections, 200µm thick, from 5 donor eyes were cut along the vertical meridian from limbus to limbus (arc length: 12mm). Backscattered SHG signals from these sections were collected as a series of overlapping 3-D images, which were concatenated to form a single 8GB+ mosaic (resolution: 0.44µm/px lateral, 2µm/px axial). Lamellar intertwining was quantified by determining branching point density as a function of stromal depth. Lamellae were manually segmented to create 3-D reconstructions using Amira software. Mechanical testing was performed on corneal flaps from 5 additional eyes. Corneas were cut into 3 layers (anterior, mid, posterior) using an Intralase femtosecond surgical laser system and then underwent compression tests to determine the elastic modulus for each layer.

Results: : 3-D reconstructions revealed a complex collagen fiber branching pattern in the anterior cornea with fibers extending from the anterior limiting lamina (ALL), intertwining with deeper fibers and reinserting back to the ALL forming ‘bowspring-like’ structures. Measured branching point density was 4 times higher in the anterior third of the cornea compared to the posterior third and decreased logarithmically with increasing distance from the ALL. Compression testing showed an 8-fold increase in elastic modulus in the anterior stroma.

Conclusions: : The axial gradient in lamellar intertwining appears to be associated with an axial gradient in effective modulus of the cornea suggesting that collagen fiber intertwining and formation of ‘bowstring-like’ structures provide structural support similar to cross-beams in bridges and large-scale structures. We hypothesize that radial and axial structural/mechanical anisotropy may determine the characteristic parabolic shape of the cornea and play a role in astigmatism and other refractive errors.

Keywords: cornea: basic science • cornea: stroma and keratocytes • imaging/image analysis: non-clinical