The influence of lamellar orientation on corneal material behavior: biomechanical and structural changes in an avian corneal disorder

Abstract

Purpose: Retinopathy, globe enlarged (RGE) is an inherited genetic disease of chickens with a corneal phenotype characterized by loss of tissue curvature and changes in peripheral collagen fibril alignment. This study aimed to characterize the material behavior of normal and RGE chicken corneas under inflation and compare this with new spatial- and depth-resolved microstructural information to investigate how stromal fibril architecture determines corneal behavior under intraocular pressure (IOP).

Methods: Six RGE chicken corneas and six age-matched normal controls were tested using trephinate inflation and their stress-strain behavior determined as a function of posterior pressure. Second harmonic generation mulitphoton microscopy was used to compare the in-plane appearance and degree of through-plane interlacing of collagen lamellae between normal and mutant corneas.

Results: RGE corneas displayed a 30-130% increase in material stiffness [E(tangent)(RGE) = 0.94 ± 0.18 MPa to 3.09 ± 0.66 MPa; E(tangent)(normals) = 0.72 ± 0.13 MPa to 1.34 ± 0.35 MPa] (P ≤ 0.05). The normal in-plane disposition of anterior collagen in the peripheral cornea was altered in RGE but through-plane lamellar interlacing was unaffected.

Conclusions: This article demonstrates changes in corneal material behavior in RGE that are qualitatively consistent with microstructural collagen alterations identified both herein and previously. This study indicates that, in general, changes in stromal fibril orientation may significantly affect corneal material behavior and thereby its response to IOP.

Figure 1.

(A) Schematic showing main components of the corneal inflation rig. (B) Photograph of apparatus.

Figure 2.

Trephinate inflation behavior of (A) six normal and (B) six RGE chicken corneas. Measured linear displacement of corneal apex (Rise) is plotted as a function of applied posterior pressure recorded during the fourth loading cycle (i.e., after preconditioning).

Figure 3.

Comparison of inflation behavior between normal and RGE chicken corneas, showing for each group pressure-rise data for the (A) highest/lowest stiffness corneas and (B) the average of all corneas. Error bars, SD. From pressure-rise data RGE corneas appeared considerably less mechanically stiff.

Figure 4.

Stress-strain behavior of the (A) normal and (B) RGE chicken corneas, derived from pressure-rise data during the fourth cycle of inflation, using mathematical back-analysis that corrects for the effects of corneal geometry and thickness. Here stress represents the average internal force per unit area acting on the tissue's cross section, while strain is defined as the ratio between the tissue's stretch and initial dimension.

Figure 5.

Average stress-strain behavior of normal and RGE chicken corneas. Error bars, SD. Once tissue thickness and geometry are accounted for, RGE corneas displayed significantly increased material stiffness compared with normal controls.

Figure 6.

Tangent modulus (E_tangent), the gradient of a tangent line drawn at a given point on the stress-strain curve, plotted as a function of applied stress for normal and RGE chicken corneas under the fourth cycle of posterior pressure loading. Error bars, SD. A 30–130% increase in E_tangent, commonly considered a direct measure of tissue stiffness, is observed in RGE corneas compared with controls.

Figure 7.

In-plane SHG collagen signals from the normal and RGE chicken cornea at stromal depths of 225 μm (posterior) and 50 μm (anterior). Bar, 50 μm. An orthogonal collagen straight latticework is evident in the posterior stroma (A, C, E, G). These structures appear wavier in the anterior stroma, while still retaining their lattice-like arrangement with 90° cross-angles (B, D). The characteristic fanlike structures in the peripheral anterior stroma of the normal cornea (F) are notably absent in RGE (H).

Figure 8.

Depth profile of in-plane SHG collagen signals from the central normal chicken cornea. Throughout the most superficial ∼3/4 of the stromal depth a 1°/μm anticlockwise rotation of fibrils is evident as the tissue is traversed in the posterior-anterior direction. In contrast, the orientation of lamellae in the deepest ∼1/4 of the stroma is fixed. Arrows denote the directions of the two principal, mutually orthogonal, fibril populations at each depth.

Figure 9.

In-plane SHG collagen signals from a 1.2 mm × 1.2 mm peripheral region of the anterior normal chicken cornea with depth, 50 μm. Bar, 100 μm. The fanlike arrangement of anterior lamellae, characteristic of the chicken eye, dominates the peripheral cornea.

Figure 10.

In-plane SHG collagen signals from a 1.2 mm × 1.2 mm peripheral region of the anterior RGE chicken cornea at a stromal depth of 50 μm. Bar, 100 μm. Anterior collagen structures in peripheral regions of mutant corneas appear straighter than those of the normal cornea.

Figure 11.

Through-plane SHG collagen signals from the normal and RGE chicken cornea. Bar, 50 μm. In the posterior stroma collagen lies in well-defined stacked layers parallel to the corneal plane (A, C, E, G). In contrast, the anterior stroma appears a more interwoven structure, with collagen fibril bundles connecting adjacent layers (B, D, F, H). No difference in the pattern of through-plane collagen arrangement was observed between normal and RGE tissue.