Collagen Macrostructure and Corneal Shape: Lessons from Different Species

Abstract

Purpose: The cornea plays a critical role both as a protective window to the eye and as a refractive lens. In aquatic vertebrates, it provides little refractive power, while in terrestrial vertebrates corneal shape needs to be precisely controlled to project a focused image on the retina. Little is known about the changes in the structural organization of corneal collagen during evolution. The purpose of this study was to begin to characterize the macrostructural organization of corneal collagen in divergent species in order to uncover basic mechanisms controlling corneal shape.

Methods: Eyes from various species (fish, shark, birds, mammals) were fixed under pressure using paraformaldehyde to control post-mortem swelling. Serial full-width (limbus to limbus) cross-sections (250μm thick) were cut using a vibratome. Sections were imaged using nonlinear optical high resolution macroscopy (NLO-HRMac) of second harmonic generated (SHG) signals. 3-D images were rendered using Amira software, and collagen fiber structures were quantified with custom-written ImageJ macros.

Results: In aquatic vertebrates stromal collagen macrostructure consisted of simplified layers (stacks) of fibers that extended from limbus to limbus as continuous sheets of collagen, much like ‘plywood’ in construction. Adjacent sheets were rotated 87°, forming orthogonal plies, with successive layers showing a continual rotation of over 360°. In birds collagen sheets were organized into distinct fibers with a uniform branching and fusing pattern similar to that of chicken wire and presented a honeycomb appearance. Fibers in the same plane appeared to extend from limbus to limbus, and successive layers showed a 270° rotation through 2/3 stromal depth, very similar yet distinct from fish and shark. By contrast, collagen fiber organization in mammals was irregular with varying degrees of branching depending on the species (human > dog > cat > rabbit). Mammals also lacked orthogonal rotational, nor were fibers constrained to extend from limbus to limbus within the same plane.

Conclusions: We have previously shown in the human cornea that collagen fiber branching and interconnectivity is associated with tissue rigidity. In this study, fiber branching was detected in corneas from terrestrial vertebrates suggesting that branching and increased corneal rigidity may play a role in the evolutionary adaptation of the cornea from a protective window to a refractive lens.

Keywords: 480 cornea: basic science • 516 evolution • 472 comparative anatomy