A Comparative Study of Vertebrate Corneal Structure: The Evolution of a Refractive Lens

Abstract

Purpose: Although corneal curvature plays an important role in determining the refractive power of the vertebrate eye, the mechanisms controlling corneal shape remain largely unknown. To address this question, we performed a comparative study of vertebrate corneal structure to identify potential evolutionarily based changes that correlate with the development of a corneal refractive lens.

Methods: Nonlinear optical (NLO) imaging of second-harmonic-generated (SHG) signals was used to image collagen and three-dimensionally reconstruct the lamellar organization in corneas from different vertebrate clades.

Results: Second-harmonic-generated images taken normal to the corneal surface showed that corneal collagen in all nonmammalian vertebrates was organized into sheets (fish and amphibians) or ribbons (reptiles and birds) extending from limbus to limbus that were oriented nearly orthogonal (ranging from 77.7°-88.2°) to their neighbors. The slight angular offset (2°-13°) created a rotational pattern that continued throughout the full thickness in fish and amphibians and to the very posterior layers in reptiles and birds. Interactions between lamellae were limited to "sutural" fibers in cartilaginous fish, and occasional lamellar branching in fish and amphibians. There was a marked increase in lamellar branching in higher vertebrates, such that birds ≫ reptiles > amphibians > fish. By contrast, mammalian corneas showed a nearly random collagen fiber organization with no orthogonal, chiral pattern.

Conclusions: Our data indicate that nonmammalian vertebrate corneas share a common orthogonal collagen structural organization that shows increased lamellar branching in higher vertebrate species. Importantly, mammalian corneas showed a different structural organization, suggesting a divergent evolutionary background.

Figure 1

Different functional requirements for aquatic and terrestrial corneas. In a terrestrial environment (top), the difference in refractive indices between air and the cornea is large enough for the cornea to affect a significant change in direction of incoming light, making it a powerful refractive element. Under water, however (bottom), the cornea is essentially index-matched to the surrounding environment and therefore has very little refractive power.

Figure 2

En face SHG images taken from great white shark cornea of two orthogonal lamellar planes (A, C) and the FFT analysis of the collagen orientation at the interface (B, D).

Figure 3

En face SHG images (1) and respective FFT analysis (2) of salmon (A), bullfrog (B), alligator (C), falcon (D), and rabbit (E). Collagenous orthogonal sheets were detected in salmon and bullfrog (A1, B1), whereas orthogonal lamellae formed ribbons in alligator (C1) and falcon (D1). Rabbit showed randomly oriented lamellar ribbons (E1). Orthogonal layers in nonmammalian vertebrate corneas were offset by 77.7° to 88.2°.

Figure 4

Three-dimensional FFTs of en face SHG stacks to highlight collagen fiber directionality in the great white shark (A), salmon (B), bullfrog (C), alligator (D), falcon (E), and human (F). Each spoke represents the predominant directionality of collagen fibers within each layer. A plot of the angular displacement with depth (G) for each species shows that nonmammalian vertebrate corneas rotate over 100° to 200°. Beginning with the alligator, the posterior layers are perpendicular, causing the rotation of adjacent layers to remain locked at fixed angles ([G], arrows).

Figure 5

Cross-sectional HRMac image of great white shark cornea showing the plywood-like organizational pattern and the representative directionality of the collagen lamellae at each band (1–16, arrows).

Figure 6

Cross-sectional HRMac image of the falcon (A) and the Atlantic sharpnose shark (B).

Figure 7

Full-thickness, cross-sectional HRMac images of different vertebrate corneas.

Figure 8

Corresponding high-resolution HRMac views of vertebrate corneas. The shark cornea (A) shows thick, straight bands of collagen lamellae interconnected by “sutural” fibers (small arrows) and occasional lamellar branching (large arrow) and anastomosis. The sturgeon cornea (B) is similarly structured but appears to lack the perpendicular sutural fibers. Fiber branching is more frequent in the bullfrog cornea (C), and even more prominent in the alligator cornea (D). The bird cornea (E) has the highest amount of branching and anastomosis of all nonmammalian corneas, but appears to be based on the same underlying structure. Human corneas (F), however, show a completely different structural paradigm, abandoning layers in favor of highly interconnected fiber bundles and transverse fibers that are most pronounced in the anterior cornea.

Figure 9

Volume density of fiber branching points by species. Fiber points were counted manually in a predefined volume. Branching is rare in sharks and other fish. The density of branching points increases in amphibians. The highest density of fiber branching is found in birds and mammals.