Abstract
The cornea is the most external layer of the eye and serves two important roles in (1) the refraction of light and (2) protection from the outside environment, both of which are highly dependent on the collagen assembly of the corneal stroma. This study sought to determine the collagen fiber arrangement of the canine corneal stroma and correlate the stromal organization with tissue stiffness in the anterior and posterior cornea. Collagen organization of the canine cornea was visualized through second-harmonic generation (SHG) imaging, and tissue stiffness of the anterior and posterior corneal stroma was determined by atomic force microscopy. Analysis of the canine anterior corneal stroma using SHG imaging documented intertwining of the collagen fibers with a high degree of fiber branching, with a more lamellar and non-branching posterior stroma. The anterior stroma had significantly higher tissue stiffness in both dogs and humans, when compared with the posterior corneal stroma (canine median: 1.3 kPa vs. 0.3 kPa; human median: 14.6 kPa vs. 2.1 kPa, respectively). There was a direct correlation between corneal collagen stromal organization and tissue stiffness in the dog, which was consistent with other mammalian species previously examined and likely reflects the need for maintenance of rigidity and corneal curvature.
Highlights
The cornea is responsible for many functions of the eye, serving as the primary refractive interface for light entering the eye, as well as forming an anterior physical barrier to the external environment.The corneal stroma comprises nearly 90% of the total corneal thickness in most domesticated mammals, and is primarily composed of stromal collagen [1]
Second-harmonic generation (SHG) imaging is a nonlinear optical imaging technique that allows for visualization of collagen fiber arrangement [3]
The use of second-harmonic generation (SHG) imaging is ideal for characterizing corneal stromal tissue due to the primarily collagen composition of the stroma and the ability of infrared light to penetrate the entire thickness of the cornea, generating an image with high axial and lateral resolution [6]
Summary
The cornea is responsible for many functions of the eye, serving as the primary refractive interface for light entering the eye, as well as forming an anterior physical barrier to the external environment.The corneal stroma comprises nearly 90% of the total corneal thickness in most domesticated mammals, and is primarily composed of stromal collagen [1]. SHG images are generated using ultrashort infrared laser pulses that result in the oscillating polarization of structures that lack central symmetry, such as collagen [5] This induced polarization of the collagen fibrils yields light emissions from a very small focal volume that are exactly half the wavelength of the incident laser beam, and the detection of these light emissions permits the visualized reconstruction of the collagen fiber arrangement within the examined tissue [5]. The use of SHG imaging is ideal for characterizing corneal stromal tissue due to the primarily collagen composition of the stroma and the ability of infrared light to penetrate the entire thickness of the cornea, generating an image with high axial and lateral resolution [6]. While there are differences among species in the extent of the interweaving of fibers, mammalian corneas generally exhibit intertwining collagen fibers of seemingly random orientation in the anterior corneal stroma with less interweaving of fibers found in the posterior stroma [7,8]
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