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Moritz Winkler, Krystel R. Huxlin, Christopher J. Murphy, Donald J. Brown, James V. Jester; Automated Measurement of Corneal Stromal Collagen Fiber Angular Distribution. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6792.
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3-Dimensional reconstructions of non-linear optical (NLO), second harmonic generated signals using high resolution macroscopy (HRMac) have shown that the anterior stroma of the human cornea contains a diverse population of bow-spring and intertwined collagen fibers that generally run transverse to the corneal surface and insert into the anterior limiting lamina (Bowman’s layer). We have hypothesized that these collagen fibers play an important role in controlling corneal biomechanical properties shape. The purpose of this study was to develop an automated and objective approach to quantify the angular distribution of collagen fibers in the corneal stroma on a large scale, which will allow investigation of the relationship between local shape and fiber angle in isolated human corneas and corneas of different species.
Human and animal corneas were fixed in 4% paraformaldehyde and cut into 250 µm sagittal cross-sections spanning the full corneal diameter using a vibratome. Sections were scanned from limbus to limbus using NLO-HRMac as previously described. An automated script based on algorithms by Rezakhaniha et al was developed to determine fiber angle relative to local corneal curvature at 10 µm steps along the full thickness of the cornea. Angles and their variance were tracked as a function of stromal depth and compared to fiber interconnectivity and structural data.
Fiber angles relative to the corneal surface decreased markedly with increasing stromal depth. Fiber angle variance in the human cornea was high in the anterior stroma and approached zero at 350 µm or 2/3 the depth from the anterior surface. In the cat cornea, fiber angle variance in the anterior cornea appeared similar to that of the human, but reached zero at 1/3 depth.
This approach generates verifiable angular distribution data consistent with corresponding SHG images and previously published measurements of fiber interconnectivity. Overall, the data indicate that there are species differences in the contribution of angled stromal collagen fibers that may be associated with differences in corneal shape and elasticity. Future studies comparing shape and 3-D fiber angle distribution may provide insight into the biomechanical mechanisms controlling corneal shape and refractive errors.
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