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C. S. Kamma Lorger, C. Boote, S. Hayes, R. D. Young, A. J. Quantock, N. White, K. M. Meek; A Three-Dimensional Evaluation of Collagen Fibril Orientation in the Human Corneoscleral Limbus. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4536.
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A circum-corneal network of tangential collagen fibrils has been demonstrated to exist in the human limbus and is thought to be important for corneal biomechanical stability, shape and curvature. However, current knowledge of limbal structure is primarily based on depth-averaged data. Here we describe a three-dimensional investigation of collagen fibril arrangement in the human limbus designed to discover if this architecture existed at any particular depth in the stroma.
Six human eye-bank corneas were examined. Serial sections, 100µm thick, parallel to the corneal surface were studied by wide angle x-ray diffraction performed in the European Synchrotron Radiation Facility (ESRF). Each section was scanned at 150 or 250µm steps with an 8 or 25µm square beam, respectively. Data analysis provided information on the direction and degree of alignment of preferentially oriented collagen fibrils, at the periphery of the cornea, where the limbus is located. Sections that showed the most aligned collagen orientation were further examined using second harmonic generation microscopy. A femtosecond titanium-sapphire laser with 800nm output was used to generate second-harmonic signals collected at 400nm from the examined sections. Three-dimensional data sets were collected at 1µm intervals and reconstructed to determine the depth of the tangential collagen structure within the limbus.
The previously well-documented circumferential collagen orientation at the limbus was mainly restricted to the posterior 30% of the stroma, and was not observed at any other depth of the examined tissue. In the more superficial layers, collagen preferred orientation tends to be isotropic with interwoven branches visible by 2nd harmonic imaging. These presumably form part of the scleral-corneal "anchoring fibril" structure that traverses the peripheral cornea and has been described in previous studies.
The degree of stromal lamellar alignment in the human limbus is depth-dependent and is in general lower in the anterior stroma. The circumcorneal collagen annulus, which is thought to be implicated in the maintenance of corneal curvature, resides in the deepest one-third of the tissue.
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