Depth-Dependant Collagen Fibrillar Architecture in Human Eye Bank Corneas Ascertained by Microfocus Synchrotron X-Ray Diffraction

Abstract

Purpose:: Collagen architecture in cornea has often been examined by x-ray diffraction, with the x-ray beam passed through an isolated, hydrated cornea parallel to the optical axis. This gives representative data on fibrillar ultrastructure and orientation as an average throughout the stroma, but cannot provide depth-profiled structural information. We sought to resolve this matter by the use of microfocus synchrotron x-ray diffraction.

Methods:: Two strips of central cornea, approximately 400µm-thick, were dissected from a left-right pair of corneas provided for research purposes. Tissues were wrapped in plastic film to limit dehydration and examined on beamline 40XU at the SPring-8 synchrotron in Japan. For data collection a circular 25µm microfocus x-ray beam was passed through the cut edge of a corneal strip in a direction parallel to the plane of the cornea, and x-ray patterns recorded at 25µm steps traversing the entire cornea.

Results:: Microfocus x-ray scans of the cut strips disclosed that the corneas were 925µm (left eye) and 875µm (right eye) thick at the time of study. Interfibrillar patterns from anterior regions of both corneas were indicative of collagen running predominantly parallel to the corneal surface, with an approximate 20° angular spread. Patterns became more intense in deeper stromal layers, with an increased circumferential extent and the occasional appearance of satellite reflections angled away from the main reflection. In the pre-Descemet’s region the x-ray pattern appeared uniaxial, indicative of collagen running mainly parallel to the corneal surface. Collagen fibril diameter remained unchanged with respect to stromal depth, measuring 35.6±0.3nm and 35.2±0.4nm (mean±SD) throughout the left and right corneas, respectively. In the anterior 100-150µm of both corneas the average collagen interfibrillar Bragg spacing was lower than in the remainder of the stroma by 9-11%.

Conclusions:: Collagen fibril diameter remains unchanged throughout the swollen human eye bank cornea at values close to normal. Interfibrillar spacing, however, is lower in superficial tissue regions. The appearance of occasional satellite reflections is suggestive of subpopulations of fibrils subtending fairly large angles with the plane of the cornea, presumably as a result of the swelling of interwoven lamellae.

Keywords: cornea: stroma and keratocytes • cornea: storage • transplantation