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J. R. Lewis, K. M. Meek, J. P. R. O. Orgel; Investigation of Fibril Ultrastructure in Optic Nerve by X-Ray Scattering. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5668.
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Fibrillar species indicative of equatorially oriented collagen were identified by wide-angle X-Ray diffraction in porcine optic nerve dissections providing proof-of-concept for X-Ray analysis of human optic nerve tissue in situ.
Sections of porcine optic nerve tissue from paired, orientated eyes were fixed in 4% paraformaldehyde and mounted in protective plastic. Medium- to wide-angle diffraction at BMC-14 BioCARS (APS, Chicago, IL) with a 200 µm diameter, 0.9 Angstrom wavelength beam and 40 cm detector distance was performed on serial sections along the optic nerve. Fibrillar and molecular packing functions were measured from 1-D slices of the X-Ray scattering patterns, after background subtraction and calibration to hydrated rat tail tendon. The degree of anisotropy and relative abundances were determined from the scattering intensity and distribution plots.
Distinct X-Ray scattering patterns were observed. Equatorially oriented fibrillar packing functions (and molecular packing functions) of the left and right eye optic nerve sections corresponding to the region of unmyelinated axons and lamina cribrosa were 39 Angstroms (15.25 Angstroms) and 41.5 Angstroms (15.25 Angstroms). The fibrillar packing function increased and the molecular packing function decreased thereafter in the distal myelinated sections. The fibrillar packing functions (and molecular packing functions) measured sequentially in these sections distal to the lamina cribrosa in the right eye were 46, 46, 44, and 44 Angstroms (and 11, 11, 10.2, and 10.6 Angstroms). In comparison, the scleral molecular packing functions were 14.6 Angstroms and 14.5 Angstroms in samples from the left and right eyes.
Wide-angle X-Ray scattering to determine the fibril and molecular packing functions and organization in the optic nerve and sclera is feasible as demonstrated here. These data indicate that the fibrillar packing function is smaller and the molecular packing function is larger at the level of the lamina cribrosa relative to distal regions along the optic nerve. Mapping the fibrillar ultrastructure of the optic nerve and surrounding sclera opens a new field of exploration. Ultrastructural changes within these connective tissues can be examined with similar X-Ray scattering techniques to correlate any changes occurring with vision loss and the effects of age, race, disease.
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