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AJ Bellezza, JF Reynaud, BA Hirons, CF Burgoyne; Digital Three-Dimensional (3D) Reconstruction of the Connective Tissues of the Monkey Optic Nerve Head . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4037.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To build high resolution, digital 3D reconstructions of the connective tissues of individual monkey optic nerve heads (ONHs) to characterize the connective tissue architecture of the normal and early glaucomatous ONH. Methods: The ONH and peripapillary sclera are trephined (6 mm), pierced with 4 to 7 alignment sutures, and embedded in paraffin. The paraffin block is mounted on a microtome with the optic disc surface visible through the block's outward face. A Ponceau S/Basic Fuchsin stain is applied to the block face, staining only the exposed connective tissues. A 1520x1080 pixel digital camera, magnified so that each pixel represents 2.5x2.5 µm, then images the stained block face. The microtome cuts a 3 µm slice from the block, leaving a new, unstained surface. Staining and imaging are repeated through the lamina cribrosa, peripapillary sclera, and into the orbital optic nerve (200 to 500 images per ONH), yielding a volume of 3D 2.5x2.5x3 µm voxel data representing the connective tissue of the entire ONH and adjacent sclera. All images are aligned and then thresholded to isolate the connective tissues. Within each thresholded image, the connective tissues are parsed into one of five objects: sclera, lamina cribrosa, optic nerve septa, pial sheath, and vasculature. Volume rendering of any or all objects is then performed using OpenDX software, and voxel data is used to build finite element model geometries within Patran finite element analysis preprocessing software. Results: Digital 3D geometries from both eyes of two early glaucoma monkeys (1 normal, 1 early glaucoma eye) have been constructed. Profound deformation of the lamina cribrosa and scleral canal wall are evident within the voxel geometry of the early glaucoma ONH compared to its contralateral normal. Software to quantify laminar position and thickness, scleral canal diameter, and peripapillary scleral thickness is in development. Continuum finite element models of each ONH are under construction. Conclusion: Digital 3D reconstruction from serial stained section images of an embedded tissue block surface represents a new technique for visualizing and measuring the load-bearing connective tissues of the peripapillary sclera, scleral canal wall, and lamina cribrosa. Within the geometries of 2 early glaucoma monkey ONHs, profound alterations in the lamina cribrosa and scleral canal wall are present relative to their contralateral normal eyes, supporting our hypothesis that the ONH connective tissues are damaged early in glaucoma. Finite element modeling will allow us to map stresses within the geometry of each ONH at varying levels of IOP.
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