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Donald Brown, Geraint Parfitt, Korey Reeid, Yilu Xie, James Jester; Pressure Induced Changes in the Human Optic Nerve Head assessed by Immunofluorescent Computed Tomography (ICT). Invest. Ophthalmol. Vis. Sci. 2013;54(15):2277.
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© ARVO (1962-2015); The Authors (2016-present)
Recent evidence suggests increasing the Intra ocular pressure to 50mm Hg, increases the anterior surface area of the lamina cribrosa by 12%. We have developed a novel method (ICT) which enables 3-D quantification of multiple antigens and distinct cell populations in large tissue volumes to validate and expand on these findings in the optic nerve head (ONH).
Pairs of fresh eyes were obtained from a local eye bank and fixed at either 15 or 50mm Hg overnight in 2% paraformaldehyde/PBS. After fixation, the ONH was carefully dissected and dehyrdrated through a series of ethanol. The tissue was embedded in butyl-methyl methacrylate and polymerised under UV light at 4oC overnight. Tissue blocks were then serially sectioned at 2µm intervals through a depth of 1mm. Each section was then imaged using non-linear optics to generate second harmonic signals (fibrillar collagen). Then, each section was sequentially immunostained for the cellular markers NCAM and GFAP and for the extracellular matrix components, elastin and type IV collagen. Prior to imaging, DAPI was applied to image cell nuclei. Images were acquired using a 20x/0.75NA objective on a Leica DMI6000B fluorescence microscope and /or a Zeiss 510 Meta coupled to a femtosecond laser. Data sets represent tissue volumes of 4mm X 4mm X 1mm with a voxel resolution of 0.45 X 0.45 X 2 μm . The alignment and 3-D reconstruction of the tissue was performed using Amira software.
ICT reconstructions reveal a layer of elastin immediately anterior to the LC collagen beams. Type IV collagen demonstrated the intricate and torturous vascular bed in the prelaminar layer and peripapillary sclera. Periodic branches from the circle of Zin-Haller were observed that directly extended into the LC layer. NCAM and GFAP staining, while strongly positive, has not yet been fully reconstructed so the distribution of of the LC cell (NCAM positive and GFAP negative) has not yet been determined. However, overall cell density dramatically drops precisely at the anterior surface of the LC.
ICT is an emerging and powerful technique to 3-D visualise distinct cell and matrix distributions in large volumes and that has enabled us to more precisely characterise the structural changes that occur in the ONH in response to IOP. Further analyses will reveal if, for example, the elastin layer and or vascular bed compresses with increased IOP.
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