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Elizabeth Martin, Kevin Curtiss, Murray A. Johnstone; Schlemm's Canal (SC) and Distal Outflow System Relationships Revealed by Immunohistochemistry (IHC) and Confocal Microscopy (CFM) Following Clarification. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3261.
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© ARVO (1962-2015); The Authors (2016-present)
Scleral opacity prevents using IHC and CFM to image important relationships distal to SC, including collector channel ostia (CCO) and intrascleral collector channels (CC) that are involved in defining outflow system resistance. We hypothesized that a clarification technique could make the sclera transparent permitting study of complex SC, CCO and intrascleral CC relationships using IHC and CFM.
Nemestrina monkey eyes (n=4), Healon GV, a viscoelastic, used to dilate SC. Radial limbal segments cut. Segment clarification with glycerol (n=3), Scale (n=3), benzyl-alcohol-benzyl-benzoate (BABB) (n=28). Optimized BABB clarification protocol took 115 min and included progressive ethanol concentrations of 30, 50, 70, 80, 96, and 100% followed by placing tissue in BABB. Differential interference contrast (DIC) and CFM were used to image segments. Native fluorescence (n=28) and collagen type I (CI)label with Alexa Fluor (AF) 555 (n=1) for sclera and trabecular lamellae imaging; DAPI (n=12) and propidium iodide (PI) (n=8) for nucleic acid labeling; CD31 with AF 647 (n=13) for vascular endothelial membrane labeling. Yellow-green fluorescent microsphere tracers, sizes 200 nm and 500 nm, were perfused into individual eyes, 3.5ml over 30 minutes, while maintaining IOP at 8 mm Hg followed by the BABB protocol.
Clarification with BABB was rapid (hours). Gycerol and Scale techniques were too slow (> 2 months) to permit rapid screening of imaging techniques. Following clarification by BABB confocal microscopy provided clear images to a mean depth of 105 µm (range 32-193). Native fluorescence, CD31, CI and PI signals remained strong while the DAPI signal was reduced. Fluorescent microspheres, clearly visible by DIC, retained no fluorescent signal. Labeling with CD31 and DAPI with a background orientation of native fluorescence permitted complex SC, CCO and intrascleral CC relationships to be effectively delineated.
The sclera was transparent following the BABB protocol permitting CFM to a tissue depth unattainable by standard CFM techniques. Clarification thus provided a unique means of imaging complex 3-dimensional SC, CCO and intrascleral CC relationships.
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