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Haiyan Gong, Thomas F. Freddo, Mark Johnson; Comparative Ultrastructural Study Of Trabecular Meshwork Between Normal And Primary Open Angle Glaucoma Using Quick-freeze/deep-etch. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4630.
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To determine structural differences in the trabecular meshwork (TM) between normal and primary open angle glaucoma (POAG) eyes using quick-freeze/deep-etch (QFDE), a technique that better preserves the extracellular matrix (ECM).
Normal (N=9) and POAG eyes (N=6) were immersion-fixed with Karnovsky’s fixative. For radial views of the TM, chopper sections (200 µm) were cut and trimmed to contain only TM, Schlemm’s canal (SC) and a narrow strip of sclera. For tangential views of the juxtacanalicular connective tissue (JCT), 1 mm TM sections were cut and SC was opened by excising the outer wall. Sections were quick frozen at -180 °C and transferred to a Cressington CFE-60 vacuum chamber. Radial sections of TM were fractured or the inner wall of SC in tangential sections was removed using a cold knife (-180°C) and then deep etched for 30 min. Sections were rotary shadowed at 20° with a mixture of evaporated platinum-carbon. The tissue was digested in 5% sodium hypochlorite containing 10-15% potassium hydroxide, and the remaining replicas were washed and examined by transmission electron microscopy (TEM).
In radial sections, no significant structural differences were found in the trabecular beams between normal and POAG eyes. Open spaces one micron in size or greater were seen in the JCT regions of both normal and POAG eyes. The ECM in the JCT regions of POAG eyes appeared more disorganized and more compacted as compared to the normal eyes. In both normal and POAG sections with the inner wall removed, greater 3-D structural detail of the ECM in the JCT was observed. The collagen fibers in the ECM of the JCT in POAG eyes appeared under tension, more disorganized and more compacted compared to normal eyes.
QFDE allows for a much greater detailed 3-D ultrastructural view of the ECM underneath the inner wall of SC than conventional TEM. Differences in the structure and appearance of the ECM were found in POAG compared to normal eyes. However, these changes did not appear to be able to explain the elevated outflow resistance characteristic of glaucomatous eyes.
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