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Cheryl R. Hann, Donald J. Brown, Michael P. Fautsch; Two-Photon and Second Harmonic Imaging of the Sheath and Tendon Network in Normal Human Trabecular Meshwork. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4647. doi: https://doi.org/.
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To determine if non-linear imaging can be used to visualize the sheath and tendon network within the juxtacanalicular tissue (JCT), adjacent Schlemm’s canal (SC) region and in collector channel (CC) walls within the normal human trabecular outflow pathway.
Representative samples of the trabecular outflow pathway from two whole perfused eyes were perfusion fixed in 3% paraformaldehyde/0.2% triton (15 minutes), incubated in 3% paraformaldehyde (30 minutes) and maintained in 1.5% paraformaldehyde. Dissected wedges of the inner wall of SC, adjacent JCT and the outer wall of SC containing CCs were examined. Wedges were evaluated with two-photon excited fluroresence (TPEF) for elastin, second harmonic generated (SHG) imaging for collagen, and Syto 59 for nuclei. Correlative immunohistochemistry was performed on similar wedges using antibodies to elastin, phalloidin for F-actin and DAPI for nuclei. Three dimensional (3-D) tissue volumes were reconstructed from collected images to examine the location and structure of the sheath and tendon structures adjacent to SC and within the JCT and CC walls.
3-D reconstructions of TPEF and SHG imaging of the sheath and tendon structures revealed a branching elastin network originating from the elongate sheath and tendons which travel adjacent to and through the trabecular beams. These elastin fibers subdivided as they extended into the JCT to form a network surrounding the JCT cells. Elastin fibers appeared to terminate adjacent to or at the cell membrane of SC and JCT cells. This elastin network was also observed in CC walls. Correlative immunohistochemistry localized elastin fibers in the same region as those found using TPEF.
Non-linear imaging allowed visualization of the sheath and tendon network within the JCT, adjacent to SC and in CC walls. This complex network of elastin fibers imparts support and unique biomechanical properties to the human trabecular outflow pathway.
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