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Jose M. Gonzalez, Jr., James C. Tan, Martin Heur, Jonathan Song; Three Dimensional In Situ Imaging and Localization of Trabecular Meshwork Extracellular Matrix Proteins. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4657.
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© ARVO (1962-2015); The Authors (2016-present)
To localize trabecular meshwork (TM) extracellular matrix (ECM) proteins in situ in relation to the tissue’s structural ECM by multiphoton optical sectioning without conventional histologic embedding and sectioning.
Tissue explants containing the aqueous drainage tissue were processed for epifluorescence (EF) microscopy. The tissue was cut into segments containing the intact TM and Schlemm’s canal, fixed, permeablized, then incubated with primary antibodies to type I, III, and IV collagens, tropoelastin and fibronectin, followed by fluorescently-labeled secondary antibodies. Multimodal 1- and 2-photon confocal microscopy was used to visualize ECM structures within the intact drainage tract by EF and autofluorescence (AF). Image reconstruction afforded a 3-dimensional (3D) appreciation of ECM proteins within the structure.
Morphologic transition of the TM’s structural ECM from uveal to corneoscleral to juxtacanalicular (JCT) portions was evident by AF imaging. Type III collagen EF coincided with the full width of AF from TM beams. Type I collagen EF coincided primarily with the peripheral regions of beam AF but could also be observed in the central portion of beam AF. Type IV collagen EF was observed entirely at the beam periphery so that it distinctly outlined the TM beams. Fibronectin EF had a similar distribution to collagen IV, and both were present in the JCT where beams are not seen. Tropoelastin EF coincided with fine fibers coursing alongside structural beams.
ECM proteins can be identified with respect to AF of the TM’s structural ECM. Our 3D image analysis located type III collagen, and less so type I collagen, to the central parts of TM beams, coinciding with the AF signal within these structures. Type IV collagen and fibronectin were observed peripheral to this, in regions consistent with the subendothelium of beams. Our observations are based on a novel approach of optically and molecularly dissecting the TM.
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