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Mini Aga, John Bradley, Janice A Vranka, Ted S Acott; A new method for en Face confocal imaging of Schlemm’s canal inner wall and juxtacanalicular trabecular meshwork. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5123. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To develop a method for en Face 3-D confocal microscopic imaging of Schlemm’s canal endothelium (SCE) and the juxtacanalicular region (JCT) of the trabecular meshwork (TM).
Human anterior segments were cut into 8 wedges and cuts made through each edge of the TM using two # 11 surgical blades clamped together by a hemostat. Excised 0.5mm wide TM/SCE strip were gently lifted out, immunostained, DAPI-stained and then placed on a coverslip. A second coverslip was mounted with antifade reagent and tissue analyzed by confocal microscopy.
Sidedness, i.e. TM outer beam or SCE side, is easily determined based on collagen and elastic fiber autofluorescence patterns. Tissue distortion is minimal. Confocal imaging from the SCE side using around 30 Z-stacks (0.35– 0.5μm) provides good imaging of the SCE inner wall and JCT. PECAM1 (CD31) or claudin 1 immunostaining was limited to SCE and ZO-1 provided excellent SCE cell-cell border visualization. Z-stack imaging was rendered in 3-D using Imaris software for better visualization. The SCE cell layer, which is much less than a micron thick, is clearly not flat but somewhat undulating. Several extracellular matrix components of interest to our studies were at relatively high levels in the SCE basement membrane and deep JCT, including: collagen IV and VI, nidogen, lumican, perlecan, fibronectin, fibrillin1, versican, SPARC, laminin γ1 and MMP14. Their staining patterns ranged from sheet-like to sporadic or highly punctate. Of course, these components were also mostly present throughout the TM, but not at the same levels nor exhibiting the same patterns. Component colocalization, as assessed by Pearson’s correlation coefficients, was predictably quite high for some of these and low or negative for others. For example, versican and fibrillin1 r = 0.84, SPARC and versican r = 0.11, and collagen VI and fibrillin1 r = - 0.24. Imaging of radial or frontal sections reinforced the molecular distribution and colocalization patterns.
This new confocal imaging method provides excellent detailed en Face 3-D views of the in situ SCE and JCT with minimal tissue disruption. Colocalization analysis of components is highly effective.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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