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J. M. Bradley, K. E. Keller, T. S. Acott; Quantum Dot Analyses of Segmental Flow Patterns in the Trabecular Meshwork. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1635.
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Studies were conducted to evaluate the feasibility of mapping segmental aqueous outflow patterns through the trabecular meshwork (TM) using fluorophore nanocrystals or quantum dots (QDots).
Porcine or human perfused anterior segment organ cultures were treated, at the end of the flow experiment, with 10 nM fluorescent QDot nanoparticles (Qtracker 655; Molecular Probes) via direct injection of 200 µl of solution into the inflow line of the flow chamber. These 10-20 nm particles were derivatized with a variant of the HIV-TAT peptide, which facilitates cellular uptake and retention. QDots are stable to prolonged light exposure and, due to their coating, relatively non-toxic to cells. After one hour exposure to QDots, anterior segments were formalin-fixed and analyzed by standard fluorescence or confocal microscopy. Anterior segment wedges were imaged en face looking from the anterior chamber toward Schlemm’s canal (SC), in radial sections or in tangential sections that vertically-bisected SC. In some instances, wedges were further evaluated immunohistochemically for versican or ADAMTS 4 proteolytically generated versican neo-epitope using antibodies and Alexa Fluor 488-conjugated secondary antibody.
Both human and porcine anterior segments exhibited considerable segmental variation in QDot labeling patterns. In human eyes QDots tended to accumulate as a gradient, with less labeling in the juxtacanalicular or SC cells and more in the uveal and corneoscleral regions. Both porcine and human segmental labeling, assessed by densitometry, exhibited as much as 20-fold regional variability. While increasing the flow rate to an eye does seem to eliminate some of this zonality, it does not eliminate it. Versican immunostaining relative to QDot distribution, gives an inverse correlation of abundance. In some cases, versican neo-epitope staining exhibited a direct correlation with QDot distribution.
Perfused QDot distributions appear to provide an efficacious and convenient indicator of relative segmental flow patterns in the outflow system. As observed by others, the degree of flow segmentation is quite high, particularly in human eyes from older or glaucoma-affected individuals. Total versican distributions appear to be higher in regions of low flow and lower in regions of high flow. The versican neo-epitope may associate with high flow areas, although more observations are necessary to establish this tentative relationship. The very high degree of flow segmentation suggests that regional analyses will be absolutely necessary to correlate specific outflow pathway properties with outflow facility.
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