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Sietse T. Braakman, A. T. Read, Darren Chan, C. R. Ethier, Darryl R. Overby; Outflow Segmentation Coincides with Regions of Greater Pore Density in Inner Wall Endothelium of Schlemm’s Canal. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2745.
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Aqueous humor (AH) drains non-uniformly through the trabecular meshwork (TM), and factors governing such "segmental" outflow patterns may be important determinants of outflow resistance generation. Downstream of the TM lies the inner wall of Schlemm’s canal endothelium (SCE) that forms micron-sized transendothelial pores. We hypothesize that pores are necessary for AH filtration across SCE and that pore location therefore coincides with outflow segmentation in the TM. To test this hypothesis, we examined the spatial relationship between local SCE pore density and outflow segmentation.
A solution of fluorescent tracer nanoparticles (20nm, sulphate coated, 0.005% w/v) was perfused at 8 mmHg into two ostensibly normal cadaveric human eyes to identify segmental outflow patterns in the TM. After immersion fixation, eyes were dissected to expose the inner wall, SCE was stained for the endothelial marker CD31, and was imaged en face with confocal microscopy to visualize CD31 and tracer distributions. The same samples were then processed for scanning electron microscopy (SEM) to visualize SCE pores. Image morphing was used to superimpose the confocal tracer image onto the SEM image, using a polynomial local weighted mean algorithm to match common landmarks between CD31 and SEM images. Four regions of interest (ROIs), 150x50 µm, were chosen in each quadrant, and we measured pore density (n: pores/mm²), pore diameter (D: µm), and tracer intensity (TI) in each ROI. To account for mapping inaccuracies, ROIs were shifted by 10µm in all directions to provide confidence intervals on TI.
We observed a significant positive correlation between TI and total pore density (p<0.005; R²=0.76) when pooling data from all quadrants. The same relationship was observed within individual quadrants, but only for paracellular "B" pores (p<0.05) and not for transcellular "I" pores (p>0.49). A strong correlation was observed for pooled data between TI and the nD -product for B-pores (p=10E-5; R²=0.99), consistent with B-pores influencing outflow segmentation via the funneling model (Johnson et al. IOVS, 1992). B-pores constituted more than 60% of the observed pores. Confidence intervals on TI were always within 10%.
Outflow segmentation in the TM coincides with regions of higher pore density in SCE and is quantitatively consistent with predictions from the funneling model. This consistency, together with the previously postulated biophysics of the funneling process, suggests that B-pores may influence local conductivity through the TM, thus indirectly contributing to outflow resistance generation, the main determinant of intraocular pressure.
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