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M.M. Civan, C.W. McLaughlin, S. Zellhuber–Macmillan, M. Karl, Z. Wang, A.D. C. Macknight; Electron Probe X–Ray Microanalyses of Cells in Schlemm’S Canal, Juxtacanalicular Tissue and Trabecular Meshwork of Intact Human Tissue . Invest. Ophthalmol. Vis. Sci. 2006;47(13):408.
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© ARVO (1962-2015); The Authors (2016-present)
To determine whether electron probe X–ray microanalysis can be used to measure the ionic compositions of the relatively inaccessible different cell types in the conventional aqueous humor outflow pathway.
Human post–mortem eyes are dissected and the outflow regions are quick–frozen in liquid nitrogen–cooled propane. Frozen tissues are freeze–dried, cryosectioned and then irradiated with electron beams to generate the X–ray spectra. The continuous background spectrum permits calculation of sample mass. The characteristic spectral signals permit measurement of the Na, K, Cl and P contents. The electrolyte contents are normalized to P content, which overwhelmingly reflects covalently–linked phosphorus.
Cells were identified by scanning electron microscopy within the inner– and outer–wall Schlemm’s canal (SC), juxtacanalicular tissue (JCT) and trabecular meshwork (TM). The P content of the SC, JCT and TM cells, normalized to mass, was 758±19 (mean ±SE, N=74), comparable to that (824±5, N=1085) of nonpigmented ciliary epithelial cells we have analyzed before. The baseline Na, K and Cl contents normalized to P were 0.18±0.02, 1.16±0.03 and 0.36±0.01 mmoles/mmole P, respectively (N=74). The high P and K contents verify that we are indeed analyzing intracellular samples within the outflow pathway. Preincubation with 100µM ouabain to block the Na+,K+–exchange pump altered the Na/P and K/P ratios by an order of magnitude. Ouabain increased the mean intracellular Na/P ratio to 1.72±0.06 and reduced the K/P ratio to 0.042±0.008, as expected (N=15).
The heterogeneous populations of cells within the conventional outflow pathway can be readily identified in freeze–dried, unstained tissue sections. Electron–probe X–ray microanalysis provides a powerful technique for measuring both baseline ionic compositions and experimentally–induced changes in intracellular composition within these cells.
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