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Jose Miguel Gonzalez, James C H Tan; Mapping abnormal elastin maintenance in the human trabecular meshwork. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3299.
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© ARVO (1962-2015); The Authors (2016-present)
Abnormal trabecular meshwork (TM) elasticity is implicated in the cellular pathogenesis and human presentations of glaucoma associated with elastic disorders such as exfoliation syndrome. Beta-aminopropionitrile (BAPN), an inhibitor of lysyl oxidase/lysyl oxidase-like (LOX/LOXL) enzymes required for collagen and elastin maintenance, was applied to the human TM to model abnormal elastin maintenance.
Human donor corneoscleral tissue containing the intact TM was incubated with 1.4mM BAPN for 5 or 13 days. Tissues were then either, (1) briefly incubated with 10mg/ml sulforhodamine that specifically labels elastin, or (2) fixed and stained with ECM-specific antibodies. 2-photon microscopy (TPM) microscopy was performed to characterize second harmonic generation (SHG; from structural collagen; Fig.1, cyan); autofluorescence (AF; Fig.1; red) and sulforhodamine epifluorescence. Tissue viability was assessed by calcein AM/propidium iodide (PI) live/dead co-labeling in situ. Elastic tissue organization was assessed qualitatively, and quantitatively by isosurface volume mapping of elastin signals in AF and SHG images (Fig.1, ovals). BAPN studies in human TM were repeated in mice.
AF revealed discrete elastic fibers corresponding to sulforhodamine epifluorescence. This was associated with broader collagen beam structures corresponding to the collagen SHG signal and collagen type I labeling. After 5 days of BAPN treatment, the elastin fibrillar organization was lost (Fig.1A, B, G, H, J, K) but collagen was unchanged (Fig.1D, E, J, K). After 13 days of BAPN, the structural organization of both elastin and collagen (asterisks) was altered, with elastin appearing amorphous and overall TM architecture unrecognizable (Fig.1C, F, I, L). Ratio of elastin to collagen in tissue-based volumetric maps was (a) 0.27 in controls; (b) 0.32 after 5 days of BAPN; and (c) 0.43 after 13 days (p=0.025; n=9). No change in cell viability was seen. Mouse TPM revealed similar structural changes to those seen in human TM.
Selectivity of BAPN effect on elastin and collagen may be titrated by varying tissue exposure to BAPN. Altered TM elastin structural changes due to inhibited LOXL/LOXL were captured by tissue-based TPEF imaging and verified by quantitative mapping. This modeling of TM elastic abnormality may be useful for understanding the pathogenesis of elastic tissue aberrations associated with glaucoma.
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