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Alex S Huang, Jose Miguel Gonzalez, Felipe A Medeiros, James C H Tan, Narsing A. Rao, David R Hinton, Robert N Weinreb; Histological Characterization of Open Angle Glaucomatous Sclera. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5688.
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To study α-smooth muscle actin (ASMA) levels and morphological changes in the sclera related to TGF-β in open angle glaucoma. TGF-β is a soluble protein known to be elevated in both primary open angle glaucoma (POAG) and pseudoexfoliation (PEX) and is implicated in trabecular meshwork (TM) pathologic changes leading to increased aqueous humor outflow resistance and elevated intraocular pressure. As the TM is not entirely occluded in OAG, TGF-β can still travel beyond the TM into the distal outflow pathway, including the intrascleral venous plexus, where it can mediate pathologic alterations such as myofibroblastic differentiation and fibrosis.
Scleral samples have been collected from a PEX glaucomatous and two (74 and 50 year-old) non-glaucomatous patients. Glaucomatous sclera was collected during canaloplasty surgery from a 77 year old female with a five-year history of PEX glaucoma with visual field defects and elevated intraocular pressures (~30 mm Hg) despite multiple aqueous suppressants and a prostaglandin. Control sclera was collected from remnant corneo-scleral rim after corneal transplantation. Tissue was fixed in 4% paraformaldehyde, cryopreserved in 30% sucrose, sectioned with a cryostat (8 microns), peroxidase quenched, blocked, permeabilized, and incubated in primary and secondary antibodies. No primary antibody controls were conducted. Signals were enhanced by ABC Elite Kit (Vector, California) and visualized with DAB chromogen. Primary antibodies included α-smooth muscle actin (ASMA; 1:200 dilution; Pierce).
Elevated levels of ASMA were observed in glaucomatous sclera compared to non-glaucomatous sclera. More myofibroblasts were seen (Figure; arrows) and collagen fibrils appeared more attenuated and possibly fibrosed (Figure; asterisk) in glaucomatous sclera. Absence of primary antibody yielded no signal. Scale bar = 5 microns.
Elevated ASMA and attenuated collagen fibrils further support TGF-β mediated alterations, scleral myofibroblastic induction, and tissue fibrosis in glaucomatous sclera. This finding is consistent with previous results we reported showing elevated ASMA protein levels comparing both POAG and PEX sclera to control sclera by Western blot.
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