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D. WuDunn, J. Haydon; Shear Stress and Interleukin–1 Induce Nuclear Factor Kappa B and Nitric Oxide Synthase in Cultured Schlemm's Canal Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1838.
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Cells lining Schlemm’s canal are subjected to shear stress forces as aqueous fluid flows through the canal. As intraocular pressure increases, Schlemm’s canal is compressed and thus shear stress within the canal may increase substantially. The purpose of this study was to determine whether shear stress applied to cultured Schlemm’s canal cells could induce the NFkB–mediated stress response that is characteristic of glaucomatous outflow tissue or induce production of nitric oxide, which may play a role in regulation of outflow facility.
Trabecular meshwork and inner wall of Schlemm’s canal were stripped off donor corneoscleral tissue and digested with collagenase. After growing the outflow cells in culture, Schlemm’s canal cells were separated from trabecular meshwork cells by using magnetic beads coupled to antibodies to CD31 (PECAM–1), a vascular endothelial marker expressed on Schlemm’s canal cells but not on trabecular meshwork cells. Cultured Schlemm’s canal cells were subjected to various levels of shear stress, in the presence or absence of interleukin–1. After fixation, cells were assayed for nuclear factor kappa B (NFkB) activation by immunofluorescence microscopy and Western immunoblotting using rabbit antisera to NFkB. Nitric oxide synthase activity was assayed by fluorescence microscopy after pre–loading cells with 10 µM 4,5–diaminofluorescein diacetate, which reacts with nitric oxide to become highly fluorescent.
Isolated Schlemm’s canal cells continued to express surface CD31 through 4 passages. When Schlemm’s canal cells were subjected to shear stress (up to 3 dynes/cm2) or to low levels of interleukin–1α (0.02 ng/ml), no significant NFkB activity was detected within 3 hours. However, the combination of 3 dynes/cm2 shear stress and 0.02 ng/ml interleukin–1α readily induced NFkB nuclear translocation. Similarly, the combination of shear stress and sub–threshold interleukin–1α, but neither separately, induced nitric oxide synthase activity.
Shear stress, in combination with low levels of interleukin–1α, can induce the stress response in isolated Schlemm’s canal cells. Nitric oxide, which may play a role in intraocular pressure regulation, is also induced by increased shear stress on Schlemm’s canal cells. Shear stress within Schlemm’s canal may play an important role in regulating trabecular outflow.
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