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S. L. Bernstein, Y. Guo; Temporal Course of RhoA Activation After Optic Nerve Stroke. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5190.
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CNS axonal regenerative failure follows either stroke or trauma, due to the release of soluble myelin-derived factors from oligodendrocyte synthesized myelin. These factors bind to the NOGO/LINGO receptor complex, activating rhoA, and result in collapse of regenerating axonal growth cones, with subsequent regenerative failure. We wanted to determine whether rhoA activation occurs after nonarteritic optic nerve stroke, and its time course.
We used the rodent model of nonarteritic anterior ischemic optic neuropathy (rAION) (Bernstein et al, IOVS 45, 1281, (2003)). Active rho selectively binds to rhotekin protein. Activated rhoA was identified by pull-down assay (Sander et al, J.Cell. Biol 143, 1385 (1998)), using a rhotekin-glutathione protein construct (kind gift of Dr. J.G. Collard; Netherlands Cancer Institute). The rhotekin construct was bound in solid phase to GST-Sepharose Beads. Total rhoA was identified by western analysis, using the appropriate antibody (sc-418; Santa Cruz). The ratio of active rhoA/total rho was determined using densitometry. Total protein loading was compared by actin levels.
High total rhoA levels are present in rodent optic nerve, but this protein is largely inactive by rhotekin assay. RhoA activation follows rAION induction, with low but detectable levels present at one day, increasing further at day 7. Little active rhoA is detectable in uninduced fellow control eyes. RhoA activation is associated with degenerating optic nerve myelin, demonstrable by transmission electron microscopy.
Similar to CNS ischemia elsewhere and optic nerve trauma, nonarteritic optic nerve stroke results in myelin degeneration and progressive rhoA activation, with a rapidly increasing temporal time course. Effective therapeutic treatment and repair of post-ON stroke tissue will need to address methods to reduce or eliminate rhoA activation, to enhance RGC axonal regeneration through the remodeling optic nerve.
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