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D. J. Long, R. M. Sappington, D. J. Calkins; Pressure-induced Retraction of RGC Axons in an ex vivo Preparation of Rat Retina. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4356.
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Glaucoma is characterized by progressive loss of retinal ganglion cells (RGCs) that is often associated with elevation of intraocular pressure. Our work has revealed that pressure-induced pathology of optic nerve is more severe closer to the brain than to the eye. Our purpose here is to test the hypothesis that elevated pressure induces retraction of RGC axons.
To evaluate pressure-induced retraction of RGC axons, we established an ex vivo preparation of rat retina optimized for axon outgrowth. Whole retinas were dissected from postnatal day 3-5 rats, flat mounted on organotypic culture inserts with or without laminin (10ng/ml)and cultured alone or co-cultured with fragments of lens or superior colliculus from adult rat. Retinal explants were cultured for 3 weeks in a media that contained basic neuronal supplements, insulin and various trophic factors. To positively identify the outgrowths as RGC axons, we used immunofluorescence against the RGC markers, SMI-32 and Thy1.1. To evaluate the effect of elevated pressure on axons, we used an automated digital imaging procedure to measure the length of individual axons prior to and after exposure to elevated hydrostatic pressure (70mmHg) for 24 hours. The automated procedure was designed such that the pre- and post-pressure images for each retinal explant were exactly aligned, allowing quantification of retraction of individual axons.
We determined that laminin-coating of the organotypic insert and co-culturing with fragments of lens procured from adult rat dramatically increased the length of RGC axons to nearly 1 mm and the rate of growth. Addition of superior colliculus did not significantly improve axon outgrowth. Under all conditions, outgrowths expressed SMI-32 and Thy1.1, confirming that these processes were RGC axons. Exposure to elevated pressure for 24 hours caused a 60% reduction in axon length, as compared to axon length prior to pressure exposure.
Our data reveals that just 24 hours of elevated pressure induces substantial retraction of RGC axons, suggesting that cues to retract axons from central targets may be a component of pressure-induced death of RGCs.
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