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Rachel Chong, Barbara Lorber, Keith Martin; The Effect of Modulators of Microtubule Stability on Dendritic Morphology in Cultured Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4941.
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Microtubule instability is strongly associated with dendritic degeneration in several neurodegenerative diseases. Simplification of retinal ganglion cell (RGC) dendritic trees and synaptic stripping have been observed as early injury responses in experimental glaucoma models. Our hypothesis is that promoting microtubule stability with Taxol may have a beneficial effect on RGC dendrites after injury. We therefore aimed to develop a model to study the effect of RGC microtubule stabilisation.
A dissociated mixed primary retinal cell culture system was used to test the effect of 3, 5 and 10 nM Taxol, a microtubule-stabiliser, and 45 nM Nocodazole, a microtubule-destabiliser, on RGCs taken from postnatal day 8 (P8) male Sprague-Dawley rats. Immunocytochemistry (ICC) was used to identify RGCs (BIII-tubulin) and label the somatodendritic compartment (MAP-2). Image processing software (ImageJ) was used to compare dendrite length, number of branch-points and Sholl analysis of RGCs exposed to the above conditions. Statistical analysis was done using the D’Agostino & Pearson test of normality to determine the distribution of dendrite characteristics from the control group, before the Kruskal-Wallis one-way analysis of variance was used to evaluate the significance of inter-group differences.
Co-localisation of MAP-2 with BIII-tubulin enabled clear visualisation of RGC dendrites in dissociated primary mixed retinal cell cultures from P8 Sprague-Dawley rats (Fig 1). Mean RGC dendrite length was significantly increased in cells that were treated with 3 nM Taxol (119.5 +/- 33.69 µm) when compared with the drug-free control group (77.48 +/- 2.08 µm, p < 0.01, Fig 2). There was no significant difference in maximum dendrite length, number of branch-points and Sholl analysis between the treated and untreated groups. Nocodazole, a microtubule destabiliser, did not have a significant effect on dendritic length in this system.
Degeneration of RGC dendrites occurs early in experimental glaucoma. This is the first study to suggest that a low concentration of Taxol increases the mean dendrite length of cultured RGCs. In on-going work we are investigating if this effect is also evident in in vivo experimental models of glaucoma, and if preservation or re-establishment of synaptic connectivity is possible.
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