Purchase this article with an account.
A. Tura, K. U. Bartz-Schmidt, S. Henke-Fahle; Efficacy of Rho-Kinase Inhibition in Reducing Glial Reactivity and Promoting Retinal Ganglion Cell (RGC) Survival in vitro. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3697.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To analyze the extent of Rho-kinase activity and the outcomes of Rho-kinase inhibition in isolated RGCs and glial cells under neurotrophin (NT) and serum deprivation, respectively.
Primary RGCs and glial cells were isolated from the retinas of newborn rats by immunomagnetic separation and cultured with or without the Rho-kinase inhibitor H-1152. The Rho-kinase activity was determined by an in vitro kinase assay and the extent of apoptosis was analyzed by immunostaining, TUNEL assay, and by measuring the neurite length. Cell proliferation in cocultures of astrocytes and Müller cells (AMC) was detected by the MTT- and BrdU-tests whereas the reactivity of these cells and the microglia seeded on fixed AMC was analyzed by immunostainings and -blotting.
NT deprivation resulted in an increase in Rho-kinase activity which was associated with a considerable degree of axon retraction and caspase-3 cleavage in the RGCs by 6h. However, the Rho-kinase inhibitor exerted a significant anti-apoptotic effect which persisted after 24h. H-1152 also suppressed the proliferation of AMC and reduced the upregulation of GFAP under serum deprivation possibly by interfering with the Rho-kinase dependent phosphorylation of this protein. Likewise, the inhibitor suppressed the upregulation of vimentin, endothelin, and fibronectin together with a decrease in Rho-kinase activity. The serum-deprived AMC stimulated the activation of microglia seeded on them as determined by the ameboid morphology the latter cell type acquired despite being grown in an optimized medium. However, the microglia seeded on serum deprived AMC treated with H-1152 exhibited a quiescent morphology, suggesting that the Rho-kinase also regulates certain extracellular changes in the activated AMC which in turn stimulate microglia activation.
The deprivation of NTs and serum results in an increase in Rho-kinase activity in the RGC and AMC cultures, respectively. Accordingly, the inhibition of Rho-kinase emerges as a promising approach for attenuating the glial reactivity and promoting RGC survival under these unfavorable conditions.
This PDF is available to Subscribers Only