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Aizhan Tapenbayeva, Aysegül Tura, Khaled Nassar, Julia Lüke, Salvatore Grisanti, Matthias Lüke; The Neuroprotective Potential Of Rho-kinase Inhibition In Promoting Cell Survival And Reducing Reactive Gliosis Under Hypoxia In Isolated Perfused Vertebrate Retina. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2650.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the neuroprotective activity of the Rho-kinase inhibitor H-1152P under hypoxia in the electrophysiological ex vivo model of the isolated perfused vertebrate retina.
Bovine retinas were isolated and perfused with an oxygen saturated serum-free nutrient solution containing either H-1152P at the concentration of 1 µM or no drug as a control. The electroretinogram (ERG) was recorded as a transretinal electrical potential using Ag/AgCl electrodes. The retinas were stimulated repeatedly until stable amplitudes were reached. The ERG was recorded at each five minutes. Hypoxia was induced for 15, 30 and 45 minutes by giving nitrogen to the nonoxygenated nutrient solution. After the hypoxic period oxygen saturation was restored. The extent of the cell damage was determined by live/dead staining. Immunohistochemistry and Western blot were performed to distinguish between apoptotic and necrotic cell death as well as to detect glial cell reactivity.
Application of nitrogen caused a reduction of the b-wave amplitude. H-1152P was not effective enough to avoid the reduction of the b-wave amplitude during hypoxia. Although the Rho-kinase inhibitor maintained higher b-wave amplitudes compared to sham, the effects did not reach statistical significance (p > 0.05). The live/dead assay showed significant reduction of cell damage in the ganglion cell layer compared to untreated retinas (p < 0.05). Hypoxia also resulted in the upregulation of the necrosis marker cathepsin-B in the untreated retinas as well as an increase in the reactivity of astrocytes, Müller cells and microglia whereas the administration of H-1152P significantly reduced the extent of these events.
Rho-kinase inhibition mediated by H-1152P exerted a neuroprotective effect against necrosis on isolated bovine retina under hypoxia together with a reduction in glial cell reactivity. However, the effects of H-1152P were limited and could not avoid the hypoxia induced retinal dysfunction. Higher concentrations of the inhibitor might be required to improve the retinal functional outcome.
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