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J. P. Wood, G. Chidlow, T. Mammone, R. J. Casson; Characteristics of Growth and Death of Adult Rat Retinal Ganglion Cells in Culture. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1887.
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© ARVO (1962-2015); The Authors (2016-present)
To perform a spatiotemporal characterisation of the growth of adult rat retinal ganglion cells in culture and to closely observe the responses of these cells to lethal influences as a prelude to assessing the protective or regenerative properties of test agents.
Retinal dissociates from 8 week old Sprague-Dawley rats were incubated in a medium containing the growth factors, BDNF, bFGF and CNTF (as previously characterised by Pang et al, BMC Neuroscience 8:11). Retinal ganglion cells were disseminated from other neurons by positive-immunolabelling for neurofilament (NFs), Thy 1.1, tau and MAP1b. Growth characteristics were determined by using a Biostation 1M live cell imaging system: measurements were recorded from these cells of neurite growth for up to 30 days. In order to determine the death characteristics of these cells in culture, they were treated with the excitotoxin, N-methyl-D-aspartate (NMDA), the apoptotic inducer, staurosporine (STSN), and with oxygen-glucose deprivation (OGD). As a test agent, the peptide factor, osteopontin (OPN) was also evaluated for influences on the latter parameters as well as on changes in immunolabelling of MAP2 and GAP43.
Retinal ganglion cells had observable neurite growth from day 1-2 in vitro (mean lengths of major neurites: days 0-3, 0-10µm; days 5-8, 25-50µm; days 12-30, 100-250µm). In most cases, an obvious axon was easily distinguishable from other neurites. Cells showed positive immunolabelling for NFs, tau, Thy 1.1 and MAP1b. Both NMDA and STSN caused a shrinking of cell perikarya which was followed by an rapid and striking retraction of major neurites just before a sudden cell body contraction characteristic of apoptotic death. OGD (60 minutes), conversely caused degeneration to occur concurrently throughout the the length of the neurites, 12-24 hours after replacing cells into normal growth conditions. The peptide factor, OPN caused cells to undertake an increased level of neurite formation and branching, an elevation in labelling for MAP2 and GAP43, and was able to delay death resulting from OGD, but not NMDA or STSN.
In the present system healthy ganglion cell growth and development was observed in culture for up to 30 days. Live imaging of induced death showed that cells died very differently depending on the lethal stimulus. These data, and those demonstrating that OPN was able to delay cell death may have implications for the treatment of diseases resulting from ganglion cell death in situ.
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