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C. Taki, Y. Shinohara, M. Hosoi, M. Nagahara, M. Nakatani, K. Otsuki, S. Nishimura; A Perfusion Culture System for Whole Retina From Adult Rat: An in–vitro Model for Neuroprotection on Retinal Ganglion Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4805.
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The present study was performed to establish a perfusion culture system for whole retinas from adult rats using gradient culture containers and to assess hypoxic damage on retinal ganglion cells (RGCs).
RGCs were labeled retrogradely with 1,1’–Dioctadecyl–3,3,3’,3’–tetramethylindocarbocyanine perchlorate (DiI), a retrograde fluorescent tracer, by injecting into both superior colliculi of adult Wistar/ST rats. Seven days after the DiI injection, the eyes were enucleated and whole retinas laid between two aluminum oxide membranes in specially–designed tissue carriers. The carriers were then placed in gradient culture containers (MINUCELLS and MINUTISSUE) and constantly perfused with Neurobasal–A medium containing B27 and glutamine on both sides of the entire tissues. Cultures were kept at 37 °C for 14 days. The control normoxic group was kept in 5% CO2 and 95% air, and hypoxic test groups were exposed to 5% oxygen for various periods (3, 7 or 14 days). RGC densities were determined at several time points within the same regions in a middle portion of cultivated retinas, by counting labeled RGCs in digital images of the retinas. As a measure of RGCs survival, total areas of DiI–labeled cells in each image were also calculated with image analysis software.
In the normoxic group, RGC density was 2,090 ± 463 cells/mm2 (mean ± SD; n=6) at Day 0 and 86.7%, 82.7% and 73.1% of them had survived at Day 3, 7 and 14, respectively. The retinal architecture was well preserved by this culture system. Hypoxia for 14 days led to a significant decrease of RGCs survival compared to that of the control normoxic condition.
This culture system resulted in a high viability of RGCs of an adult rat retina for a long term, and preservation of a whole retina. These culture systems under normoxic and hypoxic conditions are useful to examine neuroprotection on RGCs in the adult retina.
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