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C. Taki, Y. Shinohara, M. Hosoi, M. Nakatani, K. Ohtsuki, S. Nishimura; Histological Changes of Adult Rats' Whole Retinas in Perfusion Culture. Invest. Ophthalmol. Vis. Sci. 2007;48(13):48.
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The present study was performed to investigate temporal changes in histology and RGC survival of adult rats' whole retinas in perfusion culture.
RGCs were labeled retrogradely with 1,1’-Dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiI) 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. After cultivation for different periods (1, 3, 7 or 14 days), the retinas were fixed and sectioned for HE staining, TUNEL staining, and immunohistochemistry. Antibodies against Thy-1.1, phosphorylated neurofilament protein (pNF-H), and glutamine synthetase (GS) were used. As a measure of RGC survival, total areas of DiI-labeled cells were calculated with image analysis software within the same regions in a middle portion of the cultivated retinas at several time points in 14 days.
The retinal architecture was well preserved in this culture system for 14 days, although pyknotic nuclei were found in the ganglion cell layer (GCL) and inner nuclear layer (INL) and increased over time. TUNEL-positive cells were detected in the GCL, INL and outer nuclear layer (ONL) of the retinas at Day 14. Thy-1.1 immunoreactivity was found in the nerve fiber layer (NFL), GCL, and inner plexiform layer (IPL) at Day 0. Staining of the IPL decreased after 1 day of cultivation, whereas staining of the NFL and GCL continuously decreased to low levels by 3 to 14 days of cultivation. The expression of pNF-H was found in the NFL at Day 0, started to reduce after 3 days in culture, and was faint at Day 14. GS immunoreactivity was observed consistently throughout the cultivation in Müller cell bodies located in the INL and in their processes in the inner and outer limiting membranes. RGC survival decreased slowly over 14 days (compared with Day1: 97.6% at Day 3, 90.2% at Day 7, and 84.4% at Day 14).
This perfusion culture system well preserved adult rats' whole retina for 2 weeks. The expressions of the RGC markers (Thy-1.1 and pNF-H), however, decreased prior to the loss of cell bodies. These results suggest that the degeneration of RGCs in cultivated retinas occur at early stages in perfusion culture.
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