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Paul Cullen, John G Flanagan; A Novel Method Facilitates Acute Isolation of Rat Retinal Astrocytes. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4396.
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© ARVO (1962-2015); The Authors (2016-present)
To demonstrate a novel method for acute isolation of retinal astrocytes, in a rat model. The limited number of astrocytes and the overlap of their cell surface antigens with other retinal cell types has stymied previous efforts at acute isolation and lead to a reliance on cultured cells that may not accurately reflect in vivo functionality.
Eyes from 4-5 week-old female Wistar rats (N=6) were enucleated and the retinas dissected, quartered and flat mounted on filter paper substrate. A 12mm glass coverslip was placed over the retinal quarters, which were then inverted, treated with collagenase, and incubated at 37° C. After incubation, coverslips were gently removed, resulting in mechanical separation of nerve-fiber layer astrocytes and associated capillaries from the bulk of the retina. Isolated tissue was processed for immunofluorescence microscopy, with GFAP and morphology as markers of astrocyte identity and Hoechst 33342 used as a nuclear counterstain for all cells. Astrocyte yield was quantified via ImageJ using a 1mm2 region from the mid-periphery of each retina.
Mean astrocyte yield per quantification region (N=6) was 220 astrocytes/mm2 (SD ± 67) and ranged from a low of 109/mm2 to a high of 288/mm2. At the high end of the range astrocyte coverage of the region was nearly continuous, except for area occupied by blood vessels, and connections formed by astrocyte processes were preserved.
We show that it is possible to reproducibly isolate astrocytes from the inner retina, separating them from the bulk of tissue, including the Müller cells with which they share a variety of the cell surface markers commonly used to prospectively isolate brain astrocytes. This has enabled the investigation of acutely isolated cells for functional and transcriptional studies.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
Acutely isolated rat retinal astrocytes and associated blood vessels. Sample stained with anti-GFAP (in green) indicating astrocytes and Hoechst 33342 nuclear stain (blue). Capillaries are not stained, but identified by ensheathment by astrocyte processes. Scale bar = 50 µm
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