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Thomas Vincent Johnson, Ericka N Oglesby, Matthew R Steinhart, Elizabeth Cone-Kimball, Joan Jefferys, Harry A Quigley; Time-lapse retinal ganglion cell dendritic field degeneration imaged in organotypic retinal explant culture. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© ARVO (1962-2015); The Authors (2016-present)
To develop an ex vivo organotypic retinal explant culture system suitable for multiple time-point imaging of living retinal ganglion cell (RGC) dendritic arbors over a period of 1 week, and capable of detecting dendrite neuroprotection conferred by experimental treatments.
Thy1-YFP mouse retinas were explanted and maintained in organotypic culture. RGC dendritic arbors were imaged repeatedly using confocal laser scanning microscopy. Maximal projection z-stacks were traced by two masked investigators and dendritic fields were analyzed for characteristics including branch number, size, and complexity. One group of explants was treated with BDNF+CNTF added to the culture media. Changes in individual dendritic fields over time were detected using pair-wise comparison testing.
RGCs in mouse retinal explant culture began to degenerate after 3 days with 52.4% surviving at 7 days. Dendritic field parameters showed minimal change over 8 hours in culture. Intra-observer and inter-observer measurements of dendrite characteristics were strongly correlated (Spearman rank correlations consistently >0.80). Statistically significant (p<0.001) dendritic tree degeneration was detected following seven days in culture including: 40-50% decreases in number of branch segments, number of junctions, number of terminal branches, and total branch length. Scholl analyses similarly demonstrated a significant decrease in dendritic field complexity. Treatment of explants with BDNF + CNTF significantly attenuated dendritic field degeneration.
Retinal explant culture of Thy1-YFP tissue provides a useful model for time-lapse imaging of RGC dendritic field degeneration over a course of several days, and is capable of detecting neuroprotective amelioration of dendritic pruning.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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