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Sarah Anderson, Monica L Vetter; The Role of Microglia During Retinal Neurogenesis. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2225.
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© ARVO (1962-2015); The Authors (2016-present)
While microglia are present at the onset of retinal neurogenesis, their function in the developing mammalian retina is unknown. In other systems there is evidence microglia modulate neurogenesis, but the results are contradictory. To address this, we will determine whether retinal microglia regulate progenitor proliferation, differentiation, and/or survival by inhibiting microglia during embryonic retinal development.
To inhibit microglia, pregnant mouse dams were exposed to 120mg/kg minocycline by oral gavage from embryonic day e12.5-e15.5. Controls received vehicle alone. Retinas from Cx3cr1GFP/+ embryos at e16.5 were dissected, embedded, sectioned, and immunostained. Progenitors (pH3+), retinal ganglion cells (RGCs, Brn3+), and apoptotic cells (cleaved casp3+) were counted on every 4th section through the entire retina (average±SEM). RNA was isolated from e16.5 whole retina using a Qiagen RNeasy kit and reverse transcribed. Relative gene expression was determined following RT-qPCR after normalization to beta actin. For all experiments, biological replicates equaled n≥3, and an unpaired students t-test was used to determine statistical significance.
Minocycline treatment inhibited microglia, resulting in a 60% reduction in Iba1 mRNA (p<0.01). The number of pH3+ progenitors per section was decreased 30% (p<0.01), and levels of Ki67 mRNA were also reduced by 25% (p<0.01). The density of RGCs (cells/mm2) was increased 25% after minocycline treatment versus controls (p<0.01). In addition, the number of apoptotic cells was increased by 82% (p<0.01), but the majority of these were Brn3- and residing outside of the differentiated cell layer, suggesting they were not RGCs. To determine the mechanism, several candidates known to regulate neurogenesis were evaluated for differential expression. In preliminary experiments, Tnfα, Igf1, and Tgfβ1 transcripts were expressed and reduced after treatment with minocycline.
Thus, inhibition of microglia results in reduced progenitor proliferation and increased RGC differentiation, and may also cause diminished progenitor survival. While the mechanisms remain unknown, several candidate signaling systems were altered. These results suggest microglia are active participants in retinal neurogenesis, and are important in regulating the production of retinal neurons. Future studies will assess the loss of microglia on neurogenesis and test identified candidate signaling systems.
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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