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Published on: October 18, 2016
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Quantification of Proliferating and Mitotically Active Retinal Cells in Mice by Flow Cytometry
Hope K Vanzo-Sparks1, Sarah E Webster1, Mark K Webster1
1Western Michigan University, Department of Biological Sciences, Kalamazoo, MI, USA.
Bio-Protocol
|July 16, 2024
Summary
Adult mammals cannot regenerate retinal neurons. Topical application of PNU-282987, an alpha7 nicotinic acetylcholine receptor (nAChR) agonist, promotes retinal neuron proliferation and aids in developing a new flow cytometry method for retinal cell analysis.
Area of Science:
- Neuroscience
- Cell Biology
- Ophthalmology
Background:
- Adult mammalian retinas typically lack the capacity for neuronal regeneration after injury.
- Previous research indicated that topical application of PNU-282987, a selective alpha7 nicotinic acetylcholine receptor (nAChR) agonist, increases retinal neuron numbers in adult mice.
- Studies using fluorescent immunolabeling showed increased mitotic activity in retinal cells following alpha7 nAChR agonist treatment, but this method has multiplexing limitations.
Purpose of the Study:
- To develop and describe a novel flow cytometry protocol for multiplexed analysis of dissociated retinal cells.
- To enable concurrent labeling and analysis of multiple retinal cell types and progenitor cells.
- To investigate the effects of PNU-282987 on retinal cell proliferation and neurogenesis in adult mammals.
Main Methods:
- A step-by-step protocol for dissociating adult mouse retinae and preparing cells for flow cytometry.
- Utilized bromodeoxyuridine (BrdU) incorporation to label proliferating cells.
- Employed flow cytometry with multiplexed antibody labeling to analyze retinal ganglion cells, rod photoreceptors, Müller glia, and Müller glia-derived progenitor cells.
Main Results:
- Successfully established a flow cytometry method for analyzing multiple markers in dissociated retinal cells.
- Demonstrated the ability to label and differentiate various retinal cell populations, including progenitor cells.
- Confirmed PNU-282987 treatment significantly increases mitotically active and proliferating cells in the adult retina.
Conclusions:
- The developed flow cytometry protocol offers a powerful tool for high-throughput analysis of retinal cell populations and neurogenesis.
- Topical application of the alpha7 nAChR agonist PNU-282987 effectively induces proliferation of retinal progenitor cells in adult mammals.
- This approach provides a foundation for further research into retinal regeneration strategies targeting Müller glia-derived progenitors.
