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D2 Receptor Agonist Treatment Effects on Microglia Polarization Following Cerebral Ischemia in Mice

Undergraduate #63
Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology

Solana Liu - Emory University
Co-Author(s): Xiaohuan Gu, Department of Anesthesiology, Emory University School of Medicine, Atlanta, GA ; Shan Ping Yu, Department of Anesthesiology, Emory University School of Medicine, Atlanta, GA ; Ling Wei, Department of Neurology, Emory University School of Medicine, Atlanta, GA



Stroke, one of the most prevalent cause of death worldwide, affects 800,000 Americans each year and is the leading cause of long term disability with direct and indirect costs amounting to 36.5 billion annually in the United States. After early stages of stroke, a protracted inflammatory reaction occurs within a few hours. This includes the activation of microglia which respond to inflammation by producing inflammatory cytokines. Activated microglia morphologically shift and polarize on a spectrum ranging between pro-inflammatory (?M1?) and anti-inflammatory (?M2?) forms. The resultant activated phenotypes affect whether the microglia produce cytokines resulting in cytotoxic or neuroprotective effects. Thus, knowledge of M1 and M2 phenotypic ratios within appropriate time windows may provide appropriate therapeutic benefit.
Hypothesis: The purpose of this study was to investigate the effects of a highly selective dopamine 2 agonist Sumanirole on microglia polarization following cerebral ischemia in mice. We expected that dopamine would modulate microglial function during neuroinflammation.
Methods: 8 week old C57 male mice were subjected to transient distal MCA occlusion. Mice were sacrificed 3, 10, and 14 days after stroke. To investigate the effects of a D2 agonist, the D2 agonist Sumanirole was given intranasally at 1.5 mg/kg twice per day for 7 days beginning 3 days after stroke. Matching controls groups received saline. 10 days after stroke, mice were sacrificed, then their brains were perfused, flash frozen, and sliced into 10 um thick sections. Sections were stained for microglia (Iba1), D2 Receptor (D2R), iNOS (M1 marker), and CD206 (M2 marker) to note changes in microglia polarization. Cell counts were obtained from six non-overlapping images taken in the peri-infarct region of each brain section.
Results: Baseline microglia counts indicated that both M1 and M2 were observed up to 14 days after stroke. At 14 days, M1 and M2 microglia make up 27% and 22% of all microglia, respectively. We observed an increase in D2R expression in activated microglia and both M1 and M2 forms expressed D2R 3 days after stroke. Mice that received the D2 agonist treatment had increased number of M1 microglia 10 days after stroke. D2 agonist treatment did not appear to affect microglial distance from the ischemic core.
Discussion/Conclusion:
Our results indicate that prior to ischemia, microglia do not express D2R. After ischemia, both the pro-inflammatory M1 and anti-inflammatory M2 forms of microglia express D2R. D2 agonist treatment significantly increased number of M1 but not M2 microglia. This suggests that dopamine may exacerbate post-stroke inflammation.
Future Directions:
Future studies will further elucidate the immunomodulatory effects of dopamine after cerebral ischemia through infarct volume assessment and western blots, which will assess the presence of inflammatory and anti-inflammatory cytokines.

Not Submitted

Funder Acknowledgement(s): Emory University Summer Undergraduate Research Experience Program

Faculty Advisor: Ling Wei, lingwei886@gmail.com

Role: Highly trained technicians performed ischemic stroke surgeries on mice. I handled all other aspects of the project, which included transcardial perfusions, cryostat sectioning, immunohistochemistry, cell counting, and data analysis.

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This material is based upon work supported by the National Science Foundation (NSF) under Grant No. DUE-1930047. Any opinions, findings, interpretations, conclusions or recommendations expressed in this material are those of its authors and do not represent the views of the AAAS Board of Directors, the Council of AAAS, AAAS’ membership or the National Science Foundation.

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