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Research article
Annemarie Shibata
Creighton University
Corresponding Author
ORCiD: https://orcid.org/0000-0003-2451-3065
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Background: Emerging evidence suggests that microglia can support neuronal survival, synapse development, and neurogenesis in classic neurogenic niches. Little is known about the ability of microglia to regulate the cortical environment and stimulate cortical neurogenesis outside classic neurogenic niches. We used an in vitro co-culture model system to investigate the hypothesis that microglia respond to soluble signals from cortical cells, particularly following injury, to alter the cortical environment and promote cortical cell proliferation, differentiation, and maintain cortical cell survival via activation of specific cortical intracellular signaling pathways.
Results: Analyses of cell proliferation, apoptosis, protein expression, and intracellular signaling pathway activation were performed on uninjured and injured cortical cells in co-culture with EOC2 microglia. EOC2 microglia in co-culture enhanced cortical cell viability and proliferation of uninjured and injured cortical cells. Co-culture of injured cortical cells with EOC2 microglial cells significantly reduced cortical cell apoptosis. Microglial co-culture significantly increased Nestin+ and a-internexin+ cells within and outside of the injury site. NeuN+ cells increased in injured cortical cultures with microglia. Multiplex ELISA assays showed decreased levels of inflammatory cytokines in conditioned media from injured cortical cell and microglial co-culture. RTPCR analysis of microglial mRNA was performed. EOC2 microglial co-culture environment increased AKT phosphorylation in cortical cells particularly following injury. Inhibition of AKT phosphorylation in cortical cells blocked the microglial-enhanced cortical cell viability and expression of neurogenic markers in vitro.
Conclusion: The in vitro model system presented here allows for assessment of the effect of microglial-derived soluble signals, independent of cell-cell contact, on cortical cell viability, proliferation, and stages of differentiation during homeostasis or following injury. These data suggest that microglia downregulate inflammatory cytokine production following activation by acute cortical injury to enhance proliferation of new cells capable of neurogenesis. Inhibition of AKT signaling in cortical cells blocks the enhanced proliferation and expression of neurogenic markers in cortical cells. Increasing our understanding of the mechanisms that drive cortical neurogenesis stimulated by microglial cells during homeostasis and following injury will provide insight into the potential mechanisms of neuroprotective role of immune activity in the central nervous system (CNS).
Keywords
microglia, neurogenesis, PI3K/AKT
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research article
Annemarie Shibata
Creighton University
Corresponding Author
ORCiD: https://orcid.org/0000-0003-2451-3065
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
The most recent version of this article is available here.
No community comments so far
Version 2
Posted 11 Nov, 2019
No comments provided
No comments provided
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Cellular & Molecular Neuroscience
License:
This work is licensed under a CC BY 4.0 License. Read Full License
The most recent version of this article is available here.
Background: Emerging evidence suggests that microglia can support neuronal survival, synapse development, and neurogenesis in classic neurogenic niches. Little is known about the ability of microglia to regulate the cortical environment and stimulate cortical neurogenesis outside classic neurogenic niches. We used an in vitro co-culture model system to investigate the hypothesis that microglia respond to soluble signals from cortical cells, particularly following injury, to alter the cortical environment and promote cortical cell proliferation, differentiation, and maintain cortical cell survival via activation of specific cortical intracellular signaling pathways.
Results: Analyses of cell proliferation, apoptosis, protein expression, and intracellular signaling pathway activation were performed on uninjured and injured cortical cells in co-culture with EOC2 microglia. EOC2 microglia in co-culture enhanced cortical cell viability and proliferation of uninjured and injured cortical cells. Co-culture of injured cortical cells with EOC2 microglial cells significantly reduced cortical cell apoptosis. Microglial co-culture significantly increased Nestin+ and a-internexin+ cells within and outside of the injury site. NeuN+ cells increased in injured cortical cultures with microglia. Multiplex ELISA assays showed decreased levels of inflammatory cytokines in conditioned media from injured cortical cell and microglial co-culture. RTPCR analysis of microglial mRNA was performed. EOC2 microglial co-culture environment increased AKT phosphorylation in cortical cells particularly following injury. Inhibition of AKT phosphorylation in cortical cells blocked the microglial-enhanced cortical cell viability and expression of neurogenic markers in vitro.
Conclusion: The in vitro model system presented here allows for assessment of the effect of microglial-derived soluble signals, independent of cell-cell contact, on cortical cell viability, proliferation, and stages of differentiation during homeostasis or following injury. These data suggest that microglia downregulate inflammatory cytokine production following activation by acute cortical injury to enhance proliferation of new cells capable of neurogenesis. Inhibition of AKT signaling in cortical cells blocks the enhanced proliferation and expression of neurogenic markers in cortical cells. Increasing our understanding of the mechanisms that drive cortical neurogenesis stimulated by microglial cells during homeostasis and following injury will provide insight into the potential mechanisms of neuroprotective role of immune activity in the central nervous system (CNS).
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
This is a list of supplementary files associated with this preprint. Click to download.
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