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Protocol for analysis of senescent neuronal stem cells in genetic-modified embryonic mice using in utero electroporation technique.

ABSTRACT: Here, we present a detailed protocol for identification and analysis of senescent neuronal stem cells (NSCs) in the developing mouse embryonic neocortex using in utero electroporation. We describe the procedures of in utero electroporation and brain slide preparation. We then detail the procedures of senescence-associated β-galactosidase (SA-β-Gal) staining and immunofluorescence staining, followed by microscope imaging analysis. This combined approach can be used to study the in situ senescence of in utero electroporated embryonic brains. For complete details on the use and execution of this protocol, please refer to Zhu et al. (2021).

SUBMITTER: Chen L

PROVIDER: S-EPMC9201042 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Protocol for analysis of senescent neuronal stem cells in genetic-modified embryonic mice using in utero electroporation technique.

Chen Liping L Li Ying Y Wu Yan Y Li Shen S Chang Xin X Wu Haitao H

STAR protocols 20220613 3

Here, we present a detailed protocol for identification and analysis of senescent neuronal stem cells (NSCs) in the developing mouse embryonic neocortex using in utero electroporation. We describe the procedures of in utero electroporation and brain slide preparation. We then detail the procedures of senescence-associated β-galactosidase (SA-β-Gal) staining and immunofluorescence staining, followed by microscope imaging analysis. This combined approach can be used to study the i ...[more]

PMID: 35719723

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Project description:BACKGROUND:Since its inception in 2001, in utero electroporation (IUE) has been widely used by the neuroscience community. IUE is a technique developed to introduce plasmid DNA into embryonic mouse brains without permanently removing the embryos from the uterus. Given that IUE labels cells that line the ventricles, including radial fibers and migrating neuroblasts, this technique is an excellent tool for studying factors that govern neural cell fate determination and migration in the developing mouse brain. Whether IUE has an effect on microglia, the immune cells of the central nervous system (CNS), has yet to be investigated. METHODS:We used IUE and the pCIG2, pCIC-Ascl1, or pRFP-C-RS expression vectors to label radial glia lining the ventricles of the embryonic cortex and/or hypothalamus. Specifically, we conducted IUE at E14.5 and harvested the brains at E15.5 or E17.5. Immunohistochemistry, along with cytokine and chemokine analyses, were performed on embryonic brains with or without IUE exposure. RESULTS:IUE using the pCIG2, pCIC-Ascl1, or pRFP-C-RS vectors alone altered microglia morphology, where the majority of microglia near the ventricles were amoeboid and displayed altered expression signatures, including the upregulation of Cd45 and downregulation of P2ry12. Moreover, IUE led to increases in P2ry12- cells that were Iba1+/IgG+ double-positive in the brain parenchyma and resembled macrophages infiltrating the brain proper from the periphery. Furthermore, IUE resulted in a significant increase in cell death in the developing hypothalamus, with concomitant increases in cytokines and chemokines known to be released during pro-inflammatory states (IL-1β, IL-6, MIP-2, RANTES, MCP-1). Interestingly, the cortex was protected from elevated cell death following IUE, implying that microglia that reside in the hypothalamus might be particularly sensitive during embryonic development. CONCLUSIONS:Our results suggest that IUE might have unintended consequences of activating microglia in the embryonic brain, which could have long-term effects, particularly within the hypothalamus.

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Protocol for analysis of senescent neuronal stem cells in genetic-modified embryonic mice using in utero electroporation technique.

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Protocol for analysis of senescent neuronal stem cells in genetic-modified embryonic mice using <i>in utero</i> electroporation technique.

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