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Determining glioma cell invasion and proliferation in ex vivo organotypic mouse brain slices using whole-mount immunostaining and tissue clearing.

ABSTRACT: The ex vivo organotypic brain slice invasion model is commonly used to study the growth dynamics of gliomas, primary brain tumors that are known for their invasive behavior. Here, we describe a protocol where the ex vivo organotypic mouse brain slice invasion model is combined with whole-mount immunostaining, tissue clearing, and 3D reconstruction, to visualize and quantify the invasion of glioma cells. In addition, we describe an approach to determine the proliferation rate of the cells within this model. For complete details on the use and execution of this protocol, please refer to Uceda-Castro et al. (2022).

SUBMITTER: van Asperen JV

PROVIDER: S-EPMC9508478 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Determining glioma cell invasion and proliferation in ex vivo organotypic mouse brain slices using whole-mount immunostaining and tissue clearing.

van Asperen Jessy V JV van Bodegraven Emma J EJ Robe Pierre A J T PAJT Hol Elly M EM

STAR protocols 20220921 4

The ex vivo organotypic brain slice invasion model is commonly used to study the growth dynamics of gliomas, primary brain tumors that are known for their invasive behavior. Here, we describe a protocol where the ex vivo organotypic mouse brain slice invasion model is combined with whole-mount immunostaining, tissue clearing, and 3D reconstruction, to visualize and quantify the invasion of glioma cells. In addition, we describe an approach to determine the proliferation rate of the cells within ...[more]

PMID: 36136755

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Project description:BackgroundEpilepsy is a prevalent neurological disorder worldwide. It is characterized by an enduring predisposition to generate seizures and its development is accompanied by alterations in many cellular processes. Organotypic slice cultures represent a multicellular environment with the potential to assess biological mechanisms, and they are used as a starting point for refining molecules for in vivo studies. Here, we investigated organotypic slice cultures as a model of epilepsy.MethodsWe assessed, by electrophysiological recordings, the spontaneous activity of organotypic slices maintained under different culture protocols. Moreover, we evaluated, through molecular-based approaches, neurogenesis, neuronal death, gliosis, expression of proinflammatory cytokines, and activation of NLRP3 inflammasome (nucleotide-binding, leucine-rich repeat, pyrin domain) as biomarkers of neuroinflammation.ResultsWe demonstrated that organotypic slices, maintained under a serum deprivation culture protocol, develop epileptic-like activity. Furthermore, throughout a comparative study with slices that do not depict any epileptiform activity, slices with epileptiform activity were found to display significant differences in terms of inflammation-related features, such as (1) increased neuronal death, with higher incidence in CA1 pyramidal neurons of the hippocampus; (2) activation of astrocytes and microglia, assessed through western blot and immunohistochemistry; (3) upregulation of proinflammatory cytokines, specifically interleukin-1β (IL-1β), interleukin-6, and tumor necrosis factor α, revealed by qPCR; and (4) enhanced expression of NLRP3, assessed by western blot, together with increased NLRP3 activation, showed by IL-1β quantification.ConclusionsThus, organotypic slice cultures gradually deprived of serum mimic the epileptic-like activity, as well as the inflammatory events associated with in vivo epilepsy. This system can be considered a new tool to explore the interplay between neuroinflammation and epilepsy and to screen potential drug candidates, within the inflammatory cascades, to reduce/halt epileptogenesis.

Dataset Information

Determining glioma cell invasion and proliferation in ex vivo organotypic mouse brain slices using whole-mount immunostaining and tissue clearing.

Publications

Determining glioma cell invasion and proliferation in ex vivo organotypic mouse brain slices using whole-mount immunostaining and tissue clearing.

Similar Datasets

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