Article
Environmental effect on microglial cell depletion in organotypic brain slice cultures
Einfluss auf die Depletion von Mikrogliazellen in organotypischen Hirnschnittkulturen
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Authors
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Vidhya Madapusi Ravi
- Medical Center, University of Freiburg, Translational NeuroOncology Research Group, Freiburg, Deutschland; Medical Center, University of Freiburg, Department of Neurosurgery, Freiburg, Deutschland; Medical Center, University of Freiburg, Neuroelectronic Systems, Freiburg, Deutschland; Medical Center, University of Freiburg, Freiburg, Deutschland
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Kevin Joseph
- Medical Center, University of Freiburg, Translational NeuroOncology Research Group, Freiburg, Deutschland; Medical Center, University of Freiburg, Department of Neurosurgery, Freiburg, Deutschland; Medical Center, University of Freiburg, Freiburg, Deutschland
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Jürgen Beck
- Medical Center, University of Freiburg, Translational NeuroOncology Research Group, Freiburg, Deutschland; Medical Center, University of Freiburg, Department of Neurosurgery, Freiburg, Deutschland; Medical Center, University of Freiburg, Freiburg, Deutschland
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Ulrich Hofmann
- Medical Center, University of Freiburg, Department of Neurosurgery, Freiburg, Deutschland; Medical Center, University of Freiburg, Neuroelectronic Systems, Freiburg, Deutschland; Medical Center, University of Freiburg, Freiburg, Deutschland
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Oliver Schnell
- Medical Center, University of Freiburg, Translational NeuroOncology Research Group, Freiburg, Deutschland; Medical Center, University of Freiburg, Department of Neurosurgery, Freiburg, Deutschland; Medical Center, University of Freiburg, Freiburg, Deutschland
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Dieter Henrik Heiland
- Medical Center, University of Freiburg, Translational NeuroOncology Research Group, Freiburg, Deutschland; Medical Center, University of Freiburg, Department of Neurosurgery, Freiburg, Deutschland; Medical Center, University of Freiburg, Freiburg, Deutschland
| Published: | June 4, 2021 |
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Outline
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Objective: Microglia are immunocompetent cells of the central nervous system (CNS). Uncovering the role of microglia under pathologic conditions is of high importance. Chlodronate Disodium is commonly used to deplete microglia in the neural microenvironment; however, it is still unclear whether the treatment induces damages to other brain cells. Here, we have characterized the effect of microglia depletion in human ex-vivo cultures using imaging and sequencing modalities.
Methods: Non-neoplastic human neocortical tissue obtained as entry cortex during GBM surgery was sectioned and cultured for up to 10 days. A microglia depletion model was created by supplementing the medium with 11 mM clodronate-di-sodium for 72 hours. The depleted sections were cultured for an additional 7 days to characterize microglia regeneration post depletion. Finally, primary glioblastoma cell lines were inoculated into both control and depleted sections to study the role of microglia in glioblastoma proliferation.
Results: Our data shows that 72 hours of clodronate treatment is necessary for the depletion of microglia in 300 µm thick human organotypic sections. We demonstrate that depletion leads to no significant reduction in neuronal cells but results in an increase in number of astrocytes over the culture period. RNA-sequencing revealed increased enrichment of astrocytic gene expression, however without a defined reactive signature. This increase of astrocytes was unique for human tissue and not observed in murine cortical tissue. Post depletion, no regeneration or repopulation of microglia was observed. Glioblastoma growth was significantly impaired in the absence of microglia. These data clearly indicates that microglia-glioblastoma interactions help promote tumor growth and migration by maintaining astrocytic functions in addition to clearing debris within the tumor microenvironment.
Conclusion: Our data showed that clodronate-di-sodium salt sufficiently deplete microglia but also lead to increased number of astrocytes which has to be taken into consideration for further use of this method. The important role of microglia for tumour growth was confirmed by our data.
