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Updated: Aug 18, 2026
The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions
Published on: November 14, 2016
In vitro models differentiating between direct and indirect effects of ischemia on astrocytes
C K Petito1, B H Juurlink, L Hertz
1Department of Pathology (Neuropathology), New York Hospital, Cornell University Medical College, New York 10021.
Abstract:
Mouse astrocytes in primary cultures were subjected to an in vitro model of ischemia (hypoxia combined with substrate deprivation, excess potassium, or elevated glutamate) and examined with the light (phase) and electron microscope. Three hours of hypoxia alone or in combination with the other insults had little effect upon the morphology of astrocytes but did cause disaggregation of polyribosomes. With reoxygenation, polyribosomes reformed and many mitochondria changed from the orthodox to the condensed configuration. Notably, there was little swelling. Excess (50 mM) potassium, added (as KCl) to a normal isotonic medium, also caused no swelling. However, when 50 mM potassium was substituted for a similar amount of sodium, marked astrocyte swelling did occur. A morphologically similar swelling was seen when glutamate (50 microM to 1 mM) was added to the culture medium, both with or without hypoxia with or without substrate deprivation. Potassium or glutamate-induced swelling was reversible with 1 h of recovery in normal medium. These results show that alterations in postischemic astrocytic morphology in vivo to a large extent can be reproduced in astrocytes in primary cultures. In addition, they suggest that postischemic astrocyte swelling is related to alterations in extracellular milieu, including accumulation of glutamate and/or alterations in the potassium/sodium ratios with increased potassium and decreased sodium. In contrast, morphologic alterations in polyribosomes and in mitochondria appear to be a direct response to ischemia itself.
Insights
Astrocytes swell during ischemia due to changes in extracellular ions like potassium and glutamate, not just hypoxia. This swelling is reversible, mimicking in vivo conditions for studying brain injury.
Area of Science:
- Neuroscience
- Cell Biology
- Pathology
Background:
- Astrocytes play crucial roles in central nervous system homeostasis.
- Ischemic conditions can lead to significant cellular damage in the brain.
- Understanding astrocyte responses to ischemia is vital for developing neuroprotective strategies.
Purpose of the Study:
- To investigate the in vitro morphological changes in mouse astrocytes under ischemic conditions.
- To determine the specific roles of hypoxia, potassium, and glutamate in astrocyte swelling.
- To establish a reliable in vitro model for studying post-ischemic astrocyte alterations.
Main Methods:
- Primary mouse astrocyte cultures were subjected to in vitro ischemia models.
- Ischemic conditions included hypoxia, substrate deprivation, excess potassium, and elevated glutamate.
- Cell morphology was examined using light (phase) and electron microscopy.
Main Results:
- Hypoxia alone caused polyribosome disaggregation, which reformed upon reoxygenation.
- Astrocyte swelling occurred when potassium replaced sodium or when glutamate was added.
- Potassium- or glutamate-induced swelling was reversible after 1 hour of recovery.
Conclusions:
- In vitro models can effectively reproduce in vivo post-ischemic astrocyte morphological changes.
- Post-ischemic astrocyte swelling is primarily linked to extracellular milieu alterations (glutamate, K+/Na+ ratio).
- Changes in polyribosomes and mitochondria are direct responses to ischemic insults.
