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Microglia Stimulation by Protein Extract of Injured Rat Spinal Cord. A Novel In vitro Model for Studying Activated
Joaquim Hernández1, Isaac Francos-Quijorna1, Elena Redondo-Castro1
1Group of Neuroplasticity and Regeneration, Department of Cell Biology, Physiology and Immunology, Institute of Neurosciences, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Universitat Autònoma de Barcelona, Red de Terapia Celular (TerCel), Bellaterra, Spain.
Abstract:
Research on microglia has established the differentiation between the so-called M1 and M2 phenotypes. However, new frameworks have been proposed attempting to discern between meaningful microglia profiles. We have set up an in vitro microglial activation model by adding an injured spinal cord (SCI) lysate to microglial cultures, obtained from postnatal rats, in order to mimic the environment of the spinal cord after injury. We found that under the presence of the SCI lysate microglial cells changed their phenotype, developing less ramified but longer processes, and proliferated. The SCI lysate also led to upregulation of pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, downregulation of the anti-inflammatory cytokines IL-10 and IL-4, and a biphasic profile of iNOS. In addition, a latex beads phagocytosis assay revealed the SCI lysate stimulated the phagocytic capacity of microglia. Flow cytometry analysis indicated that microglial cells showed a pro-inflammatory profile in the presence of SCI lysate. Finally, characterization of the microglial activation in the spinal cord on day 7 after contusion injury, we showed that these cells have a pro-inflammatory phenotype. Overall, these results indicate that the use of SCI lysates could be a useful tool to skew microglia towards a closer phenotype to that observed after the spinal cord contusion injury than the use of LPS or IFNγ.
Insights
Spinal cord injury (SCI) lysate effectively activates microglia, inducing a pro-inflammatory phenotype. This model offers a more accurate representation of in vivo microglial responses post-injury compared to traditional methods.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia, the immune cells of the central nervous system, are traditionally classified into M1 (pro-inflammatory) and M2 (anti-inflammatory) phenotypes.
- Emerging research suggests more complex microglial activation states beyond the M1/M2 paradigm, necessitating refined models for studying their roles in injury.
Purpose of the Study:
- To develop and validate an in vitro model using spinal cord injury (SCI) lysate to mimic the microenvironment following spinal cord injury.
- To characterize the phenotypic and functional changes in microglia exposed to SCI lysate.
Main Methods:
- Primary rat microglial cultures were treated with SCI lysate.
- Phenotypic changes, cytokine expression (IL-1β, IL-6, TNF-α, IL-10, IL-4), inducible nitric oxide synthase (iNOS) expression, phagocytic activity, and flow cytometry were analyzed.
- Microglial activation was also assessed in vivo in rat spinal cords 7 days after contusion injury.
Main Results:
- SCI lysate induced morphological changes in microglia, including reduced ramification and elongated processes, alongside proliferation.
- Exposure to SCI lysate upregulated pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and downregulated anti-inflammatory cytokines (IL-10, IL-4), with a biphasic iNOS profile.
- Microglia exhibited enhanced phagocytic capacity and a pro-inflammatory profile in vitro, consistent with in vivo findings post-SCI.
Conclusions:
- SCI lysate serves as a valuable tool for inducing a microglial phenotype closely resembling that observed after spinal cord contusion injury.
- This SCI lysate model provides a more physiologically relevant approach to studying microglial activation compared to using lipopolysaccharide (LPS) or interferon-gamma (IFNγ).
