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Updated: Nov 29, 2025
Isolation and Flow Cytometric Assessment of Neuroimmune Interactions in a Mini-Stroke Murine Model
Published on: June 20, 2025
Transcriptomic characterization of microglia activation in a rat model of ischemic stroke
Wenjun Deng1,2, Emiri Mandeville1, Yasukazu Terasaki1
1Neuroprotection Research Laboratories, Departments of Radiology and Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA.
Abstract:
Microglia are key regulators of inflammatory response after stroke and brain injury. To better understand activation of microglia as well as their phenotypic diversity after ischemic stroke, we profiled the transcriptome of microglia after 75 min transient focal cerebral ischemia in 3-month- and 12-month-old male spontaneously hypertensive rats. Microglia were isolated from the brains by FACS sorting on days 3 and 14 after cerebral ischemia. GeneChip Rat 1.0ST microarray was used to profile the whole transcriptome of sorted microglia. We identified an evolving and complex pattern of activation from 3 to 14 days after stroke onset. M2-like patterns were extensively and persistently upregulated over time. M1-like patterns were only mildly upregulated, mostly at day 14. Younger 3-month-old brains showed a larger microglial response in both pro- and anti-inflammatory pathways, compared to older 12-month-old brains. Importantly, our data revealed that after stroke, most microglia are activated towards a wide spectrum of novel polarization states beyond the standard M1/M2 dichotomy, especially in pathways related to TLR2 and dietary fatty acid signaling. Finally, classes of transcription factors that might potentially regulate microglial activation were identified. These findings should provide a comprehensive database for dissecting microglial mechanisms and pursuing neuroinflammation targets for acute ischemic stroke.
Insights
Microglia activation after ischemic stroke shows complex patterns, with younger brains exhibiting a stronger inflammatory response. Most microglia adopt novel states beyond M1/M2, highlighting new therapeutic targets for brain injury.
Area of Science:
- Neuroscience
- Immunology
- Genomics
Background:
- Microglia are crucial in regulating brain inflammation following stroke and injury.
- Understanding microglial activation and diversity is key to developing stroke treatments.
Purpose of the Study:
- To profile the transcriptome of microglia after transient focal cerebral ischemia in rats of different ages.
- To investigate microglial phenotypic diversity and activation patterns post-stroke.
Main Methods:
- Transient focal cerebral ischemia was induced in 3- and 12-month-old spontaneously hypertensive rats.
- Microglia were isolated using Fluorescence-Activated Cell Sorting (FACS) on days 3 and 14 post-stroke.
- Whole transcriptome profiling was performed using GeneChip Rat 1.0ST microarray.
Main Results:
- A complex, evolving microglial activation pattern was observed from 3 to 14 days post-stroke.
- M2-like patterns were persistently upregulated, while M1-like patterns showed mild upregulation at day 14.
- Younger rats displayed a more robust microglial response compared to older rats.
- Microglia exhibited novel polarization states beyond the M1/M2 dichotomy, involving TLR2 and fatty acid signaling pathways.
Conclusions:
- Microglial activation post-stroke is dynamic and diverse, extending beyond classical M1/M2 phenotypes.
- Age influences the magnitude of microglial response to ischemic stroke.
- Identification of novel pathways and transcription factors offers potential therapeutic targets for neuroinflammation in stroke.
