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Updated: Jul 18, 2025
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Published on: May 4, 2015
M2 Microglial Extracellular Vesicles Attenuated Blood-brain Barrier Disruption via MiR-23a-5p in Cerebral Ischemic
Jia-Ji Pan1,2, Lin Qi2, Liping Wang3
1Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai 200040, China.
Abstract:
Protecting the integrity of the blood-brain barrier (BBB) is crucial for maintaining brain homeostasis after ischemic stroke. Previous studies showed that M2 microglial extracellular vesicles (EVs) played a neuroprotective role in cerebral ischemia. However, the role of M2 microglial EVs in maintaining BBB integrity is unclear. Therefore, we explored the mechanisms of M2 microglial EVs in regulating BBB integrity. To identify microglial EVs, we used nanoparticle tracking analysis, transmission electron microscopy, and western blot analysis. Adult male ICR mice were subjected to 90-min middle cerebral artery occlusion (MCAO), followed by the injection of PKH26-labeled M2 microglial EVs via the tail vein. After MCAO, we assessed brain infarct and edema volume, as well as modified neurological severity score. BBB integrity was measured by assessing IgG leakage. The effects of M2 microglial EVs on astrocytes and endothelial cells were also examined. To investigate the molecular mechanisms, we performed RNA sequencing, miR-23a-5p knockdown, and luciferase reporter assays. Our results showed that PKH26-labeled microglial EVs were mainly taken up by neurons and glial cells. M2 microglial EVs treatment decreased brain infarct and edema volume, modified neurological severity score, and IgG leakage, while increasing the ZO-1, occludin, and claudin-5 expression after MCAO. Knockdown of miR-23a-5p reversed these effects. RNA sequencing revealed that the TNF, MMP3 and NFκB signaling pathway involved in regulating BBB integrity. Luciferase reporter assay showed that miR-23a-5p could bind to the 3' UTR of TNF. M2 microglial EVs-derived miR-23a-5p decreased TNF, MMP3 and NFκB p65 expression in astrocytes after oxygen-glucose deprivation, thereby increasing ZO-1 and Claudin-5 expression in bEnd.3 cells. In conclusion, our findings demonstrated that M2 microglial EVs attenuated BBB disruption after cerebral ischemia by delivering miR-23a-5p, which targeted TNF and regulated MMP3 and NFκB p65 expression.
Insights
M2 microglial extracellular vesicles (EVs) protect the blood-brain barrier (BBB) after stroke by delivering miR-23a-5p. This microRNA targets TNF, reducing inflammation and preserving BBB integrity.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Maintaining blood-brain barrier (BBB) integrity is vital for brain recovery after ischemic stroke.
- M2 microglial extracellular vesicles (EVs) show neuroprotective potential, but their role in BBB integrity post-stroke is not fully understood.
Purpose of the Study:
- To investigate the mechanisms by which M2 microglial EVs regulate BBB integrity following cerebral ischemia.
- To identify specific molecular players involved in the protective effects of M2 microglial EVs.
Main Methods:
- Characterization of microglial EVs using nanoparticle tracking analysis, transmission electron microscopy, and western blot.
- In vivo studies involving middle cerebral artery occlusion (MCAO) in mice treated with M2 microglial EVs.
- Assessment of BBB integrity via IgG leakage, infarct volume, and neurological scores.
- Molecular analyses including RNA sequencing, miR-23a-5p knockdown, and luciferase reporter assays.
Main Results:
- M2 microglial EVs treatment reduced brain infarct/edema, neurological deficits, and IgG leakage post-MCAO.
- EVs increased expression of tight junction proteins (ZO-1, occludin, claudin-5) in brain tissue.
- RNA sequencing identified TNF, MMP3, and NFκB signaling pathways; miR-23a-5p was found to target TNF mRNA.
Conclusions:
- M2 microglial EVs attenuate BBB disruption after ischemic stroke by delivering miR-23a-5p.
- miR-23a-5p targets TNF, subsequently regulating MMP3 and NFκB p65 expression, thereby enhancing BBB integrity.
