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Updated: Dec 10, 2025
Isolation and Flow Cytometric Assessment of Neuroimmune Interactions in a Mini-Stroke Murine Model
Published on: June 20, 2025
FGF21 alleviates neuroinflammation following ischemic stroke by modulating the temporal and spatial dynamics of
Dongxue Wang1,2, Fei Liu2, Liyun Zhu2
1Department of Neurosurgery, First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325035, Zhejiang, China.
Background:
Resident microglia and macrophages are the predominant contributors to neuroinflammation and immune reactions, which play a critical role in the pathogenesis of ischemic brain injury. Controlling inflammatory responses is considered a promising therapeutic approach for stroke. Recombinant human fibroblast growth factor 21 (rhFGF21) presents anti-inflammatory properties by modulating microglia and macrophages; however, our knowledge of the inflammatory modulation of rhFGF21 in focal cerebral ischemia is lacking. Therefore, we investigated whether rhFGF21 improves ischemic outcomes in experimental stroke by targeting microglia and macrophages.
Methods:
C57BL/6 mice were subjected to middle cerebral artery occlusion (MCAO) and randomly divided into groups that received intraperitoneal rhFGF21 or vehicle daily starting at 6 h after reperfusion. Behavior assessments were monitored for 14 days after MCAO, and the gene expression levels of inflammatory cytokines were analyzed via qRT-PCR. The phenotypic variation of microglia/macrophages and the presence of infiltrated immune cells were examined by flow cytometry and immunostaining. Additionally, magnetic cell sorting (MACS) in combination with fluorescence-activated cell sorting (FACS) was used to purify microglia and macrophages.
Results:
rhFGF21 administration ameliorated neurological deficits in behavioral tests by regulating the secretion of pro-inflammatory and anti-inflammatory cytokines. rhFGF21 also attenuated the polarization of microglia/macrophages toward the M1 phenotype and the accumulation of peripheral immune cells after stroke, accompanied by a temporal evolution of the phenotype of microglia/macrophages and infiltration of peripheral immune cells. Furthermore, rhFGF21 treatment inhibited M1 polarization of microglia and pro-inflammatory cytokine expression through its actions on FGF receptor 1 (FGFR1) by suppressing nuclear factor-kappa B (NF-κB) and upregulating peroxisome proliferator-activated receptor-γ (PPAR-γ).
Conclusions:
rhFGF21 treatment promoted functional recovery in experimental stroke by modulating microglia/macrophage-mediated neuroinflammation via the NF-κB and PPAR-γ signaling pathways, making it a potential anti-inflammatory agent for stroke treatment.
Insights
Recombinant human fibroblast growth factor 21 (rhFGF21) reduces neuroinflammation and improves functional recovery in experimental stroke by modulating microglia and macrophages. This study highlights rhFGF21 as a potential therapeutic agent for stroke treatment.
Area of Science:
- Neuroscience
- Immunology
- Pharmacology
Background:
- Microglia and macrophages drive neuroinflammation in ischemic brain injury, making their modulation a therapeutic target for stroke.
- Recombinant human fibroblast growth factor 21 (rhFGF21) has known anti-inflammatory properties, but its role in focal cerebral ischemia was unexplored.
- This study investigated rhFGF21's effects on microglia and macrophages in experimental stroke.
Purpose of the Study:
- To determine if rhFGF21 improves outcomes in experimental stroke.
- To investigate rhFGF21's impact on microglia and macrophage activity and inflammatory responses.
- To elucidate the underlying molecular pathways targeted by rhFGF21.
Main Methods:
- Middle cerebral artery occlusion (MCAO) model in C57BL/6 mice.
- Administration of rhFGF21 or vehicle post-reperfusion, with behavioral assessments over 14 days.
- Analysis of inflammatory cytokine gene expression, microglia/macrophage phenotypes, and immune cell infiltration via qRT-PCR, flow cytometry, and immunostaining.
Main Results:
- rhFGF21 treatment significantly improved neurological deficits and regulated pro- and anti-inflammatory cytokine secretion.
- rhFGF21 attenuated M1 polarization of microglia/macrophages and reduced peripheral immune cell accumulation.
- The mechanism involved inhibiting nuclear factor-kappa B (NF-κB) and upregulating peroxisome proliferator-activated receptor-γ (PPAR-γ) via FGF receptor 1 (FGFR1).
Conclusions:
- rhFGF21 promotes functional recovery in experimental stroke by mitigating microglia/macrophage-mediated neuroinflammation.
- The therapeutic effects are mediated through the NF-κB and PPAR-γ signaling pathways.
- rhFGF21 demonstrates potential as an anti-inflammatory agent for stroke treatment.
