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Updated: Jan 2, 2026
Isolation and Flow Cytometric Assessment of Neuroimmune Interactions in a Mini-Stroke Murine Model
Published on: June 20, 2025
Melatonin protects against ischemic stroke by modulating microglia/macrophage polarization toward anti-inflammatory
Zong-Jian Liu1, Yuan-Yuan Ran1, Shu-Yan Qie1
1Department of Rehabilitation, Beijing Rehabilitation Hospital, Capital Medical University, Beijing, China.
Aims:
Microglia and infiltrated macrophages play important roles in inflammatory processes after ischemic stroke. Modulating microglia/macrophage polarization from pro-inflammatory phenotype to anti-inflammatory state has been suggested as a potential therapeutic approach in the treatment of ischemic stroke. Melatonin has been shown to be neuroprotective in experimental stroke models. However, the effect of melatonin on microglia polarization after stroke and underlying mechanisms remain unknown.
Methods:
In vivo, cerebral ischemia was induced by distal middle cerebral artery occlusion (dMCAO) in C57BL/6J mice. Melatonin was injected intraperitoneally (20 mg/kg) at 0 and 24 hours after ischemia. In vitro, the microglial cell line BV2 was stimulated to the pro-inflammatory state with conditioned media (CM) collected from oxygen-glucose deprivation (OGD) challenged neuronal cell line Neuro-2a (N2a). Real-time PCR was utilized to detect the mRNA expression of microglia phenotype markers. Activation of signal transducer and activator of transcription 3 (STAT3) pathway was determined by Western blot of phosphorylated STAT3 (pSTAT3). A neuron-microglia co-culture system was used to determine whether melatonin can inhibit the neurotoxic effect of pro-inflammatory microglia to post-OGD neurons.
Results:
Melatonin treatment reduced brain infarct and improved neurological functions 3 days after dMCAO, which was accompanied by decreased expression of pro-inflammatory markers and increased expression of anti-inflammatory markers in the ischemic brain. In vitro studies confirmed that melatonin directly inhibited the pro-inflammatory responses in BV2 cells upon exposure to OGD neuron CM. The microglia possessing pro-inflammatory phenotype exacerbated post-OGD N2a cells death, whereas melatonin reduced such neurotoxic effect. Further, melatonin enhanced the otherwise inhibited pSTAT3 expression in BV2 cells treated with OGD neuron CM. STAT3 blockade significantly reduced the effect of melatonin on microglial phenotype shift.
Conclusion:
Melatonin treatment ameliorates brain damage at least partially through shifting microglia phenotype from pro-inflammatory to anti-inflammatory polarity in a STAT3-dependent manner.
Insights
Melatonin reduces brain damage after ischemic stroke by shifting microglia from a pro-inflammatory to an anti-inflammatory state. This effect is mediated through the STAT3 pathway, offering a potential therapeutic strategy for stroke recovery.
Area of Science:
- Neuroscience
- Immunology
- Pharmacology
Background:
- Microglia and macrophages are key players in stroke-induced inflammation.
- Modulating microglia polarization is a potential therapeutic strategy for ischemic stroke.
- Melatonin's neuroprotective effects in stroke are known, but its impact on microglia polarization is unclear.
Purpose of the Study:
- To investigate the effect of melatonin on microglia polarization after ischemic stroke.
- To elucidate the underlying mechanisms of melatonin's action on microglia.
- To assess melatonin's therapeutic potential in a mouse model of ischemic stroke.
Main Methods:
- Cerebral ischemia induced by middle cerebral artery occlusion (MCAO) in mice.
- Melatonin administered intraperitoneally post-ischemia.
- In vitro studies using BV2 microglial cells and N2a neuronal cells under oxygen-glucose deprivation (OGD).
- Analysis of microglia phenotype markers via real-time PCR and STAT3 pathway activation via Western blot.
Main Results:
- Melatonin treatment reduced infarct volume and improved neurological function in MCAO mice.
- Melatonin inhibited pro-inflammatory responses in microglia and reduced neurotoxicity to neurons.
- Melatonin enhanced STAT3 phosphorylation in microglia, and STAT3 blockade diminished melatonin's effects.
Conclusions:
- Melatonin ameliorates brain damage following ischemic stroke.
- Melatonin shifts microglia from a pro-inflammatory to an anti-inflammatory phenotype.
- This neuroprotective effect is mediated, at least in part, by the STAT3 signaling pathway.
