Regulatory effect of neurophils in microglial polarization after permanent ischemic stroke

doi:10.16098/j.issn.0529-1356.2025.02.002

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Acta Anatomica Sinica ›› 2025, Vol. 56 ›› Issue (2) : 136-142. DOI: 10.16098/j.issn.0529-1356.2025.02.002

Regulatory effect of neurophils in microglial polarization after permanent ischemic stroke

HUANG Min-hua YE Xin-yan WU Si-yu LUO Shao-tong WU Zhi-shan CHEN Yuan* PING Su-ning*

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Abstract

Objective To investigate the effects of peripheral blood neutrophil infiltration on the polarization regulation of cerebral resident microglia under a permanent ischemic stroke model. Methods Fifty-eight C57BL/6 mice were divided into two groups. One group was sham group, and the other group of mice was subjected to permanent middle cerebral artery occlusion surgery. Mice were euthanized 48 hours, 7 days, 14 days, and 30 days after surgery for tissue collection. Western blotting was used to detect expression levels of M1 microglia markers CD16, M2 microglia marker arginase 1(Arg1), inflammatory cytokine interleukin-1β (IL-1β), and neutrophil marker myeloperoxidase (MPO) in brain tissue. Immunofluorescence histochemical staining was used to assess neutrophil infiltration and M2 microglial distribution around the infarct area in brain sections. In vitro, purified neutrophils were co-cultured with BV2 microglial cells. After lipopolysaccharide stimulation, the phagocytosis of neutrophils by BV2 cells was observed, and the expression levels of CD16 and Arg1 proteins in BV2 cells were detected. Results Western blotting showed that the levels of CD16 (P<0.05), IL-1β (P<0.001), and MPO (P<0.05) in brain tissue increased significantly 48 hours and 7 days after surgery, then decreased, with MPO expression returning to normal levels 30 days after surgery. Immunofluorescence showed a significant increase of MPO-positive cells around the infarct area of the mouse cerebral cortex 48 hours after surgery (P<0.001), followed by a decrease (P<0.05). The number of ionized calcium binding adapter molecule 1 (Iba1) and MPO double-positive cells gradually increased after surgery, and reached their peak at 14 days (P<0.05). Iba1 and Arg1 double-positive cells also increased significantly 7 days (P<0.05) and 14 days (P<0.01) after surgery. In vitro, co-culture experiments showed that after BV2 phagocytosing neutrophils, CD16 (P<0.05) significantly decreased and Arg1 significantly upregulated (P<0.05). Conclusion In a permanent ischemic stroke model, microglia transition from M1 to M2 type after phagocytosing neutrophils, and the injured brain area changes from pro-inflammatory state to anti-inflammatory state.

Key words

Ischemic stroke / Microglia / Neutrophil / M1/M2 polarization / Neuroinflammation / Western blotting / Immunofluorescence / C57BL/6 mouse

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HUANG Min-hua YE Xin-yan WU Si-yu LUO Shao-tong WU Zhi-shan CHEN Yuan PING Su-ning. Regulatory effect of neurophils in microglial polarization after permanent ischemic stroke[J]. Acta Anatomica Sinica. 2025, 56(2): 136-142 https://doi.org/10.16098/j.issn.0529-1356.2025.02.002

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