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Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry: a Novel Use for a Cell Purification Method
Published on: April 9, 2011
CD47-blocking antibody interferes with neutrophil extracellular traps formation after spinal cord injury to reduce
Yuhang Diao1, Mingyu Hao1, Minghao Xie1
1Department of Neurosurgery, Tianjin Medical University General Hospital, Tianjin 300000, China.
Objective:
Our goal was to investigate the role of neutrophil extracellular traps (NETs) in the disruption of the blood-spinal cord barrier (BSCB) following spinal cord injury (SCI) and to evaluate the therapeutic efficacy of CD47-blocking antibodies in mitigating the disruption.
Methods:
We utilized Evans blue extravasation to evaluate BSCB permeability and immunofluorescence to evaluate the formation of NETs and the expression of ZO-1, CD31, S100A8/A9, CD68, GFAP, Iba-1, and NeuN. Spinal cord edema was quantified by comparing the dry and wet weights of tissue samples. We used enzyme-linked immunosorbent assay (ELISA) to evaluate inflammatory factors, including IL-1β, IL-6, and TNF-α. Changes in genes associated with NET formation were identified by mRNA sequencing. Activation of the TLR4-NF-κB-MMP2/MMP9 signaling pathway was examined via Western blot analysis. Limb function was evaluated using the Basso Mouse Scale (BMS) to assess motor function.
Results:
We observed massive aggregation of neutrophils and the formation of neutrophil extracellular traps (NETs) after spinal cord injury. The use of CD47-blocking antibodies reduced NET formation, mitigated S100A8/A9 production, attenuated BSCB injury, decreased inflammatory cell infiltration, alleviated spinal cord edema, and minimized neuronal death at the site of injury. Furthermore, these antibodies suppressed activation of the TLR4-NF-κB-MMP2/MMP9 signaling pathway.
Conclusion:
The use of CD47-blocking antibodies post-SCI resulted in reduced NET formation. By suppressing the TLR4-NF-κB-MMP2/MMP9 signaling pathway, these antibodies contributed to the preservation of blood-spinal cord barrier (BSCB) integrity, highlighting their potential as a therapeutic strategy for SCI.
