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Identifying Microglia and Peripheral Infiltrating Macrophages in the Injured Spinal Cords Using Flow Cytometry
Published on: June 24, 2025
Ferroptosis-associated myeloid cell heterogeneity and inflammatory amplification following spinal cord injury
Jian Zhang1,2, Song Wang1, Minghang Zhang1
1Dongguan Key Laboratory of Central Nervous System Injury and Repair/Dongguan Institute of Spine and Spinal Cord Injury, The Sixth Affiliated Hospital of Jinan University (Dongguan), Dongguan, China.
Background:
Spinal cord injury (SCI) causes severe and persistent neurological dysfunction. Ferroptosis has been implicated in multiple neurological disorders, but its contribution to SCI and its relationship to myeloid-cell responses, inflammatory amplification and disturbed iron homeostasis remain unclear.
Methods:
We integrated public bulk RNA-sequencing and single-cell RNA-sequencing datasets with experiments in a rat SCI model to define ferroptosis-associated changes across the molecular, cellular and tissue levels. Differential expression, pathway enrichment, co-expression and protein-protein interaction analyses, pseudotime inference and cell-cell communication modelling were used to identify candidate molecules and relevant myeloid subpopulations, followed by qPCR, western blotting and immunofluorescence validation.
Results:
Ferroptosis-associated molecular alterations in SCI showed marked temporal dynamics and remained embedded within pathological networks linked to inflammation, oxidative stress and hypoxic responses. Single-cell analysis indicated that these signals were concentrated primarily in myeloid cells, particularly the HMOX1-high M1a and M1b subclusters. Pseudotime and cell-cell communication analyses further suggested that these subpopulations progress along a continuous trajectory towards inflammation-amplifying states and may influence the local microenvironment through MIF, TGFβ, PTN and CD99 signalling. Animal experiments further showed that sustained inflammatory activation occurs in parallel with dysregulation of ferroptosis-associated molecules, accompanied by local myeloid-cell activation and enhanced HMOX1-associated stress responses.
Conclusions:
In SCI, ferroptosis-associated signals appear to be concentrated within HMOX1-associated myeloid subpopulations and may be sustained through cell-state reprogramming and intercellular signaling networks. HMOX1 emerges as a candidate hub linking disturbed iron handling, ferroptosis and myeloid inflammatory remodeling.
Insights
Spinal cord injury (SCI) involves ferroptosis, a cell death process, concentrated in myeloid cells. Heme oxygenase-1 (HMOX1) links iron dysregulation, ferroptosis, and inflammation in SCI.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Spinal cord injury (SCI) results in significant and lasting neurological deficits.
- The role of ferroptosis, a regulated cell death pathway, in SCI pathophysiology, particularly concerning myeloid cell responses, inflammation, and iron metabolism, is not fully understood.
Purpose of the Study:
- To investigate ferroptosis-associated molecular and cellular changes in SCI.
- To elucidate the connection between ferroptosis, myeloid cell subpopulations, inflammation, and iron homeostasis following SCI.
Main Methods:
- Integration of bulk and single-cell RNA sequencing data with experimental validation in a rat SCI model.
- Utilized differential expression, pathway enrichment, co-expression, protein-protein interaction, pseudotime, and cell-cell communication analyses.
- Validated findings using qPCR, western blotting, and immunofluorescence.
Main Results:
- Ferroptosis alterations in SCI exhibit temporal dynamics and are linked to inflammation, oxidative stress, and hypoxia.
- Single-cell analysis identified ferroptosis signals predominantly in HMOX1-high myeloid cells (M1a and M1b subclusters).
- Myeloid subpopulations showed progression towards inflammation-amplifying states, potentially influencing the microenvironment via specific signaling pathways; animal models confirmed sustained inflammation and myeloid activation.
Conclusions:
- Ferroptosis signals in SCI are concentrated in HMOX1-associated myeloid cells and sustained by cell reprogramming and signaling networks.
- Heme oxygenase-1 (HMOX1) is identified as a key molecule connecting iron dysregulation, ferroptosis, and myeloid inflammatory responses in SCI.
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