A comprehensive single-cell transcriptomic atlas of hippocampal microglia in a mouse model of traumatic brain injury.

Background

Traumatic brain injury (TBI) triggers microglial activation and disrupts the inflammatory microenvironment in the hippocampus, which are key contributors to neuroinflammation and subsequent secondary brain damage. Nevertheless, the cellular heterogeneity of microglia, their pro-inflammatory activation mechanisms, and microenvironmental regulatory networks have not been fully characterized.

Methods

By integrating multi-source single-cell transcriptomic datasets and establishing a mouse TBI validation model, we constructed a high-resolution cellular atlas of hippocampal microglia. Through comprehensive re-analysis of single-cell RNA-sequencing (scRNA-seq) datasets, bioinformatic approaches, high-dimensional weighted gene co-expression network analysis (hdWGCNA), cell-cell communication profiling, and in vivo behavioral and in vivo pathological evaluations, we thoroughly explored the subpopulation diversity, functional features, and regulatory mechanisms of hippocampal microglia following TBI.

Results

TBI elicited extensive phenotypic diversification in hippocampal microglia, and five major subpopulations were identified, among which disease-associated microglia (DAM) were further classified into four distinct subclusters. A distinct inflamed DAM subset (Inflam-DAM) was found to highly express canonical pro-inflammatory signature genes. Transcriptomic profiling revealed enrichment of AP-1 family transcription factors and the IL-17 signaling pathway in this subset, confirming its robust pro-inflammatory phenotype. hdWGCNA demonstrated that the pro-inflammatory state of Inflam-DAM was tightly linked to transcriptional and translational regulation. Cell-cell communication analysis indicated that meningeal macrophages, B cells and fibroblasts acted as upstream regulators and selectively interacted with DAM subclusters in the inflammatory niche. In turn, activated DAM communicated with neurons in an injury-dependent manner, eventually impairing neurogenesis. In vivo experiments validated that TBI mice displayed severe neurological dysfunction, elevated expression of pro-inflammatory microglial markers and signaling molecules in the hippocampus, and fewer newborn mature neurons - all findings consistent with our single-cell data.

Conclusion

In summary, this work constructs a full-scale single-cell transcriptomic landscape of hippocampal microglia in TBI mice. We systematically delineated the heterogeneity of DAM subsets, the pro-inflammatory regulatory mechanism of Inflam-DAM, and the multi-layered intercellular network within the inflammatory microenvironment. Our results deepen the mechanistic understanding of TBI-associated neuroinflammation and offer valuable clues for the development of targeted anti-inflammatory therapies.