Single-nucleus transcriptomics reveals sepsis-related neurovascular dysfunction in the human hippocampus
Sepsis is increasingly recognized as a major precipitant of long-term cognitive impairment, yet the cellular mechanisms underlying hippocampal vulnerability remain elusive. Using single-nucleus RNA sequencing of human hippocampal tissues from sepsis and control patients, we identied profound neurovascular alterations involving 21 distinct cell populations. Notably, astrocytes and microglia exhibited marked polarization: Astrocyte 2 showed simultaneous upregulation of neurotoxic A1 and neuroprotective A2 gene signatures in sepsis, whereas Astrocyte 1 displayed reduced A1 activity and a relatively quiescent pro le. In parallel, Microglia 2 demonstrated a prominent M1-like in ammatory signature, including elevated expression of HLA-DRA, IL1B, and TNF, while Microglia 1 downregulated both M1 and M2 markers, suggesting a hypo-responsive state. Intercellular communication analysis revealed intensi ed astrocyte–microglia interactions in the septic hippocampus, potentially amplifying in ammatory signaling loops. Concurrently, endothelial and mural cells exhibited transcriptional signatures of blood-brain barrier (BBB) disruption, oxidative stress, and compromised vascular homeostasis. Key molecular pathways associated with antigen presentation, cytokine signaling, and vascular permeability were selectively activated across neurovascular compartments. Together, our ndings uncover a coordinated glial and vascular response to systemic in ammation, driven in part by dysfunctional astrocyte–microglia crosstalk and pro-in ammatory polarization. These changes may underlie BBB breakdown and contribute to sustained neuroin ammation and cognitive decline in sepsis survivors. Targeting glial-vascular signaling axes and modulating astrocyte or microglial polarization states may offer promising avenues for therapeutic intervention in post-sepsis neurological sequelae. Overall design: Frozen hippocampal tissue samples from three sepsis patients and three age-matched controls were processed for single-nucleus RNA sequencing. Nuclei were isolated at Novogene (San Jose, CA) by tissue homogenization, debris removal, and centrifugation. Single-nucleus 3' RNA libraries were prepared using the 10x Genomics Chromium Single Cell 3' v3.1 platform and sequenced on an Illumina NovaSeq X Plus system at a target depth of ~50,000 read pairs per nucleus. Sequencing reads were aligned to the human reference genome (GRCh38), and gene expression matrices were generated using Cell Ranger (10x Genomics) with default parameters.