Coordinated response of vascular and glial cells in a pig model of sepsis

Sepsis is a deadly syndrome arising from overactivation of the immune system in response to a pathogen. Severe sepsis can result in sepsis-associated encephalopathy (SAE) that significantly contributes to morbidity and long-term cognitive impairment. Studying the molecular underpinnings of SAE in the human brain is challenging, and rodent models have limited translational applicability due to significant neuroanatomical and immune differences. To bridge this gap, we employed a Yorkshire pig model to investigate changes in the brain cortex during the early stages of SAE using a uropathogenic strain of E. coli to model bacterial sepsis. Bulk RNAseq of cortical brain demonstrated profound global shifts in immune and inflammatory pathways, cytokine signaling, vascular dysfunction, and homeostatic dysregulation. To gain a more granular understanding of the changes in this model, we performed single-nucleus RNA sequencing (snRNAseq) on the same cortical brain tissue. This snRNAseq analysis revealed dramatic alterations in vascular cell populations, accompanied by concomitant glial cell activation. Surprisingly minimal changes were noted in neurons, likely due to the very early nature of this insult. The neurovascular unit, including vascular leptomeningeal cells (VLMCs), mural cells, endothelial cells, astrocytes, and microglia, showed strong upregulation of genes involved in TNF signaling, cellular responses to cytokine stimuli, and cytokine signaling in the immune system. Conversely, downregulated genes were enriched in pathways related to the disruption of metabolic and structural functions, including genes involved in maintaining the blood-brain barrier (BBB). Cell-cell communication analysis also identified significant rewiring of intercellular signaling in the septic brain, with an overall increase in the number and strength of cell to cell interactions in the E. coli pigs compared to saline controls. In saline controls, cell-cell communication was dominated by homeostatic signaling from microglia and astrocytes. In contrast, the septic pigs showed a significant shift toward inflammatory and immune-related signaling pathways, mainly involving microglia and astrocytes, along with the emergence of signaling pathways from mural cells and VLMCs. Our findings reveal that systemic sepsis triggers a rapid shift from homeostatic to inflammatory signaling within the neurovascular unit, driven by the emergence of pro-inflammatory crosstalk between vascular cells, astrocytes, and microglia. This porcine model effectively captures the acute transcriptional landscape of the septic brain, offering a critical window for therapeutic strategies aimed at stabilizing the BBB and preventing long-term cognitive decline in sepsis survivors.