PT/HS EC Spatial Transcriptomics

Description

A murine model of severe polytrauma-hemorrhagic shock (PT/HS) was used to compare pulmonary vascular endothelial cell (EC) responses to resuscitation with lactated Ringer’s (LR) relative to fresh frozen plasma (FFP). At 24h after PT/HS or sham procedure, lungs were fixed and processed for spatial transcriptomic profiling of pulmonary arterial, venous, and microvascular ECs using the GeoMx Digital Spatial Profiler. Differentially expressed genes and pathway analysis revealed enrichment of biological processes related to cellular bioenergetics and metabolic recovery pathways in ECs after FFP, whereas LR was associated with oxidative stress and inflammatory signaling.

Steps to reproduce

Five-micrometer–thick unstained lung sections designated for GeoMx digital spatial profiling (four sections per sham, LR, and FFP groups) were deparaffinized, rehydrated, and subjected to antigen retrieval. Sections underwent 16-hour in situ hybridization at 37°C with the GeoMx Mouse Whole Transcriptomic Atlas RNA probe mix (Bruker Corporation), followed by stringent washing and blocking in Buffer W. Tissues were then incubated with fluorescently labeled antibodies to identify ECs, epithelial cells, smooth muscle, and nuclei, respectively: AF647–conjugated anti-CD31/PECAM-1 (Bio-Techne; 1:50), AF594–conjugated anti-EpCAM (Abcam; 1:82), AF532–conjugated anti–myosin heavy chain 11 (Abcam; 1:20), and SYTO13 (Invitrogen; 1:167). After imaging on the GeoMx platform (Bruker Corporation), vascular segments were defined by anatomical criteria:28 pulmonary arteries as medium-to-large vessels adjacent to airways within bronchovascular bundles; pulmonary veins as medium-to-large vessels separate from bronchovascular bundles or at the lung periphery; and pulmonary microvasculature as alveolar-only regions. Regions of interest (ROI) were selected from ≥3 representative sections per vascular segment. RNA probes were released from ECs within each ROI by UV exposure and sequenced for transcriptomic analysis.

Institutions

• University of Alabama at Birmingham

  Alabama, Birmingham

Categories

Funders

• National Institute of General Medical Sciences

  National Institutes of Health

  Maryland, Bethesda

  Grant ID: R35GM137958

Licence