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A Novel Precision-cut lung slice stretch model using removable inflation materials [bulk RNA-seq]

ABSTRACT: Mechanical stretch is an important biomechanical stimulus that facilitates tissue development in the respiratory system by programming the epithelium, endothelium, mesenchyme, and immune cells. Lung tissue undergoes stretch-induced differentiation under normal prenatal and postnatal development, and supraphysiological or aberrant stretch responses are known mechanisms of acute lung injury and extracellular matrix (ECM) disruption. Existing in vitro and ex vivo cyclic mechanical stretch (CMS) models have well-recognized disadvantages, and agarose-inflated precision-cut lung slice (PCLS) models are hindered by retention of agarose in the tissue and by damaging methods of anchoring slices for stretch. Here we present a PCLS system that uses a removable agarose inflation medium and a supportive poly(ethylene glycol) (PEG) hydrogel for embedding, enabling application of CMS to PCLS. Bulk RNA-seq of PCLS after 24 h of cyclic stretch versus static culture, from neonatal mice exposed to room air or hyperoxia, is used to demonstrate activation of mechanotransduction pathways and validate the model transcriptomically.

ORGANISM(S): Mus musculus

PROVIDER: GSE345215 | GEO | 2026/08/31

REPOSITORIES: GEO

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Project description:Acute respiratory distress syndrome (ARDS) is a catastrophic form of acute lung injury (ALI). The necessity for mechanical ventilation (MV) renders patients at risk for ventilator induced lung injury (VILI). Exposure to repetitive cyclic stretch (CS) and/or over-inflation exacerbates injury. Reducing tidal volume (VT) is the only therapeutic strategy shown to mitigate morbidity and mortality. Cyclic stretch has been shown to differentially regulate gene expression in part through the activation of mammalian mitogen-activated protein kinase (MAPK). Although these studies have shown both molecular and cellular alterations, no unifying hypothesis to explain MV-induced lung injury has emerged. In the current study, we hypothesized that coordinated expression of cyclic stretch (CS)-responsive genes relies on the presence of common CS-sensitive regulatory elements. To identify CS-responsive genes, we undertook a comparative examination of the gene expression profile of human bronchial epithelial airway (Beas-2B) cells in response to various injurious stimuli involved in the pathogenesis of acute lung injury (ALI)/Ventilator induced lung injury (VILI): cyclic stretch, tumor necrosis factor alpha (TNF-a), and lipopolysaccharide (LPS). Experiment Overall Design: Human Bronchial Epithelial Cells (Beas-B2) cells grown on silicon elastic plates coated with Type I collagen (Flexercell International, McKeesport, PA) were exposed to six regiments for 4 h: 1) control (static, [control]); 2) mechanical stretch (25 PKa, 30 cycles per min, [stretch]); 3) LPS (1 mcg/ml [LPS]); 4) TNF-Î± (20 ng/ml; [TNF]); 5) mechanical stretch plus LPS [LPS+S], and 6) mechanical stretch plus TNF-Î± [TNF+S]. Total RNA (duplicate experiments) was extracted using TRIZOL reagent (as per manufactures specifications) and purified using Qiagen mRNA purification Kit (as per manufacturers specifications). mRNA was hybridized to Affymetrix Human U133plus2.0 chips. Probe based analysis, background reduction, and quantile data normalization was performed in MeV 4.0 of TM4 using Robust Multi-array Average (RMA).

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A Novel Precision-cut lung slice stretch model using removable inflation materials [bulk RNA-seq]

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