Preclinical Four-Dimensional Functional Lung Imaging and Quantification of Regional Airflow: A New Standard in Lung Function Evaluation in Murine Models.

The current standard for lung function evaluation in murine models is based on forced oscillation technology, which provides a measure of the total airway function but cannot provide information on regional heterogeneity in function. Limited detection of regional airflow may contribute to a discontinuity between airway inflammation and airflow obstruction in models of asthma. Here, we describe quantification of regional airway function using novel dynamic quantitative imaging and analysis to quantify and visualize lung motion and regional pulmonary airflow in four dimensions (4D). Furthermore, temporo-spatial specific ventilation (ml/ml) is used to determine ventilation heterogeneity indices for lobar and sublobar regions, which are directly compared to ex vivo biological analyses in the same sublobar regions. In contrast, oscillation-based technology in murine genetic models of asthma have failed to demonstrate lung function change despite altered inflammation, whereas 4D functional lung imaging demonstrated diminished regional lung function in genetic models relative to wild-type mice. Quantitative functional lung imaging assists in localizing the regional effects of airflow. Our approach reveals repeatable and consistent differences in regional airflow between lung lobes in all models of asthma, suggesting that asthma is characterized by regional airway dysfunctions that are often not detectable in composite measures of lung function. 4D functional lung imaging technology has the potential to transform discovery and development in murine models by mapping out regional areas heterogeneously affected by the disease, thus deciphering pathobiology with greater precision.

Comprehensive phenotyping of endothelial cells using flow cytometry 1: Murine.

The endothelium forms a selective barrier between circulating blood or lymph and surrounding tissue. Endothelial cells play an essential role in vessel homeostasis, and identification of these cells is critical in vascular biology research. However, characteristics of endothelial cells differ depending on the location and type of blood or lymph vessel. Endothelial cell subsets are numerous and often identified using different flow cytometric markers, making immunophenotyping these cells complex. In part 1 of this two part review series, we present a comprehensive overview of markers for the flow cytometric identification and phenotyping of murine endothelial subsets. These subsets can be distinguished using a panel of cell surface and intracellular markers shared by all endothelial cells in combination with additional markers of specialized endothelial cell types. This review can be used to determine the best markers for identifying and phenotyping desired murine endothelial cell subsets.

Comprehensive phenotyping of endothelial cells using flow cytometry 2: Human.

In vascular research, clinical samples and samples from animal models are often used together to foster translation of preclinical findings to humans. General concepts of endothelia and murine-specific endothelial phenotypes were discussed in part 1 of this two part series. Here, in part 2, we present a comprehensive overview of human-specific endothelial phenotypes. Pan-endothelial cell markers, organ specific endothelial antigens, and flow cytometric immunophenotyping of blood-borne endothelial cells are reviewed.

Characterization and origins of cell-free mitochondria in healthy murine and human blood.

Intact cell-free mitochondria have been reported in microparticles (MPs) in murine and human bodily fluids under disease conditions. However, cellular origins of circulating extracellular mitochondria have not been characterized. We hypothesize that intact, cell-free mitochondria from heterogeneous cellular sources are present in the circulation under physiological conditions. To test this, circulating MPs were analyzed using flow cytometry and proteomics. Murine and human platelet-depleted plasma showed a cluster of MPs positive for the mitochondrial probe MitoTracker. Transgenic mice expressing mitochondrial-GFP showed GFP positivity in plasma MPs. Murine and human mitochondria-containing MPs were positive for the platelet marker CD41 and the endothelial cell marker CD144, while hematopoietic CD45 labeling was low. Both murine and human circulating cell-free mitochondria maintained a transmembrane potential. Circulating mitochondria were able to enter rho-zero cells, and were visualized using immunoelectron microscopic imaging. Proteomics analysis identified mitochondria specific and extracellular vesicle associated proteins in sorted circulating cell-free human mitochondria. Together the data provide multiple lines of evidence that intact and functional mitochondria originating from several cell types are present in the blood circulation.