Harvest and Imaging of Pleural Mesothelium en face

In normal circumstances, visceral mesothelium is a sheet of cells with squamous morphology, apicobasilar polarity, and intercellular tight junctions that define a uniform array. With injury or transition, the mesothelium undergoes morphologic and physiologic changes that yet remain ill-defined. A direct surface view (en face) of the pleural mesothelium may provide a topographic perspective of epithelial architecture that is important in development and cancer progression. En face imaging would potentially allow for visualizing the large flat surfaces of individual mesothelial cells and intercellular adhesions. However, conventional harvest and imaging techniques pose a challenge for studying the mesothelium, since few pleural cells are captured by each slide in thin-section histology, and only a transverse view is seen. En face preparation techniques have been used extensively for vascular endothelium, but its use for visceral mesothelium has been prevented by technical challenges, including stabilizing and flattening the desired surface in a non-destructive manner. We hypothesized en face harvest and imaging techniques may be adapted for pulmonary mesothelium by carbohydrate-pectin based adhesion to the lung surface for membrane stabilization. Here, we report the development of an en face method of harvesting and imaging pleural mesothelium, resulting in successful preservation of live cells and histologic staining of intact pleura. Wild-type C57Bl/6 mice were euthanized and the pulmonary circulation was flushed with isotonic solution. Individual lobes were adhered onto wafers of high-methoxyl pectin, which maintained adherence in solution. The subpleural plane was bluntly dissected under culture medium (Fig 1A), and the specimen was gently detached and mounted on a glass slide. A calcein-ethidium live/dead assay revealed up to 91% of mesothelial cells remained alive after this process (Fig. 1B). Silver nitrate staining revealed the intact “cobblestone” pattern of intercellular tight junctions (Fig 1C). Immunofluorescence staining with mesothelial markers was largely positive (E-Caderin, WT-1, Mesothelin). Mesenchymal markers such as Vimentin were positive in some areas as well (Fig 1D). Structured illumination microscopy with automated stage-scanning and montaging was used to create topographic maps of contiguous mesothelial surface areas averaging 2.60mm2 per sample. Our en face pectin-adhesion technique, along with automated structured illumination microscopy, enables mapping of pleural mesothelium over large surface areas. This has the potential to be adapted for other organs, and will advance our ability to study the visceral mesothelium. Support or Funding Information Supported in part by NIH Grants: HL134229, HL94567, CA009535, ES000002 (A) Lung lobes were placed on a pectin wafer for adherence to surface carbohydrates, and the subpleural plane was dissected under solution. (B) Calcein-ethidium live/dead assay showed up to 91% mesothelial cells were viable. (C) Silver nitrate staining revealed characteristic epithelial “cobblestone” appearance. (D) Immunofluorescence of the mesothelial monolayer was demonstrated using Vimentin-FITC with hoechst counterstain. (A) Lung lobes were placed on a pectin wafer for adherence to surface carbohydrates, and the subpleural plane was dissected under solution. (B) Calcein-ethidium live/dead assay showed up to 91% mesothelial cells were viable. (C) Silver nitrate staining revealed characteristic epithelial “cobblestone” appearance. (D) Immunofluorescence of the mesothelial monolayer was demonstrated using Vimentin-FITC with hoechst counterstain.