Abstract
Microvascular dysfunction plays a fundamental role in the pathogenesis of salivary gland disorders. Restoring and preserving microvascular integrity might therefore represent a promising strategy for the treatment of these pathologies. The mechanisms underlying microvascular dysfunction in salivary glands, however, are still obscure, partly due to the unavailability of adequate in vivo models. Here, we present a novel experimental approach that allows comprehensive in vivo analyses of the salivary gland microvasculature in mice. For this purpose, we employed different microscopy techniques including multi-photon in vivo microscopy to quantitatively analyze interactions of distinct immune cell subsets in the submandibular gland microvasculature required for their infiltration into the surrounding parenchyma and their effects on microvascular function. Confocal microscopy and multi-channel flow cytometry in tissue sections/homogenates complemented these real-time analyses by determining the molecular phenotype of the participating cells. To this end, we identified key adhesion and signaling molecules that regulate the subset- and tissue-specific trafficking of leukocytes into inflamed glands and control the associated microvascular leakage. Hence, we established an experimental approach that allows in vivo analyses of microvascular processes in healthy and diseased salivary glands. This enables us to delineate distinct pathogenetic factors as novel therapeutic targets in salivary gland diseases.
Highlights
Saliva is an extracellular fluid produced in the head and neck by the paired parotid, submandibular, and sublingual glands as well as by minor salivary glands in the mucosa of lips, tongue, oral cavity, and pharynx [1]
In gland homogenates of unstimulated animals, low numbers of neutrophils, classical monocytes/monocyte-derived macrophages (MDMs), CD4+ T lymphocytes, CD8+ T lymphocytes, and B lymphocytes were found by multi-channel flow cytometry, whereas numbers of non-classical monocytes/tissue-resident macrophages (TRMs) were significantly higher (Figures 1A, B; n = 6)
Superfusion with tumor necrosis factor (TNF) in the dose of 1 μg per ml saline, a dose employed for analyzing the effect of TNF on leukocyte trafficking in various organ models [32,33,34], predominantly induced infiltration of neutrophils and classical monocytes/monocytederived macrophages to the inflamed tissue, whereas the numbers of non-classical monocytes/tissue-resident macrophages, CD4+ and CD8+ T lymphocytes, or B lymphocytes remained unaltered (Figure 1B; n = 6)
Summary
Saliva is an extracellular fluid produced in the head and neck by the paired parotid, submandibular, and sublingual glands as well as by minor salivary glands in the mucosa of lips, tongue, oral cavity, and pharynx [1]. White blood cells (leukocytes) start to roll on the luminal aspect of microvascular endothelial cells before they firmly adhere to it and crawl to suitable sites for extravasation These immune cells pass the endothelial barrier, breach the perivascular basement membrane, and subendothelially locomote through gaps between pericytes to migrate through the interstitial tissue to their target destination [7, 8]. The extravasation of leukocytes is associated with enhanced microvascular permeability, which leads to edema formation, reduced oxygenation, and remodeling of the underlying tissue, representing hallmarks of the inflammatory response [10] These microvascular processes are critically regulated by parenchymal sentinel cells such as tissue-resident macrophages (TRMs), which maintain microvascular homeostasis in intact and inflamed salivary glands [7, 8, 11,12,13]
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