A unifying hypothesis of the projects proposed in this grant is that reactive oxygen species (ROS) and reactive nitrogen species (RNS) play important roles in mediating both normal and pathophysiological vascular events. The purpose of the Imaging Core will be to provide resources and support to participants of the PPG for bright field and fluorescence imaging and fluorescence activated cell sorting (FACS) measurements as experimental tools to test this hypothesis using the following methods: (a) the detection and measurement of the fluorescent products of superoxide (O2.-) and hydrogen peroxide (H2O2) within intact vascular tissues and/or cultured cells from animal models; (b) the identification of ROS-producing cells in vascular tissue and cultured cells using cell-specific fluorescent probes, indirect immunofluorescence of cell-specific antibodies, or in situ hybridization of macrophage-specific markers; (c) the detection by immunocytochemistry and immunohistochemistry of subunits of NAD(P)H oxidases and signaling molecules in cells and tissues from animal models of insulin resistance; (d) the colocalization of cell types with metalloproteinases (MMPs), the major degradative enzymes in matrix remodelling associated with atherosclerosis; (e) the use of fluorescent probes to study cell viability, proliferation and apoptosis in knockout mice or in cells subjected to mechanical shear stresses similar to those found in vivo in atherosclerosis-prone regions of the vasculature; (f) the detection, measurement and histological analysis of cell proliferation, cell hypertrophy and atherosclerotic lesion size in animal models; (g) the detection of fluorescent or chromogenic products of reporter genes in transgenic animal models. The addition of the Imaging Core facility will significantly enhance the ability of project leaders to visualize the locations of ROS/RNS production under a variety of experimental or pathological conditions both in tissue culture and in intact tissue and to correlate these morphological data with other more quantitative methods that measure ROS such as electron spin resonance (ESR) spectroscopy. Specifically the Imaging Core will: 1. Provide expertise, facilities and training for individuals in performing immunocytochemistry, immunohistochemistry, and histology of vascular tissues, including microscope usage and computer-assisted analyses. 2. Assist with quantitative measurements of O2.- and H2O2 production using flow cytometry.
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