The Pathology Core (Core D) will perform all microscopic analyses needed for the four Projects of this PPG. The Core has state-of-the-art facilities and extensive experiences with virtually all pathologic analyses of cardiac tissues. This Core represents the continuation of the ongoing collaboration between Dr. Anversa's team at the New York Medical College and the Cardiology team at the University of Louisville, which have worked together for the past two years. The logistics of this arrangement have been extensively tested, and do not pose any problem. Following perfusion fixation, tissue samples will be obtained in each of the four Projects at the University of Louisville and sent to the Pathology Core at New York Medical College for analysis. The Core will perform several functions that can be summarized as follows. 1. Morphometric analysis of infarct size, myocyte volume and number, and length density of resistance arterioles and capillary network within the myocardium. Myocyte volume will be assessed both in situ and in enzymatically dissociated cells. Myocyte classes and distribution will be analyzed with confocal microscopy both in situ and in isolated myocytes. These measurements will enable us to calculate the total number of myocytes lost during post-infarction LV remodeling (Project 2) and to compare the number of lost myocytes with conventional measurements of infarct size by tetrazolium staining at 24 h of reperfusion. 2. Two-photon microscopy to perform simultaneous detection of multiple antigens without the limitations imposed by photobleaching of fluorophores. Two-photon microscopy will also be used to measure pyridine nucleotides in Project 4. 3. Confocal microscopic analysis of tissue sections obtained from perfusion-fixed hearts. The combination of two-photon and confocal microscopy will enable us to quantitate the expression of multiple antigens (e.g., iNOS, HO-1, ecSOD, nitrotyrosines, protein-HNE adducts, protein-MDA adducts, protein carbonyls) simultaneously in the same tissue section. Two-photon and confocal microscopy will be employed for quantitative analysis of fluorescence intensity of iNOS, HO-1, ecSOD, protein-HNE adducts, protein-MDA adducts, nitrotyrosines, and 8-hydroxy-deoxyguanosine. All immunocytochemistry will be done by using primary antibodies labeled directly with the fluorochromes recognized by our two-photon microscope and confocal microscope, which will avoid the complications of nonspecific staining and cross-reactivity between antibodies. 4. Quantitative measurement of apoptotic and necrotic cells with our newly-developed dual-labeling technique (hairpin-1 in situ ligation coupled with immunohistochemistry for active caspase-3, hairpin-2 ligation coupled with immunohistochemistry for active caspase-3), along with TUNEL. The dual hairpin-1/active caspase-3 labeling and hairpin-2/active caspase-3 labeling will afford a very high degree of specificity in assigning a cell to apoptosis vs. necrosis. 5. Routine histologic examination to assess inflammation, fibrosis, or any other morphologic changes.
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