Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2013 Effects of nitrite and oxygen on angiogenesis in vascular networks of the chicken embryo Michael Connery Virginia Commonwealth University Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Physiology Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/3211 This Dissertation is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. Effects of nitrite and oxygen on angiogenesis in vascular networks of the chicken embryo A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. by Michael D. Connery B.S., University of Virginia, 2005 Director: Dr. Roland N. Pittman Professor, Department of Physiology and Biophysics 1 Acknowledgements This work is the collaborative effort of an innumerable amount of people who have supported me and my education. While I have space here to recognize only a few individuals, I hope each person who has helped me in any small way knows they are a contributor in all the things I have been able to accomplish. Dr. Pittman has been a constant source of guidance, support, and inspiration. We have worked through three projects and I’m always amazed at his vast scientific knowledge and his inexhaustible patience. I credit Dr. Golub with the initial idea for my using chicken embryos as an animal model and his expertise and enthusiasm has been invaluable. He contributed significantly to nearly every piece of material developed for this research. Sami Dodhi is responsible for pushing my limits, questioning my assumptions, helping me discuss scientific methodology, and being a best friend. This would not have come together without the constant substantial, emotional, and spiritual help and support of Katarina Deshotel. Table of Contents List of Tables List of Figures List of Abbreviations Introduction . 1 Angiogenesis . 1 Angiogenic Pathology . 2 Role of Nitric Oxide in Modulating Angiogenesis . 4 NOS-independent NO . 6 Hemoglobin Catalysis . 6 Hypoxia . 8 Chicken CAM as an Angiogenesis Model . 8 Ex ovo Model . 13 Induction of Hypoxia . 14 Phosphorescence Quenching Microscopy . 16 cPTIO . 16 Endpoints . 17 Purpose of Present Study . 18 Materials Egg Sources and Transport . 20 Preincubation Chamber . 20 Overflow Incubation Chamber . 21 Incubator . 21 Poultry Dish . 23 Solution Application . 26 Plating Materials . 27 Solutions . 27 Camera for Developmental and Film Placement Images . 27 PQM Hardware . 29 PQM Software . 29 Microscopic Stage Chamber . 30 Automated Microscope Stage . 30 Stereomicroscope . 33 Stereomicroscopic Visualization Chamber . 33 Films . 35 PQM probe . 35 Methods Preincubation Conditions . 37 Plating Technique . 37 Incubation Conditions . 41 Egg Maintenance after Plating . 42 Film Placement . 42 Phosphorescence Quenching Microscopy (PQM) . 44 PQM Methodology and Scanning . 44 Determination of Film Oxygen Barrier Properties . 45 Description of Chemicals and Control Groups . 47 Angiogenic Image Acquisition . 49 Solution Application . 52 Experimental Design for Angiogenesis Measurements . 54 Image Processing for Angiogenic Variables . 54 Statistical Analysis . 55 Results PO2 under Films . 57 PO2 Transition Regions . 57 Vascular Bed Comparison . 59 Baseline Comparisons . 62 Changes in Angiogenesis at Control Sites . 65 Effects of Saline on Local Hypoxia-Induced Angiogenesis. 72 Effect of Nitrite on Angiogenesis . 72 Effect of Local Hypoxia on Angiogenesis with Nitrite Application . 77 Effect of Different Solutions in a Hypoxic Environment . 81 CAM Overgrowth . 85 Discussion Novel Materials and Methods . 86 Induction of Local Hypoxia using Gas Barrier Film . 87 PO2 Gradient around Film Boundary Interface Region . 88 Vascular Bed Comparisons . 88 Baseline Comparisons . 89 Angiogenesis at Control Sites . 90 Effect of Local Hypoxia on Angiogenesis . 91 Effect of Nitrite on Angiogenesis . 93 Nitrite and Hypoxia . 94 Conclusions . ..