Adaptation of Three Different Apoptotic Methods in Equine Bronchoalveolar Cells and Comparison of Bronchoalveolar Lavage Cell Apoptosis in Normal and COPD Affected Horses Before and After Dexamethasone Administration

(ABSTRACT) Recent studies suggest that lymphocyte apoptosis serves to regulate pulmonary inflammation. Equine COPD, an allergic disease of the lower airway, is likely due to dysregulation of the pulmonary immune response. In this study, the hypothesis tested was COPD affected horses would have less apoptotic airway lymphocytes than control horses during clinical disease. To achieve this, 3 methods of measuring apoptosis, Vindelov's propidium iodide with Triton-X (PI/Triton-X), 7-aminoactinomycin D (7-AAD), and Annexin V with propidium iodide (Annexin/PI) were evaluated in equine airway lymphocytes. A significant linear relationship was found for equine bronchoalveolar lavage (BAL) lymphocytes stained with 7-AAD and Annexin/PI. No relationship was identified with cells stained with PI/Triton-X and Annexin/PI, and 7-AAD and PI/Triton-X indicating that methods which preserve cell membrane characteristics are more comparable when measuring BAL lymphocytes apoptosis in a heterogeneous population of cells. Additionally, all stains appear to perform the same in COPD and normal horses in remission and disease. Comparison of predominately BAL lymphocyte apoptosis using the above methods were performed at baseline, after natural challenge, and after dexamethasone administration in nine horses, five of which were affected with COPD. No differences in bronchoalveolar lavage lymphocyte apoptosis between COPD and control horses were detected either before or after dexamethasone administration, although numerical trends in COPD horses identified less iii apoptosis after natural challenge indicating that defective apoptosis may play a role in equine COPD pathogenesis. Dexamethasone administration was associated with trends of improvement in the pulmonary gas exchange and increased apoptosis toward baseline in the COPD horses. for without them, this work could not have been done. We have learnt so much from them. v ACKNOWLEDGEMENTS Many grateful, heartfelt thanks go to my committee, Dr. Virginia Buechner-Maxwell, for your support, patience, and laughing with me, not at me (most of the time), Dr. Mark V. Crisman, for keeping everything in perspective, Dr. Francois Elvinger, for your great expertise and help with the many statistical evaluations, and Drs. Robert Gogal and S. Ansar Ahmed for tireless help and suggestions with the numerous manuscript revisions. I know I would still be at the starting line without all of you. Many thanks to Dan Ward whom I shared laughter, friendship, and long hours with while he imparted his statistical wisdom. More thanks to Joan Kalnitsky for flow cytometric expertise and for trying to keep me on time. Additionally, muchas gracias to Shannon McCourt and Cristi Salmon whose tremendous …