Many diseases and clinical procedures produce lysis of red blood cells that results in release of free hemoglobin in the circulation, which is often fatal. The long-term aim of the proposed research is to determine the mechanisms by which free Hb in the circulation causes intestinal tissue damage and increases microvascular permeability. The immediate goal of this proposal is to determine the roles that reactive oxygen species (ROS), produced by hemoglobin, play in causing tissue damage and disturbing macromolecular exchange in an intravital in situ preparation of the rat intestinal mucosa and mesentery. The intestine is one of the principal sites of transvascular exchange in the body, and if the selectivity of the exchange pathways is compromised, then undesirable products from the intestinal lumen will gain access to the bloodstream, possibly leading to sepsis. In this study a number of different modifications of Hb will be used, some of which produce ROS, and others which do not. The following hypothesis will be tested: The loss of selectivity of transvascular exchange barriers in the intestine and mesentery following intravascular injection of Hb, is due to the production of ROS and is catalysed by free iron, and amplified by mast cell degranulation. The role of nitric oxide in this process will also be investigated. Formation of ROS in the intestinal mucosa and mesentery, following injection of Hb, will be detected using a probe which fluoresces on contact with ROS. The effects of pretreatment with ROS scavengers, mast cell stabilizers, iron chelators and nitric oxide donors on intestinal endothelial and epithelial cell damage, and on formation of products of lipid peroxidation will be evaluated using electron microscopy and spectrophotometry, respectively. Presence or absence of endothelial junctional proteins will be evaluated using immunofluorescence microscopy. The effects of Hb on transvascular passage of bovine serum albumin (BSA) will be measured using epifluorescence microscopy and computer analysis of video images.
