Inhaled Nitric Oxide Attenuates the Adverse Effects of Transfusing Stored Syngeneic Erythrocytes in Mice with Endothelial Dysfunction after Hemorrhagic Shock

BLOOD transfusion is a lifesaving treatment for hemorrhagic shock (HS). During ex vivo storage, erythrocytes undergo numerous biochemical, structural, and functional alterations, which are collectively termed the “storage lesion.”1,2 In 2007, the average duration of storage of trans-fused units was 19.5 days in the United States and 20 ± 11 (mean ± SD) days for trauma patients.3 A number of clinical studies have documented that transfusion of blood stored for more than 14 days is associated with an increased rate of infection,4 multiorgan failure,5 extended length of stay in hospital,6 and mortality.7-10 In contrast, other clinical studies of selected patient populations, including those undergoing cardiac surgical procedures, trauma victims, and critically ill patients, reported no association between the duration of erythrocyte storage and adverse clinical outcomes.5,11-15 The precise mechanisms responsible for the adverse clinical effects seen after transfusion of erythrocytes stored for prolonged periods are uncertain. One possibility is that these adverse effects are attributable to release of free hemoglobin from stored erythrocytes, both in solution and as lipid-enclosed microparticles.16 Hemoglobin in plasma can scavenge nitric oxide more avidly than erythrocyte-encapsulated hemoglobin17 and can cause vasoconstriction,18 inflammation, and platelet activation.19 Increased plasma hemoglobin levels induce a “nitric oxide deficiency” state, contributing to the complications of various genetic and acquired hemolytic disorders such as sickle cell disease, malaria, and hemolysis-associated smooth muscle dystonia.20 Our previous studies of infusing hemoglobin-based oxygen carriers or tetrameric hemoglobin indicated that scavenging of endothelium-derived nitric oxide by plasma hemoglobin produced vasoconstriction in both mice and sheep.18 We reported that preexisting endothelial dysfunction (a deficiency of vascular nitric oxide availability commonly associated with diabetes and atherosclerosis) dramatically enhanced the susceptibility to hemoglobin-based oxygen carriers or hemoglobin-induced systemic vasoconstriction in mice.21 Moreover, we reported that breathing nitric oxide before infusing a hemoglobin-based oxygen carrier or tetrameric hemoglobin in mice prevented the vasoconstriction.18 Reproducible animal models of erythrocyte storage can provide important insights into the effects of transfusing stored blood in an HS model and aid in the development of methods to prevent stored blood toxicity. Our study had three objectives. First, we measured tissue injury, hemodynamic changes, and survival rate in mice subjected to HS for 90 min and resuscitated with erythrocytes stored for less than 24 h (fresh erythrocytes) or with erythrocytes stored for 2 weeks (stored erythrocytes). Second, we studied the impact of endothelial dysfunction (induced by feeding mice a high-fat diet [HFD-fed] for 4–6 weeks) after resuscitation from HS with fresh or stored erythrocytes. Third, we studied whether nitric oxide inhalation during and for 2 h after resuscitation could prevent or reduce the adverse effects (tissue injury, organ dysfunction, inflammation, oxidative stress, and mortality) of resuscitation with stored erythrocytes in HFD-fed mice. We report that resuscitation with stored erythrocytes induced greater tissue injury, inflammatory response, oxidative stress, mortality, and plasma hemoglobin levels than did resuscitation with fresh erythrocytes. All these adverse effects were exacerbated in HFD-fed mice resuscitated with stored erythrocytes after HS. Inhaled nitric oxide reduced tissue injury, plasma hemoglobin levels, and oxidative stress and improved the survival rate of HFD-fed mice transfused with stored erythrocytes.