R. S. Hoehn1, P. L. Jernigan1, E. F. Midura1, J. M. Sutton1, C. C. Caldwell1, M. J. Edwards1, E. Gulbins1,2, T. A. Pritts1 1University Of Cincinnati,Surgery,Cincinnati, OH, USA 2University Of Duisburn-Essen,Molecular Biology,Essen, ESSEN, Germany
Introduction: Transfusion of human packed red blood cell units (pRBCs) is ideal for resuscitation after hemorrhage. However, studies have shown that use of aged pRBCs are associated with increased morbidity and mortality compared to fresh units. Our laboratory and others have demonstrated that pRBC storage results in accumulation of microparticles that cause increased lung inflammation after transfusion. Ceramide, a sphingolipid formed by the enzyme acid sphingomyelinase (Asm), is present in cell membranes and mediates a variety of cell signaling and membrane changes in red blood cells, but its role in microparticle formation is unknown. We hypothesized that Asm inhibition would lead to decreased ceramide accumulation and microparticle formation in pRBCs, with a resultant decrease in lung injury after hemorrhage and resuscitation.
Methods: Human and murine pRBCs were obtained and treated with increasing concentrations of Amitriptyline (AT), a specific Asm inhibitor, or vehicle, then stored under standard blood banking conditions. At intervals, microparticles were isolated from pRBC units and quantified with Nanoparticle Tracking Analysis. Asm activity and ceramide concentration were measured in stored erythrocytes and microparticles. Mice underwent hemorrhage and resuscitation with equal numbers of microparticles from AT- and vehicle-treated pRBC units. Lungs were collected from shocked mice and assessed for inflammatory cytokines and histology.
Results: During storage, pRBC units demonstrated significantly increased microparticle formation, ceramide accumulation, and Asm activity. Human and murine pRBCs treated with AT formed fewer microparticles in a dose-dependent manner (Figure 1A). AT-treated pRBCs and microparticles contained less ceramide and Asm activity. After hemorrhage, mice resuscitated with microparticles isolated from vehicle-treated pRBCs had significantly elevated lung levels of pro-inflammatory cytokines including macrophage-derived chemokine (Figure 1B), IL-1b, IL-6, KC, MCP-1, MIP-1a, and MIP-2 as well as increased lung damage as determined by histology. Resuscitation with equal numbers of microparticles from AT-treated pRBCs resulted in significantly decreased lung injury.
Conclusion: Amitriptyline treatment of pRBC units leads to decreased Asm activity, ceramide formation, and microparticle accumulation. Asm inhibition caused alterations in the microparticles that led to decreased acute lung injury following hemorrhage and resuscitation. Asm inhibition represents a novel opportunity to mitigate harmful effects of resuscitation with stored pRBCs.
