On ischemia/reperfusion injury and rejection in concordant xenotransplantation to the rat

Abstract

Studies in concordant (without preformed antibodies) rodent xenotransplantation (transplantation between species) models have focused, to date, on immunological disparities. In allotransplantation (transplantation within species), there is evidence suggesting that early ischemic events may contribute to later rejection episodes and to the development of chronicrejection features. All types of xenogeneic immunological mechanisms studied thus far have more serious impacts than those seen in allogeneic transplantation.The overall objective of this thesis was to investigate the impact of standardize dischemia/reperfusion (I/RP) injury and rejection on mouse-to rat and hamster-to rat xenotransplantation models in the un-immunosuppressed situation. State of the art technology was used to explore these issues and their correlations from biochemical, immunological, histological and functional perspectives. We found that decreases in the ratio of the high-energy phosphates (HEP) phosphocreatine(PCr) and beta adenosine triphosphate (ß-ATP), as measured with in vivo 31PhosphorusMagnetic Resonance Spectroscopy (31P MRS), correlated significantly to the progressiveacute rejection process in mouse- to - rat xeno heart transplants subjected to 30 minutes ofcold ischemia (CI) at + 4° C before transplantation. By inducing singlet oxygen energy (SOE)light, a potent inhibitor of reactive oxygen species (ROS), for the first time in vivo in hamster xeno heart transplants during CI, the metabolic effects of short-term ischemia were counteracted and the bioenergetic status measured as the PCr/ ß-ATP ratio preserved for atleast 24 h. However, this had no effect on rejection time. Early onset of rejection was studiedafter 6 h reperfusion (RP) with in vitro 31P MRS, and a paradoxical increase in the PCr andthe PCr/β-ATP ratio was found in hamster xeno hearts. This was specific to the xenogeneic situation and was not the consequence of the I/RP injury itself. It was correlated to earlyrejection, probably causing mitochondrial necrosis. By 24 h, the PCr overshoot was gone inuntreated hamster hearts. When comparing the ratios of HEP between hamster and mousexenohearts with in vivo 31P MRS, an initial rise was found on day 1 in the PCr/β-ATP ratio ofmouse hearts vs. an immediate fall in hamster hearts, although they both rejected on day 3.Detailed analysis with high pressure liquid chromatography (HPLC) after 30 min of CI and 6h of RP demonstrated that mouse xeno hearts were metabolically more stressed than hamsterhearts after the same I/RP injury, as reflected by a higher PCr overshoot , leading to aninitial increase in the PCr/β-ATP ratio. This might explain the increased PCr/β-ATP ratio day1. Increasing the CI time to 120 minutes stressed the mice xenohearts further causing asublethal/moderate to severe I/RP injury (40 50% early dysfunction) and decreased the PCrconcentration. Using an ischemic preconditioning (IPC) protocol this was effectivelycounteracted and the PCr overshoot sustained. These metabolic differences might explainwhy mice xeno hearts after the same I/RP stress and rejection pace induce a higherhemagglutinating titer, leading to faster rejection in a retransplantation setting and thus aremore immunogeneic.