Early onset of rejection in concordant hamster xeno hearts display signs of necrosis, but not apoptosis, correlating to the phosphocreatine concentration

Abstract

Background: The importance of apoptosis contra necrosis for ischemia/reperfusion (RP) and acute rejection in concordant rodent xenotransplantation is largely unknown. We explored this question by comparing rodent allo and concordant xenotransplants with different morphological methods to detect apoptosis and biochemical data on the levels of high-energy phosphates obtained with in vitro 31Phosphorous Magnetic Resonance Spectroscopy ( 31P MRS). More specifically, we applied a hitherto unused method in transplantation research, apoptosis specific biotin labeled oligonucleotides designed with a 10 base pair stem region and a 20 nucleotides large loop that form a hairpin like shape. The results obtained with this method were compared to results obtained with the more widely used in situ 3′-end labeling of DNA (TUNEL) assay and extraction and gel electrophoresis of labeled DNA (DNA laddering). Methods: Cervical heart transplantations were performed between inbred Lewis (L) (RT1 1) to L, L to DA (RT1 a) rats, hamster (H) to H and H to L (X) ( n=5 for all groups except for X, n=9). All hearts were subjected to 30 min of cold ischemia (+4 °C) and 6 h of RP before explantation. In vitro 31P MRS was used to determine the phosphocreatine (PCr), beta-adenosine triphosphate (β-ATP) concentrations and the PCr/β-ATP ratio of the transplants. We correlated the biochemical data to haematoxylin and eosin (H & E) stained tissue slides scored for rejection, infiltration of antibodies and complement depositions, DNA extraction and gel electrophoresis of labeled DNA (DNA laddering), in situ 3′-end labeling of DNA (TUNEL) and the apoptosis specific hairpin probe assays scoring. Results: The rejection score of the xeno grafts differed significantly compared to their syngeneic hamster to hamster controls (2.40±0.25 vs. 1.20±0.20; P=0.005) and they had a significantly higher TUNEL score, 228±15 vs. 2.44±0.32 ( P=0.009), that correlated to changes in PCr concentration ( P<0.001) and to the PCr/β-ATP ratio ( P=0.01). The uptake was mainly (90–95%) located to 1–2 μm large extra cellular ‘granule’. A picture resembling early necrosis was seen on the H & E stainings and reflected in the Billingham rejection score above. Conclusions: After 6 h of RP the onset of acute rejection in the concordant hamster xeno hearts displayed features of early, possibly mitochondrial, necrosis, but not apoptosis, which correlated to changes in the PCr concentration and the PCr/β-ATP ratio. The mechanism for the early rejection observed is unclear and might be caused by other factors in the sera apart from cellular components, antibodies and complement factors. Identification of the underlying mechanisms could enable us to design rational therapies that prevent activation of the recipient's innate immune response.