gms | German Medical Science

Prolonged cold organ storage (CI) as a non-immunologic risk factor in the pathogenesis of chronic rejection was evaluated in the Lew to F344 rat cardiac allograft model.

Hearts were transplanted without cold storage (CR, n = 20) or subjected to 10 h of cold ischemia at 0-1 °C and then transplanted (CRI, n = 28). Both groups were compared with corresponding syngeneic control groups. After evaluation of graft function (score 0-4) hearts were retrieved at 2, 10, 40 and 60 days. Degree of vasculopathy was investigated by means of H&E histology. For investigation of underlaying mechanisms intragraft gene expression was analysed employing cDNA microarray technology („MWG Rat 10k Array", MWG^® containing 9715 cDNA sequences) and RT-PCR. A cluster of transcripts regulated in response to cold ischemia as well as chronic rejection were genes encoding for enzymes involved in mitochondrial activity. We therefore ananlysed mitochondrial function using high-resolution respirometry of permeabilised myocardial fibers. In addition, enzymatic defects were assessed in myocardial homogenates.

Cardiac score declined from score 4 at 2 days after reperfusion to score 1.6±0.5 at 60 days. Impairment of organ function was faster in the CRI when compared to the CR group and was nearly absent in isografts. Histology revealed myointimal proliferation and fibrosis after 40 and 60 days in allogeneic groups. Both findings were significantly more pronounced in the CRI group. Microarray analysis allowed for detection of a set of 82 genes regulated at 2 and 10 days in response to cold ischemia in allogeneic groups. In contrast, only 20 genes were regulated in corresponding isografts. 60 Genes were up- or downregulated during chronic rejection and 22 genes were regulated only in chronically rejected grafts undergoing 10h of cold ischemia prior to transplantation. Investigated genes mainly comprised transcripts involved in myocyte function, humoral immune response and enzymes involved in mitochondrial activity. Mitochondrial respiration declined with all substrates used, indicating a more general damage of mitochondria rather than specific defects of respiratory chain complexes. The activity of the mitochondrial matrix enzyme citrate synthase was relatively stable in syngeneic grafts, but decreased substantially (up to 80% at 60 days) in both experimental groups and correlated well with cardiac score. However, enzymatic changes were more evident in CRI group. In contrast, the activity of mitochondrial respiratory chain enzyme complex I equally declined in all groups and did not match with cardiac score. Activity of cytosolic enzyme lactate dehydrogenase was similar in CRI and CR, decreasing to 50 % of syngeneic controls at 60 days.

Prolonged CI substantially contributes to the progression of chronic rejection and loss of organ function in this model. Patterns of gene expression as well as mitochondrial respiration and enzymatic injuries reflect severe mitochondrial damage in response to CI and progression of chronic rejection.