Functional and histological comparison of rat liver preserved in University of Wisconsin solution compared with tissue preserved in a novel solution.

An isolated perfused rat liver model was used to investigate biochemical and histologic changes during 2 hours of reperfusion after 24 hours of cold storage to compare Leeds solution (LS) with University of Wisconsin solution (UW). Compared with livers stored in UW, those perfused with LS showed significantly higher bile flow and lower enzyme production (P < .05 by 1-way analysis of variance). For example, after 120 minutes, alanine aminotransferase results were: LS 38.9 U/L vs UW 66.8 U/L and bile flows were LS 10.3 µg/15 min/g liver vs UW 9.2 µg/15 min/g liver. Histologically the reticulin breakdown was greater and its reformation slower in UW-preserved livers. Liver tissue was viable in both groups, as shown by the increased glycogen content after reperfusion in both groups, but seen at a higher rate among LS, perfused livers. In conclusion, LS compared favorably with UW to prevent ischemic damage and so could offer an alternative perfusion medium to UW.

PBSH: a new improved cardiac preservation solution in comparison with three clinically proven solutions.

INTRODUCTION: A solution under development at our institute, based on phosphate-buffered sucrose, has shown good preservation for kidneys and livers. This work used a refined version of this solution (PBSH-phosphate-buffered sucrose for the heart) in heart preservation, comparing it to solutions already widely used (University of Wisconsin, St. Thomas's, and Celsior solutions). METHODS: Following an initial washout phase and control working mode on a Langendorff system, hearts were flushed with preservation solution and after 6 hours at 4 degrees C were then reperfused for 15 minutes followed by working heart mode for a further 30 minutes. Hemodynamic parameters were measured and compared with their preischemic values and expressed as percentage recovery. Enzyme measurement came from the collection of the initial 1.5 mL of the coronary effluent after the storage period. This was to test for creatine phosphokinase (CPK) and lactate dehydrogenase (LDH). Hearts were then placed immediately into liquid nitrogen for adenosine triphosphate (ATP), lactate, and creatine phosphate (CP) testing. Spectrophotometric analysis was used to assess both the release of CPK and LDH into the coronary effluent, and the level of ATP, lactate, and CP in the frozen heart tissue. RESULTS: These results show that hearts that are preserved in PBSH are hemodynamically as well preserved as the hearts preserved in other solutions tested and their enzyme and lactate content is lower while having higher levels of energy compounds in these hearts. CONCLUSION: Overall these results show that PBSH is at least as effective in cardiac preservation in the rat model of 6 hours of cold ischemia as these other widely used solutions tested.

Influence on energy kinetics and histology of different preservation solutions seen during cold ischemia in the liver.

BACKGROUND AND PURPOSE: Cold flush preservation prolongs tissue viability during ischemia. However, there is little understanding of the effects of various preservation fluids on events during this period. A study of cold ischemia in rat livers was undertaken to compare biochemical and histological changes over time, using three preservation solutions: University of Wisconsin (UW), histidine-tryptophan-ketoglutarate (HTK), and Leeds solution (LS) under development at our institution. Leeds solution is a phosphate-based sucrose solution that like UW contains the impermeant lactobionate and the metabolite allopurinol (1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one) which acts as a competitive inhibitor of xanthine oxidase, stopping the breakdown of hypoxanthine to xanthine by oxidizing it to alloxanthine, inhibiting both the conversion of hypoxanthine to xanthine and the conversion of xanthine to uric acid. MATERIALS AND METHODS: At various time points, samples were analyzed for adenosine triphospate (ATP) and metabolites by high-performance liquid chromatography as well as for histological changes. RESULTS: In all livers, ATP, ADP, and AMP degraded over 4 hours. In UW and LS groups, degradation beyond hypoxanthine was halted, and it continued in the HTK group. This blockade led to a significant reduction in the accumulation of xanthine and uric acid. Histological analysis showed protected architecture and maintenance of reticulin scaffolds in the UW and LS groups, whereas tissue breakdown was seen from earlier time points in the HTK group. Additionally, throughout ischemia, signs of pathological injury were more pronounced with UW- than with LS-preserved tissue. CONCLUSIONS: These results implied that cold ischemia in the liver is characterized by dynamic biochemical changes coincident with pathological injury which are initiated from the time of organ perfusion and influenced by the choice of the perfusion fluid. Allopurinol in UW and LS appears to be critical. We hypothesized that it may also affect the degree of subsequent reperfusion injury. The data supported the assertion that LS offerred improved preservation over UW, adding to the impetus to shorten ischemic times in clinical transplantation.

Histidine-tryptophan-ketoglutarate and delayed graft function after prolonged cold ischemia.

BACKGROUND: Several articles have compared histidine-tryptophan-ketoglutarate solution (HTK) with other preservation solutions in both liver and kidney transplantation, and the results suggest that HTK is as good or better than the criterion standard University of Wisconsin solution (UW) for short periods of cold ischemia, such as in live donation, but that it is not so efficient for longer periods of cold ischemia, causing a higher incidence of delayed graft function. OBJECTIVE: To evaluate energy levels, metabolites, and histologic findings to determine why HTK is inefficient for longer periods of cold ischemia. METHODS: Rat livers were perfused with either HTK or UW, and at various times, tissue samples were obtained for analysis of adenine triphosphate and metabolites using high-performance liquid chromatography or for histologic analysis. RESULTS: The high energy charge observed with HTK-perfused livers plateaued after 5 minutes, and by 60 minutes began to decrease, following the same trend as other samples. The plateau is due to excess available glucose; however, after 1 hour, it is beginning to be consumed. Low levels of uridine, required for glycogen synthesis, are found in HTK-perfused livers, which suggests that at reperfusion, there is none available, whereas the higher concentrations found in UW-perfused livers may be advantageous after reperfusion. This will be especially detrimental to use of HTK because glycogen is used up rapidly because of the presence of alpha-ketoglutarate in the solution, enabling continuation of the tricarboxylic acid cycle. CONCLUSIONS: Overall, HTK seems to do well for the first 2 hours, after which any advantage observed initially starts to disappear. A liver perfused in HTK and transplanted after more than 1 hour reacts like an organ from an individual who has been starved, because of the low energy charge and absence of a glycogen store or ability to synthesis glycogen because of lack of uridine. Livers perfused with UW demonstrate higher levels of uridine and do not lose their glycogen content to the same extent as HTK-perfused livers. These findings explain in part why HTK sometimes causes delayed graft function after longer periods of cold ischemia.

Addition of adenosine to University of Wisconsin solution: does it help?

Adenosine Triphosphate (ATP) precursors are sometimes added to preservation solutions in the belief that once the organ is reperfused, these precursors will build up ATP rapidly, returning it to its original metabolic state. This work studied ATP and metabolites during preservation of the rat liver using University of Wisconsin solution (UW), which contains adenosine, versus histidine tryptophan ketaglutarate solution a new phosphate-based preservation solution, or leeds solution (LS), which is under development at our institution (neither of the latter 2 contains adenosine). Tissue samples of perfused livers were analyzed for ATP and metabolites by high-performance liquid chromatography. UW did initially show the expected significant difference in overall adenosine levels, but the advantage had disappeared by 4 hours. At no time did UW show significantly higher levels of ATP; this was not seen following adenosine addition to LS. Only in living donor transplants where the cold ischemic time is short may there be some advantage to the addition of adenosine.

Ischemic epigenetics and the transplanted kidney.

The primary purpose of this investigation was to study oxidative demethylation of DNA following ischemia/reperfusion injury (I/RI) that putatively influences posttransplant gene expression in transplanted kidneys. Our hypothesis was that as a result of I/RI, oxidative damage, which is inherent in solid organ transplantation, may lead to aberrant demethylation of cytosine-guanine (CpG) sites within gene promoter regions of DNA. The methylated CpG sites normally contribute to the binding of proteins that render DNA inaccessible to transcription factors. Therefore, conversion of methylated cytosines to nonmethylated cytosines by oxidative damage in postischemic organs might facilitate enhanced gene expression in donor organs by exposing the demethylated CpG site in a gene promoter to DNA-binding proteins that enhance gene transcription. In this study, we investigated the demethylation of a specific CpG within the IFNgamma response element resident in the promoter region of the C3 gene in the rat kidney. In response to 24 hours of cold ischemia and a subsequent 2 hours of reperfusion in an isolated ex-vivo circuit, we observed a significant change in the ratio of methylated to unmethylated cytosines at this site. Epigenetic modifications to donor DNA have not been previously investigated, but our own data suggests that they have the potential to modify gene expression posttransplantation. Since epigenetic modification may become stable and heritable upon mitosis, such changes to the donor organ DNA may persist with enormous implications for transplant outcomes.

A new selective differential medium for isolation of Stenotrophomonas maltophilia.

A new selective differential medium for the isolation of Stenotrophomonas (formerly Xanthomonas) maltophilia was developed. The medium, VIA agar, contained vancomycin, imipenem, and amphotericin B as selective agents and incorporated a mannitol/bromothymol blue indicator system. Compared with Xanthomonas maltophilia Selective Medium (XMSM), VIA agar was less inhibitory to Stenotrophomonas maltophilia and was more selective than XMSM in preventing the growth of unwanted bacteria from contaminated specimens. Although vancomycin-resistant strains of Enterococcus faecium may grow on VIA agar, these can be easily distinguished from Stenotrophomonas maltophilia because of mannitol fermentation.