After the advent of the experimental evidences of positive effects of IPC in I/R injury, several clinical studies started to show conflicting results about the real benefits of this procedure in medical practice. Recently, studies with a high level of evidence, as clinical trials, systematic reviews, and meta-analyses have demonstrated beneficial effects of direct or remote IPC in clinical situations involving myocardial15,16, renal17, and neuronal ischemia18. However, it is known that different tissue and organ sensitivities to ischemia are diverse so that such benefits cannot be generalized.
In the current literature context, the real effects of IPC in hepatic I/R injury, namely in liver transplantation, are not clear. Besides, different modalities of IPC are impractical procedures that may further increase the already high complexity of transplantation surgery. So, although several experimental investigations have shown promising results of IPC in liver IRI of small animals,19-22 there are few studies with models of cold and hot ischemia in liver transplantation performed on medium-sized animals23,24 and this motivated us to evaluate such effects utilizing a model that simulates the human condition.
We have been utilizing the porcine model in our laboratory because it mimics the clinical situation of liver transplantation.9,10,25,26 In a previous study, utilizing a model similar to the present one, we observed that IPC resulted in partial attenuation of the harmful effects of I/R injury.10 In the current study, we also aimed at identifying if the positive effects of IPC remained after a longer observation time, thus providing a rationale for its use in clinical practice.
While studies of IPC are not new, still there is no consensus about technical issues such as ischemia time and number of I/R cycles needed to effectively achieve protective effects. Therefore, in our model, we used 3 alternating 5-minute cycles of ischemia followed by the same reperfusion time, to prevent severe hemodynamic repercussions in the animals, while avoiding a too short IPC period that might have less evident effects. Another objective of this experimental study was to assess RIPC as a means to protect the target organ without causing the direct stress of ischemia-reperfusion. By using the two techniques in separate groups as well as combined, in a specific group that received the two types of IPC, our goal was to ascertain if there would be a difference between preconditioning the graft or the recipient, and to check potential cumulative effects when both procedures are used concomitantly.
For RIPC, the most widely used technique is clamping one limb of a patient27 or animal.28 However, for our project we chose a different target territory, i.e. the gut, by clamping the superior mesenteric artery. This organ was chosen because it has one of the highest levels of metabolic activity and is very susceptible to oxygen pressure variations, and therefore is quickly responsive to short periods of ischemia, which should maximize the protective effect of the RIPC. Finally, it is important to stress that the gut is a territory that markedly suffers from blood stasis during the anehepatic phase of the transplant procedure.
We performed the current experiments to clarify local and systemic effects of IPC in liver transplantation and assess the potential influence of our conditioning models on common problems caused by I/R injury in organ transplants, such as acute kidney injury.29-32 Unlike previous publications, our biochemical results fail to show any benefit of DIPC or RIPC in liver transplantation. Serum AST and ALT levels are consistent with the degree of hepatocellular injury and are used as indicators of graft distress, bearing correlation with different levels of primary graft dysfunction. All groups in our study were comparable in terms of enzymatic profile. In the D+R group, AST showed high variability, with a trend towards higher median values at 12h, 18h, and 24h, suggesting a possible harmful effect of the addition of the two IPC procedures, even though the difference was not significant.
Our results differ from those of studies of liver transplantation in small animals (rats), which showed lower transaminase values and improvement of histopathological aspects in animals submitted to RIPC compared to controls after 24h of reperfusion.26 On the other hand, our results are consistent with those from human studies in which direct and remote IPC increased AST and ALT values 24h after reperfusion.25,30
We made a refinement in this current investigation, by adding some molecular analyses that could show some beneficial effects of IPC. In the liver tissue, the combined IPC approach resulted in marked positive changes in gene expression. The eNOS gene expression in the liver tissue was higher in the D+R group at 24h, and such expression is usually related to improvement in ischemia. In addition, lower expression of pro-inflammatory genes (BAX) and higher expression of anti-inflammatory genes (Bcl-XL) were observed in this same group. DIPC also had positive effects, leading to increased expression of the IL-6 and Bcl-XL genes.
The BAX/Bcl-XL ratio showed lower values for all treated groups when compared with controls at 24h. This finding may suggest an IPC-driven potential decrease in cell apoptosis secondary to I/R injury. Furthermore, in the gut, kidney, and lung tissues, some molecular changes were detected demonstrating beneficial effects of each IPC separately.
Although gene expression suggests positive effects, with results showing broad ranges probably due to the small sample size, we cannot infer that in the complex situation of liver transplantation these results would be sufficient to indicate an IPC procedure. In addition, the results of biochemical and histopathological analyses, consistent with human studies, did not confirm any benefits from IPC, which raises questions about the feasibility of extrapolating results obtained in small animals to medium-sized animals and humans. The first hypothesis to explain this difference would be a higher susceptibility of certain species, e.g. rodents, to the IPC procedure, either in situ (direct) or remote, with this propensity diminishing as we advance phylogenetically towards humans, as in the case of pigs.
Also, worth mentioning are the physiological, anatomical, and surgical variations involved in organ transplantation in different species. Despite the highly ingenious technical solutions found to overcome difficulties and enable organ transplantation in rats and mice, the surgical procedure in these animals cannot match what happens in human surgery, including technical hurdles, hemodynamic instability, surgical time, and postoperative follow-up. The human context is optimally mimicked by transplantation performed in medium-sized animals, which yields similar results.
Finally, it remains an important question: although considered a good idea, why the ischemic preconditioning in the pig model did not work out? Probably the ischemia-reperfusion represents intense stress to liver graft, that all benefits of ischemic preconditioning are covered by the real ischemic impact.
Considering the great complexity of the transplant surgery and based on the results of the current investigation, we may conclude that the three methods of IPC herein utilized may not be utilized in clinical practice.