Maintaining organ viability during preservation is essential for successful liver transplantation. With the current practice to accept extended criteria donor organs, it is imperative to improve organ preservation techniques so that the viability of these marginal organs can be enhanced. Previously, this group and others have shown that NMP is an effective method to resuscitate DCD livers [1
]. In the present study, we provide further evidence that the recovery of DCD livers with extended warm ischemia time by NMP occurs after as early as 1 h of NMP perfusion. In addition, we demonstrated that the improvement of viability and functionality is associated with restored liver tissue ATP content, providing a possible approach towards elucidating the molecular mechanisms of NMP.
As a major “energy currency,” ATP is essential to maintain biological processes in cells. There is abundant evidence based on both animal and human studies suggesting that the level of ATP in the liver prior to transplantation is an important determinant for allograft and recipient survival [15
]. In warm ischemic livers, ATP levels have been shown to decline so rapidly that they were nearly undetectable after 15 min of warm ischemia [18
]. In line with these findings, we observed a marked decrease in ATP in pig livers after 60 min of warm ischemia. Conceivably, in warm ischemic circumstances with ATP depletion of this magnitude, tolerance to the hypothermia that accompanies cold storage, as well as the subsequent re-warming phases, may be significantly compromised. In addition, although ATP is a universal cellular compound, its depletion can play a specific role in different cell-types. For instance, Doctor et al.
] have found that 60 min of warm ischemia in cholangiocytes caused striking alterations in the membrane-cytoskeleton organization, with loss of apical microvilli coinciding with selective disassociation of the linking protein “ezrin” from the microvillar cytoskeleton. Because these membrane structures are essential for differentiated epithelial function, their dysregulation may play a role in transplantation-related pathology, such as impaired biliary function or development of non-anastomotic biliary strictures after transplantation, which are more frequently observed in DCD allografts [20
]. Possibly, while a certain threshold level of ATP is likely to be crucial in regular liver transplantation, in DCD livers this threshold is not always met. Hence, restoring the ATP content might be a prerequisite for successful resuscitation of DCD livers. In fact, several strategies of modifications on hypothermic storage/perfusion aiming to increase tissue ATP levels were reported to improve DCD liver viability. In contrast to these hypothermia-based strategies, NMP provides an ideal platform for organ recovery by mimicking the physiological environment, without hypothermia-induced discontinuity in the endothelial lining and alterations in the endothelial cell cytoskeleton, which were found to be major culprits of post-ischemic liver dysfunction [21
]. We concede that in the clinical setting, a degree of cold preservation will always be incorporated. However, NMP seems able to assuage cold insults and regenerate the liver towards a state of high metabolism and energy content.
In this study, we demonstrate that during normothermic perfusion, DCD livers initially show inferior viability and functionality. Within 1 h of machine perfusion, parameters such as liver enzyme release, bile production rate, and vascular pressure improved to similar levels as the control group. These observations were accompanied by a slightly higher oxygen consumption rate and a rapidly increasing ATP content, which may indicate an active recovery process. ATP deficiency in DCD livers is linked directly to the occurrence of mitochondrial dysfunction, which has been frequently reported in ischemic livers [22
]. Concordantly, we found that both overall liver architecture and mitochondrial integrity were significantly improved after NMP. Given our results as well as the critical role of ATP in allograft viability, we propose that the improvements seen in function, morphology, and viability have an origin in the restoration of ATP levels in the mitochondria of the various cell types in the allograft. Further investigation towards the processes underlying NMP and energy restoration is warranted.
The perfusion system used for these experiments incorporates dual (portal and arterial) perfusion. Similar to the system used by Jamieson and Friend et al.
], we use a centrifugal pump to provide constant pressure on the arterial branch. The flow in the portal vein was passively driven by the gravity of the venous reservoir. To minimize pressure-injury, the organ was immersed in perfusate in a customized organ chamber, which is also designed to avoid “bruising” of the organ by its own weight, which would disrupt perfusion in the peripheral microcirculation of the organ. The stable hemodynamics demonstrated by our system shows that it is capable of avoiding such damage.
In summary, the present study provides evidence that DCD livers that have undergone 60 min of warm ischemia can be resuscitated by normothermic machine perfusion. NMP effectively restored the tissue ATP content and improved the mitochondrial integrity, which might contribute to the improvement of DCD liver viability and functionality. The information provided in study might help us to better understand the repair mechanism of NMP, which has great potential to successfully use DCD livers as an alternative resource and enlarge the total number of viable donor livers available for transplantation.