Cold or Not So Cold?—Static Organ Preservation at 10 °C May Prolong Organ Preservation and Facilitate Transplant Logistics

To achieve optimal results, particularly cardiothoracic procurement teams have pushed routinely for an optimal organization with a timed donor cross-clamp and an overlapping transplant procedure to maintain a short static cold storage (SCS). In addition to an overall functional improvement of solid organs with viability assessment, there is a great interest in prolonged preservation to facilitate logistics and potentially enable daytime transplantation. Despite economic benefits, SCS limits the ability for organ treatment and viability assessment. The main advancement in organ preservation has thus focused on novel perfusion devices and methods, whereas preservation solutions used for SCS have largely remained unchanged during the last 40 y. Preliminary studies published >30 y ago have suggested that 10 °C is the optimal lung storage temperature.1 Interestingly, most hypothermic machine perfusion studies have used temperatures between 6 and 10 °C and this higher temperature of perfusion may be one additional explanation of its beneficial effects compared with cold static preservation (at 4 °C). Investigation of this protective mechanism needs to be further explored. Ali et al from Toronto delineated the impact of prolonged SCS on lungs donated after brain death in a pig model. The authors exposed porcine lungs to 36 h of SCS at either 4 or 10 °C and evaluated them thereafter over 12 h on normothermic ex vivo lung perfusion.2 Interestingly, SCS at 10 °C demonstrated less injury and improved pulmonary function when compared with lungs cold stored at 4 °C. The concept of prolonged SCS was then tested clinically. Following standard procurement with cold flush, 5 human double lungs underwent 5–8 h of SCS at 4 °C during transport. At arrival in the recipient center, SCS was continued at 10 °C for additional 3.5–9.6 h. All lungs showed immediate function without the need of extracorporeal support after implantation.1 The work by the group from Toronto went on to investigate underlying mechanism, focusing on mitochondria as key instigators of ischemia–reperfusion injury (IRI). Higher tissue concentrations of itaconate were found after SCS at 10 °C. During hypoxia (ischemia), mammalian cells activate their own inherited protective mechanisms. Those include slowing cellular metabolisms while putting the electron flow at the respiratory chain and the tricarboxylic acid (TCA) cycle on hold. This process leads to an accumulation of succinate together with its precursors, including cis-aconitate, which is metabolized to itaconate (Figure 1).1 Tissue succinate concentrations have been shown to increase with prolonged ischemia in all solid organs.2 At reperfusion, when oxygen is reintroduced, mitochondria aim to immediately metabolize accumulating succinate molecules.2,3 Moreover, with increasing temperatures during reperfusion, the enzymatic processes of the TCA cycle and the electron flow at the respiratory chain are reestablished. Because of the initial undirected electron flow, reactive oxygen species (ROS) are released mainly from Complex I.2 A key strategy to reduce IRI with all downstream consequences has thus been to either prevent succinate accumulation during ischemia or to metabolize the existing succinate molecules at the end of SCS.3 The main actor here is the succinate dehydrogenase (SDH or Complex II), which links respiratory chain and TCA cycle. In their work, Ali et al detected higher tissue concentrations of itaconate when lungs had been preserved at 10 °C. nterestingly, accumulated itaconate during SCS at 10 °C appears as competitive inhibitor of SDH (Figure 1).1,4 Yi et al5 have recently confirmed that downstream effects of itaconate are mediated through immune-responsive Gene 1, with a loss of protection observed when the gene was knocked out. The intact gene–protein sequence, in contrast, has been shown to reduce severe IRI in hepatocytes. Other groups have also demonstrated protection through itaconate in other organs, including brain and heart.6FIGURE 1.: The key role of Complex II during ischemia–reperfusion injury in solid organ transplantation. During prolonged ischemia at cold temperatures, metabolic demands are reduced. (1) Itaconate accumulates during ischemia together with succinate, cis-aconitate, and other metabolites of the TCA cycle as a natural mechanism of protection. Accumulated itaconate inhibits Complex II (SDH) competitively, reducing oxygen consumption. (2) At the same time, TCA cycle metabolites accumulate, inducing higher concentrations of glutamate that are then metabolized to glutamine and glutathione. (3) Subsequent to reperfusion and reoxygenation, the respiratory chain aims for a rapid establishment of electron flow with subsequent ATP production to fuel the augmenting metabolic demand with increasing temperature. (4) High succinate concentration at the end of ischemia supports this mechanism and induces a rapid restart of the succinate metabolism to fumarate, at the price of undirected (retrograde and forward) electron flow with subsequent release of ROS mainly at Complex I. Accumulating itaconate may partially inhibit the SDH during initial reperfusion with a slower or reduced initial succinate metabolism. This may likely lead to a reduction of ROS molecules released through RET. This natural mechanism of protection highlights the importance of succinate metabolism by Complex II/SDH during initial ischemia/reperfusion. (5) HOPE was shown to slowly reestablish a normal directed electron flow without significant ROS release, thereby slowing the breakdown of accumulated succinate. Figure design supported by biorender.com. HOPE, hypothermic oxygenated perfusion; RET, reverse electron transport; ROS, reactive oxygen species; SDH, succinate dehydrogenase; TCA, tricarboxylic acid.Another natural protection arises with the accumulation of glutathione that shields cells through ROS quenching and covalent modification of proteins to protect their molecular structure from damage through ROS (Figure 1). The work by the Toronto group emphasizes on the importance of Complex II in the IRI cascade, providing a perfect link between underlying mechanisms of prolonged SCS at 10 °C and other organ treatments, including the more dynamic approach of hypothermic oxygenated perfusion (HOPE), also performed at temperatures between 8 and 10 °C. Two randomized controlled trials and several retrospective studies have shown clear protective effect of HOPE after SCS in liver and kidney transplantation.7,8 Although the role of cellular ATP recharging during HOPE has been established, the higher Complex II activity with subsequent succinate metabolism has recently been identified as an additional key protective mechanism.9 The slow and steady succinate reduction during HOPE leads to an amelioration of IRI with lower ROS release from Complex I and less inflammation during normothermic reperfusion (Figure 1). Further downstream effects include a reduced immune system activation that may reduce rates of acute rejections while improving immune surveillance. Interestingly, this mechanism was recently also confirmed in hearts and kidneys,10 demonstrating that the protective HOPE effect at Complex II appears in all solid organs. Although glutathione has already been added to standard perfusion solutions during HOPE treatment,7 itaconate might serve as important additional additive that could be tested in SCS solutions to achieve prolonged and improved static preservation. Clearly, more studies are required to also identify the value of itaconate in organs with higher risk profiles, for example, from donors after circulatory death. Despite the promising results presented by Ali et al, the acceptable duration of SCS in donors after circulatory death organs remains under debate and future trials will ultimately provide more data to safely accept organs after prolonged SCS, ideally making daytime transplant surgery possible. Independent of the modality used, a growing body of evidence supports the advantage of machine preservation over SCS for suboptimal organs. Nevertheless, machine perfusion adds costs and complexity. If the advantages of SCS at 10 °C will be confirmed in large randomized controlled trial for several organs, then this approach may have benefits for purely logistical purposes that have become more relevant with broader sharing. The ultimate desire for transplant surgeons is to make the procedure elective, performed during regular hours with the best quality organ. Clearly, limitation in quality of life is one of the main reasons surgical residents shy away from transplant fellowships or for practicing transplant surgeons to experience burnout. Off-hour procedures have been shown to be longer and associated with a 2-fold greater mortality rate.11 Moving transplants from the nighttime may also provide potential cost savings with reduced rates of postoperative complications and shorter length of hospital stay. In summary, the work by Ali et al provides new insight into underlying mitochondrial mechanisms to protect organs during the entire process of preservation and transplantation. Although it is expected that those mechanisms will also apply to hearts and abdominal organs, confirmation is required. Organ preservation continues to be a fast-changing, exciting, and relevant area moving our field forward. Close collaborations among experts from different specialties will therefore be critical to target the complex cascade of IRI aiming to optimize outcomes.

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