Introduction
Organ transplantation remains the ultimate treatment option for terminal organ failure. However, there is a substantial organ shortage because there is a greater demand for organs than there are available organs. Increased-risk organs from extended criteria donors (ECD) might be taken into consideration for transplantation thanks to the application of modern preservation technologies (i.e., machine perfusion). However, throughout organ collection, preservation, and transplantation, these organs are particularly vulnerable to further harm. Because multiple molecular downstream pathways are triggered, the effects of ischemia-reperfusion injury (IRI) brought on by oxidative stress and subsequent events during early reperfusion have a detrimental effect on both the short and long-term prognosis following transplantation.
IRI Is the Key Event Leading to Oxidative Stress in Organ Transplantation
Broadly, the imbalance between reactive oxygen species (ROS) generated and antioxidants present is known as oxidative stress. In the setting of solid organ transplantation, one of the most common ROS-related pathologies is IRI. IRI is inherently connected to organ transplantation. It is characterized by obstructed blood ow causing ischemia during organ retrieval and preservation, followed by a reperfusion phase when the blood ow is restored in the recipient (Figure 1). During ischemia, adenosine triphosphate (ATP) levels decrease. In turn, ATP-dependent Ca2+, H+ and Na+ pumps fail, causing accumulation of ions which contributes to cell swelling. pH levels decrease leading to acidosis. Accumulation of succinate, nicotinamide adenine dinucleotide phosphate (NADPH; resulting from NADP+ and H+) and hypoxanthine during ischemia prime for excessive ROS release after reperfusion. Additionally, in mitochondria major reactive oxygen species (ROS) generation occurs. ROS cause direct damage to biomolecules but also act as signaling molecule. Besides this, opening of the mitochondrial permeability transition pore (mPTP) during reperfusion also triggers cell death by release of cytochrome c and breakdown of ATP production.
Together with the accumulation of reductive equivalents during ischemia, the absence of ROS is responsible for reductive stress. In this regard, three of the major couples of the cellular redox network are NAD+/NADH, NADP+/NADPH and GSH/GSSG. Like oxidative stress, also reductive stress contributes to the overall redox stress resulting in impaired cellular functions.
Molecular Mechanisms Counteracting Oxidative Stress
In order to counteract oxidative stress, organisms exhibit their own enzymatic and non-enzymatic antioxidant defense systems. One of the most important transcription factors in this regard is the Keap1-Nrf2 pathway. Activation of NFκB in the liver has been shown to reduce hepatic IRI injury and facilitate orthotopic liver transplantation.
Biomarkers to Study Oxidative Stress
As direct contributors to oxidative stress, ROS should be considered as potential biomarkers. Direct detection would allow for quantification of oxidative stress. However, due to the short half-life of ROS, this is currently a very complex method. Instead of tracking ROS itself, their effects on biomolecules can be detected. Alterations in expression or formation induced by ROS can be used as valuable surrogate biomarkers for oxidative stress. Roughly, these molecules can be categorized as follows: endogenous antioxidants, lipid peroxidation, oxidative protein changes and nucleic acid oxidation.
Endogenous Antioxidants
Organisms possess defense systems against free radicals, one being facilitated by antioxidant enzymes. These can be quantified and serve as biomarkers. Catalase (CAT) is an enzyme found in almost all living organisms that are exposed to oxygen. Within the field of transplantation, it is most widely used to assess oxidative stress. Another antioxidant is SOD, an enzyme group that acts as a crucial part of the antioxidant defense against highly reactive superoxide radicals. It is responsible for splitting (dismutation) of H2O2. GPx can reduce H2O2 or organic peroxides to water and alcohol with the presence of glutathione and is subsequently converted to oxidized glutathione.
Lipid Peroxidation
Lipid peroxidation products can lead to the synthesis of, for instance, malondialdehyde (MDA). MDA and the reactive thiobarbituric acid substance (TBARS) are considered basic markers of lipid peroxidation, potentially serving as biomarkers. Additionally, isoprotanes serve as valuable markers, where F2 and F4 isoprotanes should be distinguished. F2 isoprotanes are formed by free radical catalyzed peroxidation of arachidonic acid, whereas F4 is a product of the same reaction of docosahexaenoic acid. It is also interesting to note that F4 isoprotanes exert a strong anti-inflammatory effect, which underlines the link between oxidative stress and inflammation.
Redox Modification of Proteins
When it comes to protein changes due to oxidative stress, 3-nitrotyrosin is considered as one of the most promising biomarkers. Nitration of protein-bound and free tyrosine by ROS leads to the formation of this molecule. Besides nitrotyrosines, protein carbonyls are also widely used as biomarkers for oxidative stress.
Nucleic Acid Oxidation
DNA damage caused by hydroxyl radicals occurs much less frequently than oxidative protein changes. However, the consequences of nucleic acid oxidation, such as mutations, are considerably more harmful. The selection of suitable biomarkers is depending on the study and may not rely on a single analysis method rather than on supplementary methods.
Advanced Organ Preservation: Ex Vivo Machine Perfusions
Throughout several decades, the gold standard for organ preservation has been static cold storage (SCS) at 4 °C . Cellular metabolism and oxygen consumption are reduced at hypothermia, widely preventing damage to the tissue. During the retrieval process, organs are flushed with cold preservation solution in order to deprive the organ of blood, while providing cytoprotection. In the recent years, dynamic preservation by machine perfusion has found its way into clinics, which helped to increase the donor pool for abdominal and thoracic organs. This is of specific interest for, but not limited to, ECDs. Such marginal organs are o en not considered for transplantation otherwise and predicting outcome remains dicult. Moreover, logistics and recipient-related issues are convincing reasons to opt for MP. It has been proven that MP technologies are aiding in tackling problems like IRI and downstream inflammatory processes and improving graft function early after transplantation as well as long-term survival. Different MP strategies operating at various temperatures are available and explored to different degrees (Figure 2).
Hypothermic Machine Perfusion (HMP)
Similar to SCS, HMP is carried out at 4 °C. Metabolism is reduced significantly to about 10%, which decreases energy demand and preserves ATP. Despite residual cellular function being le , oxygen supply is not routinely used in standard care. However, it could be demonstrated that the addition of oxygen carriers and providing oxygen supply to perform the so called hypothermic oxygenated machine perfusion (HOPE) exerts further beneficial effects. Superior outcome of HMP treated organs over SCS organs could be demonstrated in the past.
Subnormothermic Machine Perfusion (SNMP)
SNMP settles in between HMP and normothermic machine perfusion (NMP). Perfusion solutions rely on the physically dissolved oxygen at temperatures between 20–25 °C. Compared to HMP, partial testing of viability is possible during SNMP. However, it is not widely used and requires more research to assess feasibility.
Normothermic Machine Perfusion(NMP)
NMP is performed at 37 °C to mimic physiological conditions. Aerobic metabolism is restored in this MP modulation, therefore shortening ischemic time. Moreover, NMP enables organ assessment at a regular metabolic rate and offers the opportunity for treatment and direct manipulation of a graft prior to transplantation.
Influence of Perfusion Modalities on Oxidative Stress-Induced Tissue Damage
Temperatures, oxygenation and perfusate composition are the parameters of interest to adjust. Lower temperatures decrease the mitochondrial oxygen consumption as well as the activity of other enzyme systems. In HMP preserved DCD livers the mitochondrial redox state is altered, leading to decreased initial ROS release during reperfusion.
Conclusion
Dynamic organ preservation has increased the number of organs available for transplantation. Oxidative stress-induced IRI remains a key event during organ retrieval, preservation and reperfusion, and profound understanding of the underlying mechanisms, especially during MP, is still missing. Research in the eld of transplantation has been widely focused on in vivo studies both, in humans and animals. Of all the various MP methods mentioned above, normothermic machine perfusion of whole organs best mimics the in vivo setting by providing near physiologic conditions. However, access to human organs for research is very limited and animal models should be kept to a minimum following the 3R principles.
Additionally, complex logistics, and the need for large human and financial resources associated with MP experiments underscore the need for reductionist in vitro models, which are suitable to study particular early aspects of redox stress-associated damage and to pretest potential therapeutic interventions. Several in vitro models posing valuable alternatives are listed in this review. Depending on the research subject, there is a suitable model that allows for studying particular features, conditions and/or treatments in parallel. Isolated mechanisms can be assessed in a controlled, planned manner and in the absence of systemic influences. More o en models are combined, in order to first explore, e.g., therapeutic agents and later apply them in ex vivo MP studies. This reinforces the need for further research and development in this eld. By further deciphering of the mechanisms, novel strategies to prevent and counteract oxidative stress could be developed which may help to increase the number of organs available for transplantation.
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