Lung transplantation (LT) is the mainstay for treatment for patients with end-stage lung disease. Unfortunately, the shortage of suitable donors for transplantation remains a limiting factor and a challenge.1 Indeed, in most multiorgan-donors lungs are too deteriorated for donation and only 20–25% are acceptable for LT.2 Hence, it is critical to develop new strategies to increase the pool of organ donors. In this context, several alternatives have been investigated in recent years, including the use of so-called extended criteria donor lungs, the use of donors after cardiocirculatory death, and the implementation of ex vivo lung perfusion.3–5

Extending the accepted graft ischemia-time may increase the utility of donor lungs by enlarging the time frame to perform long distance lung procurements. Cold preservation has been the classic procedure to reduce lung injury while the organ is transported from donor to recipient. During lung procurement, lungs are flushed with a low potassium dextran solution, inflated with oxygen and stored at 4°C. The objective of hypothermic organ preservation is to decrease the cellular metabolism of the graft and thus, reduce the cell death ratio. However, ischemic insult leads to the generation of reactive oxygen species that result in cell injury.6 In this respect, it is worth noting that the limit of cold ischemia time (CIT) is poorly defined. In clinical LT, 8h is the generally accepted upper limit for CIT. Accordingly, extending the arbitrarily established ischemia time is a matter of debate because of the perceived increased rate of ischemia-reperfusion (IR) injury that could lead to primary graft disfunction (PGD).7,8

In the early post-operative period, PGD is the leading cause of morbidity and mortality, with up to 20% of recipients developing severe PGD. An important feature of PGD is the early development of inflammation as the result of the activation of innate immune mechanisms. These mechanisms involve the activation of pro-inflammatory transcription factors that induce the expression of inflammatory mediators, primarily inflammatory cytokines and chemokines, which promote the recruitment of activated leukocytes and subsequent inflammatory cytotoxic consequences.7–9 The mechanisms responsible for the triggering of innate immune response and acute inflammation in IR injury have not been elucidated so far. In this respect, several clinical series have demonstrated that the perceived detrimental effect of extended graft ischemia time on PGD might not be well founded.10–12

In view of the limited evidence in the clinical setting, the objective of the present study was to analyze the impact of extended CIT on IR injury following LT in a swine experimental model by means of determination of histologic injury, inflammatory profile and cell death in the lung graft.

This was an experimental, randomized pilot trial of parallel groups and a final blind analysis using a swine model of LT developed for the purpose of the study. Animals received humane care during experiments in compliance with the “The Guide for the Care and Use of Laboratory Animals” and were housed and handled in accordance with European law (Directive 2010/63/EU). The study was evaluated and approved by hospital and competent authority's Ethics Committee on Animal Research (no. 10325). Sixteen female commercial hybrid pigs weighing 30–35kg were used (8 as donors and 8 as receptors).

Study design

Donor animals (n=8) were submitted to organ procurement under general anesthesia. The lungs were separated into two groups, the 6h-CIT group (n=4) and the 12h-CIT group (n=4) and were respectively subjected to 6h and 12h of ischemic aerobic hypothermic static preservation. At the end of the preservation, the left lung was transplanted into a recipient animal and re-perfused for 4h (Fig. 1).

Fig. 1.

Study design. This study was comprised of 2 groups (n=4 each). Swine donor lungs were preserved at 4°C for 12h in the 12h-CIT group, and for 6h in the 6h-CIT group, respectively. The left lungs were transplanted and reperfused for 4h. Lung tissue samples were collected at different timepoints as follows: (i) CIT0: at the beginning of the CIT, (ii) CIT1: at the end of the CIT; (iii) Rp30min: 30min after the reperfusion of the left LT; (iv) Rp4h: 4h after the reperfusion of the left LT. CIT: cold ischemia time; LT: lung transplant.

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In this pilot swine model of LT after extended CIT, the performance of donor lungs that underwent 12h CIT was found to be as good as that of lungs that underwent 6h CIT. No differences were found in terms of decisive characteristics of acute lung injury14,15 such as microscopic lung injury, lung edema, inflammation or apoptosis.

The maximum safe preservation time for human LT remains undefined. However, there is a reluctance to extend the conventionally accepted <8h of cold ischemia.16 Thabut et al.16 analyzed data from 752 patients who underwent LT in seven French transplantation centers during a 12-year period. Graft ischemic time was associated with early PGD and with long-term survival in patients undergoing single or double LT, but not in patients undergoing heart-lung transplantation. More recently, a retrospective study based on United Network of Organ Sharing (UNOS) database evaluated the effect of prolonged total graft ischemia times (>6h) on long-term survival and the development of PGD.10 Interestingly, prolonged graft ischemia was not associated with an increase in 1 and 5-year mortality and was also not a negative predictor of PGF.

Bearing in mind that reproducibility in animal research and translation from bench to bedside is still a challenge,17 our large animal model study design emulated the clinical setting where lungs recovered for transplantation require a period of aerobic cold ischemic preservation to be transported back to the transplant center.

IR injury is inevitable in solid organ transplantation.18 However, the lung has to be considered differently because it contains oxygen in the alveoli which, helps to maintain aerobic metabolism and prevents hypoxia during ischemic preservation.19–21 In addition, ischemia is characterized by the absence of blood flow into the lung, which can cause oxidant injury despite the presence of oxygen. Thus, it can occur during the storage period.21,22 Our results with regards to microscopic lung injury deteriorated to a similar degree in both the 6h-CIT and 12h-CIT groups after reperfusion. Activation of the innate immune system induces the expression of inflammatory mediators which promote the recruitment of activated leukocytes that have subsequent inflammatory cytotoxic consequences.18,23,24 We assessed the development of graft inflammation through the evaluation of key proinflammatory cytokines. The exposure of lung cells to 6 and 12h of CIT followed by 4h of reperfusion resulted in enhanced proinflammatory cytokines such as TNF-α, IL-1β and IL-6. Interestingly, we observed that TNF-α protein was expressed early and then returned to baseline production at 4h of reperfusion. This early appearance of TNF-α supports the implication that it regulates the expression of other proinflammatory cytokines and affects neutrophil recruitment as early as 30min after reperfusion. We did not observe a significant IFN-γ response due to the short reperfusion period. It has been demonstrated that recipient T-cell activation occurs during the late phase of IR injury together with IFN-γ release in a lung transplant setting.25 Likewise, levels of IL-10, the only anti-inflammatory cytokine, remained barely detectable during the reperfusion period.

Unlike necrosis, apoptosis does not occur during ischemia, though it does increase during reperfusion.26 The induction of apoptosis can occur through the intrinsic or extrinsic pathway. The first is dependent on mitochondria and is activated by reactive oxygen species. The second is dependent on inflammatory molecules, such as TNF-α. In LT, it has been observed that apoptosis peaks rapidly after reperfusion corresponding to up to 30% cells of lung graft.27 In our study, caspase-3 activity was used as a hallmark indicator of apoptosis in LT.28 Although caspase-3 did increase during the reperfusion period, the increase was only slight and did not differ between the study groups. Peak expression of caspase-3 was observed in samples corresponding to 30min after reperfusion. Interestingly, this timepoint coincides with peak expression of TNF-α. In addition, our data suggest that the presence of apoptosis was not related with lung function in terms of edema development. Similar results have been reported in other studies in an experimental setting.29,30

This study has several limitations in the context of large animal experimental models. First, given its nature as a pilot study, the trial was designed as an initial test to analyze CIT in early ischemia-reperfusion injury. For this reason, it was conducted with a small number of subjects and, consequently, its statistical power to detect differences was compromised. Second, the reperfusion period was 4h, far below the 72 h-range covered by the clinical definition of PGD. However, we believe that this does not introduce significant bias as a large number of published works are limited to a 2–4-h reperfusion period.31–33 Because of the complexity of the swine LT model, the literature regarding 3 days survival after LT is scarce.34,35 Thirdly, one of the animals in the 6h-CIT group presented a pneumonia that was not detected macroscopically during lung procurement. Interestingly, this animal had the highest microscopic injury score and apoptotic cell death percentage. In fact, infection is a well-known risk factor for the development of PGD.36 The design of our study mirrors clinical practice where lungs recovered for transplantation require a period of cold ischemic preservation. However, bearing in mind the complex setting of the lung transplant procedure, a clinical trial with prolonged CITs would not be feasible for ethical reasons. It is therefore difficult to translate measures taken in an experimental context into clinical practice. In any case, because humans share more immune-system-related genes and proteins with pigs than rodents or other animals, the swine model is the most widely used large animal model in the context of lung transplantation. Although our study represents a proof of concept in pigs, its preliminary outcomes warrant further testing using a larger sample size with a longer-term survival model.

Nonetheless, there are also a number of noteworthy strengths in our study. To begin with, we believe that our work provides relevant information about the extension of the arbitrarily stablished ischemia time. Geographically distant organ procurement may be facilitated by increasing the viable time frame.

Our study additionally provides evidence to support a CIT of above 12h to obtain a model of moderate lung damage in the experimental setting. Using a histological lung injury score, as well as inflammation and apoptosis analyses, our study shows that 12h of CIT leads to mild damage. Thus, lungs that are completely healthy, or only mildly damaged, may not reflect the potential improvement provided by any tested treatment. Accordingly, the most recent experimental studies involving pig lung damage models apply CITs ranging from 18 to 24h.37–39 Finally, as our work precisely describes the technique of LT in pigs, recording not only the anesthetic and surgical protocols but also the technical information for sample processing, it consequently enables the reproducibility of these complex procedures.

In conclusion, we have demonstrated in a swine model that post-transplant lung allograft injury was equivalent in specimens that underwent 6h or 12h CIT. No statistically significant differences were found in lung injury markers between the two groups. The extent of lung injury measured using a microscopic lung injury score, proinflammatory cytokines and caspase-3 determination was mild. The implications that may arise from this study should be taken with caution due to its experimental character. Outcomes from this study warrant further testing using a bigger sample size and longer-term survival model to investigate the role of CIT in PGD.

(I) Conception and design: Ojanguren A; (II) Administrative support: Ojanguren A, Olsina-Kissler JJ; Provision of study materials: Ojanguren A, Milla L, Fraile-Olivero C, Puy S; Olsina-Kissler JJ; (IV) Collection and assembly of data: Ojanguren A, Milla L, Fraile C, Puy S, Santamaria M.; (V) Data analysis and interpretation: Ojanguren A, Gatius S, Gómez-Olles S, Boada-Pérez M, Esquinas C, Santamaria M; (VI): Manuscript writing: All authors; (VII); Final approval of manuscript: All authors.

M.B. has received speaker fees from Grifols, Menarini, CLS Behring, GSK and Boehringer Ingelheim, and consulting fees from GSK, Novartis, Boehringer Ingelheim and GebroPharma, outside the submitted work.

I.O. has received travel grants, consulting fees, speaker fees, or research grants from AstraZeneca, Bial, Boehringer Ingelheim, Chiesi, MSD, GlaxoSmithKline, Menarini, Mundipharma, Novartis, and Teva, outside the submitted work.

The authors declare that they have no conflict of interests related to this article.