One of the main causes of early graft dysfunction in kidney transplantation is ischaemia reperfusion (I/R) injury. This unavoidable event occurs immediately when oxygenated blood is re-introduced into the transplanted kidney. Its severity is influenced by many predetermined factors. However, the condition under which an organ is preserved has a significant bearing on the outcome. Traditionally, organs are preserved using hypothermic temperatures, to reduce metabolism and the requirement for oxygen. Although practical and simple, hypothermic conditions are not entirely favourable and over time the depletion of energy substrates causes substantial cellular injury. This is thought to be a particular problem in kidneys from marginal donors, which are often exposed to a period of warm ischaemia (WI) prior to retrieval. The aim of this thesis was to determine the effects of varying degrees of the combined insults of warm and cold ischaemic injury and to develop a technique of normothermic perfusion (NP) to reduce ischaemic injury. The effects were assessed using ex-vivo and in-vivo porcine kidney models before translation of NP into clinical practice for marginal donor kidneys. This research demonstrated that prolonging the hypothermic preservation period after a minimal and a substantial degree of WI injury increased the severity of acute I/R injury and graft dysfunction. A short period of NP after hypothermic preservation was able to resuscitate the kidney, replenish ATP and reverse some of the detrimental effects of cold ischaemic injury. When translated into an autotransplant model, NP was found to be a safe and feasible method of preservation. NP was then adapted for use in clinical practice for kidneys from marginal donors. This first in man clinical series of 15 cases has demonstrated the safety and feasibility of NP for marginal kidneys. Although, the high rate of initial graft function is notable, further comparative studies are required to assess the effects on delayed graft function.

Whilst the major caveat to the success of organ transplantation remains the severe lack of donor organs, rejection is still a primary confounding factor to transplant outcomes. This is an allospecific response that occurs when the recipient immune system recognises conserved proteins on donor-derived cells as 'non-self'. Currently, all immunosuppressive regimes target the recipient immune response, ignoring the large donor immune repertoire despite these cells playing a central role in acute rejection. This is likely as a result of a lack of understanding of the temporal migration of the donor compartment and its contribution to the inflammatory cascade that ensues. The development of ex vivo perfusion provides the opportunity to assess this in isolation, with no confounding factors. Furthermore, inducing the mobilisation of passenger leukocytes on an ex vivo circuit allows their removal prior to transplantation. Reducing the inflammatory burden of donor organs has the potential to impact on the clinical outcome of patients, manifesting as a reduction in the incidence or severity of acute rejection. The aim of this PhD thesis was to characterise the donor immune compartment of lungs and kidneys, to assess the impact of ex vivo perfusion on this, and determine the post-transplant impact of removing a proportion of these cells. For this purpose, donor lungs were perfused using ex vivo lung perfusion (EVLP) and the immune compartment characterised. A comparison of EVLP versus standard transplanted lungs was performed using a porcine transplant model. Clinical parameters were recorded and a histological assessment of cellular infiltration was performed to diagnose the incidence of acute rejection. To determine if these results were translatable to other organs, a porcine model of kidney ex vivo perfusion was established. In both models, a significant efflux of donor leukocytes was observed and inflammatory mediators detected. In a transplant model of EVLP, reducing the transfer of these passenger leukocytes translated into improved clinical outcomes, manifesting as a lower incidence of acute rejection, for animals receiving EVLP lungs compared to a standard transplant. Similar benefit is likely to occur following transplantation of perfused kidneys. This study describes for the first time the contribution of donor organs to the inflammatory processes that ensue following transplantation. It is clear that this untargeted population is of significant importance in clinical outcomes. Immunomodulatory strategies to alter the donor immune environment prior to transplantation therefore warrant development.

Normothermic machine perfusion (NMP) allows for ex vivo viability and functional assessment prior to liver transplantation (LT). Hyperspectral imaging represents a suitable, non-invasive method to evaluate tissue morphology and organ perfusion during NMP. Liver allografts were subjected to NMP prior to LT. Serial image acquisition of oxygen saturation levels (StO2), organ hemoglobin (THI), near-infrared perfusion (NIR) and tissue water indices (TWI) through hyperspectral imaging was performed during static cold storage, at 1h, 6h, 12h and at the end of NMP. The readouts were correlated with perfusate parameters at equivalent time points. Twentyone deceased donor livers were included in the study. Seven (33.0%) were discarded due to poor organ function during NMP. StO2 (p < 0.001), THI (p < 0.001) and NIR (p = 0.002) significantly augmented, from static cold storage (pre-NMP) to NMP end, while TWI dropped (p = 0.005) during the observational period. At 12–24h, a significantly higher hemoglobin concentration (THI) in the superficial tissue layers was seen in discarded, compared to transplanted livers (p = 0.036). Lactate values at 12h NMP correlated negatively with NIR perfusion index between 12 and 24h NMP and with the delta NIR perfusion index between 1 and 24h (rs = −0.883, p = 0.008 for both). Furthermore, NIR and TWI correlated with lactate clearance and pH. This study provides first evidence of feasibility of hyperspectral imaging as a potentially helpful contact-free organ viability assessment tool during liver NMP.

High intensity focused ultrasound (HIFU) is an emerging non-invasive thermal ablative modality that can be utilised for the treatment of solid organ tumours, including liver cancer. Acoustic cavitation is a phenomenon that can occur during HIFU and its presence can enhance heating rates. One major limitation of thermal ablative techniques in general, such as radiofrequency and microwave ablation, is the heat sink effect imparted by large vasculature. Thermal advection from blood flow in vessels &ge; 3 - 4 mm in diameter has been shown to significantly reduce heating rates and peak temperatures in the target tissue, potentially leading to treatment failure. With regards to HIFU therapy, a clearer understanding is required of the effects of blood flow on heating, cavitation and thermal tissue necrosis, which is the treatment endpoint in clinical thermal ablation. Therefore, the overall aim of this thesis project was to elucidate the effects of blood flow on HIFU-induced heating, cavitation and histological assessment of thermal ablation. A unique isolated, perfused porcine liver model was used in order to provide a relevant test bed, with physiological and anatomical characteristics similar to the in vivo human liver. The normothermic liver perfusion device used in all studies presented in this work can keep an organ alive in a functional state ex vivo for in excess of 72 hours. A further advantage of the liver perfusion device was that it allowed blood flow to be stopped completely and resumed rapidly, allowing studies to be conducted under zero flow conditions. A therapeutic HIFU system was used in order to deliver HIFU therapy to regions of hepatic parenchyma adjacent (&le; 3 mm) to large (&ge; 5 mm) blood vessels or away from vasculature (&ge; 1 cm) at either 1.06 MHz or at 3.18 MHz. Cavitation events during HIFU therapy were spatio-temporally monitored using a previously developed passive acoustic mapping (PAM) technique. The cavitation threshold at each frequency was determined through assessment of acoustic emissions acquired through PAM during HIFU exposure at a range of acoustic pressures. Real time thermal data during HIFU therapy were obtained using an implantable 400 &mu;m thermocouple, aligned with the HIFU focus, in order to assess the effect of large vessel blood flow on peak tissue temperatures. Thermal data were obtained at 1.06 MHz, in the presence of acoustic cavitation and at 3.18 MHz, in the absence of cavitation, both in the presence and complete absence of blood flow. Finally, histological assessment of cell viability and cell death was performed in order to determine whether any heat sink effect could be overcome, with the achievement of complete tissue necrosis in treatment regions directly adjacent to large vasculature. This work demonstrated for the first time that in perfused, functional liver tissue, the presence of large vasculature and physiological blood flow does not significantly affect ablative HIFU therapy, both in terms of peak focal tissue temperatures attained and histological evidence of complete tissue necrosis. Therefore, HIFU may be superior to other ablative modalities in treating tumours in tissue regions adjacent to major vascular structures, but further work needs to be performed to correlate the experimental findings with clinical outcomes.

La transplantation est la meilleure alternative en cas d'insuffisance rénale terminale. Face à la pénurie de greffons, les équipes de transplantation se sont tournées notamment vers les donneurs décédés par arrêt circulatoire (DDAC) non contrôlés. Ces greffons soumis à une période d'ischémie chaude sont plus fragiles. Des méthodes de reconditionnement chez le donneur par refroidissement in situ (RIS) et circulation régionale normothermique (CRN) se sont développées afin de réduire les lésions d'ischémie-reperfusion. Le choix de la méthode est laissé à l'appréciation de chaque équipe et il existe une grande hétérogénéité des pratiques. Après prélèvement, l'utilisation des machines de perfusion hypothermique (MPH) est généralement recommandée. L'optimisation de ces phases de reconditionnement chez le donneur et de conservation hypothermique apparait comme un enjeu majeur de santé publique. Concernant l'optimisation du mode de reconditionnement, la mise au point d'un modèle préclinique porcin parfaitement reproductible a permis de mettre en évidence une supériorité de la CRN sur le RIS. Une durée de CRN de 4 heures minimum sans dépasser 6 heures paraît optimale. Concernant la conservation hypothermique, les MPH permettent le maintien du niveau d'expression des gènes retrouvé en fin de CRN. L'ajout d'une oxygénation active en MPH ou de curcumine en solution statique améliore le devenir du greffon à court et long termes dans un modèle préclinique d'autogreffe. Ce travail pourrait s'étendre à l'étude d'autres organes, d'autres durées d'ischémie chaude et aux DDAC contrôlés afin d'élargir encore le nombre d'organes éligibles à la transplantation. / Transplantation is the best alternative to end-stage renal disease. The shortage of grafts led the transplant teams to consider uncontrolled deceased donors after circulatory death (DCDs). These grafts suffered from a period of warm ischemia and are more vulnerable. Reconditioning methods in the donor by in situ cooling (ISC) and normothermic regional perfusion (NRP) have been developed to reduce the ischemia-reperfusion injuries. Each team has the choice as to the method and there are many different practices. After removal of kidneys, the use of hypothermic perfusion machines (HPM) is generally recommended. The optimization of reconditioning in the donor and hypothermic preservation appears as a major public health challenge. About optimization of the reconditioning method, the development of a high reproducible preclinical porcine model allowed to highlight the superiority of RNP over ISC. NRP duration of 4 hours minimum without exceeding 6 hours seems optimal. About hypothermic preservation, HPM allows to maintain the level of expression of the genes found at the end of RNP. The addition of active oxygenation to HPM or curcumin in static solution improves the graft outcomes in the short and long terms in a preclinical model of auto transplantation. This work could be extended to the study of other organs, other durations of warm ischemia and to controlled DCDs in order to further increase the number of transplantable grafts.

Es wurde untersucht ob die normotherme extrakorporale Leberperfusion (NELP) als Methode geeignet ist, Lebern vor Transplantation zu konservieren, und ob sie warm ischämische Zellschäden beheben kann. Zum ersten Mal konnte experimentell gezeigt werden, daß eine erfolgreiche Transplantation nach 4 Stunden mit NELP möglich ist und sogar so zuverlässig, wie die Kaltkonservierung in der University of Wisconsin Lösung. Die NELP erhält die Leberfunktion und ermöglicht eine Regeneration warm ischämischer Schäden in Nicht-herzschlagenden Spendern. 36 Schweine der Deutschen Landrasse wurden in sechs Gruppen transplantiert. In der Gruppe 1 wurde direkt nach Organentnahme transplantiert, in Gruppe 2 nach 4 Stun-den Kaltkonservierung in der University of Wisconsin Lösung und in Gruppe 3 nach 4 Stunden NELP. In Gruppe 4 wurden die Lebern nach 60 Minuten warmer Ischämie direkt transplantiert, in Gruppe 5 nach 60 Minuten warmer Ischämie und 4 Stunden Kaltkonservierung und in Gruppe 6 nach 60 Minuten warmer Ischämie und 4 Stunden NELP. Alle Tiere deren Lebern vor Transplantation normotherm extrakorporal perfun-diert wurden (Gruppen 3 und 6) überlebten mit guter Organfunktion. Im Unterschied hierzu führte die Abfolge von 60 Minuten warmer Ischämie und 4 Stunden Kaltkonser-vierung unweigerlich zur primären Organ-Nichtfunktion innerhalb der ersten 24 Stun-den nach Lebertransplantation. Die Methode der NELP bietet die Chance eine Leber außerhalb des Körpers für Zeiträume von möglicherweise länger als 4 Stunden völlig funktionsfähig zu halten. Die NELP kann zur Organkonservierung vor Transplantation eingesetzt werden, aber auch dazu, Lebern von Nicht-Herzschlagenden Spendern zu nutzen. / Normothermic extracorporeal liver perfusion (NELP) was studied as a means to pre-serve livers for transplantation and to reverse warm ischemic injury. For the first time we provide experimental evidence that successful transplantation after 4h of normo-thermic extracorporeal liver perfusion is possible and as reliable as 4h of cold preser-vation in University of Wisconsin solution. NELP preserves liver function completely and is capable of reversing 60 min of warm ischemic injury in non heart beating do-nors. 36 German Landrace pigs were transplanted in six groups. Group 1 animals were transplanted directly, group 2 animals after 4h of cold preservation with University of Wisconsin solution and group 3 animals following 4h of normothermic extracorporeal liver perfusion. Group 4 animals sustained 1h of warm ischemia before transplantation of the liver. In group 5 animals were transplanted following 1h of warm ischemia and 4h of cold preservation, and in group 6 after 1h of warm ischemia and 4h of normo-thermic extracorporeal liver perfusion. All animals receiving livers treated by normo-thermic extracorporeal liver perfusion survived without liver failure (group 3 and 6). In contrast, all animals in group 5 developed primary graft non-function within 24 h after transplantation. The technique of NELP holds the potential to keep a mammalian liver outside the body completely functional, possibly for longer than 4h. NELP can be used for liver preservation prior to transplantation or to utilise organs from non-heart-beating donors.

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