Targeting brain death-induced injury van Erp, Anne Cornelie

Targeting brain death-induced injury van Erp, Anne Cornelie

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Targeting brain death-induced injury

Anne Cornelie van Erp

Anne Cornelie van Erp PhD-thesis

This PhD-project was financially supported by:

University Medical Center Groningen

Junior Scientific Masterclass, Faculty of Medicine University of Groningen Research Institute GUIDE The printing of this thesis was kindly supported by:

Chipsoft Noord Negentig

Research Institute GUIDE

University Medical Center Groningen

Financial support by Astellas Pharma B.V., the Dutch Kidney Foundation and the Nederlandse Transplantatie Verening for the publication of this thesis is gratefully acknowledged.

Cover and invitation: Anne van Erp Layout: Anne van Erp

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All rights reserved. No part of this publication may be reproduced, stored in a retrieval system of transmitted in any form without explicit prior permission of the author.

ISBN: 9789492597182

Proefschrift

ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen

op gezag van de

Rector Magnificus prof. dr. E. Sterken en volgens besluit van het College voor Promoties.

De openbare verdediging zal plaatsvinden op woensdag 24 oktober 2018 om 16.15 uur

door

Anne Cornelie van Erp geboren op 22 november 1988

te Hilversum

Beoordelingscommissie

Prof. dr. B.M. Bakker

Prof. dr. K. Kotsch

Prof. dr. A.J. Moshage

Chapter 2 Systematic review on the treatment of deceased organ donors.

23

Chapter 3 The effect of the β-Human Chorionic Gonadotropin- related peptide (EA230) on brain death-induced inflammation in rats.

61

Chapter 4 Anti-Apoptotic effects of 3,3’,5-Triiodo-LThyronine in the liver of brain-dead rats.

83

Chapter 5 Organ-specific responses during brain death: increased aerobic metabolism in the liver and anaerobic

metabolism with decreased perfusion in the kidneys.

105

Chapter 6 Organ-specific metabolic profile during brain death evaluated using hyperpolarized Magnetic Resonance Imaging: an initial experience.

135

Chapter 7 The crosstalk between ROS and autophagy in the field of transplantation medicine.

159

Chapter 8 Autophagy is reduced in the liver and kidney following brain death independent of mTOR activation.

185

Chapter 9 Summary Discussion

Future perspectives

211 214 218 Chapter 10 Dutch summary | Nederlandse samenvatting

Authors’ affiliations List of publications Acknowledgements About the author

223

227

228

229

234

Introduction 1

CHAPTER

1 ORGAN DONATION, PRESERVATION AND TRANSPLANTATION

Organ transplantation remains the preferred life-saving treatment for most patients with end-stage liver and kidney disease, as it can improve patients’ quality of life as well as their survival rates

. Unfortunately, not every patient on the waiting list can receive an organ because of a global shortage of suitable organ grafts. As a result, 12 people die each day while waiting for an organ transplant in Europe alone

. This problem underlines the need for a concerted action within the transplant community to increase the number of available organs. This pertains mostly to organs obtained from deceased donors to avoid ethical problems related to living organ donation as well as illegal organ trafficking. On an international level, this effort has resulted in the establishment of organizations such as the Eurotransplant International Foundation, a non-profit organization established to optimize the allocation and distribution of organs amongst a number of European countries

. On a national level, several countries including The Netherlands have adopted legislation that automatically enters their civilians as potential organ donors unless they personally object. Another approach to increase the donor pool is to increase the use of organs of suboptimal quality. However, this approach mandates an improvement of organ quality prior to transplantation in order not to risk poor transplantation outcomes. As the quality of the organ graft may be challenged during each step of the transplantation process, this approach should encompass strategies and treatments in the donor, but also afterwards during organ preservation and in the recipient. To facilitate organ-improving strategies, the first step is to understand which injuries the graft endures during the transplantation process.

ORGAN DONATION

Both chronic (aging, pre-existing medical conditions) and acute injuries (donor death,

organ retrieval strategies) in the donor are risk factors for impaired graft survival after

transplantation

. Of these injuries, donor age is considered a classical risk factor that is

associated with both short-term and long-term graft survival

However, recent studies

suggest that biological organ age and not donor age is a better predictor of transplantation

outcomes

. Halloran et al. postulated that the accumulation of aging combined with

exposure to injury and stressors impairs the ability of an organ to repair and remodel,

thereby hindering graft survival

. This might explain why we observe a clear difference

in survival dependent on the type of organ donor used. Of all donor types, organ grafts

obtained from living donors are superior when compared to those obtained from deceased

donors. However, the number of living organ grafts is limited, which means that the majority

of organs used are obtained from decreased donors. Deceased donors can be classified into

deceased brain-dead (DBD) donors or deceased cardiac death (DCD) donors. Of the two,

most organs worldwide are obtained from DBD donors. Nevertheless, the number of DCD

1

organs continues to increase, particularly since more countries are accepting organs from these donors

. In addition, many countries have started using suboptimal organs obtained from so-called extended criteria donors (ECD)

. ECD are over 60 years of age, or between 50-60 years with comorbidities including hypertension, impaired estimated GFR based on plasma creatinine (> 1.5 mg/dL), age, and gender, or death by a cerebrovascular incident.

When compared to living donation, deceased donation is associated with allograft dysfunction or delayed graft function, impaired survival or even higher mortality rates following transplantation

. This inferiority of deceased donors is caused by pathophysiological changes of death. In DBD donors, most commonly cerebrovascular incidents or traumatic brain injuries cause increased intracranial pressure. When the body fails to maintain cerebral perfusion, this results in ischemia of the brain, and brain stem. As a result, large amounts of catecholamines are released and a cascade of derailments commences that includes inflammation, hemodynamic instability, and hormonal and metabolic changes

. In DCD donors, not a cerebral event but cardiopulmonary arrest causes hypoxia, which poses a great ischemic insult to the organs. The subsequent prolonged period of warm ischemia adds additional ischemic damage to the organs and is considered another risk factor associated with impaired graft function and higher mortality rates following transplantation

. Altogether, this suggests that minimizing and targeting the accumulation of injuries in the donor could reduce cellular injury. This strategy could improve graft function and survival after transplantation and even allow more organs to be suitable for transplantation.

Organ preservation

Following this period of donor-related injuries, organ grafts suffer additional injury during the preservation period. For decades, organs have been statically preserved on ice. This strategy was based on the rate-of-life theory, which states that aging and longevity are regulated by the rate of cellular metabolism

. According to this principle, hypothermic preservation intends to lower the metabolic rate, thereby reducing hypoxic injury and preserving cellular function

. To ensure tolerance of organs to hypothermia, preservation solutions have been developed to counter negative side effects of hypothermic preservation including edema, accumulation of reactive oxygen species (ROS) production, ATP depletion, mitochondrial damage, a switch to anaerobic glycolytic metabolism, and microvascular changes

. With this in mind, prolonged duration of the cold ischemia is considered an independent risk factor for a nonfunctioning or dysfunctioning transplant, particularly in marginal or high-risk donors

.

These negative side effects of cold storage have recently led to the first clinical implementation

of hypothermic machine perfusion (HMP). This technique allows continuous, pulsatile

perfusion of the organ at 4 to 10°C, while blood and other components are flushed out

1

and the organ equilibrates with the perfusion solution

. HMP has clear benefits over static cold storage in kidney transplantation, evidenced by improved graft function and survival rates

. Despite these benefits, HMP has only recently begun to be clinically implemented in the field of kidney, liver, heart, and lung transplantation. Static cold storage currently remains the only clinical preservation method of the pancreas

. Adaptations of HMP that are currently being investigated pre-clinically are the addition of oxygen and other additives to the perfusion fluid, as well as perfusion at subnormothermic (25°C) or normothermic (37°C) temperatures. Especially normothermic temperatures would add the additional benefit of allowing viability testing of organs prior to transplantation. The key to all these preservation methods is the improvement of graft quality by limiting (the duration of) ischemic injury, preventing abrupt reperfusion and preserving or restoring cellular energy supplies.

Organ transplantation

Optimizing graft quality prior to transplantation is especially important given that the

reintroduction of warm oxygenated blood in the recipient will only amplify injury to the organ

graft. After a period of cold ischemia, graft reperfusion causes immediate ROS production

by the vascular cells of the donor allograft, followed by a subsequent hit of ROS production

originating from the recipient’s phagocytes

. Interestingly, the initial ROS produced by the

donor’s vascular cells trigger the subsequent second burst of ROS, together reducing the

capacity of the anti-oxidant machinery. Hence, this phenomenon has been described as a

vicious cycle with excess ROS production as its end result

. One of the main contributors

to this cycle are the mitochondria, which are known to increase their ROS production in

response to hypoxia

. This ROS production in turn facilitates the initiation of various cellular

death pathways, including necrosis, apoptosis, and autophagy

. It is suggested that the

amount and duration of the oxidative injury related to first ischemia and then reperfusion,

so-called ischemia-reperfusion injury (IRI), is responsible for the ischemic damage and

immune activation immediately after transplantation. This makes oxidative stress an

important factor which can ultimately result in rejection of the organ graft

. Furthermore,

the level of IRI subsequently determines the extent to which cellular death pathways such

as autophagy and apoptosis become activated. Their level of activation and interplay seems

to determine whether the end result is pro-survival or pro-death

.

1 TARGETING BRAIN DEATH-INDUCED INJURY

The potential benefit for patients with organ failure together with the global organ shortage is the driving force behind the body of research that has tried to elucidate what damages the graft during each step of the transplantation process. Recently, this has led to the clinical implementation of ex vivo hypothermic kidney perfusion as the preferred preservation method. However, the great potential of targeted treatments during ex vivo organ perfusion and the sometimes ethical difficulty of treating deceased donors, should not be a reason to refrain from investigating or targeting organ grafts in deceased donors.

Even though each step of the transplantation process may harm the organ graft, a very substantial amount of damage already occurs within the deceased organ donor. Given that many intracellular pathways such as oxidative stress and autophagy (introduced in more detail later) can be either detrimental or beneficial depending on the level of injury or stress, particularly early treatment of donor-related injuries could be of great benefit. Besides expanding on the current knowledge on donor pathophysiology, research should investigate novel injury mechanisms in the brain-dead donor, such as metabolic changes and autophagy dysregulation. Understanding what damages the organ prior to transplantation is essential to optimize organ-specific treatments in the donor but also during later stages of the transplantation process. Finally, if the outcome of a transplantation can already be predicted in the donor or during organ preservation, this would greatly benefit the success of transplantations while minimizing the impact on recipients.

Brain death physiology

Most organs world-wide are obtained from DBD donors

. Brain death is defined as a state

with irreversible absence of brain and brainstem function, in which mechanical ventilation is

required to prevent apneas while the systemic circulation remains intact

. Several common

etiologies of brain death include cerebrovascular accidents, traumatic brain injury, and

diffuse hypoxia. The common denominator in each of these injuries is a rise in intracranial

pressure. When the risen cerebral pressure cannot be overcome by a rise in blood pressure,

this results in hypoperfusion of the brain and subsequent progressive ischemia of the brain,

brainstem, and ultimately spinal cord. Herniation of the ischemic brain stem results in

sympathetic hyperactivity that is characterized by an immediate increase in systemic blood

pressure and peripheral vasoconstriction due to the release of endogenous catecholamines,

also called the catecholamine storm

. This catecholamine storm has been described as

the body’s final attempt to overcome the rise in intracranial pressure. This vasoconstrictive

response results in decreased blood flow through peripheral organs such as the liver and

kidneys

. The systemic hypertension also stimulates (to a lesser extent) parasympathetic

activity via the baroreceptors, resulting in subsequent bradycardia. These changes, together

called the Cushing response, are characteristic for the brain death condition.

1

This hypertensive period is followed by a decline in sympathetic tone, which marks the beginning of the hemodynamic instability in the DBD donor

. In addition to these changes in hemodynamics, ischemia of the brain results in failure of the hypothalamus and pituitary axis. As a consequence, a depletion of antidiuretic hormone is evident in the majority of the brain-dead donors

. The resulting diabetes insipidus causes increased diuresis and risk of hypovolemia and further contributes to the hemodynamically unstable condition. In addition, plasma levels of free thyroid hormone, thyroid stimulating hormone, and cortisol generally decline

. Besides hemodynamic and hormonal changes, BD is characterized as a systemic inflammatory state, seen by a rise in circulating cytokines, including interleukin (IL)-6, IL-10, MCP-1, and TNF-α

. This systemic inflammatory environment triggers a local response in the kidneys, liver, and lungs. The inflammatory and apoptotic response in these organs is the results of activation of the vascular endothelium, the complement and coagulation system, as well as the innate and adaptive immune response

. What triggers this pro-inflammatory environment during brain death is not well understood. Nevertheless, cerebral cytokines from the dying cerebrum

, complement activation

, translocation of bacteria from the intestines

, and organ-specific inflammation

have all been implicated. Altogether, brain death-induced pathophysiology negatively affects organ quality prior to transplantation and predisposes the recipients of these grafts to a higher risk of acute rejection, delayed graft function and lower survival rates after transplantation

. Targeting brain death-induced injury is, therefore, essential.

AIM OF THIS THESIS

The aim of this thesis is to review and expand on the current knowledge required to target brain death-induced injury (see Fig 1). The first part of the thesis (Chapters 1 – 4) focuses on interventions in the donor that target brain death-induced pathophysiological changes. The second part of this thesis (Chapters 5 – 8) expands on this knowledge by investigating novel injury mechanisms pertaining metabolic changes and autophagy (dys)regulation during brain death.

Interventions in the brain-dead donor

Currently, brain-dead donor management protocols are aimed at targeting brain death-

related pathophysiological changes. Despite considerable differences per individual

center, protocols have focused mainly on providing hemodynamic support, suppressing

the immune system, optimizing donor ventilation management, controlling donor body

temperature, and administrating hormone replacement therapy

. Providing hemodynamic

support and optimizing organ oxygenation is important, given the hemodynamic instability

that is evident within the brain-dead donor. Immunosuppressive therapy to counteract

brain death-induced inflammation is also clinically relevant, as donor plasma levels of

pro-inflammatory cytokine IL-6 were inversely correlated to recipient six-month hospital-

1

free survival

and an increased risk of recipient death following lung transplantation

. However, despite promising animal studies

, immunosuppressive therapy in brain-dead donors has not been able to improve the outcomes of liver and kidney transplantations

(see Fig 2). Finally, despite mixed reports about the effectiveness of thyroid hormone replacement therapies, its use has increased over the years

. Altogether, this highlights that despite the extensive body of preclinical and clinical research on interventions in the brain-dead donor, we are still lacking a universally accepted, optimized brain-dead donor management protocol.

Figure 1. Targeting brain death-induced injury. Brain death results in hormonal changes, hemodynamic instability, inflammation, and oxidative stress. It is unclear how these changes affect organ-specific metabolism, perfusion, mitochondrial function, and autophagy. Together these changes negatively affect organ quality, which impacts transplantation outcomes and patient and graft survival. Targeting brain death-induced injury is essential to optimize organ quality prior to transplantation, particularly given the subsequent injuries the graft endures during organ preservation and transplantation, as well as the global organ shortage.

As a starting point for this thesis in Chapter 2, we provided an update on all systematically

tested, clinical interventions that have been tested in brain-dead organ donors thus far

(see Fig 2). Only those studies were included that focused on the effects on organ quality

and graft and/or patient survival after transplantation. In Chapter 3, we tested whether

EA230, an oligopeptide of human chorionic gonadotropin with promising anti-inflammatory

properties in models of septic and haemorrhage shock, would attenuate brain death-induced

inflammation. Finally, in Chapter 4, we studied whether pre-conditioning of brain-dead

animals with free thyroid hormone T

could improve liver function and reduce apoptosic

cell death.

1

Figure 2. Overview of all systematically tested, clinical treatments or interventions in the brain-dead donor with outcome parameters pertaining the lungs, liver, heart, and kidneys. Treatments or interventions denoted in black did not affect graft function or graft and patient survival. Treatments or interventions denoted in green were beneficial, those in red were detrimental, and those in orange showed mixed results.

NOVEL INJURY MECHANISMS

Previous studies have indicated that brain death alters the plasma metabolite profile and

shifts the balance from aerobic to anaerobic metabolism

. To explain these metabolic

changes, several theories have been provided. Firstly, these changes have been attributed

to mitochondrial impairment, as this was previously observed in the muscles of brain-dead

patients

and the hearts of brain-dead pigs

. Alternatively, the hemodynamic instability

and initial organ hypoperfusion immediately following the catecholamine storm have been

implicated as the underlying cause. Regardless, the metabolic status of organs prior to

1

transplantation is of clinical importance, as these changes have been linked to transplantation outcomes following both kidney

and liver transplantation

. However, effects of donor brain death on metabolism in the individual organs has not previously been investigated.

Therefore, in Chapter 5, we investigated how brain death affects metabolism, both systemically as well as in the liver and kidney. Besides studying major metabolic pathways, we investigated whether mitochondrial function and organ perfusion were altered during brain death. In Chapter 6, we used a non-invasive imaging tool to study in vivo, metabolic pathways in the liver and kidney during and following brain death. Using hyperpolarized magnetic resonance imaging with MRI-active pyruvate molecules, we were able to visualize metabolic pathways in real time during brain death. Afterwards, we visualized glucose metabolism during ex vivo organ reperfusion using radioactively labelled glucose.

Oxidative stress, hemodynamic instability, inflammation, and hormonal perturbances

induced during brain death are each influencers of autophagy

. Autophagy is an intracellular

degradation pathway that removes, degrades, and recycles cellular constituents

. Autophagy

normally occurs at a basal level within the cells and serves as a cellular housekeeper that

removes damaged or unwanted organelles or cellular constituents

. Several types

of autophagy exist, but all types of autophagy involve the transportation of intracellular

compounds to the lysosomes for degradation. In the presence of cellular stressors such as

hypoxia, inflammation, or energy depletion, autophagy becomes stimulated and protects

the cell by removing damaged or toxic cellular products

. In this way, autophagy is

generally considered to be a protective, stress-adaptation pathway that can counter cellular

death pathways such as apoptosis

. Conversely, excessive autophagy stimulation can be

detrimental and can push the balance away from a protective towards a detrimental role for

autophagy, as is seen in disease states such as cancer, neurodegeneration, aging, and IRI

.

This is of particular importance, given the current increased use of older and extended

criteria donors. Even though autophagy has been implicated to play a role during oxidative

stress and IRI, the exact role that autophagy plays during the transplantation process is only

beginning to be understood

. In Chapter 7, we reviewed all the available knowledge on

the role that autophagy and oxidative stress play during each step of the transplantation

process. Furthermore, we covered the complex interdependency of these two pathways

and discussed several compounds that target each of these pathways. Finally, in Chapter

8 we investigated how brain death affects autophagy in the liver and kidney. Furthermore,

we studied whether stimulation of autophagy with mTOR-inhibitor rapamycin affected

autophagy, organ quality, and apoptosis.

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Systematic review on the treatment of deceased organ donors

Anne C. van Erp*

Leon F.A. van Dullemen*

Rutger J. Ploeg Henri G.D. Leuvenink

*Authors contributed equally to the manuscript

Published in Transplantation Reviews on June 22, 2018 DOI: 10.1016/j.trre.2018.06.001

2

CHAPTER

2

ABBREVIATIONS

ADH Antidiuretic Hormone AF Alkaline phosphatase AST Aspartate Transaminase ALT Alanine Aminotransferase CI Confidence Interval DBD Donation after Brain Death DCD Donation after Circulatory Death DGF Delayed Graft Function

ECD Expanded Criteria Donors ɣGT Gamma-glutamyl Transpeptidase HD Haemodialysis

HF Haemofiltration HES Hydroxyethyl Starch

INR International Normalised Ratio for Prothrombin Time (PT) IPC Ischaemic Preconditioning

IRI Ischaemia-Reperfusion Injury LDH Lactate Dehydrogenase LVAD Left Ventricular Assist Device MD Mean Difference

MTH Mild Therapeutic Hypothermia

PEEP Positive End-Expiratory Pressure

RCT Randomised Controlled Trial

ROS Reactive Oxygen Specie

T3 Triiodothyronine

TV Tidal Volume

2 ABSTRACT

Background

Currently, there is no consensus on which treatments should be a part of standard deceased- donor management to improve graft quality and transplantation outcomes. The objective of this systematic review was to evaluate the effects of treatments of the deceased, solid- organ donor on graft function and survival after transplantation.

Methods

Pubmed, Embase, Cochrane, and Clinicaltrials.gov were systematically searched for randomised controlled trials that compared deceased-donor treatment versus placebo or no treatment.

Results

A total of 33 studies were selected for this systematic review. Eleven studies were included for meta-analyses on three different treatment strategies. The meta-analysis on methylprednisolone treatment in liver donors (two studies, 183 participants) showed no effect of the treatment on rates of acute rejection. The meta-analysis on antidiuretic hormone treatment in kidney donors (two studies, 222 participants) indicates no benefit in the prevention of delayed graft function. The remaining meta-analyses (seven studies, 334 participants) compared the effects of 10 minutes of ischaemic preconditioning on outcomes after liver transplantation and showed that ischaemic preconditioning improved short-term liver function, but not long-term transplant outcomes.

Conclusions

There is currently insufficient evidence to conclude that any particular drug treatment or

any intervention in the deceased donor improves long-term graft or patient survival after

transplantation.

2

INTRODUCTION

Due to the persistent shortage of organs available for solid organ transplantation

, the transplant community has been searching for possibilities to further expand the donor pool.

One way to achieve this is by accepting organs retrieved from older and higher risk donors, without compromising good transplantation outcomes. Improving quality of suboptimal organs from donors after brain death (DBD), older expanded-criteria donors (ECD), or donors after circulatory death (DCD) mandates better assessment and optimisation prior to transplantation. These donors have all suffered cerebral injury, which leads to a profound systemic inflammatory response

. Furthermore, DBD and DCD donors face additional disturbances that threaten the quality of the future organ grafts.

In DBD donors, an increased intracranial pressure impairs brain perfusion and causes herniation of the brain stem. This results in the release of catecholamines and a cascade of derangements that lead to endothelial dysfunction and inflammation in the potential grafts-to-be

. In addition, the function of the hypothalamus and pituitary gland becomes impaired, which leads to decreased cortisol, triiodothyronine (T3), insulin, and antidiuretic hormone (ADH) plasma levels in the donor

. After herniation of the brain stem, a haemodynamically unstable state will follow that requires fluid resuscitation and often inotropic support. In DCD donors, there is no catecholamine release. Instead, withdrawal of medical support results in a significant blood pressure drop until circulatory arrest. This period of circulatory arrest is followed by a in most countries medico-legal five-minute no- touch period prior to confirmation of death, which adds extra warm ischaemic injury and threatens the quality of the potential grafts. In addition to these donor-related injuries, the grafts-to-be subsequently endure a period of preservation and cold ischaemia that are further detrimental to the organ quality.

To improve transplant outcomes in solid organ transplantation, an optimised and more organ-protective Intensive Care regimen should be adopted. In the last decade, such a strategy has increasingly become important since donor age and comorbidities have increased significantly in most countries. In deceased donor care, this has led to the consideration of numerous treatment options aspiring improvement of graft function and survival after transplantation. Unfortunately, clinical implementation has not happened, whilst a lot of controversy still exists about which treatment could actually benefit donor organs potentially improving transplant outcomes.

The purpose of this systematic review is to provide an update on all systematically tested

clinical interventions in the deceased donor and their impact on graft function and/or

survival following solid organ transplantation. This review will concern any clinical treatment

2

regimen that was carried out in either DBD or DCD donors using either specific drugs, fluids, or procedures to reduce donor organ injury prior to organ preservation and transplantation.

MATERIALS AND METHODS

Selection criteria

RCTs or quasi-RCTs (trials in which the allocation method is not truly random) were selected that compared differences in graft function and survival between pre organ retrieval-treated, deceased, adult (16 years or older) solid-organ donors (including DCD, DBD, and ECD donors) to untreated or placebo-controlled donors. Primary outcomes for this systematic review were graft function, and patient and graft survival. Secondary outcome parameters were surrogate markers of organ injury.

Exclusion criteria for this systematic review were: 1. articles not in English; 2. duplicate studies; 3. living donors; 4. average donor age <16 years old; 5. studies with pregnant participants; 6. animal studies; 7. tissue transplantation; 8. donor treatment after graft procurement; 9. ex-situ treatment of the graft; 10. treatment of the recipient; and 11. no information on organ function or survival.

Search methods for identification of studies

This systematic review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines

. Potential RCTs were identified using electronic and manual search strategies. The final electronic literature searches were performed in Pubmed (5 Nov 2016), Embase (5 Nov 2016), and the Cochrane library (7 Nov 2016). The ClinicalTrials.gov register was also searched (7 Nov 2016) to identify unpublished or ongoing trials. The search was limited to RCTs with a highly sensitive search-strategy filter. The bibliographies of identified studies and reviews were manually searched for additional trials. A qualified librarian reviewed the final search strategy. The search strategy for each consulted database is available in the supplementary data (Fig S1, Table S1 and S2).

Data extraction and validity assessment

All identified records were screened on title and abstract after removal of duplicates with an

algorithm provided by Refworks (ProQuest-LCC, USA). Full articles of selected records were

retrieved and assessed for eligibility; disagreements were resolved by consensus. Abstracts

not providing information on the study type or outcome parameters were retrieved for

full-text evaluation. Study information was extracted independently by two reviewers. The

risk of bias assessment was performed according to the Cochrane risk of bias tool

. The

assessment of study quality included random sequence generation, allocation concealment,

performance bias, detection bias, attrition bias, reporting bias, and “other” bias. Quality

2

assessments were performed independently and disagreements were resolved through discussion.

Data synthesis

Outcome parameters of interest for all transplanted solid organs were: patient and graft survival; development of primary dysfunction, sometimes subdivided in either primary non- function or initial poor function; acute rejection; Intensive Care Unit (ICU) or hospital stay;

and post-operative complications (such as post-operative infections or biliary complications, Table 1). Organ-specific parameters of interest were: creatinine clearance, serum creatinine, and delayed graft function (DGF, measured as the need for renal replacement therapy including haemodialysis (HD) or haemofiltration (HF) in the first week post transplantation) (kidney); aspartate transaminase (AST), alanine aminotransferase (ALT), albumin, alkaline phosphatase (AF), bilirubin, gamma-glutamyl transpeptidase (ɣGT), and lactate dehydrogenase (LDH) levels, and International Normalized Ratio(INR) (liver); left ventricular function, and left ventricular assist device (LVAD) and HF requirement (heart); and PaO

/FiO

ratio, compliance (plateau pressure at the end of respiration), and organ utilization rates as an indirect way of assessing graft function (lung). Qualitative assessment was performed for single studies that could not be grouped for meta-analyses. For studies that could be grouped, a forest plot was constructed to assess the heterogeneity, using Cochran’s Q test and the I

-test (considered significant when p <0.1 or I

>30%). A random-effects analysis model was applied, followed by the Mantel-Haenszel test to calculate cumulative relative risk ratios for dichotomous variables. As no more than four studies were included per meta- analysis, funnel plot analyses could not be constructed to distinguish potential asymmetry. All statistical analyses were performed with Review Manager v5.3 (The Cochrane Collaboration 2014).

RESULTS

Literature search and summary of included studies

From 7309 hits in total, 62 studies were assessed. As 29 studies failed to meet our inclusion criteria, a total of 33 articles were included in this systematic review (Fig 1, Table 1, Table S3). Even though the search strategy was aimed towards all solid organs, only studies on kidney, liver, heart, and lung transplantations were found. In addition, we identified 13 trials that were still ongoing or did not yet publish results (Table 2). As none of the included studies involved interventions in DCD donors, this systematic review describes only trials in DBD donors.

The following treatment strategies for DBD donors were identified: anti-oxidant

treatment

, enteral feeding

, organ retrieval techniques

, haemodynamic support

,

2

Figure 1. PRISMA diagram of the literature search. Flow chart summarising the search strategies and subsequent selection of trials for this systematic review and the performed meta-analyses.

mild therapeutic hypothermia (MTH)

, immunosuppressants

, ischaemic preconditioning (IPC)

, a lung protection strategy

, and T3 administration

. Table 1 shows a summary of these included studies, while Table S4 shows the risk of bias for these trials. In 16 studies, the methods for patient selection and allocation concealment were adequately performed and described. Eight studies used a placebo-controlled group, whereas the remaining studies had either non-treatment groups (n=20) or compared the intervention to a conventional treatment (n=5).

Studies that could not be included for meta-analyses

Twenty-two studies

were not included for meta-analyses because the type of

intervention or outcome parameter could not be compared to other trials included in this

review. None of the studies reported a significant effect of the treatment or intervention on

ICU or hospital stay. Four studies tested effects of anti-oxidant treatments in DBD donors on

graft function

. Treatments with N-acetylcysteine

and L-alanyl-glutamine

, respectively.

2

Sour ce Year

Number of pa

tien ts randomised Tr ea tmen t gr oup (adminis tr ation mode and time) Con tr ol gr oup Out come

(sub- gr oup)

Specific end-poin

ts (time a ft er tr ansplan ta tion) Eff ect or gan function (tr ea tmen t v s. con tr ol gr oup) ti- ts

Orban et al.

12

2015 217 600 mg N-ace tylcy st eine (bolus, 1 h be for e and 2h a fter angiogr aph y) No tr ea tmen t Kidne y function

sCr and eGFR (D1,7,14,30); DGF (HD r

equir emen t/ oliguria/ sCr >500 µmol/L , D0-7); acut e r ejection (D0-30); pa tien t and gr aft sur viv al (≤Y1); and r ecipien t hospit al s ta y

No Barr os et al.

2015 33 50 g L-alan yl-glut amine (bolus, 40 min be for e cold ischaemia) Placebo Liv er function AS T, AL T, bilirubin, INR

(D0,1,3,7,30); pa tien t and gr aft sur viv al (dur ation unkno wn)

No Kaz emi et al.

2015 40 100 mg /kg asc orbic acid (bolus, 6 h be for e pr ocur emen t) and sub sequen t 100 mg /kg /p6h (in fusion, un til pr ocur emen t)

No tr ea tmen t Liv er function AS T, AL T, bilirubin (D1,3,10) Positiv e - AS T and AL T on D3 v s. D1 (da ta not specified)

Minou et al

.13

2012 60 2.0% se voflur ane (end e xpir at or y, during pr ocur emen t) No tr ea tmen t Liv er

function (degr

ee of st ea tosis)

Peak AL T, AS T (D0-2); PNF , IPF (bilirubin ≥ 10 mg /dL , INR ≥ 1.6, AS T/ AL T ≥ 2000 IU/L , D0-7); and r ecipien t ICU/hospit al s ta y

Positiv e - P eak AS T: 792 v s.

1861 IU/L - IPF: 17 v

s. 50% ter al Her gen-

roeder et al.

14

2013 36 En ter al nutrition con tain - ing omeg a-3-PU F A, an ti-o xidan ts, glut amine (1 g pr ot ein/kg per 24 h, un til pr ocur emen t)

No tr ea tmen t Sur viv al Pa tien t and all solid or gan- gr aft sur viv al (M0-6) Not measur ed gan trie val

Chui et al.

15

1998 40 Single aortic perfusion

Double perfusion (aortic and port

al)

Kidne y and liv er function AS T, AL T, INR (D1-2); PNF; pa tien t and gr aft sur viv al (≤M3) No D ’Amic o et al.

2007 58

Double perfusion (aortic and port

al)

Single aortic perfusion

Liv er function AS T, AL T, bilirubin, INR

(D1-3,5,7,M1,3,6,9,12); PDF (PNF + IPF

, ≤D7); pa tien t and gr aft sur viv al

(≤M6); and re-tr

ansplan ta tion

Positiv e - AS T: 763 vs. 2125 IU/L , D2 - AL T: 614 v s. 1580 IU/L , D2 -PDF: 6 v s. 41% - R e-tr ansplan t: 0 v s. 5

Summar y of r andomised c on tr olled trials of in ter ven tions in deceased or gan donor s.

2

-

Benck et al.

18

2011 264 4 µg /kg /min dopamine (in fusion, a fter c onsen t un til pr ocur emen t) No tr ea tmen t

Heart function

LVF , L VAD and HF requir emen t; acut e r ejection (M0-M1); pa tien t and gr aft sur viv al (≤M3, Y1,2,3)

No Positiv and gr - 91 v - 87 v

Penne- fa 1995 24 300 µg /kg /min ar ginine ther vasopr essin (in fusion,

et al. when haemodynam - ic ally s table a fter BD con firma tion)

Placebo

Heart, kidne

y, liv er , lung function

Good initial function: - kidne

y: DGF - liv er/lung: unclear - heart: inotr opics requir emen t

No Insufficien report

Schnuelle et al.

17

2009 264 4 µg /kg /min dopamine (in fusion, a fter c onsen t un til pr ocur emen t) No tr ea tmen t Kidne y

function (in fusion

and CI time)

sCr , DGF (dialy sis r equir e- men t) (D0-7); acut e r ejection (≤M1); pa tien t and gr aft sur viv al (≤Y3)

Positiv e - DGF: 25% v s. 35% - DGF subgr oup long v s. short in fusion time: 21% vs. 36%

No (pa gr aft)

Guesde et al.

20

1998 97 1 µg desmopr essin (bolus, e ver y 2 h when diur esis <300 mL/h a fter consen t un til 2 h be for e pr ocur emen t)

No tr ea tmen t Kidne y function sCr , DGF (HD r equir emen t) (D0-15); sur viv al (≤Y5) No No Citt ano va et al.

1996 27 LMW Hy dr oxy eth - yl-s tar ch up t o 33 mL/

kg with additional fluid gela

tin when needed

Placebo Kidne y function DGF (HD/HF , D1-8), sCr (D1,2,5,10) Neg ativ e Not measur

Klein et al.

21

1999 112 500 µg pr os taglandin I2 (bolus, be for e pr ocur emen t) No tr ea tmen t Liv er function AS T, AL T, bilirubin, GLDH, AF , γG T (D0-28); PNF; in-hospit al sur viv al; vascular thr ombosis, and recipien t ICU/hospit al s ta y

Positiv e - AS T/ AL T, D0,1 - GLDH, D1-4 No (pa gr aft)

Randell et al.

23

1990 16 500 mL 6% h ydr oxy eth - yl-s tar ch and additionally

1000 mL when CVP <5 mmHg and cr

ys talloids (in fusion, be for e pr ocur emen t)

Cr ys talloids Liv er function PNF No Not measur Al-Kha faji et al.

2015 556 Pr ot oc olised r esusci - ta tion using a c onsen - sus-based pulse pr essur e varia tion alg orithm (un til pr ocur emen t)

St andar d donor man - ag emen t Recipien t sur viv al (E CD donor s)

Number of tr ansplan ted or gans per donor , r ecipien t (hospit al fr ee) sur viv al (≤M6)

Not measur ed No (pa

2

Sour ce Year

Number of pa

tien ts randomised Tr ea tmen t gr oup (adminis tr ation mode and time) Con tr ol gr oup Out come

(sub- gr oup)

Specific end-poin

ts (time a ft er tr ansplan ta tion) Eff ect or gan function (tr ea tmen t v s. con tr ol gr oup) apeutic

Niemann et al.

25

2015 394 Mild ther apeutic

hypothermia (34 - 35°C

, a fter declar ation of BD un til pr ocur emen t)

Normother -

mia (36.5

- 37.5°C) Kidne y

function (E CD donor s)

DGF (dialy sis r equir emen t D0-D7) Positiv e - DGF: 28.2% v s. 39.2% - DGF subgr oup E CD v s. SCD: 31% v s. 57% m unosup - essan ts

Kainz et al.

26

2010 306 1000 mg me th ylpr ed -

Placebo Kidne y nisolone (bolus, ≥ 3 h be for e pr ocur emen t)

function (donor ag e≤/>50)

SCr , DGF (D0-7) No Cha tt er -

rjee et al.

31

1981 50 60 mg /kg cy clo-phos - phamide (in fusion, t ≥ 4 h be for e pr ocur e- men t when possible)

No tr ea tmen t Kidne y function Gr aft f ailur e (≤Y1) Not measur ed

Soulillou et al.

27

1979 34 5 g me th ylpr ednisolone and 5 g cy clophospha - mide (in fusion, t ≥ 5 h be for e pr ocur emen t)

Placebo Kidne y function SCr , gr aft sur viv al, (M3,6,12) No Je ffer y et al.

1978 Unclear 5 g me th ylpr ednisolone and 7 g cy clophospha - mide (in fusion, t ≥ 4 h be for e pr ocur emen t when possible)

No tr ea tmen t Kidne y function sCr , r ejection, pa tien t and gr aft sur viv al (M3,6,12) No Ama tsch -

ek et al.

29

2012 83 1000 mg me th ylpr ednis - olone (bolus, be tw een 3 and 6 h be for e pr ocur emen t)

Placebo Liv er function AS T and AL T (D0-7); rejection, pa tien t and gr aft sur viv al (≤Y3); bile duct complic ations, r ecipien t ICU/hospit al s ta y

No

Kotsch et al.

30

2008 100 250 mg me th ylpr ednis - olone (bolus a t c onsen t + 100 mg /h IV un til pr ocur emen t)

No tr ea tmen t Liv er function AS T, AL T, bilirubin, AF , ɣG T (D0-10); acut e r ejection (AR), PNF (≤M6); and biliar y lesions

Positiv e - AS T: 327 v s. 1470; AL T: 461 v s. 758; AP: 127 v s. 157; ɣG T: 135 v s. 236 (D1) - AS T: (31 v s. 41; AL T: 75 vs. 115; bilirubin: 2.3 v s.

4.9 (D10) - Bilirubin: 0.6 v

s. 1.0 (M6) - AR: 22% v s. 36%, M6

on tinued. Summar y of r andomised c on tr olled trials of in ter ven tions in deceased or gan donor s.

2

ec ondi - Zapa ti Cha vir a et al.

2015 13

10 min IPC (hilar clamping

, f ollo w ed b y 10 min r eperfusion be for e pr ocur emen t)

No tr ea tmen t Liv er function AS T, AL T, bilirubin, INR (D1,3,7); PNF , IPF; pa tien t and gr aft sur viv al (M6, 24); and r ecipien t ICU s ta y

Sligh tly neg ativ e - Bilirubin: 3.5 v s. 1.6 mg / dL Cesc on et al.

2009 40

10 min IPC (hilar clamping

, f ollo w ed b y 15 min r eperfusion be for e pr ocur emen t)

No tr ea tmen t Liv er function AS T, AL T, bilirubin, INR (D1- 7, 14, 21); PNF , IPF; pa tien t and gr aft sur viv al (Y0- Y1), and r ecipien t ICU s ta y

No Fr an-

chello et al.

38

2009 75

10 min IPC (hilar clamping

, f ollo w ed b y 30 min r eperfusion be for e pr ocur emen t)

No tr ea tmen t Liv er function AS T, AL T, bilirubin, INR (D1,3,7); acut e r ejection, PNF; gr aft sur viv al (M6); in fections (M1), and recipien t hospit al s ta y

Sligh tly positiv e for subgr oup mar ginal gr afts: - AS T: 936 v s. 1268 (D1), 339 v s. 288 (D3), UI/L

Jassem et al.

37

2009 44

10 min IPC (hilar clamping

, f ollo w ed b y (on a ver ag e) 30 min reperfusion be for e pr ocur emen t)

No tr ea tmen t Liv er function AS T (D1-5); bilirubin and INR (D7, 14,30); acut e rejection Sligh tly positiv e - AS T: 410 v s. 965 (D1), 198 vs. 488 (D2), 120 v s. 216 (D3) IU/L

Koneru et al.

33

2007 101 10 min IPC (hilar clamp - ing , f ollo w ed b y median of 39 min r eperfusion be for e pr ocur emen t)

No tr ea tmen t Liv er

function (mar

ginal gr afts)

AS T, AL T, bilirubin, INR (D1-3,7,14,30); injur y sc or e (biop sy); acut e r ejection (D0-30); PNF; pa tien t and gr aft sur viv al (≤Y2); blood tr ans fusions; lung edema, and r ecipien t ICU/hospit al st ay

Neg ativ e - AS T: 385 v s. 250 IU/L , D2 - AL T: 699 v s. 520 (D1), 583 vs. 353 (D2) IU/L

Amador et al.

36

2007 60

10 min IPC (hilar clamping

, f ollo w ed b y 10 min r eperfusion be for e pr ocur emen t)

No tr ea tmen t Liv er function AS T, AL T, bilirubin, INR

(D1-10); PNF (D0-7); acut

e r ejection; pa tien t (Y2, Y4) and gr aft sur viv al (2Y); v ascular and biliar y complic ations, and r ecipien t ICU/hospit al s ta y

Positiv e - AS T: 894 v s. 1216 (D0), 918 v s. 1322 (D1), 500 v s. 756 (D2), 201 v s. 344 (D3), 120 v s. 170 (D4) U/L - AL T 671 v s. 1216 (D0), 235 v s. 304 (D7) U/L - Bilirubin 2.5 v s. 3.6 mg / dL (D1) Cesc on et al.

2006 53

10 min IPC (hilar clamping f

ollo w ed b y 15 min r eperfusion be for e pr ocur emen t)

No tr ea tmen t Liv er function AS T, AL T, bilirubin, INR (D1-D7,D14,D21); injur y sc or e (biop sy); PNF and IPF; pa tien t and gr aft sur viv al (≤Y1); r ecipien t ICU s ta y

Positiv e - AS T (D1,2) - AL T (D1-3,7) Ex act number s not giv en

Koneru et al.

32

2005 62

5 min IPC (hilar clamping follo

w ed b y > 30 min reperfusion be for e pr ocur emen t)

No tr ea tmen t Liv er function AS T, AL T, bilirubin, INR (D1,3,7); injur y sc or e (biop sy); PNF; pa tien t and gr aft sur viv al (≤M6); and recipien t hospit al s ta y

No

2

ter ven tion Sour ce Year Number of pa tien ts randomised Tr ea tmen t gr oup (adminis tr ation mode and time) Con tr ol gr oup Out come (sub- gr oup) Specific end-poin

ts (time a ft er tr ansplan ta tion)

Eff ect or gan

function (tr ea tmen t v s. con tr ol gr oup)

Eff ect on pa tien gr aft sur viv ot ection at egies W ar e et al.

2014 506 5 mg q4h albut er ol sulpha te (nebuliz ation ev er y 4 h, fr om s tudy enr olmen t un til pr ocur e- men t)

Placebo

Lung function (mar

ginal gr afts)

PaO2/FiO2, s ta tic

compliance; lung

/kidne y/heart/pan - cr eas utiliz ation r at es; pa tien t sur viv al (D30, ≤Y1); and r ecipien t hospit al s ta y

Sligh tly neg ativ e - Lung utiliz ation (mar ginal gr afts): 9% v s. 20% - Kidne y utiliz a- tion: 77 v s. 88%

No (pa Mascia et al.

2010 118 Pr ot ectiv e v en tila tion str at egy (TV 6-8 mL/kg

and PEEP 8-10 cm, during 6 h ob

ser va tional period un til or gan pr ocur emen t)

Con ven tional ven tila tion str at egy (TV 10-12

mL/kg and PEEP 3-5 cm)

Sur viv al Pa tien t sur viv al (≤M6) Not measur ed No (pa yr oid

Randell et al.

42

1992 25 2 µm/h triiodoth yr onine (in fusion, a t s tart pr ocur emen t) No tr ea tmen t Liv er function Ma x. ALA T, bilirubin,

albumin (D0-7); and r

ecipien t ICU/ hospit al s ta y

No Not mea sur ed T: Alanine Aminotr ans fer ase; AP: Alk aline Phospha tase; AR: Acut e Rejection; AS T: Aspart at e Aminotr ans fer ase; BD: Br ain Dea th; CI: Cold Ischaemia; D: tended Crit eria Donor; eGFR: Es tima ted Glomerular Filtr ation Ra te; ɣG T: gamma-glut am yl tr anspep tidase; HD: Haemodialy sis; HF: Haemofiltr ation; INR: ICU: e Unit; In terna tional Normalised Ra tio; IPF: Initial Poor Function; LV AD : Le ft Ven tricular Assis t De vice; LVF: Le ft Ven tricular Function; M: Mon th; PDF: sfunction; PNF: Primar y Non-Function; SCD: St andar d Crit eria Donor; sCr: Serum Cr ea tinine; Y : Y ear .

on tinued. Summar y of r andomised c on tr olled trials of in ter ven tions in deceased or gan donor s.

2

Trial id In ves tig at or Tr ea tmen t Partici - pan ts St art inclusion St op inclusion Title Sponsor NC T02581111 Dhar Nalo xone 250 2015 2016 Randomiz ed Placebo-c on tr olled T rial of In tr a- venous Nalo xone t o Impr ov e O xy gena tion in Hypo xemic Lung-Eligible Br ain-Dead Or gan Donor s W ashing ton Univ er sity School of Medicine, US NC T02435732 Fernande z C1 inhibit or 72 2016 2018 A Phase I, Single Cen ter , Randomiz ed, Dou - ble-Blind, Placebo-Con tr olled Study t o E valua te Toler ability of C1 Inhibit or (CINR YZE) as a Donor Pr etr ea tmen t Str at egy in Br ain-Dead Donor s Who Mee t a Kidne y Donor Risk Inde x (KDRI) Abo ve 85%

Univ er sity sin, Madison, US NC T02211053 Fr ene tt e Le voth yr oxine 60 2014 2016 Ev alua tion of the E ffic acy and Sa fe ty of Le voth y- ro xine in Br ain Dea th Or gan Donor s: a Randomiz ed Con tr olled T rial (E CHO T4) Hopit al du Sacr Coeur de Mon Canada NC T01860716 Gar cía-Gil Mela tonin 60 2013 2013 Impact of Mela tonin in the Pr etr ea tmen t of Or gan Donor and the In fluence in the E volution of Liv er T ransplan t: a Pr ospectiv e, Randomiz ed Double-blind Study

Hospit al Clínic Univ er sit Blesa, Spain NC T02907554 Ichai Cy closporine A 648 2016 2018 Eff ects of Cy closporine A Pr etr ea tmen t of Deceased Donor on Kidne y Gr aft Function: A Randomiz ed Con tr olled T rial Univ er sity Hospit Clermon t-F Fr ance NC T01939171 Jimine z Th ymoglobulin 20 2010 2013 Conditioning of the Cada ver Donor b y Th ymoglob - ulin Adminis ter ed t o R educe the Pr o-in flamma tor y St at e Aft er Br ain Dea th. Ins titut o de In tig ación Hospit Univ er sit Spain NC T01160978 Jokinen Sim vas ta tin 46 2010 2015 Donor Sim vas ta tin T rea tmen t in Or gan T ransplan - ta tion Helsinki Univ Hospit al, Finland NC T02341833 Joris 2% se voflur ane 240 2015 2017 Eff ects of Pr ec onditioning With Se voflur ane During Or gan Pr ocur emen t Fr om Br ain-Dead Donor s: Impact on E arly Function of Liv er Allogr afts Univ er sity Hospit of Lieg e, Belgium NC T00975702 Koneru Remot e ischaemic pr ec onditioning 85 2009 2014 Phase III Study of E ffic acy of R emot e Ischaemic Pr ec onditioning in Impr oving Out comes in Or gan Tr ansplan ta tion (RIPC O T) The St at e Univ of Ne w Jer NC T01515072

Koneru and W Remot e ischaemic 320 2011 2014 Remot e Ischaemic Pr ec onditioning in Neur ologic al The St at e Univ ashburn pr ec onditioning Dea th Or gan Donor s (RIPNOD) of Ne w Jer NC T01140035 Niemann In tensiv e insulin 200 2009 2011 In tensiv e Insulin Ther ap y in Deceased Donor s - t o Univ er sity of Calif tr ea tmen t Impr ov e R enal Allogr aft Function and T ransplan ted nia, San Fr Allogr aft Out comes US A

Table 2. Iden tified s tudies tha t ar e s till r ecruiting or ha ve not y et published r esults.