University of Groningen Brain death and organ donation Hoeksma, Dane

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University of Groningen

Brain death and organ donation

Hoeksma, Dane

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Hoeksma, D. (2017). Brain death and organ donation: Observations and interventions. Rijksuniversiteit

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Brain Death and Organ Donation

Observations and Interventions

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Brain Death and Organ Donation

Observations and Interventions

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 6 september 2017 om 12.45 uur

door Dane Hoeksma geboren op 14 februari 1988 te Ispingo, Zuid-Afrika Dane Hoeksma 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: University Medical Center Groningen

Research Institute GUIDE Noord-Negentig B.V. Chipsoft B.V.

Cover and invitation: Anne van Erp and Dane Hoeksma

Layout: Rens Dommerholt, Persoonlijk Proefschrift, www.persoonlijkproefschrift.nl Printing: Ipskamp printing, www.ipskampprinting.nl

Copyright: Dane Hoeksma, 2017-06-11 ISBN number: 978-94-028-0690-8

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Paranimfen M. Kirschbaum R. Mencke Promotores

Prof. dr. H.G.D. Leuvenink Prof. dr. H. van Goor

Beoordelingscommissie Prof. dr. J.L. Hillebrands Prof. dr. D.J. Reijngoud Prof. dr. B. Yard

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CHAPTER

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CHAPTER 1 INTRODUCTION

Renal transplantation is the most effective therapy for end-stage renal disease. In 2015, 442 kidneys from deceased donors, the largest donor pool, were transplanted in the Netherlands1_{. At the end of this year, 554 patients were still awaiting a kidney transplant.}

The total amount of kidneys transplanted in the Eurotransplant region was 3206 in the year 2015 while 10400 patients were waiting for a kidney transplant. In the United states, 17.107 kidney transplants were performed while 100.791 patients were still on the waiting list2,3_.

These data indicate that the demand of donor organs outweighs the supply. Therefore, an increase in donor organs is necessary to meet the pressing demand.

Kidneys can be retrieved from living donors (LD) and deceased donors. Between the two, deceased donors form the majority since living donors are scarce4_{. Deceased donors can}

be classified into deceased brain-dead (DBD) and deceased circulatory death donors (DCD)5_{. Of the kidneys transplanted from deceased donors, most are retrieved from}

brain-dead donors. However, in many countries, the number of kidneys obtained from DCD donors are increasing and in some countries, like the Netherlands, these kidneys are already transplanted as frequently as DBD kidneys. This is because increased amounts of kidneys from DCD donors who die unexpectedly are being more frequently used for transplantation. Most DCD donor kidneys used for transplantation are from donors who die expectedly in the sense that cardiac arrest is anticipated. In the future, an increased amount of DCD donors could be realized as some countries are starting to use organs from unexpected DCD donors.

The outcome of kidneys retrieved from different donor types varies. The graft survival rates of kidneys from different donor types from the University Medical Center Groningen are depicted in Figure 1. Kidneys from living donors are superior to deceased donor kidneys which can be explained by shorter warm- and cold ischemia times. DBD kidneys do not suffer warm ischemia but are usually subjected to long periods of cold ischemia during transport. In addition to cold ischemia, DCD kidneys usually suffer prolonged periods of warm ischemia which results in the worst outcomes amongst the three donor types. This effect is typically observed soon after transplantation as the incidence of primary non-function (PNF) of DCD grafts is 9%, while DBD and LD grafts show PNF rates of 5 and 1%, respectively. However, overall graft survival of DCD-, DBD- and LD transplantation is 86%, 86% and 93%, respectively. Therefore, outcomes from LD kidneys is superior to deceased donors but overall outcome is not compromised between either two deceased donor types. However, short term outcome of DCD kidneys is inferior compared to DBD kidneys with regard to, like mentioned above, PNF, but also delayed graft function (DGF; 82% and 30%, respectively). Yet, despite that deceased donation influences graft survival negatively, transplanting deceased donor kidneys represent a good solution for patients with end stage renal disease as the ten-year graft survival rate exceeds 85%.

Figure 1. Renal graft survival in transplant recipients transplanted between 1993 and 2015 in the University Medical Center Groningen. Graft survival is superior when organs are retrieved from living donors (LD) compared to deceased donors. Within the deceased donor group, organs retrieved from brain-dead donors (DBD) show superior graft survival compared to cardiac-death donors (DCD).

There are different options to meet the increasing demand for donor organs. Many countries have resorted to the use of expanded criteria donors (ECD) to meet the increased demand of donor organs6_{. ECD donors are older donors (>60 years) or donors aged 50}

to 59 years with two of the following: cerebrovascular accident as the cause of death, pre-existing hypertension, or terminal serum creatinine greater than 1.5 mg/dl. In the future, more ECD donors will be evident due to the aging population. Regarding DCD donors, an increased amount of DCD donors could be realized by increased use of organs from unexpected DCD. In Spain and Russia, kidneys from unexpected DCD donors are being increasingly used7-9_{. Unexpected DCD donors form a large group and may represent a}

potential option to decrease the demand for donor kidneys.

Transplanting kidneys from more marginal donors like ECD and DCD donors is likely to be associated with decreased rates of graft survival. Therefore, developing strategies to improve graft survival rates of transplanted kidneys could be advantageous. By doing so, these strategies could result in increased graft survival and thereby prevent patients from being relisted on the kidney waiting list. These strategies could also benefit more conventional donor types such as DBD donors as these kidneys suffer numerous insults in the donor. Furthermore, all transplants are subjected to ischemia-reperfusion (I-R) injury during the transplantation process and therefore even LD donor kidneys could benefit from such strategies. A major break-through was evident with the beneficial effect of machine perfusion on graft survival of deceased donor kidney transplants compared to cold storage10_.

As mentioned above, kidneys from brain-dead donors lead to inferior outcomes compared to kidneys from living donation4_{. This phenomenon is related to pathophysiological changes}

that take place in the brain-dead donor. Brain death (BD) leads to major hemodynamic derailments, systemic inflammation, and altered metabolism which potentially affects future donor organs11-13_{. Furthermore, brain dead donor organs suffer increased I-R}

injury 14_{. Major hemodynamic derailments are due to the catecholamine storm which is}

characteristic for the onset of BD13_{. The catecholamine storm is believed to be the bodies}

final attempt to maintain cerebral perfusion against increasing intracranial pressure. The

Graft survival 0 5 10 15 20 25 0 20 40 60 80 100 LD kidney DBD kidney DCD kidney Years Pe rc en t su rv iv al

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CHAPTER 1 INTRODUCTION

13

large amounts of secreted catecholamines lead to severe vasoconstriction and possible ischemic damage to organs15_{. Furthermore, spinal cord ischemia will result in vascular}

collapse and consequently decreased blood flow and increased ischemia. Circulating cytokines are evident soon after the onset of BD with IL-6 being the most implicated cytokine in BD. Expression of adhesion molecules and infiltration of inflammatory cells in organs is also evident soon after the onset of BD16_{. The increased production of cytokines}

could be secreted by the dying cerebrum or by organs that suffer ischemic insults due to the hemodynamic instability. Altered metabolism is observed in the kidney and liver of brain- dead animals. Most notably a switch is apparent from aerobic to anaerobic metabolism. The altered metabolism could be the result of both hemodynamic changes and inflammatory mediators. Furthermore, brain death-related processes such as the catecholamine storm are affected by the speed at which intracranial pressure (ICP) increases17_{. A faster increase}

in ICP leads to higher levels of circulating catecholamines, which is particularly detrimental for cardiac and pulmonary graft function. Indeed, traumatic brain injury, the most common cause of BD preceded by a rapid increase in ICP, is a risk factor for mortality in heart recipients18_{. In contrast, a cerebrovascular cause of death, usually preceded by a slower}

increase in ICP, is a risk factor for renal and hepatic graft dysfunction19,20_{. However, this}

phenomenon is not believed to be associated with a slower increase in ICP. Rather, donor characteristics such as obesity, old age, and the presence of cardiovascular disease are regarded as the underlying cause.

Many studies have focused on attempting to counteract BD-related pathophysiological processes. Hemodynamic, inflammatory, and metabolic changes have all been counteracted with often good experimental results. Experimental research shows that blocking the catecholamine storm has a beneficial effect on lung function parameters and histology 21_{. Treating brain-dead rats with methylprednisolone leads to improved}

renal graft survival and reduced rejection22_{. Metabolic changes have been tackled through}

administration of thyroid hormones which has differential effects on the liver and kidney

23,24_.

Many beneficial experimental interventions in brain-dead donors have been studied clinically as well. Early studies involving the administration of methylprednisolone to brain-dead donors however did not show beneficial effects25-27_{. In a later study, a reduction in}

pro-inflammatory cytokines in the donor kidney prior to transplantation was evident22_{. No}

beneficial effects of methylprednisolone on kidney function after transplantation were observed. Improved renal function after transplantation has only been observed with the administration of dopamine to brain-dead donors28-31_.

Oxidative stress has been documented in brain-dead kidneys in both experimental and clinical studies16,32_{. Several studies show that BD is associated with oxidative damage of}

cellular lipid membranes12,33_{. Lipid peroxidation leads to membrane permeabilization and}

impairment of enzymatic processes and ion pumps which results in membrane dysfunction and cell toxicity34-36_{. BD-related lipid peroxidation is correlated with DGF in renal transplant}

recipients32_{. The levels of malondialdehyde (MDA), a product of lipid peroxidation, in the}

preservation solution of kidneys retrieved from brain-dead donors correlate well with DGF. Moreover, donor serum MDA levels correlate with acute rejection and immediate and long-term renal allograft function. In expanded criteria donors (ECD), MDA levels in machine perfusion solution also correlate with DGF37_.

Reactive oxygen species (ROS) are mainly formed in the mitochondrial electron transport chain (ETC, Fig.2) and are essential for cellular homeostasis, mitosis, differentiation, signaling and survival38_{. Superoxide can be generated from complexes I and III of the}

mitochondrial electron transport chain (ETC), by xanthine and NADPH oxidase, the tricarboxylic acid (TCA) cycle enzymes aconitase and α-ketoglutarate dehydrogenase, by non-TCA cycle enzymes and by monoamine oxidases and cytochrome b5 reductase, located in the outer mitochondrial membrane39_{. Endogenous antioxidants such as}

superoxide dismutase (SOD), glutathione peroxidase (GpX) and catalase regulate the levels of ROS accurately40_{. However, certain pathological conditions increase radical}

production which can overwhelm antioxidant protection. Excessive ROS generation leads to damaged nucleic acids, proteins, and lipids. which damages enzymes in the ETC leading to mitochondrial dysfunction, decreased ATP production and increased generation of ROS.

Figure 2. Sources of cellular superoxide production and its clearance by endogenous anti-oxidants. Adapted from Wang, K. 2013. Superoxide (O2-) is formed mainly from the mitochondrial electron transport chain (ETC). Endogenous anti-oxidants such as superoxide dismutase (SOD) convert superoxide to hydrogen peroxide (H2O2) which can subsequently be converted to water (H2O) by amongst others catalase and glutathione peroxidase (GpX).

BD pathophysiology, which comprises hemodynamic, inflammatory, and metabolic changes, can lead to oxidative stress through aforementioned processes39,41,42_.

Hemodynamic changes, and the resulting ischemia, trigger mitochondrial dysfunction and the subsequent leakage of radicals from the mitochondrial respiratory chain. The influx of inflammatory cells can cause increased oxidative stress as large amounts of superoxide are released which forms part of the respiratory burst to kill pathogens. Furthermore, metabolic changes can lead to mitochondrial dysfunction and thereby increase the oxidative load. Considering all the possible sources of oxidative stress, anti-oxidative therapy could encompass many different options. Therefore, prior assessment as to what might be the most up-stream cause of oxidative stress could be useful for administration of an efficient anti-oxidative compound.

The primary aim of this thesis is to assess the detrimental effects of BD on different donor organs and to thereby put forward and test organ-specific therapies. Many studies have been conducted on the detrimental effects of brain death and many studies have focused on preventing these effects. Unfortunately, many of these interventional studies don’t show positive clinical effects. Likely, increased and more in depth knowledge about

organ-1

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CHAPTER 1 INTRODUCTION

specific effects of BD is necessary to gain advancements in treating brain-dead donors. Therefore, in this thesis we delved deeper into certain detrimental effects which were already touched upon on by previous studies, such as oxidative stress and metabolism. Furthermore, to gain more insight into the processes leading up to BD, we assessed the effects of different speeds of BD induction on donor organs. The speed of BD induction has shown to influence heart function and damage. Clinically, brain insults tend to progress to BD at different speeds. Therefore, we aimed to investigate the effects of speed of BD induction on other organs than the heart as this could also contribute to organ- and donor-specific interventions.

In Chapter 2 we focused on assessing the effects of fast and slow BD induction on the two most frequently transplanted organs, the kidney and liver. In this Chapter we aimed to assess general differences between these organs elicited by fast and slow speed BD induction. Subsequently, in later Chapters, more in depth analysis could be performed based on these initial results. In Chapter 2 we used the most clinically relevant function and damage markers, such as plasma creatinine and plasma levels of ASAT and ALAT to determine the effects of speed of BD induction on the kidney and liver. Furthermore, commonly used marker for inflammation in BD, such as IL-6 and other common markers for cell death such as caspase expression and the oxidative stress marker MDA were assessed.

In Chapter 3, similarly to Chapter 2, we assessed the effects of fast and slow BD induction on lung parameters. Experimental data shows that the heart is negatively affected by a fast increase in intracranial pressure which is attributed to a more severe catecholamine storm. Experimental research has shown that blocking the catecholamine storm has a beneficial effect on lung function. Moreover, clinical data shows that a traumatic cause of BD, associated with a fast increase in ICP, leads to decreased lung function. No experimental data has been published on the effects of speed of BD induction on the lung. Therefore, we aimed to assess the effect of the speed of BD induction on lung function and damage. This data could help in designing donor-specific management strategies and optimal organ allocation policies.

Chapter 4 of this thesis is an expansion of the observation done in Chapter 2 that slow BD leads to increased renal oxidative stress compared to fast induction. In this Chapter, we investigated oxidative and anti-oxidative processes after fast and slow speed BD induction. These processes could explain the increased renal oxidative stress observed especially after slow BD induction. This data could lead to specific anti-oxidative therapy for brain-dead donors and thereby possibly improve transplantation outcomes.

Chapter 2 and 4 show that BD leads to increased renal oxidative stress. These results were manifested despite the maintenance of hemodynamic stability. In Chapter 5, we investigated how BD affects regional renal and hepatic hemodynamics and metabolic effects in these organs and how this might affect the oxidative processes we observed in previous Chapters. Changes in metabolism elicited by brain death were shown almost thirty years ago. However, no clinical relevant interventions have evolved from this pioneering work. Therefore, in this Chapter, we looked more deeply into metabolic changes in the liver and kidney. We assessed changes in levels of glucose, fatty acids, and proteins. Moreover, mitochondrial functional changes were assessed and changes in regional hepatic and renal perfusion.

The kidney is by far the most transplanted organ. Therefore, in Chapter 6, we undertook an interventional study by treating brain-dead rats with the goal of improving renal quality. Oxidative membrane damage in brain-dead donors correlates with delayed graft function in renal transplant recipients. Therefore, anti-oxidative therapy administered to brain-dead donors could lead to improved transplantation outcomes. Based on the observations made in Chapter 2, 4, and 5, we treated brain-dead rats with MnMTPyP, a selective superoxide dismutase mimetic, after slow BD induction. In Chapter 2 we found that oxidative stress was increased more after slow BD induction so we chose this form of induction to study the effects of MnTMPyP. In Chapter 4 we showed that superoxide is probably one of the most “upstream” oxidative processes in BD. Therefore, a superoxide dismutase mimetic could potentially eliminate this process and exert beneficial downstream effects. In Chapter 5 we showed that oxidative stress is likely influence by decreased regional renal perfusion which leads to changes in metabolism and oxidative stress. At the same time this shows that in anti-oxidative therapy should comprise a compound which can exert effect in the renal tissue since the maintenance of hemodynamic stability is not sufficient.

In Chapter 6 we showed that MnTMPyP exerts beneficial effects on the kidney as shown by decreased renal and systemic oxidative stress. However, no beneficial effects on function were seen which we attributed to the fact that BD does not lead to sufficient oxidative damage to expect beneficial effects of anti-oxidative treatment. Therefore, in Chapter 7, we assessed renal function of kidneys of brain-dead rats treated with MnTMPyP in an ex vivo isolated perfused kidney system.

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CHAPTER 1 INTRODUCTION

17 1. https://www.eurotransplant.org/cms/ index.php?page=yearlystats. 2. http://optn.transplant.hrsa.gov/. 3. http://www.usrds.org/2015/view/ v2_07.aspx.

4. Terasaki PI, Cecka JM, Gjertson DW, Takemoto S. High survival rates of kidney transplants from spousal and living unrelated donors. N Engl J Med 1995 Aug 10;333(6):333-336.

5. Kootstra G, Daemen JH, Oomen AP. Categories of non-heart-beating donors. Transplant Proc 1995 Oct;27(5):2893-2894.

Rao PS, Ojo A. The alphabet soup of kidney transplantation: SCD, DCD, ECD--fundamentals for the practicing nephrologist. Clin J Am Soc Nephrol 2009 Nov;4(11):1827-1831.

7. de Gracia MC, Osorio JM, Perez-Villares JM, Galindo P, Ruiz MC, Perez-Marfil A, et al. A new program of kidney transplantation from donors after cardiac death in Spain. Transplant Proc 2012 Nov;44(9):2518-2520. 8. Pieter Hoogland ER, van Smaalen

TC, Christiaans MH, van Heurn LW. Kidneys from uncontrolled donors after cardiac death: which kidneys do worse? Transpl Int 2013 May;26(5):477-484.

9. Reznik ON, Skvortsov AE, Reznik AO, Ananyev AN, Tutin AP, Kuzmin DO, et al. Uncontrolled donors with controlled reperfusion after sixty minutes of asystole: a novel reliable resource for kidney transplantation. PLoS One 2013 May 30;8(5):e64209. 10. Moers C, Smits JM, Maathuis MH,

Treckmann J, van Gelder F, Napieralski BP, et al. Machine perfusion or cold storage in deceased-donor kidney transplantation. N Engl J Med 2009 Jan 1;360(1):7-19.

11. Novitzky D, Cooper DK, Morrell D, Isaacs S. Change from aerobic to anaerobic metabolism after brain death, and reversal following triiodothyronine therapy.

Transplantation 1988 Jan;45(1):32-36. 12. Schuurs TA, Morariu AM, Ottens PJ, ‘t

Hart NA, Popma SH, Leuvenink HG, et al. Time-dependent changes in donor brain death related processes. Am J Transplant 2006 Dec;6(12):2903-2911. 13. Bos EM, Leuvenink HG, van Goor H,

Ploeg RJ. Kidney grafts from brain dead donors: Inferior quality or opportunity for improvement? Kidney Int 2007 Oct;72(7):797-805.

14. Weiss S, Kotsch K, Francuski M, Reutzel-Selke A, Mantouvalou L, Klemz R, et al. Brain death activates donor organs and is associated with a worse I/R injury after liver transplantation. Am J Transplant 2007 Jun;7(6):1584-1593.

15. Herijgers P, Leunens V, Tjandra-Maga TB, Mubagwa K, Flameng W. Changes in organ perfusion after brain death in the rat and its relation to circulating catecholamines. Transplantation 1996 Aug 15;62(3):330-335.

16. Morariu AM, Schuurs TA, Leuvenink HG, van Oeveren W, Rakhorst G, Ploeg RJ. Early events in kidney donation: progression of endothelial activation, oxidative stress and tubular injury after brain death. Am J Transplant 2008 May;8(5):933-941.

17. Shivalkar B, Van Loon J, Wieland W, Tjandra-Maga TB, Borgers M, Plets C, et al. Variable effects of explosive or gradual increase of intracranial pressure on myocardial structure and function. Circulation 1993 Jan;87(1):230-239.

18. Cohen O, De La Zerda DJ, Beygui R, Hekmat D, Laks H. Donor brain death mechanisms and outcomes after heart transplantation. Transplant Proc 2007 Dec;39(10):2964-2969.

19. Pessione F, Cohen S, Durand D, Hourmant M, Kessler M, Legendre C, et al. Multivariate analysis of donor risk factors for graft survival in kidney transplantation. Transplantation 2003 Feb 15;75(3):361-367.

20. Feng S, Goodrich NP, Bragg-Gresham

JL, Dykstra DM, Punch JD, DebRoy MA, et al. Characteristics associated with liver graft failure: the concept of a donor risk index. Am J Transplant 2006 Apr;6(4):783-790.

21. Avlonitis VS, Wigfield CH, Kirby JA, Dark JH. The hemodynamic mechanisms of lung injury and systemic inflammatory response following brain death in the transplant donor. Am J Transplant 2005 Apr;5(4 Pt 1):684-693.

22. Pratschke J, Kofla G, Wilhelm MJ, Vergopoulos A, Laskowski I, Shaw GD, et al. Improvements in early behavior of rat kidney allografts after treatment of the brain-dead donor. Ann Surg 2001 Dec;234(6):732-740.

23. Schwartz I, Bird S, Lotz Z, Innes CR, Hickman R. The influence of thyroid hormone replacement in a porcine brain death model. Transplantation 1993 Mar;55(3):474-476.

24. Pienaar H, Schwartz I, Roncone A, Lotz Z, Hickman R. Function of kidney grafts from brain-dead donor pigs. The influence of dopamine and triiodothyronine. Transplantation 1990 Oct;50(4):580-582.

25. Chatterjee SN, Terasaki PI, Fine S, Schulman B, Smith R, Fine RN. Pretreatment of cadaver donors with methylprednisolone in human renal allografts. Surg Gynecol Obstet 1977 Nov;145(5):729-732.

26. Jeffery JR, Downs A, Grahame JW, Lye C, Ramsey E, Thomson AE. A randomized prospective study of cadaver donor pretreatment in renal transplantation. Transplantation 1978 Jun;25(6):287-289.

27. Soulillou JP, Baron D, Rouxel A, Guenel J. Steroid-cyclophosphamide pretreatment of kidney allograft donors. A control study. Nephron 1979;24(4):193-197.

28. Schnuelle P, Lorenz D, Mueller A, Trede M, Van Der Woude FJ. Donor catecholamine use reduces acute allograft rejection and improves graft survival after cadaveric renal transplantation. Kidney Int 1999 Aug;56(2):738-746.

29. Schnuelle P, Berger S, de Boer J, Persijn G, van der Woude FJ. Effects of catecholamine application to brain-dead donors on graft survival in solid organ transplantation. Transplantation 2001 Aug 15;72(3):455-463.

30. Schnuelle P, Yard BA, Braun C, Dominguez-Fernandez E, Schaub M, Birck R, et al. Impact of donor dopamine on immediate graft function after kidney transplantation. Am J Transplant 2004 Mar;4(3):419-426.

31. Schnuelle P, Gottmann U, Hoeger S, Boesebeck D, Lauchart W, Weiss C, et al. Effects of donor pretreatment with dopamine on graft function after kidney transplantation: a randomized controlled trial. JAMA 2009 Sep 9;302(10):1067-1075.

32. Kosieradzki M, Kuczynska J, Piwowarska J, Wegrowicz-Rebandel I, Kwiatkowski A, Lisik W, et al. Prognostic significance of free radicals: mediated injury occurring in the kidney donor. Transplantation 2003 Apr 27;75(8):1221-1227.

33. Rebolledo RA, Hoeksma D, Hottenrott CM, Bodar YJ, Ottens PJ, Wiersema-Buist J, et al. Slow induction of brain death leads to decreased renal function and increased hepatic apoptosis in rats. J Transl Med 2016 May 19;14(1):141-016-0890-0.

34. Jain SK, Shohet SB. Calcium potentiates the peroxidation of erythrocyte membrane lipids. Biochim Biophys Acta 1981 Mar 20;642(1):46-54.

35. Vladimirov YA, Olenev VI, Suslova TB, Cheremisina ZP. Lipid peroxidation in mitochondrial membrane. Adv Lipid Res 1980;17:173-249.

36. Tribble DL, Aw TY, Jones DP. The pathophysiological significance of lipid peroxidation in oxidative cell injury. Hepatology 1987 Mar-Apr;7(2):377-386.

37. Nagelschmidt M, Minor T, Gallinat A, Moers C, Jochmans I, Pirenne J, et al. Lipid peroxidation products in machine perfusion of older donor kidneys. J

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Surg Res 2013 Apr;180(2):337-342. 38. Poyton RO, Ball KA, Castello PR.

Mitochondrial generation of free radicals and hypoxic signaling. Trends Endocrinol Metab 2009 Sep;20(7):332-340.

39. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 2007;39(1):44-84. 40. Zhang H, Go YM, Jones DP.

Mitochondrial thioredoxin-2/ peroxiredoxin-3 system functions in parallel with mitochondrial GSH system in protection against oxidative stress. Arch Biochem Biophys 2007 Sep 1;465(1):119-126.

41. Himmelfarb J, McMonagle E, Freedman S, Klenzak J, McMenamin E, Le P, et al. Oxidative stress is increased in critically ill patients with acute renal failure. J Am Soc Nephrol 2004 Sep;15(9):2449-2456.

42. Nakayama M, Nakayama K, Zhu WJ, Shirota Y, Terawaki H, Sato T, et al. Polymorphonuclear leukocyte injury by methylglyoxal and hydrogen peroxide: a possible pathological role for enhanced oxidative stress in chronic kidney disease. Nephrol Dial Transplant 2008 Oct;23(10):3096-3102.

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2

CHAPTER

Slow induction of brain death

leads to decreased renal

function and increased hepatic

apoptosis in rats

D Hoeksma* RA Rebolledo* CMV Hottenrott YS Bodar PJ Ottens J Wiersema-Buist HGD Leuvenink

*Authors contributed equally to the manuscript

Published in Journal of Translational Medicine

Reference: J Transl Med. 2016 May 19;14(1):141 Digital object identifier (DOI):

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CHAPTER 2 BRAIN DEATH INDUCTION SPEEDS AND ABDOMINAL EFFECTS

ABSTRACT

Introduction

Donor brain death (BD) is an independent risk factor for graft survival in recipients. While in some patients BD results from a fast increase in intracranial pressure, usually associated with trauma, in others, intracranial pressure increases more slowly. The speed of intracranial pressure increase may be a possible risk factor for renal and hepatic graft dysfunction. This study aims to assess the effect of speed of BD induction on renal and hepatic injury markers.

Methods

BD induction was performed in 64 mechanically ventilated male Fisher rats by inflating a 4.0F Fogarty catheter in the epidural space. Rats were observed for 0.5 h, 1 h, 2 hrs, or 4 hrs following BD induction. Slow induction was achieved by inflating the balloon-catheter at a speed of 0.015 ml/min until confirmation of BD. Fast induction was achieved by inflating the balloon at 0.45 ml/min for 1 minute. Plasma, kidney and liver tissue were collected for analysis.

Results

Slow BD induction led to higher plasma creatinine at all time points compared to fast induction. Furthermore, slow induction led to increased renal mRNA expression of IL-6, and renal MDA values after 4 hrs of BD compared to fast induction. Hepatic mRNA expression of TNF-α, Bax/Bcl-2, and protein expression of caspase-3 was significantly

higher due to slow induction after 4 hrs of BD compared to fast induction. PMN infiltration was not different between fast and slow induction in both renal and hepatic tissue.

Conclusion

Slow induction of BD leads to poorer renal function compared to fast induction. Also, renal inflammatory and oxidative stress markers were increased. Liver function was not affected by speed of BD induction but hepatic inflammatory and apoptosis markers increased significantly due to slow induction compared to fast induction. These results provide initial proof that speed of BD induction influences detrimental renal and hepatic processes which could signify different donor management strategies for patients progressing to BD at different speeds.

INTRODUCTION

The shortage of qualitative donor organs remains a limiting factor in organ transplantation. Therefore, utilization of sub-optimal donor types to meet the increasing demand of organs is inevitable. Today, brain-dead donors form the largest donor pool worldwide for kidney and liver transplantation1,2_{. Unfortunately, transplanting kidneys from brain dead donors}

leads to a higher incidence of rejection and delayed graft function compared to living donors3_{. A cerebrovascular cause of BD is related to renal and liver graft failure indicating}

that the nature of brain insults affect graft function as well4,5_.

Brain death (BD) is a complex pathological condition, characterized by hemodynamic imbalance, hormonal impairment, and a systemic inflammatory response. Hemodynamic imbalance comprises changes elicited by brainstem herniation, the resulting catecholamine storm, and neurogenic shock due to ischemia of the spinal cord. Systemic inflammation is characterized by increased levels of circulating cytokines including interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-

α

), transforming growth factor-beta (TGF- ) and, monocyte chemotactic protein 1 (MCP-1)6-8_{. This systemic inflammatory}

environment promotes the migration of inflammatory cells into organs triggering a local inflammatory and (pro-)apoptotic response9,10_{. Furthermore, BD affects the pituitary}

function causing endocrine alterations which are considered to exacerbate the graft deterioration11-13_.

Brain death-related processes such as the catecholamine storm are affected by the speed at which intracranial pressure (ICP) increases. A faster increase in ICP leads to higher levels of circulating catecholamines, which is particularly detrimental for cardiac and pulmonary graft function14_{. Indeed, traumatic brain injury, the most common cause of BD preceded}

by a rapid increase in ICP, is a risk factor for mortality in heart recipients15_{. In contrast, a}

cerebrovascular cause of death, usually preceded by a slower increase in ICP, is a risk factor for renal and hepatic graft dysfunction. However, this phenomenon is not believed to be associated with a slower increase in ICP. Rather, donor characteristics such as obesity, old age, and the presence of cardiovascular disease are regarded as the underlying cause5,16,17_.

We aimed to assess whether the speed of BD induction affects renal and hepatic quality in brain dead donor rats.

MATERIALS AND METHODS

Sixty-four male Fisher F344 rats (270-300 g) were subjected to either fast or slow BD induction with a BD duration of 0.5 h, 1 h, 2 hrs, or 4 hrs. All animals received care in compliance with the guidelines of the local animal ethics committee according to the Experiments on Animals Act (1996) issued by the Ministry of Public Health, Welfare and Sports of the Netherlands. Animals were anaesthetized using 2-5% isoflurane with 100% O2. Two ml of saline 0,9% were administered s.c. to prevent dehydration during surgery.

Animals were intubated via a tracheostomy and ventilated (Tidal Volume: 6.5 ml/kg of body weight, PEEP of 3 cm of H20 at an initial respiratory rate of 120 and was adjusted to maintain

the ETCO2 in hypocapnic range) throughout the experiment. Cannulas were inserted

in the femoral artery and vein for continuous mean arterial pressure (MAP) monitoring and volume replacement. Through a frontolateral hole drilled in the skull, a no. 4 Fogarty catheter (Edwards Lifesciences Co, Irvine, CA) was placed in the epidural space and inflated with saline using a syringe pump (Terufusion, Termo Co., Tokyo, Japan). To prevent movements during catheter inflation, a bolus of rocuronium (0,6 mg/kg) was administered. Fast and slow induction of BD were induced by inflating the catheter at a speed of 0.45 ml/

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CHAPTER 2 BRAIN DEATH INDUCTION SPEEDS AND ABDOMINAL EFFECTS

25

min or 0.015 ml/min, respectively. For slow speed induction, inflation of the balloon was terminated when the MAP increased above 80 mmHg. For fast induction, the catheter was inflated over a period of one minute. BD was confirmed by the absence of corneal reflexes half an hour after induction after which anesthesia was discontinued. MAP was maintained above 80 mmHg. If necessary, colloid infusion with 10% polyhydroxyethyl starch (HAES) (Fresenius Kabi AG, Bad Homburg, Germany) was given in a bolus (limited to a maximum of 1 ml/h) to maintain the MAP above 80 mmHg. Unresponsiveness to HAES indicated the start of an intravenous noradrenaline (NA) drip (1 mg/mL). A homeothermic blanket control system was used throughout the experiment, maintaining the body temperature between 37 and 38 °C. At the end of the experimental period a bolus of succinylcholine (0.1 mg/kg) was administered in order to prevent movements during aortic puncture, blood and urine were collected. Animals were systemically flushed with cold saline. After the flush, organs were harvested and tissue samples were snap frozen in liquid nitrogen and stored at -80 °C or fixated in 4% paraformaldehyde. Plasma samples and urine were also snap-frozen and stored. One animal was discarded in the slow induction 2 hrs group, two animals in the fast induction group 2 hrs and one in the fast induction 4 hrs group due to unknown amounts of noradrenaline administration. One animal was discarded in the fast induction 4 hrs group due to an apnea test conducted during the BD period.

Animals were randomly assigned to one of 8 experimental groups: Fast BD induction 0.5 hrs (n = 8) Fast BD induction 1 hrs (n = 8) Fast BD induction 2 hrs (n = 6) Fast BD induction 4 hrs (n = 6) Slow BD induction 0.5 hrs (n = 8) Slow BD induction 1 hrs (n = 8) Slow BD induction 2 hrs (n = 7) Slow BD induction 4 hrs (n = 8)

Biochemical determinations

Plasma levels of alanine transaminase (ALT), aspartate transaminase (AST) and creatinine were determined at the clinical chemistry lab of University Medical Centre Groningen according to standard procedures.

Plasma IL-6 measurement

Plasma IL-6 was determined by a rat enzyme-linked immunosorbent assay (IL-6 ELISA) kit (R&D Systems Europe Ltd. Abingdon, Oxon OX14 3NB, UK), according to the manufacturer’s instructions. All samples were analyzed in duplicate and read at 450 nm.

RNA isolation and cDNA synthesis

Total RNA was isolated from whole liver and kidney sections using TRIzol (Life Technologies, Gaithersburg, MD). Samples were verified for absence of genomic DNA contamination by performing RT-PCR reactions in which the addition of reverse transcriptase was omitted, using Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers. For cDNA synthesis, 1 μl T11VN Oligo-dT (0,5 μg/μl) and 1μg mRNA were incubated for 10 min at 70 °C and cooled directly after that. cDNA was synthesized by adding a mixture containing 0.5 μl RnaseOUT® Ribonuclease inhibitor (Invitrogen, Carlsbad, USA), 0.5μl RNase water (Promega), 4 μl 5 x first strand buffer (Invitrogen), 2 μl DTT (Invitrogen), 1 μl dNTP’s and 1μl

M-MLV reverse transcriptase (Invitrogen, 200U). The mixture was held at 37 °C for 50 min. Next, reverse transcriptase was inactivated by incubating the mixture for 15 min at 70 °C. Samples were stored at − 20 °C.

Real-Time PCR

Fragments of several genes were amplified with the primer sets outlined in Table 1. Pooled cDNA obtained from brain-dead rats was used as an internal reference. Gene expression was normalized with the mean of -actin mRNA content. Real-Time PCR was carried out in reaction volumes of 15μl containing 10μl of SYBR Green mastermix (Applied biosystems, Foster City, USA), 0.4μl of each primer (50μM), 4.2μl of nuclease free water and 10 ng of cDNA. All samples were analyzed in triplicate. Thermal cycling was performed on the Taqman Applied Biosystems 7900HT Real Time PCR System with a hot start for 2 min at 50 °C followed by 10 min 95 °C. Second stage was started with 15 s at 95 °C (denaturation step) and 60 s at 60 °C (annealing step and DNA synthesis). The latter stage was repeated 40 times. Stage 3 was included to detect formation of primer dimers (melting curve) and begins with 15 s at 95 °C followed by 60 s at 60 °C and 15 s at 95 °C. Primers were designed with Primer Express software (Applied Biosystems) and primer efficiencies were tested by a standard curve for the primer pair resulting from the amplification of serially diluted cDNA samples (10 ng, 5 ng, 2.5 ng, 1.25 ng and 0.625 ng) obtained from brain-dead rats. PCR efficiency was 1.8 < < 2.0. Real-time PCR products were checked for product specificity on a 1.5% agarose gel. Results were expressed as 2− CT (CT: Threshold Cycle).

Table 1: Primer sequences used for Real-Time PCR

Gene Primers Amplication size (bp)

IL-6 5’-CCAACTTCCAATGCTCTCCTAATG-3’ 5’- TTCAAGTGCTTTCAAGAGTTGGAT-3’ 89 TNF-α 5’-GGCTGCCTTGGTTCAGATGT-3’ 5’-CAGGTGGGAGCAACCTACAGTT-3’ 79 BAX 5’-GCGTGGTTGCCCTCTTCTAC-3’ 5’-TGATCAGCTCGGGCACTTTAGT-3’ 74 Bcl2 5’-CTGGGATGCCTTTGTGGAA-3’ 5’-TCAGAGACAGCCAGGAGAAATCA-3’ 70

Tissue Malondialdehyde (MDA)

Kidney and liver tissue were homogenized with a pestle and mortar in PBS containing 5mM butylated hydroxytoluene. MDA was measured fluorescently after binding to thiobarbituric acid. For this, 100µL of tissue homogenate was mixed with 2% SDS followed by 400µL 0.1 N HCL, 50µL 10% phosphotungstic acid and 200µL 0.7% TBA. The mixture was incubated for 30 min at 97°C. To the sample 800µL of 1-butanol were added and centrifuged at 960 g. Of the supernatant 200 µL were used for fluorescence measurements at 480 nm excitation and 590 nm emission wavelengths. Samples were corrected for total amount of protein.

Immunohistochemistry

To detect caspase-3 and HIS48 positive cells in liver and kidney, immunohistochemistry was performed on 3 or 5 μm sections of paraffin embedded samples. Sections were deparaffined in a sequence of xylene, alcohol and water. As an antigen retrieval method

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CHAPTER 2 BRAIN DEATH INDUCTION SPEEDS AND ABDOMINAL EFFECTS

we used for caspase-3 samples: EDTA (1mM, pH 8.0) buffer. Next, sections were stained with Caspase-3 primary Antibody (Cell Signaling cat. nr. 9661, 100x diluted in 1% BSA/ PBS) using an indirect immunoperoxidase technique. Endogenous peroxidase was blocked using H2O2 0.3% in phosphate-buffered saline for 30 min. After thorough washing,

sections were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG as a secondary antibody for 30 min (Dako, Glostrup, Denmark. cat. nr. P0448), followed by rabbit anti-goat IgG as a tertiary antibody for 30 min (Dako, Glostrup, Denmark. cat. nr. P0449).The reaction was developed using DAB as chromogen and H2O2 as substrate.

Sections were counterstained using Mayer hematoxylin solution (Merck, Darmstadt, Germany). For HIS48, mouse monoclonal anti-rat granulocyte antibody (HIS 48; IQ products, Groningen) was dissolved in PBS (pH 7.4) supplemented with 1% bovine serum albumin (BSA). Peroxidase-labeled second antibody (rabbit anti-mouse) was diluted in 1% BSA/PBS containing 5 % normal rat serum. Peroxidase activity was visualized using aminoethylcarbazole. Sections were counterstained with Mayers hematoxylin solution. Negative antibody controls were performed. Localization of immunohistochemical staining was assessed by light microscopy. For each tissue section, positive cells per field were counted by a blinded researcher in 10 microscopic fields of the tissue at 10x magnification. Results were presented as number of positive cells per field.

Statistical Analyses

Statistical analysis was performed between both experimental groups using a nonparametric test (Mann Whitney) for every time point. Hazard function and the Mantel-Cox were used to compare time of HAES or Noradrenaline administration. All statistical tests were 2-tailed and p < 0.05 was regarded as significant. Results are presented as mean ± SD (standard deviation).

RESULTS

As an internal control we compared the catheter volume after brain death induction and blood pressure pattern during the induction phase. The final catheter volume was similar between the slow and fast induction model (0.41 ± 0.03 ml vs 0.41 ± 0.02). During BD induction, the MAP showed different characteristic patterns due to fast and slow speed induction (Figure 1). Slow speed BD induction was characterized by a period of decreased blood pressure which typically started 10 min before the end of the induction and in which the minimum pressure registered was 51.17 ± 10.76 mm Hg. In contrast, fast speed induction was characterized by a sudden and short increase in MAP which was typically observed at the end of the balloon inflation period and in which the maximum pressure registered was 167.39 ± 37.85 mm Hg.

Figure 1. Course of MAP during BD induction and during 4-h BD in fast- and slow-inducted rats. T = 0 represents the start of the BD period.

The amount of HAES needed for a stable MAP was similar after fast and slow speed induction. The amount of administered NA was significantly higher in the fast induction group compared to slow induction after 0.5 h and 1 h of BD (Table 2). We estimated the chance of noradrenaline and HAES utilization using hazard curves. Slow induction led to a 17.05% probability of NA use in the first hour of BD, while fast induction led to a 54.84% probability. Additionally, we compared both curves using the Mantel-Cox test. The curves for NA use were significantly different (p = 0.0004). HAES was used mainly in the first minutes after BD induction. Slow induction led to a 48.39% probability of HAES use in the first 10 min of BD while fast induction led to a 84.38% probability. Curve comparison was found to be significantly different using the Mantel-Cox test (p = 0.0091. Figure 2).

Table 2: Total Noradrenaline (1 mg/ml) and HAES 10% infusion requirements.

Time (hr) Fast Induction Slow Induction p Value Noradrenaline (ml) 0.5 0.35 ± 0.42 0.10 ± 0.24 0.0188* 1 0.55 ± 0.76 0.05 ± 0.14 0.0238* 2 1.1 ± 1.6 0.13 ± 0.25 0.1515 4 0.33 ± 0.58 0.23 ± 0.42 0.8564 HAES 10% (ml) 0.5 0.44 ± 0.18 0.31 ± 0.26 0.5692 1 0.56 ± 0.18 0.50 ± 0.0 0.9999 2 0.50 ± 0.0 0.38 ± 0.35 0.2000 4 0.56 ± 0.50 0.56 ± 0.42 0.9999

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CHAPTER 2 BRAIN DEATH INDUCTION SPEEDS AND ABDOMINAL EFFECTS

29

Figure 2. Hazard curves for Noradrenaline and HAES utilization in fast- and slow-inducted rats. * indicates p < 0.05.

ALT and AST plasma levels were measured as liver cell injury markers. No differences were found in ALT levels between fast and slow speed induction. The AST plasma levels were increased due to fast induction compared to slow induction groups after 0.5 and 2 hrs of BD (p = 0.0225 and p = 0.0088, Figure 3).

Plasma creatinine levels were measured in order to estimate kidney function. Creatinine was significantly higher after slow induction compared to fast induction at every time point. Plasma urea levels were increased due to slow induction compared to fast induction after 4 hrs of BD (p = 0.0308, Figure 3).

Figure 3. Plasma levels of injury markers and function markers in fast- and slow-inducted rats after 0.5, 1, 2, and 4 h BD. * indicates p < 0.05.

Plasma IL-6 levels were measured as a marker for systemic inflammation. IL-6 plasma levels were significantly increased due to slow induction compared to fast induction after 0.5 and 1 h of BD (p = 0.0014 and p = 0.0002, Figure 4).

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CHAPTER 2 BRAIN DEATH INDUCTION SPEEDS AND ABDOMINAL EFFECTS

We assessed tissue inflammation by measuring the relative expression of pro-inflammatory genes in the kidney and liver. Relative TNF-

α

gene expression in the kidney showed no differences between fast and slow speed induction. In contrast, the relative IL-6 gene expression increased significantly due to slow induction compared to fast induction after 0.5 h and 4 hrs (p = 0.0348 and p = 0.0270, Figure 5A). Hepatic TNF-

α

gene expression increased significantly due to slow induction compared to fast induction after 4 hrs of BD (p = 0.0293). No difference was found in the relative gene expression of IL-6 between fast and slow induction (Figure 5B). PMN infiltration in renal and hepatic tissue was assessed by His-48 staining. There was no difference in His-48 positive staining in the renal cortex and hepatic tissue between fast and slow induction (Figure 6).

Figure 5. Relative expression of inflammatory genes in renal and hepatic tissue in fast and slow induction BD rats after 0.5, 1, 2, and 4 h BD. The fold induction represents the relative expressions of these genes as compared to the expression level of the household GAPDH gene. * indicates p < 0.05.

In order to study apoptotic pathways in renal and hepatic tissue, we measured the ratio between the relative Bax and Bcl-2 expression. No difference was found in the expression of this ratio in renal tissue between fast and slow induction. In contrast, the hepatic gene expression of the Bax/Bcl-2 ratio was significantly higher due to slow induction compared to fast induction after 4 hrs of BD (p = 0.0293, Figure 7). Additionally, hepatic cleaved caspase-3 protein expression was significantly increased due to slow induction compared to fast induction after 4 hrs of BD (p = 0.001, Figure 8).

Figure 6. Polymorphonuclear (PMN) influx in renal and hepatic tissue after 4 h of BD. Kidneys after A) fast induction and B) slow induction. Livers after C) fast induction and D) slow induction.

Oxidative stress was assessed by measuring lipid peroxidation. MDA levels were significantly higher in renal tissue due to slow induction compared to fast induction after 4 hrs of BD (p = 0.01). Hepatic MDA levels were comparable between fast and slow induction groups after 4 hrs of BD (p = 0.48. Figure 9).

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33

DISCUSSION

The speed at which brain insults progress to BD varies greatly in ICU patients. Even donors with the same nature of brain insults progress to BD at different speeds. After infarction of the middle cerebral artery, BD can typically manifest itself anywhere between 24 hours and a week18_{. In a prospective study of patients with subarachnoid haemorrhage who}

progressed to BD, 26% were still not declared BD after one week19_{. This large range in}

time intervals is the result of the complex pathophysiology of the processes leading to BD and reflective of the speed at which ICP increases20_{. As of yet, the speed at which ICP}

increases, has not been investigated as a possible determinant of renal and hepatic graft survival.

Here we report for the first time that the speed of BD induction affects functional, immunologic, apoptotic, and oxidative stress markers in the kidney and liver. In our experimental setting, we found that a slower speed of BD induction, elicits more detrimental renal and hepatic effects compared to a faster speed of BD induction. The effect of slower speed of BD induction is especially apparent in the kidney as renal function is diminished which was measured by serum creatinine values.

Figure 8. Cleaved-caspase 3 expression in renal and hepatic tissue after 4 h of BD. Kidneys after A) fast induction and B) slow induction. Livers after C) fast induction and D) slow induction. * indicates p < 0.01

We showed that faster BD induction leads to more hemodynamic instability in the first hour after BD induction and therefore higher amounts of noradrenaline and HAES were required during this time period to maintain MAP within the physiological range. This is probably related to the higher peak of plasma catecholamine levels caused by fast BD induction as was shown by Shivalkar et al14_{. Higher levels of catecholamines lead to}

increased myocardial load and injury. Myocardial injury causes a subsequent drop in blood pressure and increases the need of hemodynamic support13_{. The negative effect of fast}

speed induction on hemodynamic stability appears to fade over time as the administered amount of HAES and NA did not differ between fast and slow induction at 2 and 4 hours of BD.

Slow BD induction leads to approximately 10 minutes of severe hypotension in rats as was observed here and in other studies24,37_{. While cerebral insults are commonly associated}

with hypertensive periods, there are a number of reports that associate cerebral insults with hypotensive periods in almost 50% of cases21,22_{and there is a particularly high risk}

for iatrogenic induced hypotension23,24_{. Even a few minutes of hypotension has been}

associated with an increased risk for acute kidney injury (AKI)38_{possibly aggravated by}

a dysfunction of the renal blood flow autoregulation39_{. The onset of AKI is reflected by}

decreased kidney function and increased systemic IL-6 release40-42_{, as observed here.}

The extent of IL-6 release can predict mortality in patients and determine the degree of kidney injury43,44_{. However, systemic IL-6 levels described in this study became comparable}

between the two BD models at 2 and 4 hrs after BD induction. Therefore, systemic IL-6 levels do not reflect the different local inflammatory responses we observed in our model40,45_{. Possibly, the combination of AKI and the second hit by BD leads to increased}

renal IL-6 production after 4 hours of BD46_{which is supported by the increased levels of}

renal IL-6 gene expression in our model. However, no concurrent increase was observed in PMN infiltration. In non-brain dead animals, others have described an infiltration at 6 hours after AKI onset47_{. However, BD also leads to induction of proinflammatory gene expression}

and infiltration of immune cells48_{and therefore, possibly no difference was found in PMN}

influx. In AKI, the infiltration correlates with MDA levels in the kidney, mediated by IL-644_{. In our model, the increased MDA levels in slowly-induced brain-dead rats did not}

coincide with an increase in PMN influx. This indicates that processes other than PMN influx affect MDA levels. It was previously shown that renal reactive oxygen species (ROS) start increasing from 2 hours of BD despite hemodynamically stable rats25_{. Therefore,}

the increase in ROS and lipid peroxidation is likely related to local changes occuring in the kidney. In AKI, oxidative processes mediate peritubular microcirculatory changes which lead to diminished renal perfusion and function35,36_{. In our model, increased lipid}

peroxidation could result from the second hit and explain the diminished renal function during the later stages of BD. No difference was observed in hepatic lipid peroxidation between fast and slow BD induction which is in line with AKI as hepatic MDA levels are the result of neutrophil granulocyte infiltration48,52_{for which we found no difference between}

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CHAPTER 2 BRAIN DEATH INDUCTION SPEEDS AND ABDOMINAL EFFECTS

Figure 9. MDA levels in renal and hepatic tissue from fast- and slow-inducted rats after 4 h of BD. * indicates p < 0.05

Plasma ALT and AST levels are known to increase in brain-dead rats6,24_{. In our experiment}

ALT levels, reflective of liver cell injury, showed no differences between both induction methods. However, AST levels were higher due to fast induction compared to slow induction after 0.5 and 2 h BD. We believe this not to be a reflection of liver damage due to no concurrent rise in ALT. Moreover, since AST is found in many tissues including the heart and lung, the early timepoints after which AST is increased, imply a causative role of the catecholamine storm and could be a reflection of lung and/or heart damage since these organs are affected by high levels of circulating catecholamines.

Hepatic IL-6 gene expression levels did not differ between models and neither was there a difference in PMN influx. However, hepatic TNF-

α

gene expression was significantly increased due to slow induction compared to fast induction after 4 hours of BD. Besides slow-induction, AKI can be responsible for this finding as it can lead to distant organ injury and increase hepatic TNF-

α

gene expression and apoptosis49,50_{. In our model, slow}

induction led to increased TNF-

α

gene and caspase-3 expression even though this group received isoflurane half an hour longer which has been shown to limit distant organ injury- induced liver apoptosis [18, 19]. TNF-

α

is a known inducer of extrinsic apoptosis and therefore signaling through death receptor mediated pathways is plausible in our model29_.

Since TNF-

α

has a major implication in hepatic ischemia-reperfusion injury, evaluating hepatic TNF-

α

levels in human donors that progress to BD at different speeds could reveal differences30-32_{. Hepatic mRNA expression of the Bax/Bcl-2 ratio was also significantly}

increased due to slow induction compared to fast induction which also suggests a possible role of intrinsic apoptosis through the permeabilization of mitochondria. The causal relationship of these processes and how they are initiated remains unclear and therefore, future investigations should focus on them in more depth. However, a possible cause could be the deposition of complement which has been shown to occur in livers of brain-dead rats and which is a known inducer of apoptosis24_{. Renal mRNA expression}

of the Bax/Bcl-2 ratio was not different between fast and slow BD induction. Moreover, there was no renal expression of caspase-3 after both fast and slow induction. This could indicate that the renal insults caused by BD are not severe enough to initiate programmed cell death or that other forms of cell death are initiated which we did not study7,21,22_.

In conclusion, the presented data provide an initial broad overview of changes elicited by the speed of BD induction. We found that a slower speed of BD induction leads to more detrimental effects in the kidney and liver. This could indicate that speed of BD induction should be taken into account when decisions about organ allocation are made. The effects of speed of BD induction are more pronounced in the kidney as renal function is diminished more due to a slower speed. Nevertheless, hepatic inflammatory and apoptotic markers are increased more due to slow induction. We believe that increased knowledge about the processes leading up to BD can be of valuable use for brain-dead donor management and thereby improve transplantation outcomes.

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37

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3

CHAPTER

Quality of donor lung grafts:

a comparative study between

explosive and gradual brain

death induction models in rats

CMV Hottenrott RA Rebolledo D Hoeksma J Bubberman J Burgerhof A Breedijk B Yard M Erasmus HGD Leuvenink In preparation

University of Groningen Brain death and organ donation Hoeksma, Dane