Energetic recovery in porcine grafts by minimally invasive liver oxygenation

j o u r n a l o f s u r g i c a l r e s e a r c h 1 7 8 ( 2 0 1 2 ) e 5 9 ee 6 3

Available online at www.sciencedirect.com

journal homepage: www.JournalofSurgicalResearch.com

Energetic recovery in porcine grafts by minimally invasive liver oxygenation Thomas Minor, MD,a,* William E. Scott III, MD,b Michael D. Rizzari, MD,b Thomas M. Suszynski, MD,b Bastian Luer,a Patrik Efferz ,a Klearchos K. Papas, PhD,b and Andreas Paul, MDc a

Surgical Research Division, University of Bonn, 53127 Bonn, Germany Department of Surgery, University of Minnesota, Minneapolis, Minnesota c Department for General, Visceral, and Transplantation Surgery, University Hospital of Essen, Essen, Germany b

article info

abstract

Article history:

Background: Gaseous insufflation of oxygen via the venous vascular system has proven to be

Received 3 November 2011

an effective tool for preventing anoxic tissue injury after extended time periods of ischemic

Received in revised form

liver preservation. Most experimental studies so far have been undertaken in rat models and

30 November 2011

include a series of pinpricks into postsinusoidal venules as an outlet for the insufflated gas.

Accepted 5 January 2012

Here, we describe a simplified technique for minimally invasive liver oxygenation in porcine

Available online 10 March 2012

grafts, representing a hassle-free access to organ oxygenation without vascular lesions. Methods: We retrieved livers from Landrace pigs and cold-stored them in histidine-tryptophan-

Keywords:

ketoglutarate solution. Subsequent to 18 h preservation, we treated some livers for an addi-

Oxygen

tional 2 h with gaseous oxygen, insufflated via silicone tubing inserted into the suprahepatic

Persufflation

caval vein. Gas pressure was limited to 18 mm Hg. We occluded the infrahepatic caval vein with

Reconditioning

a bulldog clamp. Gas bubbles left the graft via the portal vein. We assessed liver integrity by

Transplantation

energetic tissue status and by controlled in vitro reperfusion with autologous blood.

Preservation

Results: Magnetic resonance imaging demonstrated homogeneous gas distribution in the persufflated tissue without major shunting. Biochemical analyses revealed effective and homogeneous restoration of energetic homeostasis in the ischemic graft before reperfusion. Sinusoidal endothelial clearance of hyaluronic acid was significantly improved upon reperfusion, as was hepatic arterial flow. Parenchymal enzyme loss was concordantly mitigated after minimally invasive liver oxygenation. Conclusions: Our results indicate that gaseous oxygen persufflation of the porcine liver is possible without tissue trauma, and significantly enhances post-preservation recovery of the graft. ª 2012 Elsevier Inc. All rights reserved.

1.

Introduction

Oxygen persufflation (PSF) during cold preservation has been demonstrated to be an effective procedure to reduce ischemic hepatic tissue injury, and to improve recovery after liver

transplantation. Historically, the most widely applied version of the technique has involved submersing the liver in hypothermic preservation fluid and insufflating gaseous oxygen via the hepatic or caval veinsdwith small pinpricks placed into dilated venules at the margin of each hepatic lobe to allow

* Corresponding author. University of Bonn, Surgical Research Division, 53127 Bonn, Germany. Tel.: þ49 228 287 15133; fax: þ49 228 287 15199. E-mail address: [email protected] (T. Minor). 0022-4804/$ e see front matter ª 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.jss.2012.01.018

e60

j o u r n a l o f s u r g i c a l r e s e a r c h 1 7 8 ( 2 0 1 2 ) e 5 9 ee 6 3

for evacuation of the gas [1,2]. This technique has exhibited success in the treatment of livers exposed to extended warm and cold ischemia times, including livers procured from DCD animal [3,4] and human [5] donors. The main benefit of PSF has been reestablishment of cellular energy homeostasis under conditions of relatively low metabolic workload before warm reperfusion [6]. Moreover, PSF has exhibited an ability to reverse ischemia-induced breakdown of the cellular autophagy machinery, thus restoring the regenerative capacity for the cell to remove damaged cellular constituents (such as denatured proteins) upon reperfusion. A possible drawback to applying PSF in human-size livers is that the process of introducing pinpricks has proven to be cumbersome and time-consuming. Furthermore, although past experiences have indicated no obvious complications from the introduction of capsular pinpricks, it is conceivable that clinicians might be apprehensive about bleeding from the sites of pinpricks. Here, we describe a simplified technique for minimally invasive liver oxygenation that involves no pinpricks, thereby circumventing the putative risk of hemorrhaging upon reperfusion.

2.

Methods

We retrieved livers from German Landrace pigs (25e30 kg), flushed them in histidine-tryptophan-ketoglutarate (HTK) preservation solution, and stored them in the solution at 4 C for 18 h. We halted preservation in one cohort (group A; n ¼ 5); in the other cohort (group B; n ¼ 5), we subsequently connected livers to a supply of medical-grade pure oxygen gas for an additional 2 h. We performed minimally invasive liver oxygenation (MILO) via silicone tubing inserted into the suprahepatic caval vein, with the liver still submerged in cold HTK solution. The infrahepatic caval vein was temporarily clamped with an atraumatic Bulldog clamp. Oxygen gas was delivered through the caval line into the liver at a pressure limited to 18 mm Hg, to prevent barotrauma to the hepatovasculature. After a short time, we saw gas bubbles leaving the portal vein (Fig. 1A). We attempted to visualize gaseous pathways within the liver using magnetic resonance imaging (MRI) during MILO. We performed MRI using a 3.0 T magnet (MAGNETOM Trio; Siemens USA, Malvern, PA), using a total imaging matrix (TIM, a surface coil array) (Siemens USA) tuned to proton (127.728 MHz). We used a T1-weighted turbo-spin echo sequence to acquire images. We assessed the homogeneity of PSF by observing the presence of gas in the vasculature by the contrast it provided during MRI (Fig. 1B). At the end of the cold storage period (18 h in group A and 20 h in group B), liver tissue was flash frozen between precooled steel tongues and used for later analysis of hepatic energy status, as detailed elsewhere [3]. We investigated activation of the adenosine monophosphateeactivated protein kinase (AMPK) as a cellular response to tissue hypoxia or other cellular and environmental stress [7] using immunoblotting of the phosphorylated AMPKa after liver preservation. We prepared whole tissue lysates from frozen tissue obtained after reperfusion, separated them by gel electrophoresis, blotted them onto nitro-cellulose membrane, and visualized

them on x-ray film, as detailed previously [8]. Subsequently, we stripped the blots and probed them with monoclonal antiactin antibody to confirm protein loading. We quantified protein content based on the ratios of individual signal and actin, determined densitometrically with U N-SCAN-IT gel v 6.1 (Silk Scientific Corporation, Orem, UT). We used the antibodies anti-phospho-AMPK (Cell Signaling/New England Biolabs, Frankfurt, Germany) and antiactin (AB-1; Calbiochem, Darmstadt, Germany). In five additional experiments, we evaluated postpreservation recovery of the livers upon warm reperfusion in vitro for 2 h using autologous donor blood diluted with saline to a final hematocrit of 20. Details of the model are given elsewhere [9]. In brief, we pumped oxygenated blood through the portal vein at a constant flow of 1 min1 kg1, while we perfused the hepatic artery at the constant pressure of 80 mm Hg. We measured hepatic arterial flow using an ultrasonic flowmeter (HSE-Transsonic transit time flowmeter; Harvard Apparatus, March-Hugstetten, Germany) connected to an inline flow-sensor in the arterial circuit. We analyzed serum concentrations of the parenchymal lactate dehydrogenase (LDH) and mitochondrial glutamate LDH (GLDH) at the end of the experiment using commercialized standard kits (Fa. Roche, Mannheim, Germany). We approximated the integrity of sinusoidal endothelial cells by measuring serum levels of hyaluronic acid (HA) [10,11] and evaluated them spectrophotometrically using an enzymelinked immunosorbent assay test kit, according to the manufacturer’s instructions (Molecular Biotechnology, Goettingen, Germany). Values are expressed as means  standard error of the mean, unless otherwise indicated. After proving the assumption of normality and equal variance across groups, we tested differences between treated and untreated groups using the twotailed unpaired t-test. Statistical significance was set at P < .05.

3.

Results

Shortly after introducing gaseous oxygen into the caval vein, we saw gas bubbles leaving the portal vein (Fig. 1A). We assessed the intra-graft distribution of the gas using MRI, as shown in Fig. 1B. A homogeneous equilibration of the tissue was obtained throughout the liver vasculature with no major shunting of gas flow. We monitored the metabolic efficacy of liver oxygenation without pinpricks by measuring the energetic status at the end of cold storage and after subsequent treatment. During 18 h of cold storage (group A), the hepatic tissue was thoroughly depleted of ATP: concentrations fell below 0.5 mmol/g dry weight. An additional 2 h of MILO restored hepatic tissue ATP, resulting in ATP concentrations approximately 10 times higher for group B compared with group A (Fig. 2). We observed overall activation of the AMPK signaling pathway for group A, which indicated activation of this stress pathway. After 2 h of MILO (group B), a we observed significant reduction in the amount of phosphorylated AMPK, which resulted in phosphorylated AMPK levels nearly comparable to healthy liver tissue (Fig. 2).

e61

j o u r n a l o f s u r g i c a l r e s e a r c h 1 7 8 ( 2 0 1 2 ) e 5 9 ee 6 3

Fig. 1 e Minimal invasive liver oxygenation. (A) Oxygen gas bubbles leaving the portal vein. (B) Magnetic resonance threedimensional reconstruction of the paths persufflated throughout the liver with no major shunting (white indicates the pathways through which gas traveled).

9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

HA concentrations to 414  96 ng/mL, we observed a significant reduction after MILO (group B) (199  97 ng/mL; P < .05 versus cold storage). Portal vascular resistance did not differ between groups. However, post-reperfusion hepatic arterial blood flow improved after MILO (Fig. 3).

4.

Discussion

Gaseous oxygen persufflation is an easy and powerful adjunct for improved preservation of donor organs. Oxygen is the first, and by far most important, substrate that is depleted during ischemia. However, it can be sufficiently provided during hypothermic preservation by supplying gaseous oxygen via the native vasculature [12]. Gaseous oxygen is required for oxidative phosphorylation, and is thus able to provide the 5.0 4.5 4.0

*

AMPK~p (rel. U)

ATP (µmol/g)

Improved recovery of hepatic metabolism upon warm reperfusion further substantiated the functional relevance of this energetic reconditioning. Minimally invasive liver oxygenation significantly improved synthesis of cholinesterase, from 53.3  16.3 U/L to 157.0  56.6 U/L (P < .05). At the same time, hepatic bile juice secretion increased from 1.55  0.35 mL g1 h1 in untreated livers to 2.92  0.5 mL g1 h1 in treated livers (P < .05). In line with a net improvement in functional recovery, MILO also significantly reduced hepatic enzyme leakage (i.e., serum levels of LDH from 1,586  333 U/L to 1,113  123 U/L, and GLDH from 152  40 U/L to 76  3 U/L (group A versus group B, respectively). We investigated the impact on sinusoidal endothelial cell function of persufflation without pinpricks by measuring serum levels of HA upon reperfusion. Whereas static cold storage alone (group A) resulted in an elevation of

3.5 3.0 2.5 2.0

*

1.5 1.0 0.5

CS

+MILO

0.0

CS

+MILO

Fig. 2 e Energetic effect of MILO. Tissue concentrations of ATP (left) and cellular signal activation of the AMPK pathway (right) after 18 h of cold storage (CS) and after an additional 2 h of aerobic reconditioning by MILO. Values are mean ± standard error of the mean of five experiments per group (*P < .05 versus CS). The gray area represents the normal range for the respective parameters, obtained from nonischemic liver tissue.

e62

j o u r n a l o f s u r g i c a l r e s e a r c h 1 7 8 ( 2 0 1 2 ) e 5 9 ee 6 3

500

CS +MILO

HAF (ml/min)

400

* 300

200

100

0

0

15

30

45

60

75

90

105

120

min of reperfusion Fig. 3 e Hepatic arterial flow after 18 h of cold storage without (CS) or with an additional 2 h of MILO treatment. Values are mean ± standard error of the mean of five experiments per group (*P < .05 versus CS).

required substrate for resynthesis of ATP and it permits maintenance of cellular volume and morphology [13]. Since its first description for liver preservation in rats [14], oxygen persufflation has been performed via the hepatic veins in combination with pinpricks into postsinusoidal venulesd which allowed gas to escape the organ without passing through the sinusoidal capillary beds [15,16]. This was based on original findings by Isselhard in canine kidneys, having advocated for reduced driving gas pressures to spare the capillary/glomerulary network from barotrauma [16,17]. Indeed, early studies on liver persufflation in rats have shown that the gas bubbles were not able to cross the liver sinusoids at pressures that prevented the formation of lesions to parts of the liver tissue [18]. Traditionally, when persufflating larger organs, we have visualized gas bubbles exiting at both the portal vein and the pinpricks, which suggests that the pinpricks may not be necessary. Therefore, the current study attempted to determine whether eliminating pinpricks from the protocol would allow for sufficient tissue oxygenation without adversely affecting sinusoidal vascular integrity. All of the livers were readily persufflated without trauma to the dilated venules, and we observed gas exiting the portal vein within several minutes of treatment and then continuously thereafter. However, one to three additional pinpricks might help exclude fluid from the vasculature and accelerate portal gas exit. The metabolic efficiency of gas persufflation without pinpricks could be documented by the timely restoration of tissue ATP, the extent of which was comparable to the conventional method using pinpricks [9]. In addition, activation of cellular signaling pathways triggered by hypoxic exposure was significantly reduced after MILO, as we judged by measuring AMPK-phosphorylation in the liver tissue.

As an interesting side effect, we observed a dramatic reduction of the required gas flow to less than 300 mL/min compared with persufflation with pinpricks, because less oxygen gas has to be introduced into the graft to compensate for pressure drops owing to venous leaks. It was important to provide evidence that oxygen passing through the sinusoids did not adversely affect vascular patency upon orthograde reperfusion or produce gas embolism in vivo. To that purpose, We carried out three porcine transplantations out after MILO and flush-out of the liver vasculature via the portal vein with 1 L saline. We observed no adverse effects of the treatment during a 1-wk postoperative period (data not shown). Moreover, in vitro reperfusion of livers after MILO revealed no signs of vascular endothelial stress or portal flow impediments, but unveiled a significant improvement in hepatic arterial flow compared with the static cold storage controls. Early recovery of normal flow through the hepatic artery has been postulated to be an independent and powerful prognostic factor for graft viability in clinical liver transplantation [19]. Likewise, hepatic parenchymal enzyme release and synthetic function improved in our study. In conclusion, the present data indicate that MILO is an effective and timesparing alternative for aerobic reconditioning of liver grafts during hypothermic ischemia.

Acknowledgment This work was supported in part by a research grant given to T. Minor by the German Research Foundation (DFG Mi470/ 14-1).

references

[1] Minor T, Klauke H, Vollmar B, et al. Biophysical aspects of liver aeration by vascular persufflation with gaseous oxygen. Transplantation 1997;63:1843. [2] Minor T, Koetting M, Koetting M, et al. Hypothermic reconditioning by gaseous oxygen improves survival after liver transplantation in the pig. Am J Transplant 2011;11: 2627. [3] Stegemann J, Minor T. Energy charge restoration, mitochondrial protection and reversal of preservation induced liver injury by hypothermic oxygenation prior to reperfusion. Cryobiology 2009;58:331. [4] Minor T, Saad S, Nagelschmidt M, et al. Successful transplantation of porcine livers after warm ischemic insult in situ and cold preservation including postconditioning with gaseous oxygen. Transplantation 1998;65:1262. [5] Treckmann J, Minor T, Saad S, et al. Retrograde oxygen persufflation preservation of human livers: A pilot study. Liver Transplant 2008;14:358. [6] Minor T, Stegemann J, Hirner A, et al. Impaired autophagic clearance after cold preservation of fatty livers correlates with tissue necrosis upon reperfusion and is reversed by hypothermic reconditioning. Liver Transplant 2009;15:798. [7] Hardie DG, Carling D. The AMP-activated protein kinasedFuel gauge of the mammalian cell? Eur J Biochem 1997;246:259. [8] Minor T, Koetting M. Gaseous oxygen for hypothermic preservation of predamaged liver grafts: Fuel to cellular

j o u r n a l o f s u r g i c a l r e s e a r c h 1 7 8 ( 2 0 1 2 ) e 5 9 ee 6 3

[9]

[10]

[11]

[12]

[13]

homeostasis or radical tissue alteration? Cryobiology 2000; 40:182. Koetting M, Lu¨er B, Efferz P, et al. Optimal time for hypothermic reconditioning of liver grafts by venous systemic oxygen persufflation (VSOP) in a large animal model. Transplantation 2011;91:42. Xu H, Lee CY, Clemens MG, et al. Pronlonged hypothermic machine perfusion preserves hepatocellular function but potentiates endothelial cell dysfunction in rat livers. Transplantation 2004;77:1676. Yachida S, Wakabayashi H, Okano K, et al. Prediction of posthepatectomy hepatic functional reserve by serum hyaluronate. Br J Surg 2009;96:501. Minor T, Isselhard W. Synthesis of high energy phosphates during cold ischemic rat liver preservation with gaseous oxygen insufflation. Transplantation 1996;61:20. Pegg DE, Foreman J, Hunt CJ, et al. The mechanism of action of retrograde oxygen persufflation in renal preservation. Transplantation 1989;48:210.

e63

[14] Fischer JH, Fuchs M, Miyata M, et al. Hypothermic liver preservation using different flush solutions and retrograde oxygen persufflation technique. Eur Surg Res 1980;12:19 (abstract). [15] Minor T, Klauke H, Vollmar B, et al. Rat liver transplantation after long-term preservation by venous systemic oxygen persufflation. Transplant Proc 1997;29:410. [16] Isselhard W, Minor T. Gasfo¨rmiger Sauerstoff zur Protektion und Konditionierung von Organen in Ischa¨mie. Zentralbl Chir 1999;124:252. [17] Isselhard W, Berger M, Denecke H, et al. Metabolism of canine kidneys in anaerobic ischemia and in aerobic ischemia by persufflation with gaseous oxygen. Pflu¨gers Arch 1972;337:87. [18] Vollmar B, Klauke H, Minor T, et al. Gaseous pathway in venous oxygen persufflation of the liver. J Hepatol 1997;26:1429. [19] Lisik W, Gontarczyk G, Kosieradzki M, et al. Intraoperative blood flow measurements in organ allografts can predict postoperative function. Transplant Proc 2007;39:371.