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Keywords:

  • Ischemia-reperfusion injury;
  • liver regeneration;
  • liver transplantation;
  • nitric oxide;
  • organ preservation;
  • oxygen;
  • VSOP

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

The prognosis for recipients of small liver grafts is poor. The aim of this study was to determine the impact of venous systemic oxygen persufflation (VSOP) with nitric oxide (NO) gas for 30% partial liver preservation and transplantation in rats. After we determined optimal NO concentration as 40 ppm in vitro with the isolated perfused rat liver model, we assessed liver injury and regeneration in vivo at 1, 3, 24 and 168 h after transplantation in the following three groups after 3 h-cold storage (n = 20 per group): control group = static storage; VSOP group = oxygen persufflation and VSOP+NO group = oxygen with NO persufflation. The liver graft persufflation was achieved with medical gas via the suprahepatic vena cava; In comparison with control group after transplantation, VSOP+NO preservation (1) increased portal circulation, (2) reduced AST and ALT release, (3) upregulated hepatic endothelial NO synthase, (4) reduced hepatocyte and bileductule damage and (5) improved liver regeneration. These results suggest that gaseous oxygen with NO persufflation is a novel and safe preservation method for small partial liver grafts, not only alleviating graft injury but also improve liver regeneration after transplantation.


Abbreviations
ALT,

alanine aminotransferase

AST,

aspartate aminotransferase

ATP,

adenosine triphosphate

cDNA,

complementary DNA

e-NOS,

endothelial NO synthase

GAPDH,

Glyceralehyde-3-phosphate dehydrogenase

HTK,

histidine-tryptophan-ketoglutarate

i-NOS,

inducible NO synthase

IPRL,

isolated perfused rat liver

IRI,

ischemia reperfusion injury

LDH,

lactate dehydrogenase

LDLT,

living-donor liver transplantation

mRNA,

messenger RNA

NO,

nitric oxide

PVF,

portal venous flow

PVP,

portal venous pressure

RT-PCR,

reverse-transcription polymerase chain reaction

SEM,

standard error of the mean

SFSG,

small-for-size grafts

SFSS,

small-for-size syndrome

SHVC,

suprahepatic vena cava

TNF-α,

tumor necrosis factor alpha

VSOP,

venous systemic oxygen persufflation.

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Because of the severe shortage of organ donors, demand for living-donor liver transplantation (LDLT) has been increasing worldwide. Recently, some transplant surgeons have used smaller grafts in LDLT to reduce the risk for donors [1].

Venous systemic oxygen persufflation (VSOP) during the cold storage has been reported as an effective method for graft preservation, especially for marginal grafts [2, 3]. In contrast, the application of nitric oxide (NO) has shown promising results in protecting graft viability [4]. Recently, we demonstrated [5] that VSOP with NO gas reduced ischemia-reperfusion injury (IRI) and improved graft viability when compared with in vitro VSOP of warm ischemically damaged liver grafts.

The aim of this study was to evaluate the effects of VSOP in combination with NO gas for liver transplantation using small partial grafts in rats.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Experimental protocols

All experiments were conducted in accordance with the German federal law regarding the protection of animals. The principles of laboratory animal care (NIH Publication No. 85–23, revised 1985) were followed. Male Lewis rats weighing 230–270 g were used as donors and recipients. The animals were housed under specific pathogen-free conditions according to the FELASA guidelines.

Experimental groups and design

Experiment I. Determination of optimal NO concentration with the isolated perfused rat liver (IPRL) model

Rat livers were retrieved after 30 min of warm ischemia induced by cardiac arrest and were thereafter preserved for 24 h by cold storage in histidine tryptophan ketoglutarate (HTK) solution (Custodiol; Dr. Franz Köhler Chemie GmbH, Germany). During cold storage, gaseous oxygen was insufflated supplemented with NO at 400, 80, 40, 20, 10 parts per million (ppm; n = 5 each). Cold static-stored livers without persufflation served as controls. Viability of all livers was evaluated in the isolated perfused rat liver (IPRL) system as previously described [5].

Experiment II. 30% partial liver transplantation

Orthotopic partial liver transplantations with approximately 30% graft volume were performed in 60 rats following cold storage in HTK for 3 h at 5°C as described in detail previously [6]. We studied the three experimental groups (n = 20 for each): (A) control group (n = 20), cold static storage; (B) VSOP group (n = 20), VSOP during cold storage and (C) VSOP+NO group (n = 20), VSOP supplemented with 40 ppm-NO gas (optimal NO concentration from results of Experiment I) during cold storage. We excluded cases in which there was any technical failure; anhepatic time was more than 20 min (one in control); blood loss during operation was more than 2 mL (one in VSOP). At 1, 3, 24 and 168 h after portal reperfusion (n = 5 for each), we measured the microcirculation of the graft and portal circulation. Prior to euthanasia, we collected the systemic venous blood and samples of liver graft tissue.

In situ liver perfusion with 20 mL of HTK at a hydrostatic pressure of 20 cm H2O was achieved through the portal vein using a 16-gauge catheter (Vasofix 16G; B. Braun, Germany). The liver graft was stored for 3 h in 125 mL of HTK at 3–4°C.

Gaseous persufflation

In the VSOP (O2 0.2 L/min) and VSOP+NO group (O2 0.2 L/min, NO 22 mL/min; 40 ppm-NO) persufflation of the liver was achieved with medical-grade gaseous oxygen via the suprahepatic vena cava (SHVC) at a pressure limited to 18 mmHg. The HTK, in which the liver was immerged, was supplemented with 20 mM N-acetylcysteine (NAC, Hexal AG, Germany). At the margin of each liver lobe, 2–3 small pinpricks were made into the dilated postsinusoidal venules using a fine acupuncture needle (0.18 × 30 mm, Seirin Corporation, Japan), which allowed the gas to escape from the liver microvasculature.

Evaluation of hepatic microcirculation and portal circulation (Experiment II)

We evaluated hepatic microcirculation and portal circulation at the time of euthanasia before collecting blood or tissue samples as described previously [6]. As a reference (baseline), we measured hepatic microcirculation and portal circulation immediately after laparotomy.

Aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase enzyme release

Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) were measured by a standard enzymatic method.

Lipid peroxidation (Experiment I)

In order to assess the impact of oxygen free radicals after reperfusion, we measured malondialdehyde (MDA) concentrations as described in detail previously [6].

Tumor necrosis factor ELISA assays (Experiment II)

Tumor necrosis factor (TNF-α) concentrations were measured to in both systemic venous blood using rat ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

TNF-α, endothelial NO synthase and inducible NO synthase messenger RNA (mRNA) detection by reverse-transcription polymerase chain reaction (Experiment II)

For reverse-transcription polymerase chain reaction (RT–PCR) analyses, RNA was isolated from liver graft tissue samples using TRI reagent. 200 ng of RNA ware used for complementary DNA (cDNA) synthesis using reverse transcriptase (Applied Biosystems, Germany). The following primers and probes were selected from the Applied Biosystems Assays-on-demand gene expression products: Glyceralehyde-3-phosphate dehydrogenase (GAPDH) (Rn_01775763_gl), endothelial NO synthase (e-NOS) (Rn_02132634_s1), inducible NO synthase (i-NOS) (Rn_00561646_m1) and TNF-α (Rn_99999017_m1).

Ki-67 staining (Experiment II)

Tissue sections were subjected to immunohistochemical testing using monoclonal mouse Ki-67 (MIB5, 1:10; DACO, Denmark) for assessing cell proliferation. The Ki-67 labeling index represented the percentage of hepatocytes with Ki-67-positive nuclei relative to the total number of hepatocytes in randomly selected sections (under ×400 magnification, three fields in each rat).

Electron microscopy (Experiment II)

Tissue samples were examined under electron microscopy (EM 400 T/ST, Philips, Amsterdam, the Netherlands) as described previously [5].

Statistical analysis

Results are expressed as the mean ± standard error of the mean (SEM) for each group. The comparison among groups were performed using Kruskal–Wallis test and Bonferroni's post hoc correction. A p-value < 0.05 was considered statistically significant. Calculations were made using Prism 5 computer software (GraphPad Software Inc. San Diego, CA, USA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

(1) Experiment I: optimal NO concentration with the IPRL system

After 45 min of IPRL, AST (p = 0.0078; posttest: 40 ppm vs. 10 ppm: p < 0.01), ALT (p = 0.0125; posttest: cold static storage vs. 40 ppm: p < 0.01), PVP (p = 0.0164; posttest: cold static storage vs. 40 ppm: p < 0.01) and MDA (p = 0.0016; posttest: cold static storage vs. 40 ppm: p < 0.01) in hepatic tissue was the lowest in 40 ppm-NO. Accordingly, we determined 40 ppm-NO is optimal concentration of NO for gaseous persufflation during cold storage.

(2) Experiment II: 30% partial liver transplantation

All rats survived until sacrifice. There was no difference among the three groups for liver graft weight, the ratio between liver graft weight to recipient's whole liver weight, the cold ischemic time or anhepatic time.

Portal venous circulation and graft microcirculation

Figure 1 shows the serial changes over time in portal venous circulation; PVP (Figure 1A), PVF (Figure 1B), PVF/PVP (Compliance) (Figure 1C) and liver graft microcirculation (Figure 1D). Due to severe adhesion in the hepatic hilum, we could not measure the PVF at 168 h after reperfusion. The PVP in VSOP+NO group was the lowest at 24 h (p = 0.0124; posttest: control vs. VSOP+NO, control vs. VSOP: p < 0.05). PVF increased gradually until 24 h after reperfusion and was the highest in the VSOP+NO group at 1 and 3 h (1 h: p = 0.0053, 3 h: p = 0.0442). In addition, PVF/PVP (compliance) in the VSOP+NO group was the highest at 1 and 3 h (1 h: p = 0.0104, 3 h: p = 0.0070). The liver graft microcirculation measured by laser Doppler in the VSOP+NO group was the highest at 1, 3 and 168 h after reperfusion (1 h: p = 0.0065, 3 h: p = 0.0059, 168 h: p = 0.0092).

image

Figure 1. Portal venous circulation and liver graft microcirculation. (A) Time course of portal venous pressure (PVP). (B) Time course of portal venous flow (PVF). (C) Time course of compliance (PVF/PVP). (D) Time course of mean value of hepatic microcirculation measured by laser-Doppler flowmetry in each lobe. The difference among the groups by Kruskal–Wallis test: Bonferroni's posttest: *p < 0.05, **p < 0.01 (mean ± SEM, n = 5 each). Dotted line = baseline values after laparotomy.

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AST, ALT and LDH enzyme release

The level of AST (Figure 2A) was the lowest in the VSOP+NO group at 1 and 168 h (1 h: p = 0.0128, 168 h: p = 0.0263). The ALT level (Figure 2B) was the lowest in the VSOP+NO group at 1 and 3 h (1 h: p = 0.0021; posttest: control vs. VSOP+NO: p < 0.05, 3 h: p = 0.0185; posttest: control vs. VSOP+NO: p < 0.05). The LDH level (Figure 2C) was also the lowest in the VSOP+NO group at 1, 24 and 168 h (1h: p = 0.0052; posttest: control vs. VSOP+NO: p < 0.01, 24 h: p = 0.0098; posttest: control vs. VSOP+NO: p < 0.01, 168 h: p = 0.0207; posttest: control vs. VSOP+NO: p < 0.05).

image

Figure 2. (A) Time course of aspartate aminotransferase (AST), (B) alanine aminotransferase (ALT) and (C) lactate dehydrogenase (LDH). The difference among the groups by Kruskal–Wallis test: Bonferroni's posttest: *p < 0.05, **p < 0.01 (Mean ± SEM, n = 5 each).

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TNF-α protein in hepatic tissue

TNF-α protein level in hepatic tissue by ELISA has a tendency to be highest in VSOP group and lowest in VSOP+NO group at 3 h (p = 0.1931).

TNF-α, e- NOS and i-NOS mRNA expression in hepatic tissue

TNF-α relative mRNA expression of the liver graft at 1 h after reperfusion (Figure 3A) was the lowest in the VSOP+NO group (p < 0.0001, Posttest; VSOP vs. VSOP+NO: p < 0.001, control vs. VSOP+NO: p < 0.001). Relative e-NOS mRNA expression (Figure 3B) of the liver graft at 1 h after reperfusion was the highest in the VSOP+NO group (p = 0.0011, posttest; VSOP vs. VSOP+NO: p < 0.01, control vs. VSOP+NO: p < 0.01). In contrast, i-NOS relative mRNA (Figure 3C) of the liver graft at 1 h after reperfusion was the lowest in the VSOP+NO group (p = 0.0040, posttest; VSOP vs. VSOP+NO: p < 0.01, control vs. VSOP: p < 0.05).

image

Figure 3. (A) TNF-α, e-NOS (B) and i-NOS (C) relative mRNA expression of the liver graft at 1 h after reperfusion. The difference among the groups by Kruskal–Wallis test: Bonferroni's posttest: *p < 0.05, **p < 0.01.

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Electron microscopy

The ultrastructure of the liver graft parenchyma at 24 h after reperfusion in each group (Figure 4) was evaluated using electron microscopy. Regarding hepatocytes (Figure 4A, D, G), significant vacuolization was shown in hepatocytes in the control and VSOP groups; however, these changes were almost completely abrogated in the VSOP+NO group. Regarding the bile ductule (Figure 4B, E, H), microvilli in the control group (Figure 4C) appeared to be shorter and dissociated; however, the VSOP (Figure 4F) and VSOP+NO (Figure 4) groups were longer and well preserved. Therefore, the VSOP+NO group maintained almost normal ultrastructural features at 24 h after reperfusion.

image

Figure 4. Ultrastructual analysis using electron microscopy. (A, B, C) control group. (D, E, F) VSOP group. (G, H, I) VSOP+NO group. Liver sections show hepatocytes (H), bile ductules (B) and microvilli (arrow).

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Liver regeneration

At 168 h, the liver weight to the recipient's whole liver weight ratio was the highest in VSOP+NO group (control vs. VSOP vs. VSOP+NO: 76 ± 3 vs.87 ± 6 vs. 96 ± 4%; p = 0.0263; posttest: control vs. VSOP+NO: p < 0.01). The Ki-67 labeling index at 24 h was also the highest in VSOP+NO group (4.3 ± 1.5 vs. 14.6 ± 1.8 vs. 41.0 ± 5.6%; p < 0.0001; posttest: control vs. VSOP+NO: p < 0.001, VSOP vs. VSOP+NO: p < 0.05).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Small partial liver grafts with a graft weight/standard liver volume ratios of less than 40% have been considered small-for-size grafts (SFSG), and functional impairments such as prolonged cholestasis, ascites and coagulopathy secondary to SFSG have been clinically described as small-for-size syndrome (SFSS; [7, 8]. In previous studies, a 20–40% partial liver graft without arterial reconstruction has been reported as SFSG in rats [9-11]. However, because SFSS depends not only on the graft weight but also on recipient status, anhepatic time and IRI, which was the primary aim of this study, the definition for SFSS in rats is still unclear [12]. Therefore, in this study we used the term “small partial liver graft” for a 30% partial graft.

Because the characteristic microscopic findings of SFSS, including hepatocyte ballooning and cholestasis, are thought to be due to microcirculatory disturbance [13], we consider that improvement of the graft microcirculation is a key to overcome the small partial liver grafts issue. In this study, the PVF and microcirculation of the graft and liver graft function were the highest in the VSOP+NO group, that suggests VSOP with 40 ppm-NO is effective to increase the liver graft microcirculation. Although the PVF increased at 24 h, microcirculation of the graft decreased at the same time point; we hypothesize that due to liver regeneration, tissue flow/volume (microcirculation) decreased in spite of elevation of the PVF at 24 h. Nevertheless we could successfully show significantly lower enzyme results with better graft microcirculation as well as better histological findings in VSOP+NO group with lower mean values and SEM than control group. We consider lower SEM in VSOP+NO group suggests stable graft preservation and perfusion compared to control group.

VSOP during cold storage has been reported to be an effective method for graft preservation especially for marginal grafts. In experimental studies [2, 3]; VSOP reduced apoptosis; and improved graft viability by increasing tissue adenosine triphosphate (ATP) concentration, vascular endothelial function and ultrastructure in several models. In a clinical pilot study for deceased-DLT, Treckmann et al. [14] reported that the VSOP during cold storage increased ATP more than twofold and, thus, improved early aerobic metabolism; therefore, the VSOP could potentially improve primary organ function after liver transplantation.

NO is a free-radical diatomic gas of low molecular weight with an unpaired electron, which is highly lipophilic, allowing it to permeate quickly across the cell membranes. Its half-life in vivo is a few seconds, and it is rapidly converted to stable nitrites and nitrates [15]. Thus, NO can act as a toxic or protective agent, depending on the surrounding conditions [16]. Murakami et al. [17] showed that 30 ppm-NO, administered immediately or in a delayed manner during reperfusion, decreases IRI in lung transplantation in rats. In our in vitro study (Experiment I), 40 ppm was the most effective concentration for reduction of IRI. Recently, we also reported the effectiveness of VSOP with NO gas (40 ppm; [5] for warm ischemically damaged graft in vitro, which showed less IRI and better graft viability by IPRL system. The present proof of principle study demonstrated the safety of VSOP supplemented 40 ppm-NO gas during cold storage in vivo because no animal treated with VSOP+NO died within 168 h after liver transplantation or showed any other adverse effects.

NO in combination with oxygen down-regulated TNF-α mRNA expression, which was upregulated by sole oxygen persuffulation. This is in line with results from Dong et al. [18] who reported that NO ventilation of rat lungs from cardiac death donors downregulated TNF-α mRNA, which was upregulated by sole oxygen ventilation. Because NO act as a free radical scavenger [5, 15, 18], we speculate that addition of NO gas might suppress TNF-α that was upregulated by oxygen persufflation (VSOP).

Small partial liver graft injuries are related to microcirculatory disorders due to an imbalance of vasoconstricting and vasorelaxing mediators such as endothelin-1/NO [11, 19]. Because e-NOS-derived NO prevents hepatic IRI [20], endogenous e-NOS activation and e-NOS-derived NO may be a promising approach to limiting organ injury. In our study, VSOP with NO gas during cold storage upregulated hepatic e-NOS expression, down-regulated hepatic i-NOS expression 1 h after reperfusion and protected graft microcirculation and viability, even through 168 h after reperfusion. Because e-NOS is induced in response to specific extracellular stimuli such as shear stress, we hypothesize that better microcirculation in the VSOP+NO group could upregulate e-NOS expression. In addition, exogenous NO, as well as activated e-NOS-derived NO, could also play an important role in the reduction of IRI and in the protection of graft viability in the VSOP+NO group for this study. Kuriyama et al. [11] demonstrated that the e-NOS-derived NO decreases hepatic levels of endothelin-1, improved hepatic microcirculation and significantly attenuated TNF-α hepatic expression and, remarkably, reduced the activation of caspase-8 and caspase-3 in a nonarterialized 20% partial liver transplantation in rats. In this study, we used NO only during the cold-ischemic time because we avoid possible side effects of NO in vivo.

We are aware of the mechanistic shortcomings in this study. However, we can conclude from the results that VSOP supplemented with NO represent a feasible combination of medical gases for increasing graft viability and possible to expand the donor pool.

In conclusion, VSOP supplemented with NO gas is a novel, easy and safe method for small partial liver transplantation, not only alleviating liver graft injury but also improving regeneration. For the first time, we demonstrated biological safety in vivo for the application of a gas mixture of NO and oxygen. Further experiments with larger animals as well as mechanistic investigation are necessary for the application of this novel organ preservation technique in clinical marginal grafts and small partial liver grafts.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

The authors thank Pascal Paschenda, Mareike Schulz and Martyna Wojcieszak for their skillful technical assistance.

Disclosure

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References