Dual versus single vessel normothermic ex vivo perfusion of rat liver grafts using metamizole for vasodilatation

Felix Claussen; Joseph M G V Gassner; Simon Moosburner; David Wyrwal; Maximilian Nösser; Peter Tang; Lara Wegener; Julian Pohl; Anja Reutzel-Selke; Ruza Arsenic; Johann Pratschke; Igor M Sauer; Nathanael Raschzok

doi:10.1371/journal.pone.0235635

. 2020 Jul 2;15(7):e0235635. doi: 10.1371/journal.pone.0235635

Dual versus single vessel normothermic ex vivo perfusion of rat liver grafts using metamizole for vasodilatation

Felix Claussen ¹, Joseph M G V Gassner ¹, Simon Moosburner ¹, David Wyrwal ¹, Maximilian Nösser ¹, Peter Tang ¹, Lara Wegener ¹, Julian Pohl ¹, Anja Reutzel-Selke ¹, Ruza Arsenic ², Johann Pratschke ¹, Igor M Sauer ^1,^*, Nathanael Raschzok ^1,³

Editor: Michael Bader⁴

PMCID: PMC7332079 PMID: 32614897

Abstract

Background

Normothermic ex vivo liver perfusion (NEVLP) is a promising strategy to increase the donor pool in liver transplantation. Small animal models are essential to further investigate questions regarding organ preservation and reconditioning by NEVLP. A dual vessel small animal NEVLP (dNEVLP) model was developed using metamizole as a vasodilator and compared to conventional portovenous single vessel NEVLP (sNEVLP).

Methods

Livers of male Wistar rats were perfused with erythrocyte-supplemented culture medium for six hours by either dNEVLP via hepatic artery and portal vein or portovenous sNEVLP. dNEVLP was performed either with or without metamizole treatment. Perfusion pressure and flow rates were constantly monitored. Transaminase levels were determined in the perfusate at the start and after three and six hours of perfusion. Bile secretion was monitored and bile LDH and GGT levels were measured hourly. Histopathological analysis was performed using liver and bile duct tissue samples after perfusion.

Results

Hepatic artery pressure was significantly lower in dNEVLP with metamizole administration. Compared to sNEVLP, dNEVLP with metamizole treatment showed higher bile production, lower levels of transaminases during and after perfusion as well as significantly lower necrosis in liver and bile duct tissue. Biochemical markers of bile duct injury showed the same trend.

Conclusion

Our miniaturized dNEVLP system enables normothermic dual vessel rat liver perfusion. The administration of metamizole effectively ameliorates arterial vasospasm allowing for six hours of dNEVLP, with superior outcome compared to sNEVLP.

Introduction

Liver transplantation is still the only curative treatment option for end stage liver disease. However, there has been an increasing mismatch of organ supply and demand in recent years [1]. The reasons for this development are manifold including progressive aging of the population and increased prevalence of non-alcoholic fatty liver disease in many countries [2, 3]. The severe organ shortage makes the use of marginal organs from so-called extended criteria donors (ECD) necessary [4]. ECDs are donors that do not meet the usual criteria for liver transplantation and are for example old or severely overweight [5, 6]. Data of the United Network for Organ Sharing show an increasing number of donors that are of high age or severely overweight [7]. Indeed, in Germany, up to 75% of liver grafts transplanted fulfil at least one Eurotransplant criterion for extended criteria donors [8]. However, it has been shown, that marginal organs from ECDs perform significantly worse and show higher rates of post-transplantation complications than organs from donors matching the usual transplantation criteria [9, 10]. This is commonly attributed to their increased susceptibility to ischemia-reperfusion injury after cold storage and consecutive warm reperfusion [10, 11].

The necessity of using ECD liver grafts, resulting in poorer patient outcome after cold static storage and transplantation, calls for new strategies for organ preservation. In recent years normothermic ex vivo liver perfusion (NEVLP) has been proven to be a useful alternative to static cold storage of liver grafts [12]. Several studies have demonstrated that NEVLP improves preservation of both fit and marginal liver grafts compared to static cold storage [13–15]. Besides its beneficial effects on the preservation of donor organs NEVLP, unlike subnormothermic or hypothermic ex vivo liver perfusion, also offers the opportunity to metabolically characterize the graft prior to transplantation and to perform pharmacological interventions that rely on a fully functional metabolism and near-to-physiological conditions. Such interventions could improve the quality of marginal liver grafts that are otherwise not acceptable for transplantation [12, 16, 17].

In order to develop and investigate organ recovery strategies based on NEVLP, animal models are needed. Since porcine models are costly and require highly developed infrastructures, small animal models seem more feasible for this task. Several models for ex vivo machine perfusion of rodent livers have been introduced in recent years. The majority of these models propose single vessel perfusion through the portal vein (PV). However, considering the physiological situation, a dual vessel approach, realizing perfusion through both the PV and the hepatic artery (HA), seems more coherent. Also, a dual vessel small animal NEVLP model would correspond to the clinical situation more adequately since NEVLP devices for human liver grafts also employ dual vessel perfusion [18–20].

We developed a dual vessel normothermic ex vivo liver perfusion (dNEVLP) model for rat livers that would sustain stable perfusion conditions for a perfusion period of six hours. We developed a flow-controlled perfusion system using metamizole to mitigate arterial vasospasm and control arterial perfusion pressure. Amongst other qualities, metamizole is known to have spasmolytic effects and has been shown to effectively ameliorate arterial vasospasm of the HA of the rat [21, 22]. Subsequently we compared our previously established single vessel NEVLP model (sNEVLP) to our new dNEVLP model [23].

Methods

Perfusion setup

The laboratory-scaled sNEVLP setup, as initially described by Gassner et al. [23], consisted of a custom-made glass perfusion chamber with multiple inlets (Glass Gaßner GmbH, Munich, Germany). A flow-controlled roller pump provided a continuous flow through the portal vein. Flow was set to 1 mL/min/g liver weight through the portal vein. A pressure sensor ensured continuous monitoring of the portal venous pressure. Average initial pressure was 5.65 mmHg. Portovenous pressures up to 9 mmHg were considered physiological. Pressures were continuously recorded with BDAS 2.0 software (Harvard Apparatus, Holliston, MA, USA). Gas exchange was ensured by a silicon membrane oxygenator (Radnoti, Dublin, Ireland) with a priming volume of 10 mL and 90% oxygen atmosphere. A glass bubble trap prevented air embolization and worked as a Windkessel to ensure laminar flow through the portal vein. A dialysis circuit was diverted from the main perfusion circuit directly after the perfusion chamber. Another roller pump brought perfusate to the dialysis cartridge with a constant flow of 10 mL/min. 500 mL of Ci-Ca dialysate K2 (Fresesnius Kabi, Bad Homburg, Germany) substituted with 12 mM glycin were used. Dialysate flow of 10 mL/min was generated by two roller pumps, one up- and one downstream from the dialysis cartridge, allowing flow adjustments to counteract volume shifts. 1000 IE/h Heparin (Rotexmedica, Trittau, Germany) and 500 μL/h of 1.2 M glycin (45 mg/h) were continuously infused using a syringe driver (Perfusor®, B. Braun Melsungen, Melsungen, Germany, Fig 1A).

Fig 1 — (A) Technical drawing of the sNEVLP setup consisting of the perfusion chamber with inlets for the portal vein (PV) and outlets for the vena cava (VC) and bile duct (BD), an oxygenator, a bubble trap (BT), a dialysis circuit and four roller pumps (P1, P3-5). Several outlets within the perfusion circuit allowed for sample collection of the arterial (A) and venous (V) perfusate and dialysate (d) and for measurement of the portovenous pressure (PV). (B) Technical drawing of the dNEVLP setup with an additional inflow for the hepatic artery (HA), a fifth roller pump (P2) and another two outlets for metamizole bolus application (B) and measurement of the arterial pressure (AP). (C) Liver in the perfusion chamber (dNEVLP) with cannulated bile duct, hepatic artery, portal vein and infrahepatic vena cava (top to bottom). (D) Close-up on cannulated hepatic artery (yellow cannula) witch patch from abdominal aorta.

For dual vessel NEVLP (dNEVLP) a second flow-controlled roller pump, installed downstream to the first one, diverted a defined volume from the main circuit, generating a pulsatile flow through the hepatic artery. Flow was set to 1.1 mL/min/g liver weight for the first roller pump and 0.1 mL/min/g liver weight for the second roller pump, generating a flow of 1 mL/min/g liver weight through the portal vein and 0.1 mL/min/g liver weight through the hepatic artery. A second pressure sensor allowed continuous monitoring of the arterial pressure. Average initial arterial pressure was 48.8 mmHg and pressures up to 110 mmHg were considered physiological. The rest of the perfusion circuit was set up in the exact same manner as described for sNEVLP (Fig 1B).

In metamizole treatment groups, 100 mg boluses of metamizole sodium (Winthrop Arzneimittel GmbH, Frankfurt am Main, Germany) were administered into the hepatic artery either hourly or on demand at a pressure cutoff of 110 mmHg.

Animals and group protocols

Male Wistar rats were purchased from Janvier (Le Genest-Saint-Isle, France). To allow for adequate acclimatisation, animals were kept on a 12-hour light-dark circle for a minimum of one week. All procedures were conducted within a weight range of 280–350 g and with the approval of the local authorities for animal welfare and testing (LaGeSo Berlin, O0365/11). Rats were randomly assigned to 4 groups: dNEVLP without metamizole treatment (dNEVLP^–M, n = 4), dNEVLP with hourly administration of metamizole beginning one hour after perfusion start (dNEVLP^MH, n = 4), dNEVLP with administration of metamizole on demand at a pressure cut-off of 110 mmHg (dNEVLP^MP, n = 4) and sNEVLP (sNEVLP, n = 4) as baseline control. In a first step the three dNEVLP groups were compared. Subsequently the best dNEVLP group was compared to the sNEVLP group.

Surgical procedures

General anaesthesia was performed on animals using isoflurane inhalation and subcutaneous injections of 100 mg/kg metamizole (Winthrop Arzneimittel GmbH, Frankfurt am Main, Germany) and 12 mg/kg ketamine (CP-Pharma, Burgdorf, Germany). Subsequently, the abdominal cavity was opened. The liver was freed from its ligaments and the bile duct was catheterized using a custom-built catheter. The gastroduodenal, left gastric and splenic artery were ligated. 1 mL of Ringer solution supplemented with 500 IE Heparin (Rotexmedica, Trittau, Germany) was injected into the abdominal vena cava inferior. The abdominal aorta and the portal vein were cannulated for blood collection and later flushing. In the dNEVLP groups the hepatic artery was cannulated through an aortic patch (Fig 1C and 1D). The thoracic cavity was opened, and the thoracic aorta clamped. The liver was flushed via both, the aorta and the portal vein with 20 mL of 4°C cold HTK-solution (Dr. Franz Köhler Chemie GmbH, Bensheim, Germany), supplemented with 12mM glycine. Time between blood collection and cold flushing of the liver (warm ischemia time, WIT) did not exceed 15 minutes. The right suprarenal vein, the oesophageal veins and the suprahepatic vena cava were ligated. A custom-made cannula was inserted into the infrahepatic vena cava. The liver was completely mobilised and then transferred into a pre-weighed container filled with cold HTK-solution supplemented with 12 mM glycine.

Perfusion procedure

The HTK solution was flushed out of the liver via the portal vein using 20 mL ringer solution. The liver was placed on a silicon mat in the perfusion chamber exposing the hilum (Fig 1C). The portal vein, vena cava and hepatic artery (only in dNEVLP groups, Fig 1D) were connected to the perfusion circuit. The bile duct catheter tube was inserted into a pre-weighed collection tube to allow for free bile outflow. Cold ischemia time did not exceed 60 minutes. After connection to the perfusion circuit, the flow rates were slowly increased over a rewarming period of 15 minutes. T0 was set when full flows were reached. Subsequently the liver was perfused for six hours.

Composition of the perfusate

Erythrocytes and plasma were separated by centrifugation at 4°C and 3200 RPM for 15 minutes. The plasma phase was collected, and the buffy coat was withdrawn by suction. 10 mL of the erythrocyte concentrate were suspended in 35 mL of Dulbecco’s Modified Eagle’s Medium as used by Gassner et al. [23]. 5 mL of strain specific rat plasma were added generating a total perfusion volume of 50 mL with a calculated haematocrit of 20%. The perfusate was additionally supplemented with 1000 IE heparin and 12mM glycine.

Measurement of biochemical markers and blood gas analysis

Perfusate samples were taken at the start, after 3 and 6 hours of perfusion and centrifuged at 3200 RPM and 4°C for 10 minutes. The supernatant was collected for analysis. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and Urea were photometrically measured by Labor Berlin–Charité Vivantes GmbH. Additionally, blood gas analysis was performed on samples from the in- and outflowing perfusate (ABL800 FLEX, Radiometer GmbH, Berlin, Germany). Oxygen uptake was calculated according to Tolboom et al. [24]

Tissue sampling and histological analysis

After perfusion livers were removed from the perfusion chamber and flushed with 20 mL of Ringer solution. At least four tissue samples were collected from each liver lobe and preserved in formaldehyde and at -80°C for later histological analysis. Haematoxylin and eosin (H&E) staining (AppliChem, Darmstadt, Germany) was performed on 2 μm and 5 μm thick paraffin sections. A pathologist (R.A.) examined H&E-stained slices from a minimum of four different lobes from each perfused liver, blinded to the treatment groups. Levels of hepatocellular ballooning, loss of nucleus, and cellular fragmentation were assessed as markers for necrosis and sinusoidal dilatation was determined. Additionally, the hilar part of the extrahepatic bile duct, proximal to the catheter, was removed from the liver, frozen in liquid nitrogen and stored at -80°C for later analysis. H&E staining was performed on 8 μm cryo sections. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) for special tissues (F. Hoffmann-La Roche AG, Basel, Switzerland) and 4′,6-diamidino-2-phenylindole (DAPI) staining (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) was performed on 8 μm cryo sections according to the manufacturer’s instructions. TUNEL-positive areas were measured using NIH ImageJ (Version: 2.0.0-rc-68/1.52f).

Bile collection and analysis

The bile was continuously collected, weighed hourly, frozen in liquid nitrogen and then stored at -80° C for later analysis. In order to assess epithelial cell death in the extrahepatic bile duct, lactate dehydrogenase (LDH) and gamma glutamyltransferase (GGT) were determined in the collected bile. LDH was determined by photometric measurement (LDH activity assay kit, Sigma-Aldrich Chemie GmbH Munich, Germany) and GGT was determined by an ELISA (Elabscience, Houston, USA) according to the manufacturer’s instructions.

Statistical analysis

Data is presented as median in the text and as median and interquartile range in the tables. Categorical variables were analysed using the chi-squared test. After testing for normality using the Shapiro-Wilk test, group variables were analysed with two-way ANOVA or Kruskal-Wallis test and Bonferoni post-hoc test, accordingly. Data is presented as 95% confidence interval (CI). Overall, a p value < 0.05 was considered significant. Calculations were carried out using IBM SPSS Statistics Version 24.0 for macOS (IBM Corp., Armonk, NY, USA). Graphs were generated using GraphPad Prism Version 8.11 (GraphPad Software, La Jolla, CA, USA).

Results

Stable perfusion conditions were achieved for all groups

Animal and liver weight did not differ significantly between all four groups (p = 0.47 and p = 0.34, respectively). Surgical procedures were performed in the same manner for all experiments apart from the arterial preparation in the dNEVLP groups. Cold ischemia time and macroscopic flush after explanation did not differ significantly. Initial pH was within physiological range and decreased to slightly acidic conditions in all experiments without significant differences (pH_T0 p = 0.22, pH_T3 p = 0.66, pH_T6 p = 0.22). Sodium, potassium and chloride stayed within physiological ranges in all four groups. Throughout perfusion, PV pressure did not significantly differ between all groups (PVP_T0 p = 0.26, PVP_T3 p = 0.63, PVP_T6 p = 0.87) and did not fall below or exceed the physiological range of 4–9 mmHg in all four groups throughout perfusion. Histopathology did not show relevant edema in any of the four groups after perfusion. Oxygen consumption remained high throughout perfusion in all groups without significant differences (dNEVLP^MP: VO2_T0 = 0.03 ml/min/g, VO2_T3 = 0.04 ml/min/g, VO2_T6 = 0.03 ml/min/g; dNEVLP^MH: VO2_T0 = 0.03 ml/min/g, VO2_T3 = 0.04 ml/min/g, VO2_T6 = 0.05 ml/min/g; dNEVLP^-M: VO2_T0 = 0.04 ml/min/g, VO2_T3 = 0.05 ml/min/g, VO2_T6 = 0.04 ml/min/g; sNEVLP: VO2_T0 = 0.02 ml/min/g, VO2_T3 = 0.04 ml/min/g, VO2_T6 = 0.03 ml/min/g).

Administration of metamizole significantly decreased arterial pressure and increased bile production

Initial portovenous and arterial pressures did not significantly vary between all dNEVLP groups (PVP_T0 p = 0.09, aP_T0 p = 0.67, Fig 2A and 2B). In the dNEVLP^-M group, the arterial pressure exceeded the physiological cutoff of 110 mmHg after three hours of perfusion leading to severely high pressures of up to 190 mmHg after six hours of perfusion. A significant decrease in arterial pressure was observed after both the hourly and the pressure dependent administration of metamizole in the metamizole treatment groups. This effectively prevented an increase of the arterial pressure above 110 mmHg (aP_T3 p = 0.007, aP_T4 p = 0.01, aP_T5 p = 0.02, aP_T6 p = 0.03, Fig 2B). Arterial pressures did not significantly differ between the two metamizole treatment groups.

Bile production significantly increased in the dNEVLP groups with metamizole application. In the second hour of perfusion bile production in the dNEVLP^MH group was significantly higher than in the dNEVLP^MP group, in which metamizole had not yet been administered, but not significantly higher than in the dNEVLP^-M group (B_T2 p = 0.02, Fig 3A). In the fourth hour of perfusion, bile production in the dNEVLP^MH group was significantly higher than in the dNEVLP^-M group (B_T4 p = 0.04, Fig 3A). In the fifth and sixth hour of perfusion bile production in the dNEVLP^MP group was significantly higher than in the dNEVLP^-M group (B_T5 p = 0.04, B_T6 p = 0.03, Fig 3A).

Fig 3 — Comparison of the three dNEVLP groups: (A) amount of bile production, (B) bile gamma-glutamyl transferase, (C) lactate dehydrogenase within bile, (D) liver parenchyma necrosis, (E) liver parenchyma sinusoidal dilatation, (F) bile duct necrosis in TUNEL staining. * indicates p ≤ 0.05, ** p ≤ 0.01 and *** p = 0.001. Data shown as median and interquartile range.

Administration of metamizole on demand (dNEVLP^MP) achieved lowest markers of liver and bile duct damage

Between all three dNEVLP groups perfusate urea levels and pH did not differ significantly throughout perfusion (U_T0 p = 0.79, U_T3 p = 0.1, U_T6 p = 0.32, pH_T0 p = 0.23, pH_T3 p = 0.78, pH_T6 p = 0.14, Fig 2C and 2D). In the dNEVLP^MH and dNEVLP^MP groups lactate levels after six hours of perfusion were lower than in the dNEVLP^-M groups. However, differences did not reach statistical significance (L_T6 p = 0.14).

Initial ALT levels were significantly lower in the dNEVLP^MP group than in the dNEVLP^-M group (ALT_T0 p = 0.048; Fig 2E). In the dNEVLP^MP group, ALT and AST levels trended to be lower than in dNEVLP^MH and dNEVLP^-M groups throughout perfusion (Fig 2E and 2F)

HE staining showed considerably less sinusoidal dilatation in the dNEVLP^MP group than in the dNEVLP^-M group and significantly less sinusoidal dilatation than in the dNEVLP^MH group after six hours of perfusion (p = 0.01, Figs 3E and 4A–4C). Tissue necrosis was also lowest in the dNEVLP^MP group (Figs 3D, 4A–4C). However, no statistical significance could be shown (p = 0.9).

Fig 4 — (A-D) HE staining of liver parenchyma, (E-H) HE staining of bile duct, (I-L) TUNEL & DAPI staining of bile duct, (A, E, I) dNEVLP^-P, (B, F, J) dNEVLP^MH, (C, G, K) dNEVLP^MP, (D, H, L) sNEVLP.

Measurement of bile LDH showed similar levels for all three dNEVLP groups during the first four hours of perfusion. After five and six hours of perfusion bile LDH levels were significantly lower in both metamizole treatment groups compared to the dNEVLP^-M group (p = 0.02, Fig 3C).

Throughout perfusion bile GGT levels in the metamizole treatment groups were lower than in the non-treatment group. After two, three, four and six hours of perfusion, GGT levels in the dNEVLP^MH group were significantly lower than in the dNEVLP^-M group (Fig 3B). GGT levels did not differ significantly between the two metamizole treatment groups.

HE and TUNEL staining of the bile duct clearly showed lower necrosis in the dNEVLP^MP group compared to the other two groups (Figs 3F, 4E–4G and 4I–4K). However, no statistical significance could be shown (p = 0.06).

dNEVLP with administration of metamizole on demand (dNEVLP^MP) showed better liver function and lower markers of liver and bile duct damage, compared to sNEVLP

Between the two groups, perfusate pH and PV pressure did not show significant differences throughout perfusion (Fig 5A and 5B). Urea levels were higher in the dNEVLP^MP group, but did not reach significance (Fig 5C). Lactate levels after six hours of perfusion were lower in the dNEVLP^MP group than in the sNEVLP group even though differences did not reach statistical significance. Bile production was significantly higher in the dNEVLP^MP group from three hours of perfusion on until the end (B_T4 p = 0.03, B_T5 p = 0.03, B_T6 p = 0.03, Fig 6A).

Fig 6 — Comparison of dNEVLP^MP and sNEVLP: (A) amount of bile production, (B) gamma-glutamyl transferase within bile, (C) lactate dehydrogenase within bile, (D) liver parenchyma necrosis, (E) liver parenchyma sinusoidal dilatation, (F) bile duct necrosis in TUNEL staining. * indicates p ≤ 0.05 and *** p = 0.001. Data shown as median and interquartile range.

ALT levels were significantly lower in the dNEVLP^MP group after three hours and still lower after six hours of perfusion (ALT_T3 p = 0.03; ALT_T6 p = 0.11; Fig 5E). AST levels in the dNEVLP^MP group were lower after three and significantly lower after six hours of perfusion (AST_T3 p = 0.06, AST_T6 p = 0.03, Fig 5D).

H&E staining showed significantly lower sinusoidal dilatation and necrosis in the dNEVLP^MP group after six hours of perfusion (necrosis p = 0.02, sinusoidal dilatation p = <0.001, Figs 6D–6E, 4C and 4D).

Bile LDH levels showed similar developments in both groups in the first four hours of perfusion. After five and six hours, LDH levels in the dNEVLP^MP group were considerably lower than in the sNEVLP group. However, no statistical significance could be shown (Fig 6C). As well, GGT levels trended to be lower in the dNEVLP^MP group from three hours of perfusion on (Fig 6B).

H&E and TUNEL staining of the bile duct showed significantly lower necrosis in the dNEVLP^MP group (p = 0.03, Figs 6F, 4G–4H and 4K–4L).

Discussion

Normothermic ex vivo liver perfusion (NEVLP) is regarded as a beneficial alternative to static cold storage in liver transplantation, especially when using marginal liver grafts. Small animal NEVLP models are needed to foster the development of strategies for organ preservation and reconditioning. In current literature, laboratory-scaled NEVLP models usually utilize single vessel perfusion, which means the organ is only perfused through the PV. Tolboom et al. could show that single vessel normothermic liver perfusion is a feasible strategy for organ preservation of rat livers with high survival rates after transplantation [24]. However, when it comes to metabolic reconditioning, there are many arguments for a dual vessel approach: 9–12% of rat liver parenchyma is supplied by the arterial blood flow only [25]. The extrahepatic bile duct obtains at least 49% of its blood supply exclusively from the HA and its epithelial cells are known to be especially vulnerable to ischemia [26, 27]. Mora et al. showed, that in porcine extracorporeal liver perfusion as a method to provide temporary liver support for patients with severe liver failure, dually perfused livers performed better than such solely perfused trough the PV [28]. Generally, a dual vessel perfusion model corresponds to the physiological situation better than a single vessel perfusion model. Most importantly, it more directly reflects the clinical situation, since NEVLP devices for human liver grafts also employ dual vessel perfusion [18–20].

Despite these considerations, there are only a few studies reporting on dual vessel small animal NEVLP. Op den Dries et al. and Schlegel et al. presented dual vessel rat NEVLP models in their recent works, realizing perfusion periods of three and four ours, respectively [15, 29]. However, for ex vivo organ reconditioning, perfusion periods of more than four hours may be necessary to reflect the clinical situation, where perfusion time frequently exceeds such durations. Moreover, in our opinion, longer perfusion periods could be necessary to effectively conduct pharmacological organ reconditioning.

Although there are several arguments for dual vessel NEVLP, single vessel small animal NEVLP models are still commonly used in basic research. However, there is no evidence in literature for the equivalence of the single vessel and the dual vessel approach or even the superiority of one over the other. t´Hart et al. compared single and dual vessel NEVLP but were unable to accomplish perfusion periods of longer than 90 minutes without witnessing a severe escalation of arterial perfusion pressure and transaminase levels [30]. Reasons for that could include the lack of a dialysis circuit and the utilization of a cell free perfusate, as previous findings of our work group show [31, 23]. Brüggenwirth et al. recently published a comparative study on dual and single vessel end-ischemic normothermic reperfusion of the rat liver following six hours of cold storage and one hour of subnormoythermic reperfusion [32]. They showed similar outcomes for dual and single vessel perfused livers. In our opinion, short end-ischemic reperfusion after prolonged cold storage might be a feasible strategy for organ preservation but not the right option for metabolic reconditioning of rat liver grafts.

The first aim of this work was to develop a dual vessel NEVLP rat model that would maintain near-to-physiological conditions for a perfusion period of six hours. The development of such a model comes with many difficulties. The most serious problem we observed was a severe increase of vascular resistance in the arterial flow area. We attributed this phenomenon to progressive vasospasm in the small arterial vessels of the rat liver and assumed that sufficient vasodilatation would be necessary for successful dual vessel NEVLP. We identified metamizole as a possible agent to accomplish that. Kaya et al. have shown that metamizole is able to effectively ameliorate arterial vasospasm of the hepatic artery, when applied topical [22]. We investigated, whether the direct administration of metamizole into the HA could effectively decrease arterial vascular resistance in the liver and ensure sufficient vasodilatation. In many countries, including Germany, metamizole is inexpensive, widely available and therefore often used in animal research and veterinary medicine. It has a low risk of causing acute liver failure and has been shown to have no toxic effects on hepatocytes [33, 34]. Agranulocytosis, which is a known severe adverse effect of metamizole treatment, has led to its ban in several countries. Although we do not propose using metamizole for clinical NEVLP, the risk of myelotoxic effects during leukocyte-free NEVLP should be low. Since metamizole has not been shown to accumulate in the liver and the liver would be flushed before transplantation, the risk of adverse effects after transplantation should also be low.

Our results confirm our previous observation of increasing vascular resistance occurring from three to four hours of perfusion onward. As presumed, the subsequent unphysiologically high arterial pressures resulted in poor perfusion outcome, as parameters of liver and bile duct damage were considerably elevated. Interestingly, the high arterial pressures did not lead to relevant development of edema. We could show, that the application of metamizole into the HA ensured sufficient vasodilatation and kept arterial pressures in the physiological range. This led to better organ preservation as both metamizole groups showed lower markers of liver damage as well as better results in the histopathology of both liver and bile duct tissue. Notably, the application metamizole significantly increased bile production and lowered levels of bile duct damage markers.

The comparison between the two metamizole groups indicated, that the administration of metamizole on demand–as opposed to a static hourly administration–was altogether more beneficial for the organs. Interestingly, the hourly administration of metamizole even led to significantly more sinusoidal dilatation than the pressure dependent administration on demand. This suggests, that metamizole itself does not have an intrinsic positive effect on the liver but develops its beneficial effect through vasodilatation, when vasodilatation is needed.

Furthermore, the on-demand-protocol accomplished more stable perfusion conditions, as results in this group were the most consistent ones between the three dNEVLP groups.

Our results show that dual vessel NEVLP is only beneficial if sufficient vasodilatation is performed during perfusion. We propose Metamizole as one possible agent to accomplish that. However, other vasodilators (e.g. Epoprostenol or Verapamil) might show similar results [35].

The second aim of this work was the evaluation of our newly developed dNEVLP system by comparing it to our well-established sNEVLP system. Our results indicate that dNEVLP leads to lower liver damage and better liver function. Although oxygen uptake did not show a significant difference, histological architecture was better preserved, as necrosis and sinusoidal dilatation in the liver parenchyma were significantly lower. We attribute this not only to the improved perfusion of areas that are supplied only by the HA, but also to a general improvement of the microcirculation of the liver. Histopathology also showed significantly better preservation of the extra hepatic bile duct by dNEVLP compared to sNEVLP. Since the extra hepatic bile duct obtains a great part of its blood supply only from the hepatic artery, these results seem coherent. Chemical markers of bile duct epithelial damage show the same trend, but results did not reach statistical significance. One reason for this is the high standard deviation of the results in the sNEVLP group. Another reason might be, that bile duct damage in the sNEVLP group was more frequently located in the deeper layers of the bile duct than in the epithelium, as histopathology showed. Again, it is important to mention that dNEVLP achieved more stable perfusion conditions, as results were more consistent than in the sNEVLP group.

Our results suggest, that dNEVLP with on demand application of metamizole for vasodilatation leads to superior organ preservation after six hours of perfusion compared to sNEVLP in our miniaturized rat liver perfusion system. Judging from our oxygen consumption analysis, liver function tests, and histological analyses, the organs were well perfused and viable throughout the entire perfusion period. Moreover, all livers in our experiments met the clinical criteria for viability and transplantability as established by Mergental et al. (bile production, stable flow rates and homogenous perfusion) [36]. However, these criteria were established for human livers and thus have limited validity in the assessment of rat livers. It remains to be seen, if dually perfused rat livers also perform better after transplantation. This is currently being investigated in our work group.

In conclusion, we here present a dual vessel small animal NEVLP model. We introduce metamizole as a potent agent to mitigate arterial vasospasm, thus allowing for perfusion periods of six hours and more. Furthermore, we present a comparison between single and dual vessel NEVLP in a small animal model. We show, that dNEVLP with sufficient vasodilatation by metamizole application on demand seems to be superior to sNEVLP in terms of organ preservation.

Supporting information

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Acknowledgments

The Authors would like to thank Steffen Lippert, Kirsten Führer and Dietrich Polenz for their instruction and support in the methods.

Abbreviations

ALT: alanine aminotransferase
AST: aspartate aminotransferase
dNEVLP: dual vessel normothermic ex vivo liver perfusion
ECD: extended criteria donor
GGT: gamma glutamyltransferase
H&E: haematoxylin and eosin
HA: hepatic artery
HTK: histidine-tryptophan-ketoglutarate solution
LDH: lactate dehydrogenase
NEVLP: normothermic ex vivo liver perfusion
PV: portal vein
sNEVLP: single vessel Normothermic ex vivo liver perfusion
TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling

Data Availability

All relevant data are within the paper and its Supporting Information file.

Funding Statement

This work was funded by institutional financial support of the Charité – Universitätsmedizin Berlin, the German Research Foundation (grant number: RA 3044/3-1) and by a Kickbox Seed Grant of the Einstein Center for Regenerative Therapies. Nathanael Raschzok is fellow of the BIH Charité Clinician Scientist Program funded by the Charité – Universitätsmedizin Berlin and the Berlin Institute of Health.

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PLoS One. doi: 10.1371/journal.pone.0235635.r001

Decision Letter 0

Michael Bader

30 Mar 2020

PONE-D-20-06139

Dual versus single vessel normorthermic ex vivo perfusion of rat liver grafts using metamizole for vasodilatation.

PLOS ONE

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5. Review Comments to the Author

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Reviewer #1: This study was performed in a rat model of ex vivo liver perfusion using dual versus single vessel perfusion. The authors evaluate these effects after the use of metamizole.

Although the authors showed a relationship between the use of metamizole and the protection against the arterial vasospasm with is important because one of the main complications associated with liver transplantation is related to bile duct ischemia.

Despite these, I sent my comments:

The heterogeneous etiologies for liver transplantation and methodology were problematic. In this study, the time of cold ischemia and warm ischemia is very short. One of the main problems of this work is that it is barely reproducible since cannulating a rat's hepatic artery is technically demanding. I suggest placing an image in the article.

During this study, the liver was flushed via the aorta and the portal vein with HTK solution supplemented with glycine. Given the variability of liver preservation solutions, will this finding be transversal to other preservation solutions? UW or Celsior?

The underlying molecular mechanism was not well suggested in this study. What is the effect in the mitochondrial function, the principal cellular source of energy? For example, the content of ATP?

How do you think it could be used in liver transplantation?

Reviewer #2: This paper provides a novel technique at delivering metamizole through the hepatic artery during single or dual vessel ex vivo perfusion of the rat liver to improve perfusion outcomes. Those livers perfused with metamizole, whether through pressure control or hourly administration, appeared to have less ischemic injury as evidenced by greater urea production and lower AST and ALT levels as well as more physiologic pH values. My critique of this paper is that this medication appears to be banned in the United States for human use by the FDA. Therefore, I recommend a major revision of your conclusion portion of the manuscript. Namely, I caution you in your statement that "metamizole is a widely used drug in human medicine" as well as your notations qualifing its limited risks of agranulocytosis. The main limitation of this paper, while well designed, is that the drug used has limited clinical utility.

Reviewer #3: In this study, the authors present a comparison between dual vessel vs. single vessel normothermic liver perfusion in a small animal model. The authors also focused on a known perfusion pressure issue associated with hepatic artery perfusion by introducing metamizole as an agent to mitigate arterial vasospasm. They hypothesized based on previous research, that since metamizole has been shown to decrease vasospasm in the rat femoral artery, that these findings may also decrease pressure related issues during hepatic artery perfusion. As a result, the authors conclude dual vessel liver perfusion to be superior to single vessel perfusion in the presence metamizole for use in arterial pressure control for perfusions that last 6 or more hours.

This is a well-designed and rigorous study with one main exception: The work is persuasive for the use of metamizole improving arterial pressures during dual vessel perfusion in small animal models; however there appears need of significant data and likely another experimental group to back up the claims that dual vessel perfusion is superior to single vessel perfusion in this small animal model. There are also some questions regarding the methods employed in liver recovery and perfusion which may need clarifications or corrections.

It is unclear as to how a definitive conclusion can be reached that dual vessel perfused livers perform better than single vessel perfusion. First, unless I am missing it there was no single vessel perfusion with metamizole group, which makes comparisons difficult. It would have been a better comparison if single vs dual perfusion groups compared without metamizole, as it would be an apples to apples comparison. Still, a question that rises is if metamizole would have had similar beneficial effects in single vessel perfusion, and if the results would then be comparable to dual+ metamizole group. In addition, oxygen consumption and oxygen saturation levels at inlet and effluent would be important to evaluate, since providing oxygen via both vessels is a key benefit of dual perfusion based on human liver perfusion literature. Supplemental data shows some lactate clearance, but it is unclear if the dialysis cartridge used removes lactate from the perfusate. Other indicators that come to mind are edema and ATP levels; another suggestion is to use some of the clinical perfusion viability criteria already used, such as the one published by Mergental et al to compare the viability of the perfused grafts.

A more technical concern that could potentially influence the perfusion metrics and overall outcome was ligating the SHVC and cannulating the IVC. This can restrict the outflow of the perfusate and can increase the overall pressure within the liver. Research has shown that even partial occlusion of veins in the liver can lead to diffused hepatic congestion and enlargement (ie. sinusoidal thrombosis). This effect may have several different downstream consequences, all of which could alter the evaluation of the grafts. The results of the graft assessment given (pressure (both arterial and portal), histological analysis, biochemical markers ALT/AST), could be influenced by complications arising from the methodology of the experimental design itself. Have the authors considered such issues, and if so how did they avoid it affecting their results?

The final technical concern is the flow rates that were used in this study. For dual vessel perfusion the flow was set to 1.1mL/min/g (paragraph 201-202) generating a flow of 1ml/min/g liver through the portal and 0.1 ml/min/g liver through the hepatic artery. These flow rates are very low, compared with other papers in literature which listed the arterial flow from 0.21 +/- 0.02 to 3.5 +/- 0.2 mL/min/g liver, and portal flow 1.53 +/- 0.19 to 32.1 +/- 1.6 mL/min/g liver. The flow rates stated in the manuscript are consistent with mouse liver perfusions. With such low flow rates, the concern is about the availability of oxygenated perfusate to the liver and if the flow rates were high enough to fully oxygenate the organ. Without data on the oxygen consumption and saturation values, it is not possible to ascertain if the organ was oxygenated sufficiently.

Minor issues in the manuscript:

1. 1 mL Lactated Ringers that is supplemented with 500 IE Heparin is injected into the IVC. This concentration of Heparin in rats weighing between 280-350g will subsequently euthanize the rat exposing the liver to warm ischemic time (WIT). The portal and aorta are then cannulated and flushed after with 20 mL of 4C HTK, however the liver is still within the body cavity, and in the subjected to room temperature while the hepatic artery SHVC, right suprarenal vein, esophageal veins are ligated and the IVC is cannulated. Even though the WIT time is likely minimal, it should be clarified in the manuscript.

2. Average initial pressure was 5.65 mmHg. This pressure seems awfully high given that the temperature of the liver is close to 4C at the time of connection? This could cause damage to the endothelial layer within the vasculature of the liver and subsequently cause further complications during the length of perfusion.

3. There are various spelling and grammatical errors throughout the manuscript, which could use a thorough review by the authors.

**********

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Reviewer #2: Yes: Corey Eymard

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PLoS One. 2020 Jul 2;15(7):e0235635. doi: 10.1371/journal.pone.0235635.r002

Author response to Decision Letter 0

14 May 2020

Dear Dr. Heber,

Thank you very much for giving us the opportunity to submit a newly revised version of our manuscript. We hereby submit our revised manuscript as clean copy (Claussen_Manuscript_PlosOne_R1.docx) and additional copy with the changes being done highlighted (Claussen_Manuscript_PlosOne_R1_marked.docx).

Please find below our specific answers to the questions and suggestions of the reviewers as a point-to-point response. We hope that our manuscript is now appropriate for publication in PLOS ONE.

Yours sincerely,

Reviewer #1

“The heterogeneous etiologies for liver transplantation and methodology were problematic. In this study, the time of cold ischemia and warm ischemia is very short. One of the main problems of this work is that it is barely reproducible since cannulating a rat's hepatic artery is technically demanding. I suggest placing an image in the article.”

Thank you for your suggestions. It is true that the cannulation of a rat’s hepatic artery is technically very demanding. We therefore developed a technique that makes this task considerably easier for the veterinary surgeon. Instead of directly cannulating the hepatic artery, the surgeon will cannulate the celiac trunk via a patch that is cut out of the aorta and then push the cannula forward into the hepatic artery. This ensures a much better and more secure grip on the vessel and makes the whole procedure much easier and quicker. Employing this technique, it does not take the veterinary surgeon more than 5-10 minutes to cannulate the hepatic artery, which considerably shortens warm ischemia time in our experiments (max. 15 minutes). Speaking from our experience, it will take an inexperienced trainee between 10 and 15 surgeries to securely learn this technique.

To keep the cold ischemia time (i.e. time between cold flushing of the liver and connection to the perfusion circuit) short, it is important to have the perfusion circuit primed as soon as the liver is procured. If everything has been prepared thoroughly, a CIT of 60 minutes is practicable.

The cannulation of the hepatic artery is definitely one of the more challenging steps of our perfusion experiments. However, with the right technique, a decent amount of practice and thorough preparation, a WIT of 15 minutes and a CIT of 60 minutes will be reproducible.

We have adjusted the “Surgical procedures” part of our manuscript to make this more apparent and thank you for your question to clarify this.

“Methods” part, p. 10: “In the dNEVLP groups the hepatic artery was cannulated through an aortic patch (Fig. 1C/D).”

We have also included two pictures of the liver lying in the perfusion chamber, in which the cannulated artery (yellow cannula) and the aortal patch can be seen (Fig1 C, D).

“During this study, the liver was flushed via the aorta and the portal vein with HTK solution supplemented with glycine. Given the variability of liver preservation solutions, will this finding be transversal to other preservation solutions? UW or Celsior? The underlying molecular mechanism was not well suggested in this study. What is the effect in the mitochondrial function, the principal cellular source of energy? For example, the content of ATP? How do you think it could be used in liver transplantation?”

Glycine has been shown to have cytoprotective effects on hepatocytes as well as to ameliorate Kupffer cell activation in the liver (1, 2). These effects are mainly achieved by temporary hyperpolarization through glycine-gated chloride channels (3, 4). We assume that this mechanism and therefore the beneficial effects of glycine are independent of the preservation solution and would work in a similar way in UW or Celsior solution. We decided to use HTK instead of the other available preservation solutions because it is the gold standard for liver and kidney static cold storage in Germany.

The metabolic mechanisms of glycine treatment have extensively been investigated elsewhere (3, 4). Furthermore, the underlying mechanisms and beneficial effects of glycine treatment on ex vivo perfused livers have been investigated and discussed by our work group in previous publications (5). Analyzing the molecular mechanisms of glycin as part of our perfusion protocol was not an aim of this work and we relied on these previous findings when we chose to use glycine as a supplement to our preservation solution and perfusion medium.

HTK-N, a modified HTK solution including glycine, is already in clinical use and has been shown to have beneficial effects on organ preservation (6).

Given the fact, that the aim of our work was to develop a small animal model for dual-vessel normothermic liver machine perfusion, which should be used for preclinical studies in a field with already well-established and well-working clinical systems, we did not aim to develop a solution that is immediately transferrable to clinical practice. We added this to the Discussion section. This is stated several times in the Introduction and Discussion section, e.g. page 7 (“In order to develop and investigate organ recovery strategies based on NEVLP, animal models are needed. … We developed a dual vessel normothermic ex vivo liver perfusion (dNEVLP) model for rat livers …”) or page 19 (“The first aim of this work was to develop a dual vessel NEVLP rat model…”). We also added this specific point to the Conclusion of the abstract (“Our miniaturized dNEVLP system enables normothermic dual vessel rat liver perfusion.”).

Reviewer #2

“My critique of this paper is that this medication appears to be banned in the United States for human use by the FDA. Therefore, I recommend a major revision of your conclusion portion of the manuscript. Namely, I caution you in your statement that "metamizole is a widely used drug in human medicine" as well as your notations qualifing its limited risks of agranulocytosis. The main limitation of this paper, while well designed, is that the drug used has limited clinical utility.”

Thank you very much for your remarks on our manuscript and for your appreciation of our study design. As requested, we removed the statement that "metamizole is a widely used drug in human medicine" and our statement regarding the limited risks of agranulotcytosis. Instead, we emphasize in the revised version of our Conclusion section that the aim of our work was to develop a dual-vessel model for normothermic rat liver perfusion for basic, preclinical research.

The Discussion section, p. 20 was modified from “However, agranulocytosis has only been observed very rarely and metamizole is still regarded as a safe drug in literature and for example in regular use in Germany. Also, when using Metamizole in our NEVLP model, the risk of agranulocytosis should be very low because the perfusion circuit is leukocyte-free.” to “Although we do not propose using metamizole for clinical NEVLP, the risk of myelotoxic effects during leukocyte-free NEVLP should be low. Since metamizole has not been shown to accumulate in the liver and the liver would be flushed before transplantation, the risk of adverse effects after transplantation should also be low.”

We showed that sufficient vasodilatation is key to successful dual-vessel rat liver perfusion. We propose metamizole as a feasible agent to accomplish this. If there are specific concerns regarding the use of metamizole due to availability or other reasons, other vasodilators may be used. We altered our manuscript to clarify this. (“Discussion” part, p. 21: “Our results show that dual vessel NEVLP is only beneficial if sufficient vasodilatation is performed during perfusion. We propose Metamizole as one possible agent to accomplish that. However, other vasodilators (e.g. Epoprostenol or Verapamil) might show similar results.”)

Reviewer #3

“It is unclear as to how a definitive conclusion can be reached that dual vessel perfused livers perform better than single vessel perfusion. First, unless I am missing it there was no single vessel perfusion with metamizole group, which makes comparisons difficult. It would have been a better comparison if single vs dual perfusion groups compared without metamizole, as it would be an apples to apples comparison. Still, a question that rises is if metamizole would have had similar beneficial effects in single vessel perfusion, and if the results would then be comparable to dual+ metamizole group. “

The aim of this work was to develop a dual vessel perfusion model for rat liver grafts that reflects the in vivo situation of portal and arterial blood supply to the liver, and that would work as well as or even better than the commonly used single vessel model. We started of by performing dual vessel perfusion in analogy to our single vessel perfusion (Dual vessel w/o metamizole). We observed potential beneficial effects of the dual vessel approach e.g. lower levels of transaminases up to four hours of perfusion and higher bile production throughout perfusion compared to our single vessel perfusion experiments. However, these potential beneficial effects were outweighed by negative results, e.g. higher transaminase levels after six hours of perfusion, higher or similarly high levels of bile duct damage especially from four hours of perfusion onward and no improvement in tissue preservation/histopathology. We attributed these negative results to damages done by unphysiologically high arterial pressures that occurred from after three to four hours of perfusion.

From our point of view it was crucial to ensure near to physiological conditions including physiological arterial pressures before drawing a comparison between the two perfusion models. A comparison between dual vessel NEVLP w/o vasodilation and single vessel NEVLP would have been a comparison between a well-established and well working perfusion model (single vessel NEVLP) and an unready new perfusion model that was unable to sustain near-to-physiological conditions. In our opinion such a comparison would be of no consequence.

The question, whether a single vessel NEVLP + metamizole group would be necessary to draw a more direct comparison and investigate similar beneficial metamizole effects on single vessel perfusion is understandable and we ourselves have considered including such a group in this work. However, in our opinion, there are several reasons that make a single vessel NEVLP + metamizole group unnecessary:

Regarding the potential positive effects of metamizole, our results show that the use of metamizole for vasodilatation is beneficial in dual vessel perfusion. However, the application mode plays an important role. Overall, the on-demand protocol showed better and more stable results than the static hourly protocol. Static hourly metamizole application led to higher levels of transaminases and worse histopathology. This indicates that metamizole itself does not have an absolute positive effect on the perfused liver, but should only be used, when vasodilatation is required. A single vessel perfusion model does not require vasodilation. In our experiments metamizole application did not have an effect on vascular resistance and pressure in the portovenous flow area. The use of a vasodilator like metamizole does not seem logical in such a model and we do not think that the addition of metamizole to our single vessel NEVLP model would have a beneficial effect. We altered the manuscript accordingly (“Discussion” part, p. 21: “This suggests, that metamizole itself does not have an absolute positive affects on the liver but develops its beneficial effect through vasodilatation, when vasodilatation is needed.”)

Regarding the comparability of a sNEVLP + metamizole group and the dNEVLP + metamizole on demand group, we have two concerns. The first one is the application route. Establishing our dual vessel perfusion model we found that vasodilatation was most sufficient, when the agent was administered directly into the hepatic artery. The application into both vessels usually led to insufficient vasodilatation and poorer perfusion outcome. We therefore decided to administer metamizole directly into the hepatic artery in all experiments of the two metamizole groups. Naturally, this would not be possible in our single vessel NEVLP model, since there is no artery. In a sNEVLP + metamizole group metamizole would thus have to be administered into the portal vein. This would lead to considerably higher concentrations of metamizole in the portovenous flow area than after injection into the heaptic artery, where the metamizole bolus is diluted by the perfusate before reaching the portal vein. An alternative approach would be to administer the bolus into the effluent of the liver, thus mimicking the metamizole outflow after arterial injection. However, the bolus would be diluted instantly and metamizole concentrations in the portal vein would likely be too low to have any effect. In our opinion, neither of the two strategies would improve the quality of the comparison between the single vessel NEVLP model and the dual vessel NEVLP + metamizole model.

The second concern regards the scheme of application. In our optimized dual vessel NEVLP model we perform vasodilatation by on-demand metamizole application at a certain arterial pressure cut-off at different points in time. As mentioned above, in a single vessel NEVLP model there is no demand for vasodilatation and therefor no such cut-off. In a single vessel NEVLP + metamizole group metamizole would thus have to be administered at completely random points in time without any underlying principle. This does not seem reasonable and would, again, not improve the quality of the comparison.

We agree that we cannot completely rule out any effects metamizole might have besides its properties as a vasodilator. However, if the vasodilator used does have effects other than vasodilatation, these effects are an intrinsic part of this perfusion model and add to its qualities and deficits as a whole. We cannot distinctly determine, whether the dual vessel approach or the vasoldilatation by metamizole has greater beneficial impact. However, we show that a perfusion model that combines both accomplishes successful perfusion and then seems superior to the common single vessel model.

We revised the “Discussion” part of our manuscript to make this clearer. (“Discussion” part, p. 22: “We show, that dNEVLP with sufficient vasodilatation by metamizole application on demand seems to be superior to sNEVLP in terms of organ preservation.”)

I our opinion, the above mentioned facts and considerations make the addition of a sNEVLP + metamizole group unnecessary. In compliance with the 3R-principles for responsible animal experiments we therefore decided, not to include this group in our work.

“In addition, oxygen consumption and oxygen saturation levels at inlet and effluent would be important to evaluate, since providing oxygen via both vessels is a key benefit of dual perfusion based on human liver perfusion literature.”

Thank you for your remark. Oxygen saturation was measured in our experiments. We calculated the oxygen consumption and included the results in our manuscript

“Methods” part, p. 11: “Oxygen uptake was calculated according to Tolboom et al.”

“Results” part, p.14: “Oxygen consumption remained high throughout perfusion in all groups without significant differences (dNEVLPMP: VO2T0 = 0.03 ml/min/g, VO2T3 = 0.04 ml/min/g, VO2T6 = 0.03 ml/min/g; dNEVLPMH: VO2T0 = 0.03 ml/min/g, VO2T3 = 0.04 ml/min/g, VO2T6 = 0.05 ml/min/g; dNEVLP-M: VO2T0 = 0.04 ml/min/g, VO2T3 = 0.05 ml/min/g, VO2T6 = 0.04 ml/min/g; sNEVLP: VO2T0 = 0.02 ml/min/g, VO2T3 = 0.04 ml/min/g, VO2T6 = 0.03 ml/min/g; VO2T0 p = 0.68, VO2T3 p = 0.19, VO2T6 = 0.91).“

„Discussion“ part, p. 20: „Although oxygen uptake did not show a significant difference...“).

Our data show that oxygen consumption was high throughout perfusion in all four groups without significant differences. Overall, the dual vessel groups show a trend towards higher oxygen consumption, however, no statistical significance could be shown. As mentioned in the manuscript, only 9-12% of rat liver parenchyma are solely supplied by the hepatic artery. Therefore, differences in oxygen consumption between sNEVLP and dNEVLP can be expected to be very small and difficult to detect. Since oxygen saturation in the arterial and portovenous flow area is the same in our perfusion model, we do not expect higher oxygen supply to be the key beneficial factor of dNEVLP. We much more think that it`s positive effects rely on the perfusion of areas that are not reached by the portal vein, such as the bile duct. These areas may be small (and might therefore not significantly contribute to the livers total oxygen consumption) but are of great importance for the organs viability. Our results show that dNEVLP does make a difference as both liver and bile duct tissue were significantly better preserved.

“Another suggestion is to use some of the clinical perfusion viability criteria already used, such as the one published by Mergental et al to compare the viability of the perfused grafts.”

Mergental et al. (7) proposed two major and three minor criteria for the viability of human livers after two hours of NMP: Major viability criteria were perfusate lactate levels lower than 2.5 mmol/L (= 22.52 mg/dL) and the presence of bile production. Minor viability criteria were pH of greater than 7.3, homogenous perfusion with soft consistency of the parenchyma and stable arterial and portovenous flows of 150 ml/min and 500 ml/min, respectively. For the human liver with a total weight of around 1500 g these flow rates result in 0,1 ml/min/g arterial and 0.33 ml/min/g portovenous flow. Mergental et al. suggested that perfused livers should be judged viable if they met one or more major and two or more minor criteria.

Eventhough lactate clearance is difficult to assess due to our dialysis circuit, our (supplementary) data show that even after six hours of normothermic perfusion, all perfused livers met at least one major criterion (bile production) and 2 minor criteria (stable flows and homogenous perfusion). Therefore, all livers in our experiments had to be estimated as viable and transplantable. We added this to our discussion. However, a comparison between the human system used by Mergental et al. and ours is difficult, especially due to the fact that using criteria developed for human livers to assess rat livers is questionable and implicates numerous limitations. (“Discussion” part: p. 22: “Moreover, all livers in our experiments met the clinical criteria for viability and transplantability as established by Mergental et al....”)

“A more technical concern that could potentially influence the perfusion metrics and overall outcome was ligating the SHVC and cannulating the IVC. This can restrict the outflow of the perfusate and can increase the overall pressure within the liver. Research has shown that even partial occlusion of veins in the liver can lead to diffused hepatic congestion and enlargement (ie. sinusoidal thrombosis).”

Ligating the SHVC and cannulating the IHVC is an essential step in creating a closed perfusion circuit. This technique is also used in human normothermic machine perfusion of the liver (e.g. described by Ravikumar et al. using the Organox Metra Device)(8). Moreover, the diameter of the cannula that we used for the IHVC outflow was large enough not to downsize the lumen of the vessel. In our experiments, histopathology showed no signs of sinusoidal congestions, thrombosis or any other signs of pressure damage other than those caused by excessive arterial pressures in the dNEVLP-M group. Also, we never witnessed any problems using this technique in our previous publications (5, 9).

“The final technical concern is the flow rates that were used in this study. For dual vessel perfusion the flow was set to 1.1mL/min/g (paragraph 201-202) generating a flow of 1ml/min/g liver through the portal and 0.1 ml/min/g liver through the hepatic artery. These flow rates are very low, compared with other papers in literature which listed the arterial flow from 0.21 +/- 0.02 to 3.5 +/- 0.2 mL/min/g liver, and portal flow 1.53 +/- 0.19 to 32.1 +/- 1.6 mL/min/g liver. The flow rates stated in the manuscript are consistent with mouse liver perfusions. With such low flow rates, the concern is about the availability of oxygenated perfusate to the liver and if the flow rates were high enough to fully oxygenate the organ. Without data on the oxygen consumption and saturation values, it is not possible to ascertain if the organ was oxygenated sufficiently.”

Thank you for your remark on this important aspect. The flow rates were chosen based on our previous experiences with rat liver perfusion (5, 9) In our previous works we have shown that a portovenous flow rate of 1 ml/min/g liver accomplishes sufficient liver perfusion. Establishing our dual vessel NEVLP model, we found that the arterial flow rate of 0.1 ml/min/g liver achieved sufficient arterial perfusion without causing notable damage. Since there is no literature on dual vessel NEVLP of rat livers for more than four hours, we chose the flow rate that worked best for six hours of dNEVLP in our preliminaries. Other publications on dual vessel NEVLP either show arterial pressures but lack data on flow rates (10) or vise versa (11.). Our data on oxygen consumption show that sufficient oxygenation was accomplished for all organs. These results support our results in bile production, urea production and histopathology that show sufficient organ perfusion.

“1 mL Lactated Ringers that is supplemented with 500 IE Heparin is injected into the IVC. This concentration of Heparin in rats weighing between 280-350g will subsequently euthanize the rat exposing the liver to warm ischemic time (WIT). The portal and aorta are then cannulated and flushed after with 20 mL of 4C HTK, however the liver is still within the body cavity, and in the subjected to room temperature while the hepatic artery SHVC, right suprarenal vein, esophageal veins are ligated and the IVC is cannulated. Even though the WIT time is likely minimal, it should be clarified in the manuscript.”

Thank you for your remark. The rat was euthanized by blood withdrawal immediately after the injection of Lactated Ringer´s and heparine. We clarified the warm ischemia time (WIT) in our manuscript. (“Methods” part, p.10: “Time between blood collection and final mobilization of the liver (warm ischemia time, WIT) did not exceed 15 minutes.”)

“Average initial pressure was 5.65 mmHg. This pressure seems awfully high given that the temperature of the liver is close to 4C at the time of connection? This could cause damage to the endothelial layer within the vasculature of the liver and subsequently cause further complications during the length of perfusion.”

Thank you for giving us the chance to clarify this point. After the liver was connected to the perfusion circuit flows were slowly increased over a rewarming phase of 15 minutes. T0 was set when full flow was reached and “initial pressures” were then measured. We altered the manuscript to clarify this (“Methods” part, p. 11: “After connection to the perfusion circuit flows were slowly increased over a rewarming period of 15 minutes. T0 was set when full flows were reached.”)

“Supplemental data shows some lactate clearance, but it is unclear if the dialysis cartridge used removes lactate from the perfusate.”

Thank you for your remark. Average lactate levels in the dialysate rose from 2,3 mg/dL to 5,4 mg/dL without significant differences between the groups. We therefore attribute differences in perfusate lactate levels to differences in lactate clearence. However, since this is difficult to assess reliably, no conclusions regarding lactate clearance were drawn.

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PLoS One. doi: 10.1371/journal.pone.0235635.r003

Decision Letter 1

Michael Bader

8 Jun 2020

PONE-D-20-06139R1

Dual versus single vessel normorthermic ex vivo perfusion of rat liver grafts using metamizole for vasodilatation.

PLOS ONE

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Reviewers' comments:

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Reviewer #3: Yes

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6. Review Comments to the Author

Reviewer #1: I was satisfied with the answers given by the authors that meet my questions. The placement of the figures values the work. Congratulations.

Reviewer #2: (No Response)

Reviewer #3: The authors did address some key comments that were raised in the original manuscript, and the detail provided in the responses is appreciated. I also appreciated the nuances observed in human vs rat comparisons.

I have two minor comments: First, the question re. ATP from two reviewers was not addressed. I do appreciate that performing such analyses may require repeat of most if not all experiments which in my opinion is not necessary and would likely be unnecessary duplication that would not justify the use of research animals; one reason the Mergenthal criteria was recommended as an alternative to ATP is it can be evaluated practically. Since the authors have already done this in the revision, I would have recommended the authors to note this in their rebuttal so it does not appear like an ignored comment to the reviewers.

My second comment would be if the authors could shed some light on if they noticed any difference in relation to edema (or lack thereof) between the groups? Noting that pressures in dNEVLP in the metamizole on-demand group were better, I would be interested to see if this correlates in the context of edema as well.

Overall, I consider the manuscript is acceptable for publication with very minor revisions.

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Reviewer #1: Yes: Rui Miguel Martins

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PLoS One. 2020 Jul 2;15(7):e0235635. doi: 10.1371/journal.pone.0235635.r004

Author response to Decision Letter 1

17 Jun 2020

Dear Dr. Heber,

Thank you very much for giving us the opportunity to submit a newly revised version of our manuscript. We hereby submit our revised manuscript as clean copy (Manuscript.docx) and additional copy with the changes being done highlighted (Revised Manuscript with Track Changes.docx). Please find below our specific answers to the remaining questions and suggestions raised by Reviewer #3 as a point-to-point response. We hope that our manuscript is appropriate for publication in PLOS ONE.

Yours sincerely,

Igor M. Sauer.

Reviewer #3

Thank you for pointing this out. We apologize for the negligence.

Thank you for your remark and we apologize for not addressing this aspect in our first rebuttal. The assessment of edema in our work was limited to the histopathological examination of the livers. The pathologist did not find relevant edema in any of the four groups. We now mention this in the manuscript (“Results” part, p. 13: “Histopathology did not show relevant edema in any of the four groups after perfusion.” and “Discussion” part, p. 19: “Interestingly, the high arterial pressures did not lead to relevant development of edema.”)

The assessment of edema by lyophilisation is currently being established in our workgroup. Preliminary results correlate with the results from our histopathological examination and do not show significant edema after neither single vessel nor dual vessel perfusion when compared to native livers. These results indicate that the development of edema is not a relevant problem in our small animal NEVLP model. However, we will further investigate this aspect in our future projects and thank you for your constructive feedback.

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PLoS One. doi: 10.1371/journal.pone.0235635.r005

Decision Letter 2

Michael Bader

19 Jun 2020

Dual versus single vessel normothermic ex vivo perfusion of rat liver grafts using metamizole for vasodilatation.

PONE-D-20-06139R2

Dear Dr. Sauer,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0235635.r006

Acceptance letter

Michael Bader

24 Jun 2020

PONE-D-20-06139R2

Dual versus single vessel normothermic ex vivo perfusion of rat liver grafts using metamizole for vasodilatation.

Dear Dr. Sauer:

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Data Availability Statement

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PERMALINK

Dual versus single vessel normothermic ex vivo perfusion of rat liver grafts using metamizole for vasodilatation

Felix Claussen

Joseph M G V Gassner

Simon Moosburner

David Wyrwal

Maximilian Nösser

Peter Tang

Lara Wegener

Julian Pohl

Anja Reutzel-Selke

Ruza Arsenic

Johann Pratschke

Igor M Sauer

Nathanael Raschzok

Roles

Abstract

Background

Methods

Results

Conclusion

Introduction

Methods

Perfusion setup

Fig 1. Perfusion setups.

Animals and group protocols

Surgical procedures

Perfusion procedure

Composition of the perfusate

Measurement of biochemical markers and blood gas analysis

Tissue sampling and histological analysis

Bile collection and analysis

Statistical analysis

Results

Stable perfusion conditions were achieved for all groups

Administration of metamizole significantly decreased arterial pressure and increased bile production

Fig 2. Comparison of dNEVLP groups 1.

Fig 3. Comparison of dNEVLP groups 2.

Administration of metamizole on demand (dNEVLPMP) achieved lowest markers of liver and bile duct damage

Fig 4. Histopathology.

dNEVLP with administration of metamizole on demand (dNEVLPMP) showed better liver function and lower markers of liver and bile duct damage, compared to sNEVLP

Fig 5. Comparison of dNEVLPMP and sNEVLP groups 1.

Fig 6. Comparison of dNEVLPMP and sNEVLP groups 2.

Discussion

Supporting information

Acknowledgments

Abbreviations

Data Availability

Funding Statement

References

Decision Letter 0

Michael Bader

Roles

Author response to Decision Letter 0

Decision Letter 1

Michael Bader

Roles

Author response to Decision Letter 1

Decision Letter 2

Michael Bader

Roles

Acceptance letter

Michael Bader

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Administration of metamizole on demand (dNEVLP^MP) achieved lowest markers of liver and bile duct damage

dNEVLP with administration of metamizole on demand (dNEVLP^MP) showed better liver function and lower markers of liver and bile duct damage, compared to sNEVLP

Fig 5. Comparison of dNEVLP^MP and sNEVLP groups 1.

Fig 6. Comparison of dNEVLP^MP and sNEVLP groups 2.