Research Article
Minimal portal vein stenosis is a promising preconditioning in living donor liver transplantation in porcine model E. Gregoire1,⇑, P. Brige2, L. Barbier1, C. Buffat3,4, A. Coppola2, J. Hardwigsen1, Y.P. Le Treut1, V. Vidal2, P.H. Rolland2 1
Aix-Marseille University, Department of General Surgery and Liver Transplantation, Hôpital de la Conception Marseille, France; 2Aix-Marseille University, Experimental Interventional Imaging Laboratory, European Center for Medical Imaging Research, Marseille, France; 3Aix-Marseille University; URMITE, CNRS UMR 6236-IRD 198, Marseille, France; 4Laboratoire de Biochimie et de Biologie Moléculaire, Hôpital de la Conception Marseille, France
Background & Aims: The main hindrance in promoting living donor liver transplantation remains the morbi-mortality risk for the donor. Considering the opposed remodeling influence of portal and hepatic artery flows, our working hypothesis was to identify a lobar portal vein stenosis capable of inducing a contralateral liver mass compensatory enlargement, without the downstream ipsilateral atrophic response. Methods: Twenty-four pigs entered this study. Six of them were used to establish hemodynamic changes following a progressive left portal vein (LPV) stenosis, in blood flow, pressure and vessel diameter of the LPV, main portal vein and hepatic artery. Sixteen pigs were divided into 4 groups: sham operated animals, 20% LPV stenosis, 50% LPV stenosis, and 100% LPV stenosis. Daily liver biopsies were collected until post-operative day 5 to investigate liver regeneration and atrophy (Ki67, STAT3, LC3, and activated caspase 3) according to the degree of LPV stenosis. Finally, changes in liver volumetry after 20% LPVS were investigated. Results: A 20% LPV stenosis led to dilatation of the hepatic artery and a subsequent four-fold increase in hepatic arterial flow. Concomitantly, liver regeneration was triggered in the non-ligated lobe and the cell proliferation peak, 5 days after surgery, was comparable to that obtained after total LPV ligation. Moreover, 20% LPV stenosis preconditioning did not induce left liver atrophy contrary to 50 and 100% LPV stenosis. Conclusions: A 20% LPV stenosis seems to be the adequate preconditioning to get the remnant liver of living donor ready to take on graft harvesting without atrophy of the future graft. Ó 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Received 4 October 2013; received in revised form 10 February 2014; accepted 22 February 2014 ⇑ Corresponding author. Address: Aix-Marseille Université 13284, Service de Chirurgie Générale et Transplantation Hépatique, APHM – Hôpital de la Conception, 147, Boulevard BAILLE, 13005 Marseille, France. Tel.: +33 4 91 383 658; fax: +33 4 91 381 655. E-mail address: [email protected] (E. Gregoire). Abbreviations: HABR, hepatic artery buffer response; LPV, left portal vein; ALAT, alanine aminotransferase; ASAT, aspartate aminotransferase; GGT, Gammaglutamyl transferase; ALP, Alkaline Phosphatase; TB, total Bilirubin; FFPE, formalin-fixed, paraffin-embedded; HA, hepatic artery; LLW, left liver weight; TLW, total liver weight; TLV, total liver volume; BW, body weight.
Introduction The shortage of deceased donor organs compared to the number of patients on the waiting list for liver transplantation requires the use of other sources of grafts. The main hindrance in promoting living donor liver transplantation (LDLT) remains the morbi-mortality risk for the donor. This risk is correlated to the amount of resected parenchyma and to the volume of the donor remnant liver [1,2]. Unilateral portal vein ligation and percutaneous transhepatic portal vein embolization procedures can be performed prior to major hepatectomy. Both of these techniques occlude a lobar portal vein aiming to inducing the atrophy of the ipsilateral liver and thus the hypertrophy of the future remnant liver before hepatectomy. Nevertheless, the atrophic consequences of these techniques on the future liver graft preclude inclusion of these practices in the living donor preconditioning. The rationale for this study is that the hepatic artery buffer response (HABR) maintains a constant hepatic blood flow-to-liver mass. Indeed, by this mechanism, reduced portal flow leads to accumulation of adenosine into the space of Mall and hepatic arterial dilatation, thereby serving to buffer the impact that changes in portal flow have on total hepatic blood flow [3–5]. Based upon passive, reflex, and active mechanisms, HABR highly contribute to the liver proliferative response following portal flow changes. We also noted, from clinical and CT-scan personal observations in our patients, that a tumor-induced portal vein stenosis is frequently associated with substantial enlargement of the contralateral liver. Moreover, Belghiti et al. recently published an interesting control matched study of consequences following a right hepatectomy for living donation and benign liver lesions [6]. They conclude that right hepatectomy in LDLT induces a more severe deprivation of liver volume that could represent ‘‘inherent limitation’’ in healthy donors that makes them more vulnerable for postoperative complications [6]. Our working hypothesis was to identify a lobar portal vein stenosis in the living donor liver capable of inducing the basic contralateral liver mass compensatory enlargement (i.e., to
Journal of Hepatology 2014 vol. xxx j xxx–xxx
Please cite this article in press as: Gregoire E et al. Minimal portal vein stenosis is a promising preconditioning in living donor liver transplantation in porcine model. J Hepatol (2014), http://dx.doi.org/10.1016/j.jhep.2014.02.034
Research Article Liver living donor preconditioning (human)
R fu igh tu t re lo gr be af t
Left lobe future remnant liver donor
PRECONDITIONING right portal vein stenosis
C
Right liver harvesting
Animal model of liver living donor preconditioning (pig)
Right liver donor remnant liver
Left liver future graft
VII IV
VIII
II
VI
C
PRECONDITIONING left portal vein stenosis
V
Left liver harvesting Fig. 1. The human and porcine model for living donor liver preconditioning. Our hypothesis: minimal left portal vein stenosis enhance regeneration in the right liver (donor remnant liver) without atrophy of the left liver (future graft). Animal model: the right liver represents the future remnant liver of the donor and the left liver represents the future graft in the porcine model.
prepare the future remnant liver of the donor), without the downstream atrophic response (i.e., protect the future liver graft) (Fig. 1). 2
We here report our results of a new liver preconditioning based on moderate changes in the portal venous flow in a porcine model, and their consequences on hepatic remodeling.
Journal of Hepatology 2014 vol. xxx j xxx–xxx
Please cite this article in press as: Gregoire E et al. Minimal portal vein stenosis is a promising preconditioning in living donor liver transplantation in porcine model. J Hepatol (2014), http://dx.doi.org/10.1016/j.jhep.2014.02.034
JOURNAL OF HEPATOLOGY Materials and methods
lateral lobe were collected (Needle core biopsy BARD, 18 G). At day 7 animals were euthanized and the whole liver was removed, weighted, and separated into left (segment II, III, IV) and right liver (segment V, VI, VII, VIII, and I).
Animal model Because of its similarities in terms of human anatomy and hepatic metabolism, we have chosen the porcine model to experiment our hypothesis [7]. However, it was not possible to work on the right liver because of the absence of a unique right portal vein. Indeed, the main portal vein is divided in two veins for the right liver, which immediately penetrate in the liver parenchyma making them very difficult to control. The left portal vein, however, is unique and includes a proximal portion of a few centimeters which is easy to control. As the right and the left lobe are of equivalent size, we decided to reverse the human model using the right liver as the future remnant liver of the donor and the left liver as the future graft in the porcine model (Fig. 1). Animals Twenty four, six-month-old pigs (Pietrin), weighting 40 to 50 kg were used for these studies. After a 5-days period of acclimatization, all surgical procedures or samples procurements were performed between 8:00 AM and 12:00 AM. The experiments were terminated by intravenous injection of 15 mg midazolam and 25 mg chlorpromazine in 20 ml KCl (15%). The experiments described in this study were conducted according to the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes and after approval of the Ethical Committee of Provence. Phase A – hemodynamics study Six pigs were fasted 12 h before surgery with free access to water. After sedation by 30 mg/kg ketamine and 0.03 mg/kg acetopromazin, a venous catheter was inserted in a large vein in the ear for initial blood sampling and intravenous administrations. Induction of anesthesia was obtained by 2 mg/kg propofol. Anesthesia was maintained with gaseous sevoflurane (1.2%) by mechanical respiration (Zeus Dräger Inc.) and constant intravenous infusion of sufentanil (1 lg/kg/hour). Hemodynamic measurements was were carried as previously described in details [8–10]. Briefly, a midline abdominal incision was performed and ultrasound transit time flowmeter probes were positioned around the portal vein, the left portal vein and the hepatic artery for continuous measurement of blood flow (20, 7, and 3 RS probes, Transit Time Flow Meter, Transonic, Ithaca, NY, USA). Pressure-measurement catheters were placed into the main portal vein and the hepatic artery downstream of the flowmeter probes so as not to disturb flow measurements. Piezoelectric sensors were placed around the portal vein and the hepatic artery next to the flowmeter probes for continuous measurement of vessel diameter. All probes and sensors were recorded on a multichannel LabView based PXi system (National Instrument). Then, a portal vein band was placed around the left portal vein upstream of the flowmeter probe including an external rigid ring (polypropylene) and an internal inflatable balloon (silicone) in order to progressively reduce left portal flow. The ‘‘Zero’’ calibration was checked for each animal. All parameters were recorded simultaneously, a 15 min stabilization, under basal conditions and after each left portal flow modification during 15 min. The balloon was inflated milliliter by milliliter and the degree of the stenosis was verified on successive portography in order to obtain 10, 20, 30, 50, 60, 65, 70, 75, 80, 90, and 100% left portal vein stenosis (LPV). At the end of the experiments, the animals were euthanized. Phase B – biological, cellular and molecular consequences of LPVS Surgery and samples collection Twelve pigs were divided in 4 groups: sham operated animals (group 0, n = 3), 20% LPV stenosis (group 20, n = 3), 50% LPV stenosis (group 50, n = 3), and 100% LPV stenosis (group 100, n = 3). In each group, the animals were anesthetized as described above. In each animal, the left portal vein was isolated (day 0). Deep Liver biopsies were performed on the right and left lobes and blood samples were collected. The circumference of the left portal vein was directly measured by passing a silk thread around the vessel. A non-stretch stripe of polypropylene whose length was calculated to induce a 20, 50, or 100% stenosis was sewed around the left portal vein. Sham operated animals underwent only portal dissection and basal portography. Every day, from day 1 to day 5, animals were anesthetized (ketamine 1 mg, acepromazine 10 mg, IM). Blood samples and USguided percutaneous deep liver biopsies from the left lateral lobe and the right
Samples analysis Biological consequences of LPVS Serum alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), gamma-glutamyltransferase (GGT), alkaline phosphatase (ALP) and total bilirubin (TB) were measured with the biochemical multi-analyser in the hospital biochemistry department. Prothrombin time was measured with STA-R (diagnostica stago Asniere France) in the hospital hematology department. Cellular consequences of LPVS Cell proliferation: Ki67 immunohistochemistry using MIB-1 antibody was performed on 5-micron-liver sections formalin-fixed, paraffin-embedded (FFPE) tissues from right and left liver daily biopsies. The Leica BOND-III fully automated IHC stainer was used with high-efficiency de-waxing processes, EDTA-based Ag unmasking procedures and IHC with appropriate NovoCastra antibodies and final DAB-horseradish peroxidase stainings and hematoxylin counterstaining. Proliferation index was determined by the percentage of cells that display nuclear staining reported to the total number of nuclei (ten fields per slide, 400). Nuclear density and lobar area: morphometric analysis were performed using the Nikon Eclipse Ni H600L microscopic and image analysis systems. Molecular consequences of LPVS Bilateral liver biopsies 3 and 24 h after surgery for Group 20 were analyzed for the IHC detection of STAT3 by the technique described above using Leica IHC Bond-III stainer and Nikon Eclipse image analyzer. Analysis of LC3-II was made of two ways: IHC analysis on liver biopsies from D0 to D5 for 4 groups and analysis of mRNA expression by PCR on liver biopsies from D0 D3 and D7. The presence of activated caspase 3 was analyzed on biopsies IHC at D5 for all 4 groups. The quantification of the labeling was performed by counting the number of labeled nuclei on the total number of nuclei. Phase C – hepatic remodeling after 20% LPV stenosis Six pigs were used for this phase. All animals were weighed 3 days before and 14 days after surgery. An abdominal CT scan was performed for each animal under general anesthesia, 3 days before (D-3) and 11 days after surgery (D11). The apparatus used was a veterinarian scanner monobaret (Cabassu Clinic, Marseille) of the General Electric Company (GE). The acquisition was started 80 s after the IV injection of iodinated contrast (50 ml Telebrix). Three millimeters slides were done every 2 mm. Volumetric reconstruction was performed using the ADW server software (GE). Surgical procedure was similar to the Phase B. The animals were divided into 2 groups: a ‘‘test’’ group with a 20% LPVS (n = 3) and a control group (n = 3), consisting of 2 sham operated animals ( = Group 0) and an animal with a ligature of the LPV ( = Group 100). The animals were euthanized 14 day after surgery. Statistics Data treatment and statistical analyses (Kruskall-Wallis non-parametric anova and Mann-Whitney U test) were performed by using Systat12 (SPSS Inc, Chicago, IL) and Prism 5.0 (Graphpad Prism, La Jolla Inc). A p value less than 0.05 was considered significant. A non-parametric kernel density estimator was preferred to rule out a functional form on the distribution function curves. Results were expressed as mean ± SD.
Results Hemodynamic study according to an increasing left portal vein stenosis We did not observe any significant modification of hemodynamic parameters (blood flow, blood pressure and diameter in the main portal vein, left portal vein and hepatic artery (HA) when a 10%
Journal of Hepatology 2014 vol. xxx j xxx–xxx
3
Please cite this article in press as: Gregoire E et al. Minimal portal vein stenosis is a promising preconditioning in living donor liver transplantation in porcine model. J Hepatol (2014), http://dx.doi.org/10.1016/j.jhep.2014.02.034
Research Article Table 1. Hepatic artery hemodynamics according to the degree of left portal vein stenosis.
% LPVS Hepatic artery diameter (mm) Mean Max Min Pulse Hepatic artery blood pressure (mmHg) Mean Max Min Pulse Hepatic artery blood flow (L/min) Mean Max Min Pulse Portal vein blood pressure (mmHg) Mean Max Min Pulse Portal vein blood flow (L/min) Mean Max Min Pulse LPV blood flow (L/min) Mean Max Min Pulse
0%
10%
20%
100%
4.38 ± 0.04 4.44 ± 0.02 4.35 ± 0.02 0.09 ± 0.01
4.32 ± 0.05 4.38 ± 0.03 4.29 ± 0.02 0.09 ± 0.01
5.20 ± 0.04§* 5.26 ± 0.03§* 5.08 ± 0.02§* 0.16 ± 0.01§*
7.25 ± 0.54§ 9.52 ± 0.36§ 7.12 ± 0.42§ 2.12 ± 0.25§
72 ± 03 97 ± 03 65 ± 04 32 ± 02
75 ± 04 99 ± 04 62 ± 03 35 ± 03
102 ± 09§ 118 ± 010§ 71 ± 08§ 47 ± 05§
112 ± 10§ 131 ± 14§ 90 ± 13§ 41 ± 09§
0.41 ± 0.08 0.57 ± 0.09 0.27 ± 0.06 0.30 ± 0.05
0.45 ± 0.06 0.55 ± 0.05 0.30 ± 0.02 0.27 ± 0.03
1.65 ± 0.08§* 1.95 ± 0.09§* 1.25 ± 0.06§* 0.70 ± 0.03§*
0.95 ± 0.09§ 1.12 ± 0.12§ 0.87 ± 0.11§ 0.25 ± 0.04§
4.33 ± 0.15 4.95 ± 0.10 4.09 ± 0.12 0.86 ± 0.05
4.34 ± 0.15 4.95 ± 0.10 4.12 ± 0.12 0.86 ± 0.05
4.80 ± 0.61 4.95 ± 0.54 4.85 ± 0.31 0.10 ± 0.05
4.85 ± 0.70 4.95 ± 0.53 4.81 ± 0.29 0.14 ± 0.06
0.56 ± 0.10 0.72 ± 0.11 0.47 ± 0.09 0.26 ± 0.06
0.60 ± 0.10 0.73 ± 0.11 0.48 ± 0.09 0.25 ± 0.06
0.68 ± 0.08 0.75 ± 0.05 0.61 ± 0.07 0.14 ± 0.05
0.40 ± 0.09 0.44 ± 0.08 0.28 ± 0.05 0.16 ± 0.03
0.39 ± 0.09 0.45 ± 0.08 0.28 ± 0.05 0.15 ± 0.03
0.33 ± 0.06 0.36 ± 0.06 0.31 ± 0.04 0.05 ± 0.01
0.80 ± 0.12 0.85 ± 0.13 0.61 ± 0.11 0.24 ± 0.09 n.d. 0 0 0 0
§
p
Minimal portal vein stenosis is a promising preconditioning in living donor liver transplantation in porcine model E. Gregoire1,⇑, P. Brige2, L. Barbier1, C. Buffat3,4, A. Coppola2, J. Hardwigsen1, Y.P. Le Treut1, V. Vidal2, P.H. Rolland2 1
Aix-Marseille University, Department of General Surgery and Liver Transplantation, Hôpital de la Conception Marseille, France; 2Aix-Marseille University, Experimental Interventional Imaging Laboratory, European Center for Medical Imaging Research, Marseille, France; 3Aix-Marseille University; URMITE, CNRS UMR 6236-IRD 198, Marseille, France; 4Laboratoire de Biochimie et de Biologie Moléculaire, Hôpital de la Conception Marseille, France
Background & Aims: The main hindrance in promoting living donor liver transplantation remains the morbi-mortality risk for the donor. Considering the opposed remodeling influence of portal and hepatic artery flows, our working hypothesis was to identify a lobar portal vein stenosis capable of inducing a contralateral liver mass compensatory enlargement, without the downstream ipsilateral atrophic response. Methods: Twenty-four pigs entered this study. Six of them were used to establish hemodynamic changes following a progressive left portal vein (LPV) stenosis, in blood flow, pressure and vessel diameter of the LPV, main portal vein and hepatic artery. Sixteen pigs were divided into 4 groups: sham operated animals, 20% LPV stenosis, 50% LPV stenosis, and 100% LPV stenosis. Daily liver biopsies were collected until post-operative day 5 to investigate liver regeneration and atrophy (Ki67, STAT3, LC3, and activated caspase 3) according to the degree of LPV stenosis. Finally, changes in liver volumetry after 20% LPVS were investigated. Results: A 20% LPV stenosis led to dilatation of the hepatic artery and a subsequent four-fold increase in hepatic arterial flow. Concomitantly, liver regeneration was triggered in the non-ligated lobe and the cell proliferation peak, 5 days after surgery, was comparable to that obtained after total LPV ligation. Moreover, 20% LPV stenosis preconditioning did not induce left liver atrophy contrary to 50 and 100% LPV stenosis. Conclusions: A 20% LPV stenosis seems to be the adequate preconditioning to get the remnant liver of living donor ready to take on graft harvesting without atrophy of the future graft. Ó 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Received 4 October 2013; received in revised form 10 February 2014; accepted 22 February 2014 ⇑ Corresponding author. Address: Aix-Marseille Université 13284, Service de Chirurgie Générale et Transplantation Hépatique, APHM – Hôpital de la Conception, 147, Boulevard BAILLE, 13005 Marseille, France. Tel.: +33 4 91 383 658; fax: +33 4 91 381 655. E-mail address: [email protected] (E. Gregoire). Abbreviations: HABR, hepatic artery buffer response; LPV, left portal vein; ALAT, alanine aminotransferase; ASAT, aspartate aminotransferase; GGT, Gammaglutamyl transferase; ALP, Alkaline Phosphatase; TB, total Bilirubin; FFPE, formalin-fixed, paraffin-embedded; HA, hepatic artery; LLW, left liver weight; TLW, total liver weight; TLV, total liver volume; BW, body weight.
Introduction The shortage of deceased donor organs compared to the number of patients on the waiting list for liver transplantation requires the use of other sources of grafts. The main hindrance in promoting living donor liver transplantation (LDLT) remains the morbi-mortality risk for the donor. This risk is correlated to the amount of resected parenchyma and to the volume of the donor remnant liver [1,2]. Unilateral portal vein ligation and percutaneous transhepatic portal vein embolization procedures can be performed prior to major hepatectomy. Both of these techniques occlude a lobar portal vein aiming to inducing the atrophy of the ipsilateral liver and thus the hypertrophy of the future remnant liver before hepatectomy. Nevertheless, the atrophic consequences of these techniques on the future liver graft preclude inclusion of these practices in the living donor preconditioning. The rationale for this study is that the hepatic artery buffer response (HABR) maintains a constant hepatic blood flow-to-liver mass. Indeed, by this mechanism, reduced portal flow leads to accumulation of adenosine into the space of Mall and hepatic arterial dilatation, thereby serving to buffer the impact that changes in portal flow have on total hepatic blood flow [3–5]. Based upon passive, reflex, and active mechanisms, HABR highly contribute to the liver proliferative response following portal flow changes. We also noted, from clinical and CT-scan personal observations in our patients, that a tumor-induced portal vein stenosis is frequently associated with substantial enlargement of the contralateral liver. Moreover, Belghiti et al. recently published an interesting control matched study of consequences following a right hepatectomy for living donation and benign liver lesions [6]. They conclude that right hepatectomy in LDLT induces a more severe deprivation of liver volume that could represent ‘‘inherent limitation’’ in healthy donors that makes them more vulnerable for postoperative complications [6]. Our working hypothesis was to identify a lobar portal vein stenosis in the living donor liver capable of inducing the basic contralateral liver mass compensatory enlargement (i.e., to
Journal of Hepatology 2014 vol. xxx j xxx–xxx
Please cite this article in press as: Gregoire E et al. Minimal portal vein stenosis is a promising preconditioning in living donor liver transplantation in porcine model. J Hepatol (2014), http://dx.doi.org/10.1016/j.jhep.2014.02.034
Research Article Liver living donor preconditioning (human)
R fu igh tu t re lo gr be af t
Left lobe future remnant liver donor
PRECONDITIONING right portal vein stenosis
C
Right liver harvesting
Animal model of liver living donor preconditioning (pig)
Right liver donor remnant liver
Left liver future graft
VII IV
VIII
II
VI
C
PRECONDITIONING left portal vein stenosis
V
Left liver harvesting Fig. 1. The human and porcine model for living donor liver preconditioning. Our hypothesis: minimal left portal vein stenosis enhance regeneration in the right liver (donor remnant liver) without atrophy of the left liver (future graft). Animal model: the right liver represents the future remnant liver of the donor and the left liver represents the future graft in the porcine model.
prepare the future remnant liver of the donor), without the downstream atrophic response (i.e., protect the future liver graft) (Fig. 1). 2
We here report our results of a new liver preconditioning based on moderate changes in the portal venous flow in a porcine model, and their consequences on hepatic remodeling.
Journal of Hepatology 2014 vol. xxx j xxx–xxx
Please cite this article in press as: Gregoire E et al. Minimal portal vein stenosis is a promising preconditioning in living donor liver transplantation in porcine model. J Hepatol (2014), http://dx.doi.org/10.1016/j.jhep.2014.02.034
JOURNAL OF HEPATOLOGY Materials and methods
lateral lobe were collected (Needle core biopsy BARD, 18 G). At day 7 animals were euthanized and the whole liver was removed, weighted, and separated into left (segment II, III, IV) and right liver (segment V, VI, VII, VIII, and I).
Animal model Because of its similarities in terms of human anatomy and hepatic metabolism, we have chosen the porcine model to experiment our hypothesis [7]. However, it was not possible to work on the right liver because of the absence of a unique right portal vein. Indeed, the main portal vein is divided in two veins for the right liver, which immediately penetrate in the liver parenchyma making them very difficult to control. The left portal vein, however, is unique and includes a proximal portion of a few centimeters which is easy to control. As the right and the left lobe are of equivalent size, we decided to reverse the human model using the right liver as the future remnant liver of the donor and the left liver as the future graft in the porcine model (Fig. 1). Animals Twenty four, six-month-old pigs (Pietrin), weighting 40 to 50 kg were used for these studies. After a 5-days period of acclimatization, all surgical procedures or samples procurements were performed between 8:00 AM and 12:00 AM. The experiments were terminated by intravenous injection of 15 mg midazolam and 25 mg chlorpromazine in 20 ml KCl (15%). The experiments described in this study were conducted according to the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes and after approval of the Ethical Committee of Provence. Phase A – hemodynamics study Six pigs were fasted 12 h before surgery with free access to water. After sedation by 30 mg/kg ketamine and 0.03 mg/kg acetopromazin, a venous catheter was inserted in a large vein in the ear for initial blood sampling and intravenous administrations. Induction of anesthesia was obtained by 2 mg/kg propofol. Anesthesia was maintained with gaseous sevoflurane (1.2%) by mechanical respiration (Zeus Dräger Inc.) and constant intravenous infusion of sufentanil (1 lg/kg/hour). Hemodynamic measurements was were carried as previously described in details [8–10]. Briefly, a midline abdominal incision was performed and ultrasound transit time flowmeter probes were positioned around the portal vein, the left portal vein and the hepatic artery for continuous measurement of blood flow (20, 7, and 3 RS probes, Transit Time Flow Meter, Transonic, Ithaca, NY, USA). Pressure-measurement catheters were placed into the main portal vein and the hepatic artery downstream of the flowmeter probes so as not to disturb flow measurements. Piezoelectric sensors were placed around the portal vein and the hepatic artery next to the flowmeter probes for continuous measurement of vessel diameter. All probes and sensors were recorded on a multichannel LabView based PXi system (National Instrument). Then, a portal vein band was placed around the left portal vein upstream of the flowmeter probe including an external rigid ring (polypropylene) and an internal inflatable balloon (silicone) in order to progressively reduce left portal flow. The ‘‘Zero’’ calibration was checked for each animal. All parameters were recorded simultaneously, a 15 min stabilization, under basal conditions and after each left portal flow modification during 15 min. The balloon was inflated milliliter by milliliter and the degree of the stenosis was verified on successive portography in order to obtain 10, 20, 30, 50, 60, 65, 70, 75, 80, 90, and 100% left portal vein stenosis (LPV). At the end of the experiments, the animals were euthanized. Phase B – biological, cellular and molecular consequences of LPVS Surgery and samples collection Twelve pigs were divided in 4 groups: sham operated animals (group 0, n = 3), 20% LPV stenosis (group 20, n = 3), 50% LPV stenosis (group 50, n = 3), and 100% LPV stenosis (group 100, n = 3). In each group, the animals were anesthetized as described above. In each animal, the left portal vein was isolated (day 0). Deep Liver biopsies were performed on the right and left lobes and blood samples were collected. The circumference of the left portal vein was directly measured by passing a silk thread around the vessel. A non-stretch stripe of polypropylene whose length was calculated to induce a 20, 50, or 100% stenosis was sewed around the left portal vein. Sham operated animals underwent only portal dissection and basal portography. Every day, from day 1 to day 5, animals were anesthetized (ketamine 1 mg, acepromazine 10 mg, IM). Blood samples and USguided percutaneous deep liver biopsies from the left lateral lobe and the right
Samples analysis Biological consequences of LPVS Serum alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), gamma-glutamyltransferase (GGT), alkaline phosphatase (ALP) and total bilirubin (TB) were measured with the biochemical multi-analyser in the hospital biochemistry department. Prothrombin time was measured with STA-R (diagnostica stago Asniere France) in the hospital hematology department. Cellular consequences of LPVS Cell proliferation: Ki67 immunohistochemistry using MIB-1 antibody was performed on 5-micron-liver sections formalin-fixed, paraffin-embedded (FFPE) tissues from right and left liver daily biopsies. The Leica BOND-III fully automated IHC stainer was used with high-efficiency de-waxing processes, EDTA-based Ag unmasking procedures and IHC with appropriate NovoCastra antibodies and final DAB-horseradish peroxidase stainings and hematoxylin counterstaining. Proliferation index was determined by the percentage of cells that display nuclear staining reported to the total number of nuclei (ten fields per slide, 400). Nuclear density and lobar area: morphometric analysis were performed using the Nikon Eclipse Ni H600L microscopic and image analysis systems. Molecular consequences of LPVS Bilateral liver biopsies 3 and 24 h after surgery for Group 20 were analyzed for the IHC detection of STAT3 by the technique described above using Leica IHC Bond-III stainer and Nikon Eclipse image analyzer. Analysis of LC3-II was made of two ways: IHC analysis on liver biopsies from D0 to D5 for 4 groups and analysis of mRNA expression by PCR on liver biopsies from D0 D3 and D7. The presence of activated caspase 3 was analyzed on biopsies IHC at D5 for all 4 groups. The quantification of the labeling was performed by counting the number of labeled nuclei on the total number of nuclei. Phase C – hepatic remodeling after 20% LPV stenosis Six pigs were used for this phase. All animals were weighed 3 days before and 14 days after surgery. An abdominal CT scan was performed for each animal under general anesthesia, 3 days before (D-3) and 11 days after surgery (D11). The apparatus used was a veterinarian scanner monobaret (Cabassu Clinic, Marseille) of the General Electric Company (GE). The acquisition was started 80 s after the IV injection of iodinated contrast (50 ml Telebrix). Three millimeters slides were done every 2 mm. Volumetric reconstruction was performed using the ADW server software (GE). Surgical procedure was similar to the Phase B. The animals were divided into 2 groups: a ‘‘test’’ group with a 20% LPVS (n = 3) and a control group (n = 3), consisting of 2 sham operated animals ( = Group 0) and an animal with a ligature of the LPV ( = Group 100). The animals were euthanized 14 day after surgery. Statistics Data treatment and statistical analyses (Kruskall-Wallis non-parametric anova and Mann-Whitney U test) were performed by using Systat12 (SPSS Inc, Chicago, IL) and Prism 5.0 (Graphpad Prism, La Jolla Inc). A p value less than 0.05 was considered significant. A non-parametric kernel density estimator was preferred to rule out a functional form on the distribution function curves. Results were expressed as mean ± SD.
Results Hemodynamic study according to an increasing left portal vein stenosis We did not observe any significant modification of hemodynamic parameters (blood flow, blood pressure and diameter in the main portal vein, left portal vein and hepatic artery (HA) when a 10%
Journal of Hepatology 2014 vol. xxx j xxx–xxx
3
Please cite this article in press as: Gregoire E et al. Minimal portal vein stenosis is a promising preconditioning in living donor liver transplantation in porcine model. J Hepatol (2014), http://dx.doi.org/10.1016/j.jhep.2014.02.034
Research Article Table 1. Hepatic artery hemodynamics according to the degree of left portal vein stenosis.
% LPVS Hepatic artery diameter (mm) Mean Max Min Pulse Hepatic artery blood pressure (mmHg) Mean Max Min Pulse Hepatic artery blood flow (L/min) Mean Max Min Pulse Portal vein blood pressure (mmHg) Mean Max Min Pulse Portal vein blood flow (L/min) Mean Max Min Pulse LPV blood flow (L/min) Mean Max Min Pulse
0%
10%
20%
100%
4.38 ± 0.04 4.44 ± 0.02 4.35 ± 0.02 0.09 ± 0.01
4.32 ± 0.05 4.38 ± 0.03 4.29 ± 0.02 0.09 ± 0.01
5.20 ± 0.04§* 5.26 ± 0.03§* 5.08 ± 0.02§* 0.16 ± 0.01§*
7.25 ± 0.54§ 9.52 ± 0.36§ 7.12 ± 0.42§ 2.12 ± 0.25§
72 ± 03 97 ± 03 65 ± 04 32 ± 02
75 ± 04 99 ± 04 62 ± 03 35 ± 03
102 ± 09§ 118 ± 010§ 71 ± 08§ 47 ± 05§
112 ± 10§ 131 ± 14§ 90 ± 13§ 41 ± 09§
0.41 ± 0.08 0.57 ± 0.09 0.27 ± 0.06 0.30 ± 0.05
0.45 ± 0.06 0.55 ± 0.05 0.30 ± 0.02 0.27 ± 0.03
1.65 ± 0.08§* 1.95 ± 0.09§* 1.25 ± 0.06§* 0.70 ± 0.03§*
0.95 ± 0.09§ 1.12 ± 0.12§ 0.87 ± 0.11§ 0.25 ± 0.04§
4.33 ± 0.15 4.95 ± 0.10 4.09 ± 0.12 0.86 ± 0.05
4.34 ± 0.15 4.95 ± 0.10 4.12 ± 0.12 0.86 ± 0.05
4.80 ± 0.61 4.95 ± 0.54 4.85 ± 0.31 0.10 ± 0.05
4.85 ± 0.70 4.95 ± 0.53 4.81 ± 0.29 0.14 ± 0.06
0.56 ± 0.10 0.72 ± 0.11 0.47 ± 0.09 0.26 ± 0.06
0.60 ± 0.10 0.73 ± 0.11 0.48 ± 0.09 0.25 ± 0.06
0.68 ± 0.08 0.75 ± 0.05 0.61 ± 0.07 0.14 ± 0.05
0.40 ± 0.09 0.44 ± 0.08 0.28 ± 0.05 0.16 ± 0.03
0.39 ± 0.09 0.45 ± 0.08 0.28 ± 0.05 0.15 ± 0.03
0.33 ± 0.06 0.36 ± 0.06 0.31 ± 0.04 0.05 ± 0.01
0.80 ± 0.12 0.85 ± 0.13 0.61 ± 0.11 0.24 ± 0.09 n.d. 0 0 0 0
§
p
