Functional shift with maintained regenerative potential following portal vein ligation

Selective portal vein ligation (PVL) allows the two-stage surgical resection of primarily unresectable liver tumours by generating the atrophy and hypertrophy of portally ligated (LL) and non-ligated lobes (NLL), respectively. To evaluate critically important underlying functional alterations, present study characterised in vitro and vivo liver function in male Wistar rats (n = 106; 210–250 g) before, and 24/48/72/168/336 h after PVL. Lobe weights and volumes by magnetic resonance imaging confirmed the atrophy-hypertrophy complex. Proper expression and localization of key liver transporters (Ntcp, Bsep) and tight junction protein ZO-1 in isolated hepatocytes demonstrated constantly present viable and well-polarised cells in both lobes. In vitro taurocholate and bilirubin transport, as well as in vivo immunohistochemical Ntcp and Mrp2 expressions were bilaterally temporarily diminished, whereas LL and NLL structural acinar changes were divergent. In vivo bile and bilirubin-glucuronide excretion mirrored macroscopic changes, whereas serum bilirubin levels remained unaffected. In vivo functional imaging (indocyanine-green clearance test; 99mTc-mebrofenin hepatobiliary scintigraphy; confocal laser endomicroscopy) indicated transitionally reduced global liver uptake and -excretion. While LL functional involution was permanent, NLL uptake and excretory functions recovered excessively. Following PVL, functioning cells remain even in LL. Despite extensive bilateral morpho-functional changes, NLL functional increment restores temporary declined transport functions, emphasising liver functional assessment.


Conventional liver lobe analysis -weight and volume
In the sample harvest group, after bile collection and exsanguination, ligated lobes (LL) and non-ligated lobes (NLL) of the liver were harvested, and their wet weights were determined gravimetrically (AG 245, Mettler-Toledo LLC, Columbus, OH; confidence: 0.01mg/0.1mg).
Another group of animals were subjected to serial hepatobiliary scintigraphy (HBS) and magnetic resonance imaging (MRI) volumetry [coronal T1-weighed gradient echo sequencing, 128 axial slices of 0.4 mm thickness] (nanoScan PET/MRI; Mediso Ltd., Budapest, Hungary). In vivo liver lobe volumes were determined following manual delineation of LL and NLL on each axial slice and three-dimensional reconstruction in a 160x160 matrix. Both parameters (weight and volume) were ultimately expressed as a fraction of body weight (bw).

Hepatocyte isolation and generation of cell cultures
For hepatocyte isolation, a three-step retrograde perfusion procedure was applied, since the portal vein branches of the median-, left lateral-and caudate lobes had been ligated. A glass cannula was inserted into the suprahepatic inferior vena cava, and the liver was first flushed with 300 ml of Ca 2+ -free Earle's balanced salt solution (EBSS) (self-produced) containing EGTA. The liver was then perfused with the same buffer without the chelating agent and finally with EBSS containing Ca 2+ and type IV collagenase (prepared from Clostridium histolyticum, Sigma-Aldrich, St. Louis, MO) for approximately 5 min. The portal vein was incised to allow efflux. The flow rate was 30 ml/min and all perfusion solutions were preoxygenised and temperature-controlled at 37°C. Perfusion was considered successful when the whole organ was completely blanched and the endpoint when the tissue was visibly digested and the capsule started to separate from the liver surface after approximately 5 mins.
3 Following the precise separation of the LL and the NLL, cell viability was determined by trypan blue exclusion. From the separate suspensions of the LL and the NLL, only hepatocytes from preparations with more than 90 % viability were plated at a density of 2.0 x 10 6 cells/well on 6-well plates, and 0.36 x 10 6 cells/well on 24-well plates, in William's Medium E containing 5% of fetal calf serum, 0.1μM insulin, 0.05 μM glucagon, 0.05 mg/ml gentamicin, 30 nM Na2SeO3, and 0.1μM dexamethasone. Calf serum was present for the first 24h and then omitted. Cells were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2. Twenty-four hours after plating, cells were overlaid with matrigel basement membrane matrix (Matrigel Matrix; SoftFlow Hungary, Pecs, Hungary) at a concentration of 0.25 mg/ml in 2 ml of ice-cold William's Medium E supplemented with insulin, glucagon, gentamicin, dexamethasone, and Na2SeO3 to achieve sandwich configuration. The culture medium was replaced every 24h.

In vitro Ntcp, Bsep and ZO-1 immunofluorescence staining
For immunofluorescence (IF) staining of isolated hepatocytes, the cells 72h in culture were fixed and permeabilised with 4% paraformaldehyde and 0.1% Triton X-100 in phosphate

In vitro bilirubin transport experiments
Bilirubin transport experiments were performed 72h after culturing as described previously. 1 Briefly, cells were incubated with 10 μM bilirubin for 5 min in standard Hanks' Balanced Salt Solution (HBSS) at 37°C. After washing off the uptake medium, the cells were incubated with either standard or Ca 2+ /Mg 2+ free HBSS containing 1 mM EGTA. Efflux was allowed for 10 min, after which the cells were lysed with an acetonitrile/water solution [30% (v/v)]. The amounts of bilirubin and its mono-and diglucuronide conjugates (BMG, BDG) in the efflux medium and in the cell lysates were analysed by high-performance liquid chromatography (HPLC). In standard HBSS, the bile canalicular networks maintain their integrity, whereas in calcium-free HBSS the tight junctions are disrupted and the content of the canaliculi leaks into the efflux medium. The biliary transport was determined by subtracting the amount of bilirubin and its conjugates in standard efflux medium from that in Ca 2+ /Mg 2+ free efflux medium, and was expressed as nmol/mg protein. Sinusoidal transport was determined in the standard efflux medium. The intracellular accumulation was measured in the lysate of cells incubated in Ca 2+ /Mg 2+ free medium. Assays were run using three wells as one set from at least three cell preparations. The HPLC method for analysing bilirubin and conjugated bilirubin (BG) was performed as described previously 1 . 5 In vitro taurocholate transport experiments Taurocholate (TC) uptake experiments were performed 24h after plating, as described previously. 2 Briefly, the wells were washed once with HBSS. The uptake experiment was started by the addition of HBSS containing 1 μM 3 H-TC and lasted for 1 min at 37°C. The uptake was terminated by the removal of the substrate-containing buffer, and the wells were washed three times with ice-cold HBSS. Then the cells were lysed with 0.5% Triton X-100 solution. The intracellular radioactivity was determined by liquid scintillation counting.
Assays were run using four wells in one set; all experiments were carried out with hepatocytes from three independent cell preparations. TC efflux experiments were performed similarly to that of with bilirubin transport 72h after plating. 3 H-TC was applied at 1 μM. The uptake period was 1 min and the efflux lasted for 10 min. Following the efflux period, the cells were lysed with 0.5 % Triton X-100 in PBS. The amount of TC in the efflux medium and the cell lysates were determined by liquid scintillation counting. Assays were run using three wells as one set. All experiments were carried out with cultures from at least three independent cell preparations. Data were normalised for the protein content analysed in wells where the cells were incubated with standard HBSS (Pierce BCA Protein Kit, Thermo Scientific).

In vivo bilirubin transport experiments
The amount of bilirubin, and BG metabolites [BMG: bilirubine monoglucuronide; BDG: bilirubine diglucuronide] in the bile and serum samples, collected as described in the article, was determined by HPLC with the same method as in case of the in vitro experiments. The bile samples were diluted hundredfold with distilled water, and the protein content of the serum was precipitated with 3x volume of acetonitrile before injection.
In vivo Ntcp and Mrp2 immunofluorescence staining 6 For in vivo immunohistochemistry, liver samples of identical anatomical loci of the LL and NLL of control, 72h and 336h survivors were excised after liver weight measurements, and immediately frozen in liquid nitrogen. Tissue sections were cut by cryomicrotome, fixed in pre-chilled methanol (-20°C) for 10 min, and blocked with the same blocking buffer as described with in vitro IF. The samples were subjected to K4 rabbit anti-Ntcp antibody

Indocyanine-green clearance test
The indocyanine-green (ICG)-clearance test was performed similarly to the previous study of our workgroup 3 . The medial side of the left upper thigh was shaved, and a neonatal laser probe (PV50200 disposable sensor for neonates; PULSION Medical Systems, Feldkirchen, Germany) of a commercially available analysing device (PC5000 LiMON; PULSION Medical Systems) for ICG densitometry was fixed with an elastic bandage. Following automatic calibration and test initiation, 1 ml/bwkg of 1.5 mg ICG/ml distilled water was injected into the lateral tail vein. Results were displayed 5-6 minutes later as plasma disappearance rate (PDR) and 15-minute retention (RT15) values. Ltd., Budapest, Hungary) was acquired as projections from four separate angles in a resolution of 256 x 256 using Ultrahigh Resolution (UHR) parallel septal collimator (NanoSPECT-UHR, Mediso Ltd, Budapest, Hungary). A dynamic protocol of three different phases was used including 20/6/2 frames per minutes for 2/4/35 minutes, respectively, to monitor the rapid uptake and the canalicular elimination of the tracer. Recordings were evaluated with manual allocation of elliptic regions of interest (ROI) to the anteroposterior projection corresponding to the blood pool, as well as the LL and NLL. With respect to its more elusive position, the duodenal ROI was placed considering projections from all four angles. The characteristic parameters derived from the kinetics curves included the blood halflife (B1/2), first duodenal appearance (DSTART), as well as regional-specific information of LL and NLLtime of maximum, tracer half-life, and relative ratio of LL or NLL peak counts (PC) to corresponding blood counts.

Confocal laser endomicroscopy
Following laparotomy, the inferior right lateral lobe of the liver was mobilised, and carefully placed on a fastened plastic foil, elevated to the level of about the median axillary plane while preserving portal inflow, to attenuate respiration-associated movement. The endoscopic wire probe of the laser unit (Cellvizio, MaunaKea Technologies, Paris, France) was harmlessly pressed against and stabilised on the medial surface of the inferior right lateral lobe. After an injection of ICG (1 ml/bwkg of 1.5 mg ICG/ml distilled water) into the tail vein, a 40-minute time lapse was acquired. Video evaluation was performed by manual ROI allocation to liver 8 acini with the exclusion of large vessels. Exponential growth and decay were used to determine time of signal maximum (TMAX) and ICG half-life (T1/2) values, respectively. were also absent from 336h NLL sections, whereas liver acini were notably enlarged (f).

Supplementary
Summarizing, the course and characteristics of histopathological alterations pertaining to presence of necroapoptotic lesions and cellular-and liver acini size were diverging between LL and NLL. Observed changes are in accordance with previously described histological alterations following PVL. Furthermore, these are in great corroboration with the current observations of immunofluorescence staining of ex vivo liver specimens for sodiumtaurocholate cotransporting polypeptide (Ntcp) and canalicular multispecific organic transporter (Mrp2) immunofluorescence (see Figure 4) showing the same pattern of cellular and liver acini size changes, and similar characteristics of necroapoptotic lesions in the LL at