Female Wistar rats (Rattus norvegicus, Charles River Italy Inc.), raised in GR900 cages (5 animals per cage) (Tecniplast) with JELUXYL HW 300/500 (JELU) bedding, under constant environmental conditions (room temperature 21-23˚C, humidity 40-60%, 12-hour light/dark cycle, air changes 25 volumes/hour) and ad libitum access to VRF 1 (P) feed (SDS) and tap water.
ISOLATION AND PERFUSION OF THE LIVER
Method and ethics
The surgical procedure is according to 7, whereas the perfusion of the isolated liver with HBBS and PBS is new. Obtain the prior approval of animal experimentation from the competent bioethical authority. Justify the use of animals, by applying reasons for the 3R principles, e.g.:
§ There are no in vitro experimental models preserving the bile flow through the biliary system 6 (no available replacement).
§ The procedure causes no sufferance to the anaesthetized rat (maximum achievable refinement).
§ Repeated tests of BR (up to 12) can be performed in the same liver preparation, thus increasing the number of observations in the same liver, thus limiting the animal number (reduction by a factor of 10).
Anesthetize animals (> 220 g b.w.) by an intraperitoneal injection of 0.5 mL containing Zoletil (2 mg/kg body weight) and Xylazine (8 mg/kg body weight).
§ Perform a horizontal laparotomy in the lower abdomen, by lifting the abdominal wall with the tweezers. Start the cut from the median point, continue towards each lateral side, and then turn vertically upwards, up to the lowest rib. Take care not to cut the diaphragm, the chest and its respiratory muscles at this stage.
§ Displace the intestine to the left side to expose the hepatic hilus, with the portal vein and the common bile duct.
§ Prepare a ligature around the inferior vena cava, above the insertion of the right renal vein, and a second one around the portal vein (about 1 cm distally from the hilus), using a two-strand sewing cotton thread.
§ At this stage, consider to prepare the common bile duct for cannulation, if planned in the experiment (not described in this protocol).
§ To prevent blood clotting, inject a bolus of heparin (500 U in 0.25 mL PBS) into the inferior vena cava by an insulin syringe.
§ Activate the peristaltic pump at 1 mL/min in order to fill the silicon tubing with the perfusion solution. Allow it to leak dropwise.
§ Insert the inlet catheter (20 G winged & ported) into the portal vein and secure it by tightening the ligature around the portal vein.
§ Connect the inlet catheter to the “perfusion tubing terminal” (see above, Labware).
§ Perfuse the liver at a flow rate of 4 ml/min and at a constant temperature of 37 °C.
§ Incise the inferior vena cava caudally to the ligature, to allow blood and perfusion buffer to outflow from the liver, so to maintain normal intrahepatic pressure.
§ Perform a thoracotomy starting from the diaphragm up to the neck, along two lateral lines.
§ Insert the 18 G catheter through the right atrium into the inferior vena cava, secure it with a tight ligature.
§ Connect the screw cap on the PEEK tubing (OD 2 mm, ID 1.8 mm) to this catheter, to collect the liver perfusion effluent.
§ Close the inferior vena cava, above the tributary renal vein, by tightening the ligature, to prevent retrograde perfusion of the liver.
§ Accelerate the peristaltic pump to deliver the intra-portal perfusion HBSS solution at 6 mL/min for 20 min (110 mL), to exsanguinate the liver and prepare it for BR uptake tests (read Tip 6).
§ Switch the liver perfusion buffer from HBSS to PBS and perfuse until the effluent is clear (read Tip 7).
BR AND BRG UPTAKE TESTS
Sinusoidal administration of BR and BR uptake inhibitors
§ Prepare a set of at least 120 vials (1.5 ml) and 12 tubes (10 mL) for the collection of the effluent fractions.
§ Insert a 1 mL-syringe, filled with 0.2 mL BR solution, into the bolus delivery port on the tubing connected to the inlet catheter (read Tips 8-9).
§ Inject the BR bolus (0.2 mL) in 2 seconds and keep the syringe in place.
§ Collect 10 effluent fractions (0.4 mL each; 4 sec/fraction) into 1.5 ml vials (read Tip 10).
§ Remove the 1 mL-syringe from the portal cannula and continue the liver perfusion for 1 min (6 mL) before the next BR bolus and collect this inter-bolus fraction in a 15 mL-tube.
§ Inject the second BR bolus (0.2. mL), as described above.
§ Continue the liver perfusion for 1 min (6 mL), as above.
§ Repeat BR boli injections up to 12 times.
HEPATIC VENOUS EFFLUENT ANALYSIS
Bilirubin and Bilirubin glucuronide
Perform the fluorometric analysis of BR by means of the Help-UnaG fusion protein (HUG) 8, after fluorescence calibration with a BR standard solution. Check for interference of BR-specific fluorescence by the compounds added with BR. Analyze BRG as BR equivalents, after hydrolysis by β-Glucuronidase.
Calibrate fluorescence intensity emitted by the HUG•BR complex by incubating a HUG solution at a fixed concentration with serial bilirubin standard solutions. HUG must be in molar excess of the highest BR concentration.
§ Prepare 5 mM BR in DMSO (Solution A).
§ Use Solution A to prepare 0.01 mM BR (Solution B) in PBS:DMSO (99.7:0.3, vol/vol) (read Tip 11).
§ Use Solution B to prepare the series of standard BR solutions (0-0.05 µM in PBS:DMSO, 99.7:0.3, vol/vol), as in Figure 2.
§ Add 0.2 mL of each standard BR solution in a 96 well-plate containing 0.01 mL HUG solution (0.4 mg/mL; 6.62 µM, 66.2 pmoles) solution.
§ Record fluorescence intensity in the microplate reader (λex = 485 nm; λem = 528 nm).
Analysis of assay interferences
When planning to co-inject BR with other compounds via intra-portal boli, perform a prior check for their possible interference with the BR fluorometric assay (read Tip 12).
§ Test the compounds at 1/10 their concentration in the bolus, to take account of their dilution in the sinusoids (read Tip 13).
§ Use the concentrated solutions of compounds in DMSO, as described above (section bolus preparation): E17G, Indomethacin and Ketoprofen (50 mM); C3G, M3G, P3G, TC (100 mM); Pravastatin (300 mM).
§ Dilute 10 µL of these concentrated solutions in 5 mL of standard BR as in the table below, to obtain BR standard solution containing 0.1 mM E17G or Indomethacin or Ketoprofen, 0.2 mM C3G or M3G or P3G or TC, and 0.6 mM Pravastatin, as in Figure 3.
The final solvent composition is PBS:DMSO (99.5:0.5, vol/vol). This has no effect on the HUG BR assay performance, while simplifying the preparation protocol. Fluorescence intensity of BR standard solutions ranges 0-26000 ± 2700.
§ Prepare a 96-well plate, pre-filled with 0.010 mL HUG solution, setting aside 20 wells for serial dilutions of at least 5 standard BR solutions (0-50 nM range).
§ Add 0.2 mL of each fraction to this 96-well plate. Keep it covered.
§ Incubate at 25°C for 1 h.
§ Record fluorescence in the microplate reader (λex = 485 nm; λem = 528 nm), to obtain fluorescence reading 1 (F1), corresponding to sample [BR].
§ Withdraw the plate from the instrument and add 0.01 mL (8 IU) β-Glucuronidase solution to each well (including those with standard BR solutions).
§ Incubate at 25°C for 2 h and record the increase of fluorescence, as above. Or, store the covered plate at 4°C overnight.
§ Repeat fluorescence recording, as above, to obtain fluorescence reading 2 (F2), corresponding to sample [BR] + [BRG].
§ Calculate [BRG] = F2 – F1.
Liver functional tests
Assess the liver functional integrity of the liver preparation by analyzing the hepatic venous effluent for biomarkers of microcirculation integrity (albumin-FITC), of possible cytolysis (LDH), membrane permeabilization (ATP) and lipoperoxidation (MDA).
Albumin - FITC
§ Inject a 0.2 mL bolus of BSA-FITC solution (5.15 g/L, or 77.5 μM, containing 3% BSA-FITC, w/w) into the portal vein (read Tip 14).
§ Collect 15 fractions, 0.7 mL/each.
§ Prepare a 96-well plate, setting aside 10 wells for serial dilutions of a standard BSA – FITC solution (ranging 0.0036 μM - 0.45 μM).
§ Transfer 0.1 mL of each perfusion effluent fraction in a black multiwell plate.
§ Record FITC-related fluorescence in the microplate reader (λex = 485 nm; λem = 525 nm).
§ Calculate the concentration of BSA-FITC in each fraction by using the standard curve (mg/mL). Sum the amount (mg) of BSA-FITC of all fractions and express this value as percent of the injected dose (% recovery).
Lactate dehydrogenase activity
§ Prepare a 96-well plate pre-filled with 0.10 mL of In Vitro Toxicology Assay Kit solution.
§ Add 0.05 mL of inter-boli samples (as indicated in assay kit protocol) and incubate up to 30 min.
§ Add 0.015 ml of the stop solution (1 M HCl).
§ Record absorbance (OD490-690) in the microplate reader.
§ LDH (EC 184.108.40.206) activity assay is based on the reduction of INT to INT-formazan (e490 = 1.42 x 104 M-1 cm-1). Express LDH activity as U/L of liver perfusion effluent (1 U = µmol of INT-formazan min-1).
§ Prepare a 96-well plate pre-filled with 0.1 mL of In Vitro Toxicology Assay Kit solution, setting aside 16 wells for serial dilutions of a standard ATP solution (ranging 0.00001 - 0.01 mM in PBS).
§ Add 0.10 ml of effluent fractions to estimate the concentration of ATP in the effluent throughout the period of the liver perfusion.
§ Incubate 10 min at RT.
§ Record luminescence in the microplate reader at λex = 485 nm and λem = 528 nm.
§ Express the tissue ATP concentration as picomoles/g of wet tissue.
Analyse malondialdehyde (MDA) by the method of Agarwal 12, with some modifications, on an HPLC instrument, equipped with a GP50 quaternary pump, an AD25 UV-Visible detector, a GF2000 in-line fluorescence detector, and Chromeleon software, version 6.8, for data analysis (Dionex Corporation; Sunnyvale, CA, USA).
8.34 µM TEP. Dilute 0.2 mL 1,1,3,3-tetraethoxypropane (TEP, CAS: 122-31-6) in 1 L EtOH:H2O (40:60, vol/vol). Then, dilute 1 mL of the latter in a 100 mL EtOH:H2O (40:60, vol/vol).
Prepare the following standard solutions in 5 mL glass flasks, as shown in Figure 4.
0.05% (w/vol) BHT ethanolic solution: dissolve 50 mg Butylated hydroxytoluene (CAS 128-37-0) in 100 mL 95% ethanol.
0.44 M phosphoric acid solution: dilute 86 mL concentrated H3PO4 (85%, 5.102 M) to the final volume of 1L.
42 mM TBA solution: dissolve 6.054 g 2-thiobarbituric acid (CAS 504-17-6) in 1 L water and stir at T = 50 °C.
§ In screw cap micro tubes, 1.5 ml (Sarstedt, code 72.692), add 200 µL of either sample or standard, 80 µL of BHT and 136 µL of 0.44 M phosphoric acid solution. Vortex and wait 10 min at room temperature.
§ To each tube, add 232 µL TBA and 110 µL water. Vortex and incubate at 100°C in the oven for 1 h. Then, chill tubes on ice for 5 min.
§ Spin at 14000 rpm for 3 min.
§ Filter the supernatant on Durapore 0.45 µm (Durapore ®, Millipore) in vials.
§ Inject 25 µL sample or standard into the HPLC column (Water Symmetry C18 column; 5 μm and 250×4.6 mm).
§ HPLC conditions. Flow rate: 0.8 mL/min; mobile phase: 50 mM phosphate buffer pH 6.8: methanol (60:40, vol/vol); T = 30 °C; Fluorescence detection: λex = 515 nm, λem = 553 nm; UV-vis detection: λ = 532 nm.