Experimental animals.
Three-week-old, female, DPPIV-deficient Fischer 344/DuCrlCrlj rats were purchased from Charles River Laboratories Japan, Inc. (Kanagawa, Japan). DPPIV-positive Fischer 344/NSlc rats were purchased from Japan SLC, Inc. (Shizuoka, Japan). All animal experiments were performed according to the ethical guidelines of the Animal Research Center in Medical College of Yokohama City University. The institutional animal care use committee of Yokohama City University approved all our animal studies (approval no. 17–25). We confirm that the study was carried out in compliance with the ARRIVE guidelines.
Induction of liver cirrhosis in rats.
Before transplantation, 5-week-old DPPIV-deficient Fischer 344 rats were injected into peritoneal cavity with 10 mg/kg DMN (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for three consecutive days for two weeks. Transplantation was performed after three days of wash out period. Starting the day after transplantation, the rats were injected DMN into peritoneal cavity for three days again and dissected three weeks after the transplantation.
Methods for detachment of the serous membrane.
Five detachment methods were used in the present study: needle, once-used electric scalpel, twice-used electric scalpel, adhesive, and ultrasonic homogenizer. In the needle method, 2–3 mm of serous membrane of the left lobe of the liver was detached using an 18G needle (Terumo, Tokyo, Japan) to achieve a depth of 1 mm. In the electric scalpel methods, an area with a diameter of 2–3 mm was cauterized on the surface of the left lobe of the liver using an electric scalpel (Braintree Scientific, MA, USA) either once or twice, and the cauterized area of serous membrane was detached by tweezers. In the adhesive method, 2 µL Vetbond™ tissue adhesive (3M, MN, USA) was dropped on the surface of the left lobe of the liver and the solidified portion was detached by tweezers. In the ultrasonic homogenizer method, several drops of saline were dropped on the surface of the left lobe of the liver and 2–3 mm of serous membrane was detached using an ultrasonic homogenizer (Yamato Scientific, Tokyo, Japan) to achieve a depth of 1 mm. All detachment methods were performed on two separate places of the left lobe of the liver for each rat.
Transplantation of rat fetal livers.
Fetal liver tissues were harvested from 14-day-old embryos of pregnant DPPIV-positive Fischer 344 rats and used as donors for transplantation. To harvest the donor tissue, pregnant rats were euthanized, fetuses were collected, and whole fetal livers were isolated under a microscope. The collected whole fetal livers were stored in phosphate-buffered saline (PBS) on ice until transplantation. For transplantation, DPPIV-deficient Fischer 344 rats underwent laparotomy under anesthesia and portal blood flow was blocked with a vascular clamp. Following the detachment of serous membrane on the surface of left lobe of the liver by the indicated method, bleeding was stopped using pressure with cotton swabs and the whole fetal livers were transplanted. The left lobe was covered with the middle lobe, followed by removal of the vascular clamp and abdominal closure.
Measurement of bleeding.
The weight of cotton swabs were weighed before and after their use for hemostasis following serous membrane detachment, as described above. The cotton swabs were stored in tubes containing moistened tissue papers until measurement to prevent drying. The bleeding amount (mg/min) was determined by subtracting the weight after hemostasis from the weight before hemostasis.
Assessment of liver function.
Concentrations of AST, ALT, NH3, and T-Bil in plasma were measured by a DRI-CHEM 7000V analyzer (FUJIFILM, Tokyo, Japan).
Enzyme histochemistry for DPPIV.
Frozen tissue sections, 7 µm in thickness, were fixed in a mixed solution of acetone (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan):chloroform (FUJIFILM Wako Pure Chemical Corporation) at 1:1 ratio for 10 min at 4 °C. Next, the sections were air-dried and incubated in a staining solution prepared by mixing 8 mg glycyl-prolyl-4-methoxy-β-naphthylamide hydrochloride (Sigma-Aldrich, MO, USA) dissolved in 1 mL dimethyl sulfoxide (FUJIFILM Wako Pure Chemical Corporation) and 1 mg Fast Blue BB Salt hemi zinc chloride salt (Sigma-Aldrich) dissolved in 1 mL PBS (pH = 7.0) at a 1:20 ratio. After incubation for 20 min at room temperature, the tissue sections were incubated for 5 min in 2% CuSO4 and rinsed with MQ and fixed with 10% formalin. After washing with MQ, the sections were counterstained with Carazzi’s hematoxylin (Muto Pure Chemicals, Tokyo, Japan). Images were acquired using a V120 Virtual Slide microscope (Olympus, Tokyo, Japan). Areas positive for DPPIV were quantified using ImageJ.
Immunohistochemical staining.
Unfixed frozen tissue sections were fixed in 1:1 acetone/methanol solution for 30 min at − 30 °C. After washing with PBS with 0.05% Tween20, the slides were blocked with the serum-free Protein Block reagent (Dako) for 1 h at room temperature. Next, the slides were incubated overnight at 4 °C with primary antibodies diluted in the blocking reagent. The following antibodies were used: anti-CD26 (559639, 1:100; BD Biosciences, San Jose, CA, USA), anti-CD31 (550300, 1:50; BD Biosciences), anti-CK19 (61029, 1:200; Progen Biotechnik, Heidelberg, Germany), anti-albumin (NBP1-32458, 1:200; Novus Biologicals, Littleton, CO, USA), anti-HNF-4α (clone H-1, sc-374229, 1:200; Santa Cruz, CA, USA). Next, the slides were incubated for 1 h at room temperature with appropriate Alexa Fluor 488-, 555-, or 647-conjugated secondary antibodies and counterstained with DAPI (4’,6-diamidino-2-phenylindole dihydrochloride, 1:1000; FUJIFILM Wako Pure Chemical Corporation). Images were acquired using the V120 Virtual Slide microscope. Areas positive for CD26, CD31, HNF-4α, or albumin were divided by the entire area of transplanted fetal liver to calculate areas positive for the indicated marker.
Statistical analysis.
Statistical analyses were performed by non-repeated measures analysis of variance with Bonferroni correction. Data were presented as means ± standard error of the mean. P values < 0.05 were considered to indicate statistical significance.