Nembutal

Cultures of mouse hepatocytes were prepared as previously described [23 (link)]. Briefly, C57BL/6 male mice or Nrf-2 deficient mice (B6/SJL background), 6–8 weeks old, were anesthetized with Nembutal (Dainippon Sumitomo Pharma, Japan) intraperitonealy and their livers were perfused with 40 ml of Liver Perfusion Medium (Gibco, Grand Island, NY) followed by 30 ml of Liver Digestion Medium (Gibco), both at a flow rate of 5 ml/min. Hepatocytes were dispersed in Hepatocyte Wash Medium (Gibco) supplemented with 1 % penicillin/streptomycin by dissection and gentle shaking. After filtration through a 100 μm nylon mesh filter, hepatocytes were purified by repeated centrifugation (three times) at 50 × g for 2 min. A typical yield was about 4–5 × 10⁷ hepatocytes/mouse with >80 % cell viability as determined by trypan blue exclusion assay. The purity of hepatocytes was determined by flow cytometry (FACSCanto II, BD Bioscience, San Jose, CA). Hepatocytes and non-hepatocytes were separated based on cell size using forward scatter (FSC) and side scatter (SSC). Data were analyzed with Diva Software (BD Bioscience), and 85 % of the cells were found to be hepatocytes. The isolated hepatocytes were resuspended in DMEM (Gibco) supplemented with 10 % FBS, and 1 % penicillin/streptomycin, and cultured in type-1 collagen-coated 12-well plates at a cell density of 2 × 10⁵ cells/well.

After follow-up angiography, the rabbits were euthanized using pentobarbital (Nembutal, Dainippon Sumitomo Pharma Co., Ltd., Tokyo, Japan), and their embolized kidneys were removed. The specimens were fixed in 10% formalin, embedded in paraffin, cut into 3.5 µm sections, and stained with hematoxylin–eosin for microscopic examination. Pathological images were captured using an Olympus IX83 microscope (Olympus).

The animals were anesthetized with a lethal dose of sodium pentobarbital (Nembutal, Dainippon Sumitomo Pharma, Osaka, Japan; P28 and P56 animals, 100 mg/kg, i.p.) and transcardially perfused with ice-cold 0.01 M phosphate buffer (PB) for 2 min, followed by 4% paraformaldehyde and 0.2% picric acid in 0.1 M PB, pH 7.4, for 10 min. The brains were dissected and postfixed overnight in the same fixative at 4°C. They were then cryoprotected by incubation in 15% sucrose for 7 h followed by 30% sucrose for 20 h at 4°C. The brains were then frozen in O.C.T. compound (Tissue-Tek, Sakura Finetek, Tokyo, Japan) by freezing in dry ice-cold normal hexane. Serial 40 μm coronal sections were prepared on a cryostat (CM-1900, Leica, Wetzlar, Germany) at −20°C. Every section at the level of the PFC was collected and placed in ice-cold phosphate-buffered saline (PBS).

A total of 66 7‐week‐old mice were divided into two groups of 51 (group 1) and 15 (group 2) mice, and were pretreated with NNK (2 mg/0.1 mL saline; mouse i.p.)⁴ at weeks 0 and 1 (Figure 1). At week 3, the 51 mice of group 1 underwent left pulmonary ligation. The left lung, but not the right, was chosen for ligation because the left lungs of mice consist of one lobe and a prominent pulmonary hilum. The procedure was as follows. Each mouse was given an i.p. injection of 0.2 mL pentobarbital sodium (Nembutal, Dainippon Sumitomo Pharma Co., Osaka, Japan) diluted 10 times (0.06–0.1 mL/10 g body weight). Under deep anesthesia, a skin incision (approximately 7 mm long) was made in the left axilla. After confirmation of the location of the thoracic wall, thoracotomy was completed with an incision (approximately 5 mm long) between the ribs¹⁶ (Figure 2a). The left lung was observed directly through this incised opening to confirm atelectasis. The left pulmonary hilum was tied up using a clipping device, Hemoclip manual‐load appliers (No. 17‐085‐01, Mizuho Corp., Tokyo, Japan) and Hemoclip‐cartridge (No. 17‐088‐01; Mizuho Corp.). Finally, the opened chest wall was clipped to close the thorax. The experiment was terminated after 12 weeks and all mice of each group were killed under deep anesthesia.

The process was referenced in the previous study [30 (link)]. Log-phase cells were harvested with 0.05% trypsin–0.02% EDTA in MC38 cells Hanks Balanced Salt Solution (HBSS), washed three times with PBS, and suspended in PBS at a final concentration of 5*10⁷ cells/ml. The C57BL/6 mice were anesthetized with an intraperitoneal injection of pentobarbital (Nembutal, Dainippon Sumitomo Pharma Co., Ltd., Osaka) regulated to 75 mg/kg. Then the mice were incised about 10 mm on the left subcostal, the spleen was confirmed under the peritoneum, the peritoneum was opened for about 8 mm, and the spleen was exposed over the peritoneum. Next, a needle injected the cell suspension of 5*10⁶/100ul of human colon cancer cells into the spleen. After 5 min, the spleen was resected, the peritoneum was sutured with one stitch, and the wound was closed with a clip [31 (link)]. The mice were killed two weeks after inoculation with tumor cells, and the liver sample was resected for evaluation. The tumor sections were paraffin-embedded and preserved for immunohistochemical analysis. All mice experiments were performed with the approval of the Animal Ethics Committee of Tongji University (approval number: SHDSYY-2021-2965a).