Sevoflurane

For three out of the five animals in each experimental group, a 3D micro-CT scan was conducted hours to days prior to dissection at each designated observation time. The X-ray 3D micro-CT system used for this purpose was the CosmoScan GX, manufactured by Rigaku Co., Tokyo, Japan. The system was operated under specific conditions, including a scanning time of 4.0 min, an average whole-body exposure of 161.9 mGy per scan, a tube voltage of 90 kV, a tube current of 88 µA and a chest CT with a field of view (FOV) measuring 60 mm × 40 mm (voxel matrix: 512 µm × 512 µm × 512 µm; voxel size: 120 µm × 120 µm × 120 µm). The rats were positioned in the prone posture during the scanning process, and sevoflurane (supplied by Pfizer Japan, Tokyo, Japan) and oxygen were administered through a nose cone. The acquired images were retrospectively gated at both respiratory phases, encompassing both inspiration and expiration.

Microminipigs were bred and maintained at the Fuji Micra Co., Ltd. (Shizuoka, Japan). Experiments using the pigs were performed at Jichi Medical University according to the guidelines provided by the Institutional Animal Care and Concern Committees, which also approved all protocols. Invasive procedures were carried out under general anesthesia by inhalation of sevoflurane (Pfizer, Inc., Tokyo, Japan), with vital-signs monitoring conducted in accordance with the stipulated guidelines. Vector solutions were injected into the cervical vein. Microminipigs with low NAb titers (≤56×) were used in this study. To evaluate luciferase expression in pigs, 7 days after vector injection, 15 mg/ml of D-luciferin solution (OZ Biosciences, San Diego, CA, USA) was injected into the unused cervical vein. Animal #1 was then imaged using the IVIS Spectrum CT imaging system (Caliper Life Sciences, Hopkinton, MA, USA).

Anesthesia was induced with 2 mg/kg propofol (Fresenius Kabi, Lake Zurich, IL, USA), or 5 mcg/kg fentanyl (Hospira, Lake Forest, IL, USA) if the patient had an indwelling intravenous catheter. Otherwise, inhalational induction with sevoflurane 5% (Abbott Laboratories, Abbott Park, IL, USA) was performed. Anesthesia was maintained with sevoflurane and dexmedetomidine (Pfizer, New York, NY, USA; 1 mcg/kg/h). Following anesthetization and intubation, a Foley catheter was placed along with an arterial line and a double lumen central venous line.

Lipopolysaccharides (LPS) from Escherichia coli O26:B6, fluorescein isothiocyanate (FITC)-labeled wheat germ agglutinin (WGA) lectin from Triticum vulgaris, and tetramethylrhodamine (TMR)-labeled dextran (average molecular weight, 75 kDa [TMR-dex75]) were purchased from Sigma-Aldrich Co. (St Louis, MO). The mouse-soluble syndecan-1 (Sdc-1) enzyme-linked immunosorbent assay (ELISA) kit from Diaclone SAS (Besancon Cedex, France) was purchased. Ketamine hydrochloride, xylazine hydrochloride, and rhodamine 6G were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Sevoflurane was purchased from Pfizer Japan Inc. (Tokyo, Japan), and used for anesthesia using a small animal anesthetizer (MK-A110D, Muromachi Kikai Co., Ltd., Tokyo, Japan).

Doses of 0.2 mg (0.8 mg/kg BW) and 1.0 mg (4.0 mg/kg BW) of Non-CL-PAA and CL-PAA suspended in 0.4 mL distilled water were administered to the lungs of rats (12 weeks old) in single intratracheal instillations. Rats were intratracheally instilled under anesthesia by sevoflurane (Pfizer Japan, Tokyo, Japan) inhalation. Briefly, a laryngeal extension was performed using a laryngoscope blade (MAC1, Rudolf Riester GmbH, Jungingen, Germany), an animal feeding needle (KN-348, Natsume Seisakusho Co., Ltd., Tokyo, Japan) was inserted directly into the trachea, and the suspension was manually injected. Then, 3 mL of air twice with a syringe from the animal feeding needle was inserted into the trachea. The rats were then allowed to awaken spontaneously and were observed periodically. Single intratracheal instillations of 0.2 mg and 1.0 mg of Non-CL-PAA and CL-PAA were performed at different times, and the single intratracheal instillations were conducted a total of two times. The control group received distilled water, and a control group was established for each intratracheal instillation. Dosages of 0.2 and 1.0 mg/rat were used in the intratracheal instillation of the PAAs. This maximum dose was set assuming human exposure and to avoid overload in the lung [10 (link)].

All canines were anesthetized with intramuscular injection of ketamine (10 mg/kg; Daiichi Sankyo, Tokyo, Japan), xylazine (0.25 mg/kg; Bayer Yakuhin, Osaka, Japan), and atropine sulfate (0.05 mg/kg; Mitsubishi Tanabe Pharma, Osaka, Japan) and intubated for mechanical ventilation. Anesthesia was maintained via the inhalation of 2–3% sevoflurane (Pfizer, New York, NY, USA) and O₂. All donor and recipient canines were ventilated with an inspired O₂ fraction of 1.0 at a tidal volume of 20 mL/kg, a respiratory rate of 15 breaths/min, and a positive end-expiratory pressure of 5 cmH₂O.