Forane

A previously described rat model of pulmonary aspergillosis was used for the in vivo experiments [22 (link)]. The introduction of A. fumigatus spores into animals, injection of radiolabeled siderophores and small animal imaging were carried out under 2% isoflurane anesthesia (FORANE, Abbott Laboratories, Abbott Park, IL, USA) to minimize animal suffering and prevent animal motion. Infection in the lung of immunodeficient rats was established by intratracheal inoculation of 100 μL of A. fumigatus spores (10⁹ CFU/mL A. fumigatus ATCC 46645; American Type Culture Collection) using the TELE PACK VET X LED system equipped with a flexible endoscope (https://www.karlstorz.com/cps/rde/xbcr/karlstorz_assets/ASSETS/3386919.pdf, accessed on 20 August 2021; Karl Storz GmbH & Co. KG, Tuttlingen, Germany). Depending on the development of the infection, experimental animals underwent an ex vivo biodistribution study or PET/CT imaging, typically 2–4 days after inoculation.

Mouse hepatocytes were prepared by the collagenase perfusion method through the postcaval vein [35 (link)]. Briefly, 7- to 10-week-old male C56BL/6 J mice (Charles River, Kanagawa, Japan) were anesthetized by isoflurane (Forane^®; Abbott) inhalation, and the liver was perfused through the postcaval vein with 30 ml of Liver Perfusion Medium (Gibco, Grand Island, NY, USA), followed by 30 ml of Liver Digest Medium (Gibco), at a flow rate of 3–4 ml/min. The perfusion medium was incubated at 37 °C. After perfusion, the liver was removed and immersed in ice-cold Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco), supplemented with 1% antibiotic–antimycotic mixture (Gibco). The hepatocytes were released by gentle pipetting and then filtered using a 100-µm pore size mesh nylon filter (Corning, NY, USA). The cells were washed thrice with DMEM, supplemented with 1% antibiotic–antimycotic mixture by centrifugation (30×g, 3 min, 4 °C), followed by suspension in 10 ml of cell culture medium.

At the start of the experiment, all animals were examined by echocardiography to exclude rats with any heart abnormalities. Transthoracic two-dimensional echocardiography was performed under inhalation anesthesia at the beginning of the experiment and on the day of sacrifice. Rats were lightly anesthetized with a mixture of 1.5% isoflurane (Forane, Abbott Laboratories, Hungary) and 98.5% oxygen. The chest of animals was shaved, acoustic coupling gel was applied and warming pad was used to maintain normothermia. Rats were imaged in the left lateral decubitus position. Cardiac dimensions and functions were measured from short- and long-axis views at the mid-papillary level by a VEVO 770 high-resolution ultrasound imaging system (VisualSonics, Toronto, Canada) equipped with a 25 MHz transducer. LV fractional shortening (FS), ejection fraction (EF), LV end-diastolic volume (LVEDV), LV end-systolic volume (LVESV), and the thickness of septum and posterior wall were determined. FS (%) was calculated by 100x((LVID_d-LVID_s)/LVID_d) (LVID: LV inside dimension; d: diastolic; s: systolic), EF (%) was calculated by 100x((LVEDV-LVESV)/LVEDV), relative wall thickness (RWT) was calculated by (PW thickness + interventricular septal thickness)/LVID_d.

After treatment with isoflurane [2% (v/v); Forane; Abbot, Tokyo, Japan] to induce deep anesthesia, the mouse underwent laminectomy at the T9–10 vertebral level to expose the spinal cord, with use of a surgical microscope (Vanox; Olympus Optical, Tokyo, Japan). An Infinite Horizon Impacter (Precision Systems and Instrumentation LLC, Fairfax, VA, USA) was used to produce a contusion SCI model, using an impact force of 60 kilodynes. For sham SCI, laminectomy only was performed at T9-T10, with no SCI. The wound and surrounding skin were sutured with 5–0 silk.

At the end of the experimental period, after an overnight fast, blood was collected from the retro-orbital plexus, under 2% isoflurane anaesthesia (Forane, Abbot Laboratories Ltd, Illinois, USA), into tubes containing 0.1% (w/v) EDTA and plasma was separated after centrifugation for 10 min at 5900× g at 4 °C. The mice were then sacrificed under general anaesthesia with 2% isoflurane and the blood was removed by perfusion with phosphate-buffered saline (PBS). The aorta was rapidly dissected from the aortic root to the iliac bifurcation, and as much periadventitial fat and connective tissue were removed as much as possible. The aorta was then longitudinally opened, pinned flat on a black wax surface in ice-cold PBS and photographed unstained50 (link)51 (link) for plaque quantification (see En face analysis). For histological/immunohistochemical analysis, the hearts were removed, fixed in 10% formalin for 30 min and transferred into PBS containing 20% sucrose (w/v) overnight at 4 °C before being embedded in OCT compound (Sakura Finetek, The Netherlands) and stored at −80 °C.
The apex of the heart, liver, stomach, duodenum, jejunum, ileum, large intestine, kidney, brain, abdominal white adipose tissue, lung, inguinal lymph node, spleen, testis and ovary were immediately snap-frozen in liquid nitrogen for subsequent analyses.