Aestiva 5

As soon as we transferred the uncooperative patients to the dental chair, we administered 8 vol% inspired sevoflurane gas with 4.0 L/min of oxygen and 4.0 L/min of nitrous oxide to the patients using a full facial mask. After achieving loss of consciousness, we placed nasal cannula (HUDSON RCI: Teleflex, NC, USA) incorporated with capnography line into patients' nostril. Then, we delivered sevoflurane anesthetic gas to the patients through the nasal cannula. We adjusted sevoflurane vaporizer setting and delivered 100% oxygen at gas flow of 2 L/min to the patients.
For monitoring and sevoflurane administration, anesthesia machine (Aestiva/5; Datex Ohmeda, WI, USA) was used. We monitored electrocardiogram, oxygen saturation, noninvasive blood pressure, end-tidal carbon dioxide concentration (ETCO₂) and end-tidal sevoflurane concentration (ETS). We adjusted inspired sevoflurane gas concentration to maintain ETS in the range of 1 to 1.5 vol% as much as possible. After the treatment, sevoflurane was discontinued and 8 L/min of 100% oxygen gas was maintained until the patients recovered their consciousness. Then patients were transferred to a recovery room and complications were observed.
Age, gender, treatment type, duration of sedation, duration of treatment, ETCO₂, ETS, recovery time, and post treatment complications was recorded.

An anesthesia machine (Aestiva 5) and gas monitor (F-MCI) were purchased from GE Healthcare UK (Little Chalfont, UK). A digital eclipse modular confocal microscope (C1) was obtained from Nikon (Tokyo, Japan). A BCA protein-assay kit was purchased from Bio-Rad Laboratories (Hercules, CA, USA).
GAPDH monoclonal antibody and Bax monoclonal antibody were obtained from Cell Signaling Technology (Danvers, MA, USA). LC3 monoclonal antibody and p62 monoclonal antibody were from Abcam (Cambridge, UK). Drp1 monoclonal antibody was purchased from Wanleibio (Beijing, China). Bcl2 monoclonal antibody was obtained from R&D Systems (Minneapolis, MN, USA). Nissl staining kits (cresyl violet method) were from Solarbio (Beijing, China).

After SPIO labeling for 12 h, we transplanted the labeled PBMCs. To avoid the destruction
of implanted cells caused by the injection pressure and tight junctions of myocytes, we
decided to inject all the labeled cells in small amounts at each site separately but as
closely together as possible. Four regions of the gastrocnemius of cynomolgus monkey were
transplanted i.m. with a 1-ml suspension of 2.7 × 10⁷ SPIO-labeled PBMCs using
a 22-gauge needle. Thereafter, an 11-ml suspension of 2.7 × 10⁸ PBMCs was
injected into the saphenous vein using a 22-gauge intravenous cannula (Terumo, Tokyo,
Japan). The administered volume of cells was increased by mixing the PBMCs labeled with
the two types of SPIOs. These procedures were accomplished under general anesthesia that
was induced with ketamine hydrochloride (Ketalar, 10 mg/kg; Sankyo, Tokyo, Japan) as
described above and maintained with isoflurane (Aestiva/5; GE Healthcare, Madison, WI,
USA). The monkey exhibited no clinical signs or symptoms after transplantation.

All patients and controls had undergone CMR at 1.5-T MR field strength (Intera, Philips Healthcare, Best, the Netherlands). Electrocardiography (ECG)-gated two-dimensional (2D) steady-state free precession images were acquired for functional assessment. To assess fibrosis, late gadolinium enhancement images (inversion recovery turbo fast low-angle shot) were acquired after 10 min. An ECG-gated 3D steady-state free precession sequence with T2 and fat saturation prepulses was used to visualize the coronary arteries in a whole-heart approach. Imaging was performed under general anesthesia, if necessary, with continuous intravenous infusion of remifentanil with the use of a CMR safe anesthesia delivery system (Aestiva/5; GE Healthcare, Waukesha, Wisconsin, USA) with controlled ventilation (routine protocol for young or uncooperative children at our institution).