Isoflurane

TG693 metabolism and tissue bioavailability were assessed in mouse plasma and muscle tissue. To collect plasma, mice were anesthetized with isoflurane (Mylan, Osaka, Japan) at the indicated time point. Whole blood samples were collected, allowed to clot, and then centrifuged to isolate serum. TG693 or TG003 serum levels were analyzed by LC/MS with an Agilent 6420 Q-TOF mass spectrometer (Agilent Technologies) and ZORBAX HILIC Plus (TG693) or ZORBAX Eclipse Plus C18 (TG003) columns (Agilent Technologies). For muscle tissue bioavailability, mice were sacrificed by cervical dislocation and the tibialis anterior (TA) muscles, heart, and diaphragm dissected. TA homogenates were prepared using a Bead Crusher μT-12 shaking machine (Taitec, Kyoto, Japan) in saline and used to assess TG693 levels by LC/MS with a Poroshell 120 PFP (Agilent Technologies).

In vivo experiments were conducted on 4-week-old female athymic BALB/c nu/nu mice obtained from CLEA Japan (Tokyo, Japan). The animal experimental protocol was approved by Ethical Committee of the Osaka City University, Osaka, Japan. All studies on mice were conducted in accordance with the National Institute of Health (NIH) ‘Guide for the Care and Use of Laboratory Animals’. The mice were housed in a standard animal laboratory with ad libitum access to food and water. MDA-MB-231 cells (10⁷ cells) were suspended in 100 μL of DMEM, and injected into the backs of the mice. After a week, the mice were randomized into two groups: (1) control (saline alone) and (2) DFO (20 mg/kg/day, 5 days/week). For evaluation of the combination therapy, the mice were randomized into four groups: (1) control (saline alone), (2) DFO alone, (3) eribulin alone (0.8 mg/kg/day, 5 days/week), and (4) DFO plus eribulin.
Both DFO and eribulin were dissolved in DMEM without FBS. DFO was directly injected into the tumor, and eribulin was intravenously administered. The resultant tumor volumes (length × width) were measured weekly. Animals were euthanized via isoflurane (Mylan, PA, USA) (1 ml per one mouse) and cervical dislocation, and autopsied at 6 weeks after cell inoculation [17 (link)]. In sacrifice, animals were unconscious.

The mice were anesthetized with 2% isoflurane (Mylan, Osaka, Japan). After tracheotomy, the jugular vein was cannulated for fluorescent dye injection and infusion of maintenance fluid, which consisted of 1.0 μL/min of 2% albumin in phosphate-buffered saline. The left kidney of each mouse was exteriorized through a small flank incision and attached to a coverslip. The microscope stage and animals were warmed using a heating pad during all experimental procedures. Intravital multiphoton microscopy was performed using an Olympus FV1000MPE multiphoton confocal fluorescence imaging system powered by a Chameleon Ultra-II MP laser at 860 nm (Coherent, Inc., Santa Clara, CA, USA). The microscope objective was a 25x water immersion lens with a 1.05 numerical aperture. The imaging settings for the microscope (gain and offset for all three channels; blue, green, and red) were fixed throughout the experiment. After a 30 min recovery period, rhodamine B-70 kD dextran was injected intravenously, and 3-D z-stack images were taken at depths of 5–50 μm (2 μm apart) from the kidney surface.

All the rats were placed on an acrylic wheel (11 cm in width and 40.8 cm in diameter) that was revolving at 3.5–4 rpm and trained to keep walking on it for 2 min a day, 3 times a week prior to the experiments. On the day prior the experiment, each rat was made to walk for 1 min on the revolving wheel. On the day of the experiment, each rat was anesthetized with isoflurane (Mylan, Canonsburg, PA, USA), followed by intra‐articular injection of a yeast suspension. The walking behaviour was video‐recorded from the bottom of the wheel with a high‐speed camera (GT‐03‐01; Noveltec Inc., Kobe, Japan). Gait was evaluated visually and graded semi‐quantitatively on a scale of 0–3, as shown in Table 1, as a consensus score between two experienced examiners. The evaluation was conducted in a blinded manner and always carried out by the same experimenter, to minimize variability. The mean value of the two data‐sets collected by the two experimenters was used as the visual gait score for each rat.