Isoflurane gas

Pregnant C57BL/6J mice and six-week-old male ICR mice (Charles River Japan, Tokyo, Japan) were maintained at the Kirin Company Ltd. Nine-week-old male C57BL/6N mice and ICR mice (retired breeders) (Japan SLC, Shizuoka, Japan), which were used in the experiments to assess SDS, were maintained at Kobe University or Kirin Company Ltd. Seven-week-old Sprague–Dawley (SD) rats (Charles River Japan) were maintained at Sekisui Medical Ltd. All experiments using mice and rats were approved by the Animal Experiment Committee of Kobe University, Kirin Company Ltd., and Sekisui Medical Ltd., and conducted in strict accordance with each of their guidelines since 2016 to 2017 (Approval ID; AN10145-Z00 AN10200-Z00, AN10253-Z00). Mice were euthanized by placing them in a chamber filled with isoflurane gas (Wako, Tokyo, Japan), and rats anesthetized with isoflurane were euthanized by exsanguination from the abdominal aorta. Mice were fed a standard rodent diet (CE-2, CLEA Japan, Tokyo, Japan) and maintained at room temperature (23 °C ± 1 °C) under constant 12 h light/dark cycles (light period from 8:00 am to 8:00 pm). All efforts were made to minimize suffering.

Wistar rats were orally administered 2.5 mL/day of ethanol (25% concentration) on gestational day 15, and postnatal fetuses were used as the FASD model. The rats were anesthetized with a mixture of air and 3% isoflurane gas (Wako Pure Chemical Industries, Ltd., Osaka, Japan), and then orally administered ethanol using the sonde method. The female rats were sacrificed 2 weeks after the birth of the fetuses, which were weaned at 3 weeks, with three male rats per gauge. Four groups were used: three FASD model groups that received one (2.5 × 1), two (2.5 × 2), and four doses (2.5 × 4) of alcohol in utero, respectively, and a non-treatment group. The four groups were compared using MRI at 4 and 8 weeks of age (non-treatment: n = 11, 2.5 × 1: n = 11, 2.5 × 2: n = 7, and 2.5 × 4: n = 11).
Weights were postnatally recorded every other week from 2 to 8 weeks, and averages were recorded. Controls were groups in a range of 6~19 animals per group for weight measurements, with female rats included in the measurements being 2 weeks old.

The mice were divided into two groups with similar mean body weights (stroke, n = 8, 22.1 ± 0.3 g; sham, n = 6, 21.8 ± 0.3 g). Mice in the induced stroke group were confined in a stereotaxic apparatus (SR-6 M-HT, NARISHIGE Group, Tokyo, Japan) and anesthetized using isoflurane gas (3% for induction, 1–2% for maintenance; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The occipital skin was incised and the occipital bone, 1 mm caudal to lambda, was drilled through to the dura (URAWA Corporation CO., Ltd, Saitama, Japan). We minimized the stress to the mice by keeping them warm; we monitored rectal temperatures and heart rates. Rose Bengal dye (30 μg/ g body weight; Sigma-Aldrich) was injected via the tail vein. Then, a 1 mm diameter probe was placed over the surface of the drilled region, the location of lobules IV and V, for illumination by a green laser (532 nm, 100 mW, 10 min; MGL-III-532-100mW, Changchun New Industries Optoelectronics Technology CO., Ltd, China). After illumination, the occipital bone was covered by bonewax and the skin was sutured. Sham animals were submitted to the same surgical procedure as described above, but without laser illumination. The mice were given a 4-day recovery period prior to locomotion tests. This interval was previously shown to be after the peak of expansion of cerebellar infarction induced by photothrombotic ischemia^{28 (link)}.