Male icr mice

ICR male mice (eight weeks of age, n = 18; Japan SLC, Hamamatsu, Japan) were used. Mice were randomly assigned to two groups: a control group (Con; n = 9) and a lactate group (Lac; n = 9). They were injected with 50 µL of 50% v/v glycerol into the bilateral tibialis anterior (TA) muscles, as previously described [54 (link),55 (link)], under anesthesia by isoflurane inhalation. Then, sodium lactate (500 mg/kg) or the equivalent volume of saline was administered daily by intraperitoneal injection for seven days to the mice in the Lac and Con groups, respectively. At day 7, 14, and 28 after glycerol injection, mice were sacrificed (three mice for each time point) and TA muscles were harvested. Muscle tissues were immediately frozen and cryosectioned (7 µm thickness) at the mid-belly region, followed by staining with hematoxylin and eosin. Twelve representative fields per animal (four fields from three sections) were examined and the myofiber areas in the samples, determined at day 14 and 28 after glycerol injection, were analyzed using a digital microscope (BZ-X700; Keyence, Osaka, Japan). All animal procedures were approved by the Animal Ethics Committee of Ochanomizu University (Approval No.: 17021R, Approval date: 4/8/2017) and performed in accordance with the Japanese Act on Welfare and Management of Animals.

ICR male mice were obtained from Japan SLC Inc. (Hamamatsu, Japan) and housed under specific pathogen-free conditions. In total, we used 32 ICR mice aged 7.5–10 weeks. The animal experimentation protocol was approved by the Animal Research Committee of Osaka Prefecture University (approval number: 19–49, 20–32, 21–26). All experiments were performed following the relevant guidelines of the Osaka Prefecture University. The mice were euthanized by cervical dislocation, and the blood was removed by intracardiac perfusion with Ca-/Mg-free Hanks’ Balanced Salt Solution (HBSS; H6648, Sigma-Aldrich, St. Louis, MO, USA), supplemented with 50 U/mL heparin (224122485, Mochida Pharmaceutical, Tokyo, Japan) when necessary.

Eight-week-old ICR female mice (Japan SLC) received 7.5 U of serotropin (ASKA Animal Health) by intraperitoneal injection. Forty-eight hours after serotropin treatment, mice were injected with 7.5 U of gonadotropin (ASKA Pharmaceutical) and then mated with ICR male mice (Japan SLC). Two-cell fertilized eggs were collected by perfusion with mWM medium and maintained in KSOM medium to obtain blastocysts. After injection of six to ten ESCs, the injected blastocysts (22–26 blastocysts/mouse) were transplanted into the uterus of pseudopregnant ICR female mice (Japan SLC). A single ESC line for each genotype was used in this study.

To measure the effect of background noise on ABR measurement accuracy, 10 randomly selected ICR male mice (Japan SLC, Inc., Shizuoka, Japan) weighing 20-30 g, were used at the age of 8 weeks.
The animals were singly housed in individually ventilated cages (Lab Products, Inc., Seaford, DE, USA), in a room with controlled temperature (22℃ ± 2℃), humidity (60% ± 5%), and light (12/12 h light/dark cycle with lights on at 08:00). The light intensity at the surface of the cages was approximately 100 lux. All mice were on a standard rodent diet (AIN-93M, Research Diets, Inc, USA), and food and water were provided ad libitum.
Next, to identify sources of mouse model candidates for congenital hearing loss, we selected 10 male mice from each of the following strains: C57BL/6JJmsSlc (Japan SLC, Inc. Shizuoka, Japan), C57BL/6JJcl (CLEA Japan, Inc., Tokyo, Japan), C57BL/6NCrl (Charles River Laboratories Japan, Inc., Tokyo. Japan), Balb/c (Japan SLC, Inc.), CH3 (CLEA Japan, Inc.), ICR (Japan SLC, Inc.), and ddY (Japan SLC, Inc.).
Mice were tested at 8 weeks of age, and their body weights ranged between 20 and 30 g. Mice were randomly selected from each strain.
All mice remained in the same environmental conditions as described.
In admission, this experiment was approved by the Waseda University laboratory animal ethical review board.

Blastocysts collection: 8-week-old ICR female mice (Japan SLC) were treated with 7.5 U of serotropin (ASKA Animal Health) by intraperitoneal injection. After 48 h of serotropin treatment, mice were injected 7.5 U of gonatropin (ASKA Pharmaceutical). These mice were then mated with ICR male mice (Japan SLC). Plug check was performed the next morning. Two days later, these female mice were sacrificed by cervical dislocation and operated to pick out oviducts. Two-cell stage fertilized eggs were collected by perfusion with M2 medium (Sigma) and maintained in KSOM medium. Two days later, the obtained blastocysts were used for microinjection. For microinjection, ESCs were treated with trypsin and pipetted 15 times to dissociate them into single cells. The single cells were cultured in a gelatin-coated 10-cm dish with 10 mL ESC medium for 30 min to attach MEFs onto the dish. Three milliliters of supernatant containing ESCs was collected. Three to five ESCs were injected into the ICR blastocysts under an OLYMPUS IX71 microscope. Twenty to 25 injected blastocysts were transplanted into the uterus of pseudopregnant ICR female mice (Japan SLC).