All animals were anesthetized with 1% pentobarbital sodium (35 mg/kg, Dainippon Sumitomo Pharma) and fixed on a 60° inclined board with the help of rubber bands. The tongue of the mice was pulled and held using blunt forceps. Under the head mirror, endotracheal intubation was performed using an epidural anesthesia catheter with a connector attaching it to a 1-ml syringe that was inserted into the trachea to the point of the tracheal bifurcation. The crystalline particles were suspended in normal sterile saline and the suspension (50 mg/ml) was vigorously mixed using a Stuart® vortex shaker (Cole-Palmer) prior to instillation. For pathological tissue extraction, the rats were anesthetized with 1% pentobarbital sodium (35 mg/kg, Dainippon Sumitomo Pharma) and fixed on the plate. After the bronchoalveolar lavage fluid (BALF) was collected, the rats were euthanized by bloodletting. Then the pathological organs were collected immediately after animal euthanasia.
Pentobarbital sodium
Manufactured by Sumitomo Pharma
Sourced in Japan
Pentobarbital sodium is a barbiturate compound used as a sedative and hypnotic agent in laboratory settings. It is a white, crystalline powder that is soluble in water. Pentobarbital sodium acts as a central nervous system depressant, producing a calming effect and inducing sleep.
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19 protocols using pentobarbital sodium
1
Intubation and Pathological Tissue Extraction
2
Blood and Tissue Collection in Fasted Mice
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At the end of the experiment, mice fasted for 3 h were anesthetised with intraperitoneal injections of pentobarbital sodium (Dainippon Sumitomo Pharma, Osaka, Japan). Blood samples were drawn from the inferior vena cava and treated with EDTA-2Na, and were centrifuged (900 ×g, 4°C, 10 min) to separate plasma, which was then frozen at -80°C until analysis. The liver, white adipose tissue and pancreas were excised. Tissue samples were weighed, frozen in liquid nitrogen, and stored at -80°C until analysis.
3
Quantifying Hemorrhagic Transformation in Ischemic Stroke
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At 21 h after reperfusion, mice were given an overdose of pentobarbital sodium (Dainippon Sumitomo Pharma, Osaka, Japan) and transcardially perfused with cold saline. Brains were immediately removed and cut into 5 serial 2-mm-thick coronal block slices. In our preliminary study, we found that the reproducibly largest infarct size was in the cortical region of the left hemisphere on the anterior fourth slice. Therefore, we analyze the extent of hemorrhagic transformation in this area. After adding 10 ml/g of saline to individual samples, they were homogenized, followed by 30-min of centrifugation (13,000 rpm). A 200 µl volume of reagent (QuantiChrom Hemoglobin Assay Kit; BioAssay Systems, CA, USA) was then added to 50 µl of supernatant. After 15 min, optical density was measured at 400 nm with a spectrophotometer (Skan It RE for Varioskan 133 Flash 2.4; Thermo Fisher Scientific, Waltham, MA, USA). The total hemoglobin content was expressed as micrograms per sample (Control; n = 11, Diabetes; n = 12).
4
Xenograft Tumor Development in C57BL/6 Mice
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C57BL/6 mice were purchased from Jinan Xingkang Biotechnology Co., Ltd. (Jinan, China). The animal house was maintained at a temperature of 22 ± 2°C with relative humidity of 50% ± 15% and 12 h dark/light cycle. Xenograft tumors were established by subcutaneous injection of H1299 cells into 6-week-old male C57BL/6 mice. Sh-USF1 group mice were transfected with sh-USF1 tail-vein injection. The tumor was monitored by Vernier caliper while the tumor volume was indicated by a × b2/2 (a for long diameter; b for short diameter) every 5 days after injected 10 days. After 30 days, mice were anesthetized with 1% pentobarbital sodium (35 mg/kg, Dainippon Sumitomo Pharma) and fixed on the plate. The tumors were then removed, weighed and analyzed after the mice were euthanized by slow release of carbon dioxide in a box. All experimental procedures were approved by the Ethics and Scientific Committees of our institution (No. 2017440) performed following the Guide for the Care and Use of Laboratory Animals.
5
Stereotactic Lesions of the Accumbens Core
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Stereotactic lesions of the AcbC were made using a standard stereotaxic apparatus (David Kopf Instruments). Rats were first injected with pentobarbital sodium (i.p.; 50 mg/kg body weight, Dainippon Sumitomo Pharma Co., Ltd) dissolved in saline and then maintained on anesthesia with isoflurane (Foren; Abbott Japan Co., Ltd.) for the surgical procedure. Bilateral lesions were made by infusing ibotenic acid (0.6 µl/hemisphere of 5 mg/ml ibotenic acid in PBS; Sigma Aldrich, Japan) through a stainless steel needle (200 µm outer diameter) attached to the stereotaxic device. Coordinates for injection sites were as follows: AP, +1.9 mm rostral to bregma; ML, ±1.7 mm lateral to the midline; V, −6.3 mm ventral to the dura mater. Sham-lesioned rats received injections of the same volume (0.6 µl/hemisphere) of PBS at these coordinates. The needle was left in place for 15–20 min to allow the toxin to diffuse sufficiently within the target sites. Rats were allowed to recover for 7 days following the lesion surgery. For the first 4 days after surgery, rats were given daily i.p. injections of antibiotic solution (0.4 ml/day of 1 mg/ml gentamicin in saline; Schering-Plough).
6
Abdominal Fat Estimation by CT Scan
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Radiographic estimations of abdominal composition were performed by computed tomography (CT) for experimental animals using the mouse mode of the CT scanner (La Theta LCT100; ALOKA, Tokyo, Japan). At the end of the experiment, mice fasted for 3 h were anesthetized with intraperitoneal injections of pentobarbital sodium (Dainippon Sumitomo Pharma, Osaka, Japan) before scanning. Abdominal compositions of visceral and subcutaneous fats were estimated by fat slice images at 2 mm intervals between the second lumbar vertebra (L2) and L4 using La Theta software (version 2.10).
7
Pulmonary Fibrosis in C57BL/6 Mice
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For the mouse model of pulmonary fibrosis, C57BL/6 male mice (4–6 weeks of age) were purchased from the Shanghai Laboratory Animal Center (SLAC, Shanghai, China). A total of 48 C57BL/6 mice were randomly divided into 6 groups (n = 8 in each group): day 7, 14, and 28 saline groups (control) and day 7, 14, and 28 silica groups. The mice were anesthetized using pentobarbital sodium (Dainippon Sumitomo Pharma, Osaka, Japan) through intraperitoneal injection. The mice were instilled with 50 mg/kg of silica (Sigma Aldrich, USA) in 0.05 ml sterile saline or 0.05 ml sterile saline intratracheally. The mice were sacrificed on day 7, 14 or 28 after instillation, and the lungs were harvested and stored at −80 °C immediately for further analysis.
8
Histopathological Analysis of Hippocampus and Hypothalamus
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After the behavioral experiment, the mice were anesthetized (1% pentobarbital sodium, 35 mg/kg, intraperitoneal injection; Dainippon Sumitomo Pharma) and decapitated. The brain was quickly removed, and the hypothalamus and hippocampus were stripped. Briefly, the hippocampal and hypothalamic tissues were fixed in 10% formalin (cat. no. 0-10-02; Beijing Yili Fine Chemicals Co., Ltd.) for 24 h at 37°C. Subsequently, the fixed tissues were embedded in paraffin and cut into 5-µm sections with a microtome. After regular dewaxing using 50% xylene for 20 min and dehydration with 80% ethanol for 20 min at 37°C, the sections were stained with hematoxylin for 5 min at 37°C, and eosin for 4 min at 37°C. Finally, histopathological changes in the hippocampus and hypothalamus were observed under a light microscope (magnification, х200).
9
Focal Cerebral Ischemia by MCAO in Rats
10
Silica-Induced Pulmonary Fibrosis Model
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C57BL/6 male mice (4‐6 weeks of age) were purchased from SLAC Laboratory Animal Co., Ltd., (Shanghai, China). All experimental protocols were approved by the Animal Care and Use Committee at Nanjing Medical University. The mice were anaesthetized with an intraperitoneal injection of 1% pentobarbital sodium (Dainippon Sumitomo Pharma, Osaka, Japan). A 0.05 mL sterile saline containing silica (50 mg/kg, Sigma‐Aldrich, St. Louis, MO, USA) was directly instilled intratracheally. The control group was instilled with 0.05 mL saline. The lung tissues were harvested on day 7, 14 or 28 after silica instillation. The miR‐503 up‐regulation mouse model was conducted by a co‐instillation of 200 nmol/kg miR‐503 agomir (RiboBio Co., Ltd., Guangzhou, China) with silica instillation. Subsequently, 120 nmol/kg miR‐503 agomir was injected via the tail vein weekly. The lung tissues were harvested at day 28.
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