Nembutal

At 7 days post-notexin injury, both injured and uninjured mice were anesthetized with sodium pentobarbitone (Nembutal; 60 mg/kg; Sigma-Aldrich) via i.p. injection. The methods for assessment of the contractile properties of the mouse tibialis anterior muscle in situ have been described in detail previously [31 (link)]. At the conclusion of the contractile measurements in situ, the right and left TA muscles were carefully excised, blotted on filter paper, weighed on an analytical balance, and frozen in thawing isopentane for later histological examination.

Dexamethasone, glucagon, indomethacin, methacholine, nembutal, and OVA (Grade V) were purchased from Sigma Chemical Co (St. Louis, USA). Pancuronium bromide and sodium thiopental were purchased from Cristália (São Paulo, Brazil). Dexamethasone, glucagon, OVA, Pancuronium bromide, nembutal, and sodium thiopental were dissolved in 0.9% NaCl sterile solution. methacholine was diluted in phosphate-buffered saline (PBS). indomethacin was prepared in DMSO 0.3%. All of the solutions were freshly prepared immediately before use.

Ad libitum fed mice (8 C57BL/10 and 8 mdx, weighing 30.9±0.5 and 34.7±0.4 gram respectively, P<0.0001) were anesthetized at 9 AM with sodium pentobarbitone (Nembutal, 60 mg/kg, Sigma-Aldrich Co., Castle Hill, NSW, Australia) via i.p. injection in our laboratory, such that they were unresponsive to tactile stimuli. Blood was collected from the abdominal aorta in EDTA-containing tubes and centrifuged at 1000 g and 4°C for 10 min. Plasma was frozen in liquid nitrogen and stored at –80°C. After careful excision of the tibialis anterior (TA), plantaris (PLAN), gastrocnemius (GAST), soleus (SOL) and quadriceps (QUAD) muscles, the liver, and epididymal fat, mice were killed by cardiac excision while still deeply anesthetized. Tissues were blotted on filter paper and weighed on an analytical balance. The right TA muscle was mounted in embedding medium and frozen in thawing isopentane, while the other muscles were frozen directly in liquid nitrogen and stored at −80°C for subsequent analyses.

Surgical procedures are described in Woldeit et al. (2012 (link)). Briefly, gerbils were anesthetized with Nembutal (initial 50 mg/kg intraperitoneally, Sigma-Aldrich, St. Louis, MO, USA) and mounted into a stereotaxic frame. A custom-made surface electrode array (4 × 4 stainless steel electrodes, 100 μm single contact diameter, impedance range: 0.2–0.6 MOhm) was placed on the dura above the right auditory cortex as described in Ohl et al. (2000 (link)). For the array placement the vascularization pattern of the inferior cerebral vein and the middle cerebral artery were used as topographic landmarks (cf. Ohl et al., 2000 (link)).
A depth electrode array (4 bundles of two twisted micro wires, stainless-steel, 50 μm diameter per single wire, impedance range: 0.4–0.7 MOhm) was stereotaxically lowered into the ventral striatum of the same hemisphere (anterio-posterior: +0.5 mm, medio-lateral: −1.3 mm, dorso-ventral: −4.1 mm from bregma). A stainless steel screw in the frontal bone served as reference electrode for both arrays. Dental resin and further anchoring screws were used to secure the wiring and fix electrical connectors (Molex, USA) to the skull. Following surgery, animals were allowed at least 5 days for recovery.

A mouse model of segmental (70%) hepatic warm ischemia was established using a previously reported method46 (link). Mice were fasted for 24 h and placed on a sterile table after receiving an intraperitoneal injection of 1.25% Nembutal (Sigma-Aldrich). All mice underwent midline laparotomy. All structures in the portal triad were blocked for 45 min with a metal microvascular clamp. After 45 min, the clamps were loosened to begin liver reperfusion. After reperfusion, the abdominal incision was closed with surgical thread and the mice were placed in a warm environment until they were awake. A total of 72 mice were randomly divided into four groups as follows:
Group I, normal control group (n = 18): mice were intraperitoneally injected with saline 2 h before laparotomy without I/R.
Group II, I/R group (n = 18): mice were intraperitoneally injected with saline 2 h before laparotomy with I/R for 45 min.
Group III, low dose group (n = 18): mice were intraperitoneally injected with 7.5 mg/kg shikonin 2 h before laparotomy with I/R for 45 min.
Group IV, high dose group (n = 18): mice were intraperitoneally injected with 12.5 mg/kg shikonin 2 h before laparotomy with I/R for 45 min.
Six mice from each group were randomly selected and killed at 3, 6, and 24 h after hepatic I/R. All serum and liver tissues were immediately collected and stored for further experiments.