Beuthanasia d

After the experiment was finished, all lambs were euthanized with Beuthanasia-D (1 mL/10 lb; Schering-Plough, Union, NJ). During necropsy, the LV was opened from the apex incision, along left side of ventricular septum, across aortic valve to ascending aorta. The LV, aortic valve, and ascending aorta intima were visualized at necropsy for thrombosis and tissue damage.

The same timeline was followed as previously described (Vermeer, 2014 (link)). Briefly, rats were given one week to acclimate following arrival and then were subjected to two days of presurgical baseline testing. Animals then underwent the cannula implantation surgery and were given a further week to recover. Finally, rats were given two days of postsurgical baseline testing prior to dural IS application. As previously described, behavioral experiments using IS applications and estradiol exposure were performed every third day for seven total applications. Rats were injected with estradiol 30 min prior to IS application and behavioral testing was completed 30 min following dural stimulation by the IS (Oshinsky and Gomonchareonsiri, 2007 (link)). Following recommendations of the Panel on Euthanasia, American Veterinary Medical Association, rats were dosed with 60 mg/kg pentobarbital sodium (Beuthanasia D, Schering-Plough, Summit, NJ, USA) and decapitated approximately 1 hr after the final behavioral experiment. The skull, brain, and cervical spinal cord were immersed in RNALater (Ambion, Foster City, CA, USA) and stored for up to 3 days at 4°C. The ipsilateral trigeminal nucleus, trigeminal ganglion, and dura mater were later dissected as previously described (Stucky, et al., 2011 (link)).

This study was approved by our institutional animal care and use committee. All experiments were performed under general anesthesia with mechanical ventilation. Animals were anesthetized with intramuscular injections of tiletamine and zolazepam (Telazol; Zoetis, Kalamazoo, MI) and xylazine (AnaSed; Shenandoah, IA). Anesthesia was sustained with inhaled isoflurane gas (1.5–2.5%; Halocarbon Laboratories, River Edge, NJ). Tidal volumes were maintained between 370–400 mL. A veterinary monitoring device (Bionet America; Tustin, CA) assessed the swine’s vital signs continuously during the procedure. Prior to treatment, animals were shaved, placed in a supine position, and fixed to a degassed water bath by an adhesive drape (loban; 3M Company, St. Paul, MN). Following treatment and imaging, animals were euthanized by an intravenous administration of phenytoin pentobarbital sodium (Beuthanasia-D; Schering-Plough, Kenilworth, NJ).

To assess the impact of tryptophan on wound healing we utilized an experimental murine skin wound model, previously established in our laboratory (44 (link), 45 (link)). All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Wisconsin-Madison. Eight to twelve week old BALB/c mice were housed individually in a temperature-controlled facility with a standard light/dark cycle. Environmental enrichment, food, and water ad libitum were provided to all mice. Mice were anaesthetized with isoflurane (Piramal Healthcare, Bethlehem, PA) using an induction chamber. Buprenorphine (0.1 mg/kg) was administered for pain control. The cranial thoracodorsal region was shaved and aseptically prepared for surgery. Silicon O-rings (McMaster-Carr®) were secured with tissue glue (Tissumend II) to the skin 4 mm caudal to the base of the ears on each side of the dorsal midline and were further attached to the skin by six 5-0 interrupted nylon sutures. Two symmetrical wounds within each O-ring were made using a 6mm biopsy punch. Body weights of mice were recorded on post-operative day 1, and every 2–3 days until the end of the study. Upon completion of each study, mice were euthanized by intra-peritoneal injection of Beuthanasia®-D (Schering-Plough) solution (0.5 ml/mouse) after induction of anesthesia with isoflurane.

After successful completion of the five day post-hyperthermia survival period, all sheep were euthanized with Beuthanasia-D (1 mL/10 lb body weight, Schering-Plough, Union, NJ) and subjected to a full necropsy. The brain, lungs, heart, liver, kidney, gastrointestinal tract, spleen, adrenals, and bladder were grossly examined and samples of these tissues were preserved in 10% buffered formalin. The tissue sections were paraffin-embedded and stained with hematoxylin-eosin. Slides were examined by a veterinary histopathology lab (Antech Diagnostics, Louisville, KY).