Sevoflurane

Performance of the EPC was tested in awake behaving monkeys. Following initial training monkeys were implanted with a head holder, eye coil, and recording chambers above V1 under general anesthesia and sterile conditions. For the anesthesia animals were initially sedated with a 0.1 ml/kg ketamine intra-muscular injection (100 mg/ml). Thereafter, bolus injections of propofol were administered intra-venously to allow for tracheal intubation (0.05–0.1 ml). Prior to surgery a bolus injection of dexamethosone sodium phosphate was administered i.v. (0.33 mg/kg). During surgery anesthesia was maintained by gaseous anaesthetic (1–3% sevoflurane) combined with continuous i.v. application of an opioid analgesic (Alfentanil, 156 μg/kg/h). The animal's rectal temperature, heart rate, blood oxygenation and expired CO₂ were continuously monitored during surgery. Immediately after surgery (and during the following 3–5 days) the animals were given antibiotics (Cephorex 0.5 ml/kg or Synolux 0.25 ml/kg) and analgesics (Metacam 0.1 ml/kg).
Following surgery the recording chambers were regularly cleaned under sterile conditions and 5-fluoro-uracil treatment was performed three times per week (Spinks et al., 2003 (link)). Despite 5-fluoro-uracil treatment it was necessary to perform dura scrapes every 6–8 weeks for the removal of fibrous scar tissue above the craniotomy.
All animal and surgical procedures were in accordance with the European Communities Council Directive 1986 (86/609/EEC), the National Institutes of Health guidelines for care and use of animals for experimental procedures, the Society for Neurosciences Policies on the Use of Animals and Humans in Neuroscience Research, and the UK Animals Scientific Procedures Act.

The in vitro cytotoxicity assay of the purified protease of S. typhimurium against HT29 human adenocarcinoma cell line (Merck, Darmstadt, Germany) was performed on a 96-well plate by incubation for 24 h. Per 1 × 10⁵ HT29 cells per milliliter, fifteen micrograms of the enzyme were used. By the end of incubation, the percent of cell death of HT29 was assessed by the standard MTT assay^{16 (link)}. For negative blanks, physiological saline was used instead of the active protease preparations, while for blanks, a medium without cells was used. The idea of this assay is that the remaining surviving cells can convert the yellow tetrazolium MTT reagent into the purple formazan complex with a characteristic absorption at A₅₄₀ nm, therefore indicating HT29 viability. The intensity of the purple color is in direct relation to the number of surviving cells after exposure to the UcB5.
Furthermore, an assay of cell-damaging activity against RBCs (hemolytic activity) due to the purified enzyme was done. This was achieved by vortexing identical volume sizes of 15 µg protease/mL and 4% (v/v) washed human RBCs suspended in 0.1 M borate buffer, pH 7.5. Incubation was done at 37 °C for 90 min, thereafter, the quantity of released hemoglobin was measured colorimetrically. For comparison, a complete hemolysis treatment was done by mixing RBCs suspension with 1% (v/v) triton X-100 solution.
Also, an in vivo screening of cell-damaging activity was done and the LD₅₀ value was calculated. For this, BALB/c mice weighing 22–25 g were acclimatized to the laboratory conditions for one week and retained at relatively fixed nutritional and physical conditions. They were then divided into six groups of six per cage. The first group was represented as the universal blank group. Mice in this group were intraperitoneally inoculated with an equal volume of a heat-denatured enzyme preparation at a concentration of 60 µg/body weight. While the other five groups were intraperitoneally injected with the active protease preparation at various concentrations (60, 30, 15, 8, and 4 µg protease/body weight) in a total volume of 1 ml solution. Animals were then observed at time intervals throughout 48 h for the LD₅₀ calculation according to the method of Karber. Livers of affected and blank mice were removed instantly after death and fixed in 5% (v/v) glutaraldehyde then 1% (w/v) OsO₄ solution. Before dissection of a blank mouse, it was anaesthsized by the inhalant gas sevoflurane. Ultrathin sections of 70 nm were sliced by RMC ultramicrotome and loaded on standard-grade TEM support grids made-up of copper for examination under JEOL 1010 TEM.