Ketamine xylazine solution

Animals were anesthetized with ketamine/xylazine solution (80 and 12 mg/kg, respectively; Sigma-Aldrich) and positioned in a stereotaxic frame. A scalp incision along the sagittal midline was performed to access the skull and the bone was perforated with a drill in the following coordinates: 0.5 mm posterior and 1.4 mm lateral from Bregma (Paxinos and Watson, 2007 ). NA from Clostridium perfringens (Roche Diagnostics, Basel, Switzerland, ref. 11 585 886 001) dissolved in 0.9% sterile saline was administered by a single injection 3.5 mm below the dura mater into the right lateral cerebral ventricle. With the aid of a pump, 500 mU (in 20 μL) of NA were perfused for 10 min with a rate of 2 μL/min.

All injections were given i.p., except for the hydrodynamic intravenous (i.v.) tail injection of the DNA plasmid. Injection volumes were always adapted to the bodyweight of the mice. In lethality experiments, mice were monitored by measuring rectal body temperature. Mice with body temperature below 28°C were euthanized using cervical dislocation. Blood was taken via cardiac puncture after sedation of the mice with a ketamine/xylazine solution (Sigma-Aldrich N.V.) or via retro-orbital eye bleeding after sedation with isoflurane. To obtain mouse plasma, blood samples were collected in EDTA-coated tubes, and samples were centrifuged at 3.000 rpm for 15 minutes at 4°C. Plasma samples were stored at -20°C for biochemical analysis. For sampling of liver, mice were killed by cervical dislocation at indicated time points.