Rompun 2

Detailed description of anesthetic and monitoring procedures is available in Hohlbaum et al. [17 (link)]. In brief, a dosage of 80 mg/kg ketamine and 16 mg/kg xylazine (Ketavet^® 100 mg/mL, Zoetis Deutschland GmbH, Berlin, Germany; Rompun^® 2%, Bayer Vital GmbH, Leverkusen, Germany) was administered intraperitoneally at a volume of 10 μL/g body weight using 27 ¾ gauge needles [17 (link)]. After the loss of the righting reflex, mice were placed in abdominal position on a heating pad. The temperature of the heating pad could be adjusted from 30–42 °C. Artificial tears (Artelac^® Splash MDO^®, Bausch & Lomb GmbH, Berlin, Germany) were administered to the eyes [17 (link)]. Reflexes and vital parameters were carefully monitored during anesthesia, as reported in Hohlbaum et al. [17 (link)].
The mice were either anesthetized once (13 females, 13 males) or six times (i.e., every three to four days; 13 females, 12 males). In the latter case, images generated after their last anesthesia were examined. Control animals (7 females, 6 males) did not receive anesthesia. Since the effects of the different anesthesia regimes on the well-being of the animals had already been investigated in Hohlbaum et al. [17 (link)] and are outside the focus of this article, we do not differentiate between the treatment groups in the present article.

Seven calves served as non-infected controls.
At the age of 3 months, BALF was sampled from all animals for flow cytometric analysis. Within the next four months, BALF was again sampled up to three times from each animal and BALF cells were stored for quantitative real time reverse transcription PCR (RT-PCR) of BALF cells at -80°C. The 17 BALF samples from non-infected controls originated from 7 animals, four animals were sampled three times, two animals were sampled twice and one animal was sampled once. For bronchoscopy, animals were sedated with xylazine (Rompun 2%, Bayer Vital GmbH, Leverkusen, Germany) and bronchoalveolar lavage was performed endoscopically in the standing animal, fluid used and further preparation have been described previously [16 ].
From two animals, lung tissue was sampled by transbronchial lung biopsy [19 ], and from another two animals, lung tissue was sampled at necropsy as described [16 ]. Tissue samples were stored at -80°C until RT-PCR analysis.
All animals remained clinically healthy during the time they were included in the study and the two animals that underwent necropsy showed no lesions or other pathological signs either.

For injection anesthesia a stock solution with 160 μL Ketavet^® 100 mg/mL (Zoetis Deutschland GmbH, Berlin, Germany), 160 μL Rompun^® 2% (Bayer Vital GmbH, Leverkusen, Germany), and 1680 μL physiologic saline solution was prepared in a syringe. A dosage of 80 mg/kg ketamine and 16 mg/kg xylazine [34 ], warmed to body temperature, was administered intraperitoneally at a volume of 10 μL/g body weight using 27 $\frac{3}{4}$ Gauge needles. Then the mouse was transferred to a Makrolon Typ III cage placed on a heating pad. After the loss of righting reflex, the mouse was transferred to a heating pad. Body temperature was measured using a rectal probe during anesthesia and the heating pad temperature, that could be adjusted from 30-42°C, was increased or decreased accordingly to keep body temperature constant. Artificial tears (Artelac^® Splash MDO^®, Bausch & Lomb GmbH, Berlin, Germany) were administered to both eyes to protect them from drying out. Overall duration of anesthesia was 60–84 minutes. Pedal withdrawal and lid reflex were regularly tested and vital parameters (i.e., respiratory rate, heart rate, and oxygen saturation) were monitored during anesthesia. According to the design of our previous study, mice were anesthetized either once or six times at an interval of three to four days [31 (link)].

Ketamine–xylazine was administered at surgery onset and throughout the acute experiment to maintain a steady level of anesthesia. Infusion of 20% v/v ketamine (ketavet or ketabel) (50 mg ml − 1, Ratiopharm GmbH, Ulm, Germany), 5% v/v xylazine (Rompun 2%, Bayer Vital GmbH, Leverkusen, Germany), and 75% v/v of isotonic sodium chloride solution (154 mmol 1–1, B. Braun AG, Melsungen, Germany) was given intraperitoneally for an initial dose of 4 mL per 1 kg of body weight. A needle was placed subcutaneously or intraperitoneally to maintain anesthetic status with an infusion rate of ~0.4 ml per 1 kg of bodyweight per 1 h during the experiment. Anesthetic status was regularly checked (every 7.5–10 min) by paw withdrawal reflex, tail pinch, and breathing frequency. Body temperature was kept stable at 37°C.

All experiments were carried out according to the guidelines stated in Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and were approved by the local authorities in Baden-Württemberg (X-16/10R). New Zealand white rabbits were used in this study (n = 18, age ≈ 10 weeks, both sexes, weight ≈ 1700 g). Rabbits were anesthetized via intramuscular injection of esketamine hydrochloride (Ketanest S 25 mg/mL, Pfizer Pharma PFE GmbH, Berlin, Germany; 12.5 mg/kg body weight) and xylazine hydrochloride (Rompun 2%, Bayer Vital GmbH, Leverkusen, Germany; 0.2 mL/kg body weight). During anesthesia, 1000 units of heparin (Heparin Sodium 5000 I.U./mL, B. Braun Melsungen AG, Melsungen, Germany) and 5 mg of esketamine hydrochloride were given intravenously. Thiopental (Thiopental Inresa 0.5 g, Inresa Arzneimittel GmbH, Freiburg, Germany; 12.5 mg/mL) was then injected intravenously until apnea.