Medetomidine

After 24 hours of surgery, the animals were euthanized with an intraperitoneal injection of 75 mg/kg body weight of ketamine (Zoetis inc.) and 1 mg/kg body weight of medetomidine (Zoetis inc.). The abdominal cavity was opened, and a heparinized sharp 25-gauge syringe was used to puncture the heart for blood collection, followed by cervical dislocation. Afterwards, the incision was widened along the chest wall to the trachea. To flush the lungs, a 25-gauge needle was punctured into the trachea and a 19-gauge syringe containing 1.1 ml of phosphate-buffered saline (PBS) was inserted. The lungs were flushed with 1.1 ml of PBS and 800 µl of bronchoalveolar lavage fluid (BALF) were collected. BALF was centrifuged at 1164 x g for 5 min at 4°C and the supernatant was frozen at -80°C for subsequent analysis. The upper trachea was closed with a blunt clamp followed by perfusion of 20 ml of PBS through the heart using a 21-gauge blunt-tipped syringe to ensure systemic perfusion of the mouse. The left lung was ligated and removed, rapidly frozen in liquid nitrogen, and stored at -80°C. Furthermore, 10 ml of 4% buffered Zn-Formalin was perfused using a 21-gauge syringe for the right lung lobe, which was then removed and fixed overnight for subsequent (immuno)histological analyses.

Simultaneously, groups of female and male wildtype BALB/c mice were exposed to either: (i) a s.c. injection of 500 L3 in 100 µl RPMI-1640 medium isolated from Chrysops flies; (ii) 10,000 MF via the tail vein in 100 µl RPMI-1640 medium isolated from human peripheral blood [22 (link)], or (iii) 10 L4; (iv) 10 L5; and (v) 10 adult worms all isolated from infected-BALB/cRAG2γc^−/− mice. Whereas L4 were injected s.c. in 100 µl of RPMI-medium, L5 and adult stages were implanted as previously described [23 (link)]. In short, mice were anesthetized with ketamine (Ketaset, 70 mg/kg; Zoetis, Parsippany-Troy Hills Township, New Jersey, USA) and medetomidine (Domitor, 0.8 mg/kg; Zoetis), flanks were shaved, dosed with betadine and following a small incision, L5 or adult worms were implanted into the mice. The incised area was then sutured and a s.c. shot of penicillin (12.06 mg/ml) was administered based on mouse weight (i.e. 100 µl/20 g) at the back of the neck followed by 200 µl antiserdan to wake the mouse up.

Once an animal was identified, four staff members experienced in working with CFSs individually estimated the weight of the animal. An average between these four estimates was calculated. Animals were darted according to this weight with 0.03 mg/kg medetomidine (20 mg/mL; Kyron Laboratories, Benrose, South Africa), 0.2 mg/kg midazolam (50 mg/mL, Dazonil; Wildlife Pharmaceuticals, White River, South Africa) and 0.2 mg/kg butorphanol (50 mg/mL; Kyron Laboratories, Benrose, South Africa). The medetomidine and butorphanol were antagonised intramuscularly using atipamezole 0.15 mg/kg (5 mg/kg, Antisedan; Zoetis, Sandown, South Africa) and naltrexone 0.4 mg/kg (50 mg/mL, Trexonil; Wildlife Pharmaceuticals, White River, South Africa), respectively, both administered intramuscularly. In one animal there was a shortage of naltrexone, and naloxone was substituted (0.4 mg/mL, Narcan; Fresenius-Kabi, Midrand, South Africa) at 0.03 mg/kg, then repeated once at 0.015 mg/kg 7 min later, and a final dose of 0.005 mg/kg 15 min after the first reversal, all administered intramuscularly. As none of the animals were weighed, all dosages were based on estimated weights. Vital parameters including respiratory rate, heart rate, capillary refill time and depth of sedation were monitored where possible.

For anesthesia, medetomidine (0.5 mg/kg, Zoetis, Berlin, Germany), midazolam (5 mg/kg, Ratiopharm, Ulm, Germany), and fentanyl (0.05 mg/kg, Albrecht, Aulendorf, Germany) were administered via intraperitoneal injection. The depth of anesthesia was assessed using toe pinch observing no withdraw reflex (stage of surgical tolerance). Peri-interventionally, mice were placed on a heated pad (Witte & Sutor, Murrhardt, Germany) for the maintenance of homeostasis. For endoscopy and endoscopy-guided tumor cell implantation, the Coloview^® system (Storz, Tuttlingen, Germany) with a zero-degree optic (diameter 1.9 mm) was utilized (Figure 6A). The colon was then washed gently with 2 mL of DPBS at 37 °C using a soft pipette (Nerbe plus, Winsen/Luhe, Germany). For endoscopy without cell implantation, a 7 Fr. examination sheath was used. The valve of the Luer lock adapter (Figure 6A, black asterisk) was adjusted to create a slow constant air flow which made the mucosa just become flattened after gentle trans-anal insertion of the endoscope. This enabled a clear 360-degree view without inflating the gastrointestinal tract too severely. The extent of insertion was controlled endoscopically by using gradations on the endoscope (Figure 6A, red asterisk).