Isoflurane

Animals were transcardially perfused with 50–100 ml of a fixative composed of 4% paraformaldehyde (PFA) in 0.1M phosphate buffer (PB; pH 7.4), the brains dissected out, and post-fixed in the same fixative overnight. Alternatively, animals were deeply anesthetized (5% isoflurane (AbbVie) in 1L/min air flow), their brains rapidly dissected, and immersion-fixed in 4% PFA in phosphate-buffered saline (PBS, 0.05M, pH 7.4) at 4 °C for 24 h. Tissues were then extensively washed in PB, cryoprotected by immersion in 30% sucrose in distilled water at 4 °C 24–48h, and processed as described below.

Control (SHAM) and duodenojejunal omega switch (DJOS) surgeries were performed as described in the work of Stygar et al. [18 (link)].
Oxygen flow of 2 L/min and 2% isoflurane (AbbVie, Ludwigshafen, Germany) under spontaneous breathing was used to maintain the anesthesia. Xylazine (5 mg/kg, ip; Xylapan, Vetoquinol Biovet, Puławy, Poland) was used to achieve analgesia. Gentamicin (gentamycin 40 mg/mL, Krka, Warszawa, Poland) was used as the antibiotic prophylaxis.
A 3–4 cm midline incision was made to access the abdomen. The duodenum was separated from the stomach slightly distally to the pyloric sphincter. The proximal part of the small bowel and the duodenum was excluded from the bowel content passage and closed using PDS 6/0 (Ethicon, Cincinnati, OH, USA). The end-to-side anastomosis (duodeno-enterostomy) was positioned at one-third of the small intestine total length to restore the bowel content passage. The mesentery was closed with PDS 6/0.
During SHAM surgery, the gastric tract was cut at the site analogous for duodenum separation during DJOS surgery, and it was immediately reattached to the stomach at the same position, and thus the intestinal food passage was restored.
Carprofen (4 mg/kg, sc; Rimadyl, Pfizer, Zürich, Switzerland) was used to achieve analgesia in the post-operative period for three consecutive days.

Mice were euthanized with an overdose of isoflurane (AbbVie, Chicago, USA) 5, 10, 15, 35, and 60 days post infection. The thoracic cavity was flushed with 1 ml of RPMI 1640 (PAA Laboratories, Pasching, Austria) and adult worms or L3/L4 larvae were collected and counted, the sex determined of L4 as well as adult worms and the lengths measured. Measurements of larvae were made using one randomly selected worm per mouse for 15 dpi and 10 female worms per mouse, if present, at 30 dpi. For measurements, the microscope analysis software Diskus 4.6 (Buero Hilgers, Königswinter, Germany) was used.

Dorsal root ganglions were harvested from naïve male Wistar rats following their sacrifice by an overdose of isoflurane (5%) (AbbVie, Wiesbaden, Germany). The thoracic and lumbar spinal regions were exposed and all DRGs were collected in a working medium (DMEM/HAM’s F-12 without penicillin/streptomycin). A total of 1.25% collagenase was added just before incubation. Following their incubation at 37^°C for 60 min, DRGs were washed three times with PBS and incubated in the working medium digestive solution with trypsin for 10 min at 37^°C. Thereafter, the tissue was further triturated using plastic pipette tips and filtered through a 40 μl filter. The filtrate was centrifuged and the pellet was resuspended in 1 ml culture medium (DMEM/HAM’s F12 supplemented with 1% penicillin/streptomycin and 10% horse serum). Neurons were then seeded onto poly-L-lysine coated plastic culture dishes (35 mm) and placed in an incubator (5% CO₂ at 37^°C). One hour later, 2 ml of culture medium were added to the cell culture and neurons were incubated for 24–48 h until patch clamp recordings or calcium imaging, as previously described (Nockemann et al., 2013 (link); Dembla et al., 2017 (link)).

Experiments were performed in male Wistar rats (200–300 g, Janvier, France) and were approved by the State animal care committee (Landesamt für Gesundheit und Soziales, Berlin). All experiments were performed in accordance with the relevant guidelines and regulations. The sample size was calculated with the G*Power 3.1.2 program (80% power and 0.05 level of significance). Animal experiments were stopped when specific termination criteria were reached (approved by the State animal care committee). The experimenter who performed experiments and analyzed the results was blinded to the interventions. Rats were kept on a 12 h dark-light cycle in groups of 3 in cages lined with ground corncob bedding with free access to food and water ad libitum and constant room temperature and humidity of 22 ± 0.5 °C and 60–65%, respectively. Before nociceptive testing, handling was performed once per day for 1–2 min. Animals were habituated to the test cages once or twice a day for 15 min, starting 4 days prior to the experiments. Statistical power calculations were performed to obtain the minimal number of animals for the experiments. After termination of the experiments, rats were killed by an overdose of isoflurane (AbbVie, Ludwigshafen, Germany).

All animal experiments were conducted according to Israeli law and were approved by the ethics authority (Weizmann Institute IACUC application number: 11400614-4).
Analyses of primary tumor growth and lung metastases were performed in 10–12 week-old female BALB/c mice. One-day pre-injection, mice were anesthetized using 2 mg/10 gr body weight Sedaxylan, 10 mg/10 gr body weight Ketamine, and shaved using a shaving machine and Orna 19 (hair removal cream, Alpha Cosmetics, Israel).
Primary tumor injection: 2.5*10⁴ 4T1-luciferase (shCon or shEcad) cells in 50 μl PBS were injected into the mammary fat pad.
Intravenous injection: 1*10⁵ 4T1-luciferase (shCon or shEcad) cells in 150 μl PBS were injected into the tail vein. For visualizing tethers in lung metastases, 1*10⁶ 4T1-GFP cells were injected intravenously, animals were sacrificed, and small metastatic nodules in the lungs were immediately imaged, using 2-photon microscopy (LSM 880, Zeiss; 20× objective).
Bioluminescence imaging (BLI): Prior to mice imaging, 50 μl of 30 mg/ml luciferin (Regis Technologies, Inc., Morton Grove, IL, USA) were injected intraperitoneally. After 7 minutes, mice were anesthetized using isoflurane (AbbVie, Inc., North Chicago, IL, USA), delivered with oxygen from a precision vaporizer. Non-invasive BLI was performed using a Xenogen IVIS Spectrum optical imaging system (PerkinElmer, Inc., Waltham, MA, USA).