Zoletil mixture

Sham and the other groups received a sham surgery or RE induction surgery 1 h after the last pretreatment, respectively [22 (link)]. Rats were anesthetized with Zoletil mixture™ (25 mg/kg, i.p.; Virbac, France). For RE induction, the abdomen was opened by a 2 cm median incision. The transitional region between the forestomach and corpus and the pylorus portion were ligated with a 2–0 silk thread (B. Braun Surgical SA, Spain) (Figure 1). A 1 cm longitudinal cardiomyotomy was performed across the gastroesophageal junction. The incised region was immediately sutured. For the sham, the abdomen was opened in the same manner as the RE induction and closed immediately without ligation and cardiomyotomy. All animals were sacrificed after collection of 1 ml of blood from the orbital plexus at 8 h after surgery and samples of esophagus and stomach tissue; gastric contents were collected.

Pigs (70–90 kg) were anesthetized using Zoletil mixture (1 mg/10 kg; Virbac, Denmark) with intravenous supplement of propolipid (Fresenius Kabi, Sweden). Pigs were placed on their side and injected in the neck region with 50 μl of phosphate buffer (10 mM; pH 7.4) supplemented with niacinamide (170 mM; adjusted to isotonic with 85 mM NaCl) or 1-methyl-niacinamide (170 mM). Tissue samples measuring ~2 x 2 cm were excised at 0 min (acute), 2, 5, 15 or 60 min after injection and immediately frozen in 2-methyl-butane. To evaluate whether niacinamide and 1-methyl-niacinamide were entirely confined within the excised tissue, 1 cm thick sections along the four edges of the 0 min (acute) sample were also excised. Following euthanasia by overdose of pentobarbital (Lundbeck, Denmark), tissue samples measuring ~10 x 10 cm were excised and injected to obtain ex vivo 0 min (acute) and 60 min depots.

The laboratory animals were treated according to the rules of Saratov State Medical University (Ethics Committee Protocol No. 7, dated 2 February 2021) and the Geneva Convention of 1985 (International Guiding Principles for Biomedical Research Involving Animals). All experimental procedures were performed on white Balb/c male mice 6–8 weeks old (20–25 g weight) using general anesthesia (Zoletil mixture (40 mg per kg, 50 $\mathsf{\mu }$ L, Virbac SA, Carros, France) and 2% Rometar (10 $\mathsf{\mu }$ L and 10 mg per kg, Spofa, Czech Republic)) via intraperitoneal injection. At the end of the experiment, the animals were euthanized by an overdose of anesthesia.