The study was performed on 12 juvenile (8–12 weeks old; 15–20 kg bodyweight (b.w.)) female Large White Polish pigs. The animals were kept under standard laboratory conditions. They were fed standard fodder (Grower Plus, Wipasz, Wadąg, Poland) and had free access to water. To ensure adequate acclimatization, the pigs were transported from the breeder to the animal quarters 5 days before the scheduled procedure. The pigs were divided into two groups. Six pigs served as controls (CTR) and were treated with intravesical instillation of a 5% aqueous solution of ethyl alcohol (60 mL). Another group of 6 pigs was treated with RTX (product code: AG-CN2-0534, AdipoGen Life Sciences, Hamburg, Germany) by intravesical instillation of the toxin (500 nmol per animal in 60 mL of 5% aqueous solution of ethyl alcohol) to mimic the dose and route of its administration used in humans. Both the alcohol and RTX solutions were given under anesthesia. First, all the animals were pretreated with atropine (Atropinum Sulfuricum, Polfa, Warsaw, Poland, 0.05 mg/kg b.w., s.c.) and azaperone (Stresnil, Janssen Pharmaceutica, Beerse, Belgium, 2.5 mg/kg b.w., i.m.). After that, buprenorphine (Bupaq, Richter Pharma AG, Wels, Austria, 20 µg/kg b.w., m.c., i.m.) was given to abolish the visceral pain sensation. Thirty minutes later, anesthesia and analgesia were induced by intramuscular injection of ketamine (Bioketan, Vetoquinol, Gorzów Wielkopolski, Poland, 10 mg/kg b.w.) and xylazine (VetaXyl, Vet Agro, Lublin, Poland, 0.15 mg/kg b.w.) and by propofol (Propofol-Lipuro, B. Braun Melsungen AG, Vienna, Austria, 10 mg/kg b.w.,) given intravenously in a slow, fractionated infusion. The depth of anesthesia was monitored by testing the corneal reflex.
One week after the administration of the aqueous solution of ethyl alcohol or RTX, all the pigs were once again premedicated and deeply anesthetized (the drugs, their doses, and routes of administration were analogous to those previously used), and a midline laparotomy was performed. The urinary bladder was gently exposed in each animal and cut out for transcriptome analysis (the samples of the urinary bladder wall used in this experiment were collected from the middle part of the ventral side of the bladder). The samples were taken in vivo to ensure the acquisition of high-quality and -purity RNA for transcriptome sequencing. Next, all the pigs were euthanized with an overdose of sodium pentobarbital (Euthasol, LeVet B.V., Oudewater, Holland, 140 mg/kg).
One week after the administration of the aqueous solution of ethyl alcohol or RTX, all the pigs were once again premedicated and deeply anesthetized (the drugs, their doses, and routes of administration were analogous to those previously used), and a midline laparotomy was performed. The urinary bladder was gently exposed in each animal and cut out for transcriptome analysis (the samples of the urinary bladder wall used in this experiment were collected from the middle part of the ventral side of the bladder). The samples were taken in vivo to ensure the acquisition of high-quality and -purity RNA for transcriptome sequencing. Next, all the pigs were euthanized with an overdose of sodium pentobarbital (Euthasol, LeVet B.V., Oudewater, Holland, 140 mg/kg).
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