301 Intraperitoneal cytostatic-loaded supramolecular hydrogel and intestinal anastomotic healing studies described by Wintjens et al., the rats received 20 mL/kg of hydrogel corresponding to a single intraperitoneal injection of 5 mL for female rats (±250 g) or 8 mL for male rats (±400 g). Anesthesia, Surgical Procedure and Analgesia A subcutaneous injection of 0.05 mg/kg buprenorphine (Richter Pharma AG, Wels, Austria) was given one hour prior to surgery as an analgesic. The surgical procedure was performed by experts (A.J. and N.B.) certified for performing anastomotic models in laboratory animals. All animals underwent general anesthesia using 4–5 vol.% isoflurane supplied with air (IsoFlo, Zoeties B.V., Rotterdam, The Netherlands) for induction which was maintained with 2–3vol.%. The body temperature was maintained by placing the animals on a heated plate with a temperature of ca. 36 °C. A 5 cm craniocaudal midline incision of the skin and abdominal musculature was performed with a scalpel, after removing the abdominal fur with electric clippers and local injection of bupivacaine (Aurobindo Pharma BV, Baarn, The Netherlands). The cecum and additional intestines were taken outside the abdomen onto sterile gauzes hydrated with sterile saline solution to prevent dehydration. The site for colon–colon anastomosis was identified at ca. 4 cm ab ani, whereafter the colon was fully transected with scissors. An end-to-end anastomosis was created using at least 9 interrupted polypropylene sutures (Prolene 6-0, Ethicon, Johnson & Johnson; Supplementary Figure S1). After the creation of a sufficient anastomosis, it was tested for leakage of water by injection of NaCl via the rectum. In case of water leaking through the anastomosis, additional sutures were placed until the anastomosis remained dry. Thereafter, the intestines were repositioned in the abdomen, and the abdomen was closed with a running suture for the muscle layer (Prolene 4-0, Ethicon, Inc., Johnson & Johnson) and interrupted sutures for the skin (Monocryl 4-0, Ethicon, Inc., Johnson & Johnson). Subsequently, the animals received a single intraperitoneal injection with 5 or 8 mL (F/M) saline, unloaded hydrogel or MMC-loaded hydrogel, corresponding to a volume-to-weight ratio of 20 mL/kg. Postoperatively, a saline + 3% glucose solution (3–5 mL) was administered subcutaneously to prevent dehydration. General anesthesia using isoflurane was maintained for at least 20 min after intraperitoneal administration of either one of the three interventions, conforming previous experiments with a comparable hydrogel formulation 12. Subcutaneous injections of 0.03 mg/kg buprenorphine were continued every six hours for 48 h for all animals, as most post-operative discomfort was expected in the first 48 h. In addition, 200 mg/kg paracetamol (Dafalgan, UPSA, France) was given in a separate drinking bottle during the entire experiment. If animals showed signs of discomfort based on the welfare scoring sheets, additional pain medication by subcutaneous injections of buprenorphine was administered and/or saline + 3% glucose solution in case of dehydration signs. After seven days, all animals were euthanatized via CO2 asphyxiation. Afterwards, the intraabdominal cavity was inspected via laparotomy. If needed, blood samples were taken from the vena cava. 13
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