These experiments were performed as a pilot of a project aiming to explore the effects of a new endotoxin scavenger as a randomized placebo-controlled trial, the results of the main experiments are not published in this manuscript.
The experiment was reviewed and approved by the Animal Care and Experimentation Committee of the Canton of Bern, Switzerland (26169 BE6/15) and followed the Swiss national guidelines for the performance of animal experiments. Male Wistar rats aged 8–9 weeks, weighing 380 g in average were obtained from Janvier Labs (Le Genest-Saint‐Isle, France) and kept in individually ventilated cages (IVC) with controlled 12 hours light/dark cycles at 22 ± 2 °C at the Central Animal Facility of the University of Bern. Food and water were provided ad libitum. A total of 56 animals were included (4 groups: 20 animals sham surgery ± scavenger/placebo, 36 animals CASP ± scavenger/placebo).
Anesthesia was induced with 6% sevoflurane (Sevorane, AbbVie, Baar, Switzerland) in oxygen in an induction chamber, and 50 µg/kg fentanyl (Janssen-Cilag, Zug, Switzerland) was added intraperitoneally. The animals were intubated using a modified 2.0 mm Angiocath (BD, Allschwil; Switzerland) and mechanically ventilated with a small animal ventilator (KTR-4 Rodent Ventilator, Hugo Sachs, March, Germany). Anesthesia was maintained with sevoflurane 2.5–3.5%, and analgesia with additional 50 µg/kg fentanyl administered subcutaneously 30 minutes later. The animal was placed on an operation table with an in-built feedback controlled heater system (TCAT-2, Harvard Apparatus, Hugo Sachs, March, Germany) aiming for a constant body temperature of 37 °C.
First, we incised the right groin, and then tunneled a Redon needle/wound drainage trocar from the neck to the groin. Two polyurethan catheters (PU 40, SAI Infusion Technologies, Lake Villa, IL, USA) were inserted in the subcutaneous tunnel. The catheters were then equipped with a 1.5 to 2 cm tip made of PE-50 for vessel cannulation, and filled with heparinized sterile normal saline solution (5000 I.U. heparin in 1000 ml NaCl 0.9%) via a blunt needle and luer lock syringe on the cranial part. The femoral artery and vein were microsurgically exposed, cannulated and secured with a suture. Before skin closure, the catheters were additionally bend as a loop and secured twice with sutures. The animals were turned in prone position and the catheters secured with a subcutaneous loop and suture at the neck. We channeled the catheters trough a tethered harness (SAI Infusion Technologies, Lake Villa, IL, USA) and connected them via a two channel swivel with 2 purge systems (syringe drivers) to guarantee free movement of the animal and integrity of the catheters. After drawing a first arterial and venous blood sample, both purge lines were continuously flushed with 1.5 ml/h Glucose 5% in H2O 2:1 and heparin (5000 I.U. heparin in 1000 ml GS), to the arterial line purge we added buprenorphine (Temgesic, Reckitt Benckiser Switzerland, Wallisellen) to achieve a concentration of 1 µg/ml. The animals were weaned from the ventilator, extubated and placed for recovery from surgery in a cage equipped with a swivel mount. Duration of surgery was 50–60 minutes.
Colon Ascendens Stent Peritonitis
6 hours after finishing the preparation the animals were re-anesthetized with sevoflurane 6% in oxygen in the induction chamber, supplemented with 20 µg/kg buprenorphine subcutaneously. The animals were placed on the heated operation table, and anesthesia was maintained with 2.5 to 3.5% sevoflurane in oxygen via facemask. A median laparotomy was performed, and the coecum and the ascending colon identified and exposed. A small incision was made about 1 cm distal the iliocoecal valve at the anti-mesenterial side, and a 12 French PU catheter of 1.5 cm length was inserted. This stent was additionally fixed to the colon by a suture. Patency was tested by instillation of saline into the stent, with spontaneous emptying of feces afterwards, or gentle squeezing the coecum until intestinal content was visible in the opening of the stent. The animals in the sham group received the same surgery, omitting colostomy, with the stent only sutured at the outer side of the colon ascendens. The intestine was packed back into the abdominal cave and the abdominal wall was closed in two layers. Surgical time was below 20 min. The animal was placed back into its cage equipped with the swivel mount. Volume replacement was started, the buprenorphine dosing was resumed. Additionally the animals were provided with water and nourishment ad libitum.
Surgical repair and treatment
16 hours after peritonitis induction and randomization, animals were again re-anesthetized as described before using induction chamber and facemask. The abdominal sutures were re-opened, the stent identified, which could necessitate exploration with sterile cotton swabs to remove fibrin and detritus. Cultures from the abundant purulent ascites and detritus were taken at this time point for the main experiment. Once the stent was identified, the additional suture was cut, the stent removed and the intestinal wall closed with simple interrupted stitchings. The abdominal cavity was then rinsed with warm Ringer’s solution, and the abdominal wall closed with sutures. Surgical time was below 20 minutes.
Then the rats were placed in their cages with the swivel mount. All animals received an intravenous infusion of meropenem (Meronem, Pfizer Switzerland, Zurich, 75 mg per kg BW, diluted to 5 mg/ml, in 15 min) followed by a continuous infusion via the central venous line (225 mg/kg BW/24 hours). Analgesia was provided continuously with buprenorphine administered via the intraarterial purge line.
Blood pressure was measured continuously with a standard transducer system linked to a BIOPAC MP100 analog-digital-converter and was acquired and analyzed with the Biopac AcqKnowledge Data Acquisition Software (BIOPAC, Goleta, CA 93117, USA). Heart rate was calculated with the blood pressure tracing.
Blood samples were drawn 16 and 40 hours post peritonitis induction, and after the last blood sampling. The animals were euthanized with by intravenous infusion of pentobarbital (150 mg/kg BW, Esconarkon, Streuli Pharma AG, Uznach, Switzerland).