Animals
In this study, 12 healthy three-months-old domestic pigs (a crossbreed between Swedish country breed, Hampshire and Yorkshire) of both sexes were used, with mean body weight 28 kg (21-37). The pigs were housed at room temperature at a farm, with free access to standard porcine fodder before the experiment. They were kept in a 16-hour day and 8-hour night cycle. The experiment was approved by the Regional Animal Ethics Committee in Linköping (Dnr 174-3). The study was conducted in accordance with the guidelines of the European Union for the protection of animals used for scientific purposes. The animal experimentation in this study is reported according to the ARRIVE guidelines (25).
Anesthesia, fluid administration, ventilation and euthanasia
As premedication at the farm the pigs were given Azaperone (200 mg, i.m.; Elanco, Herlev, Denmark). On arriving at the laboratory, anesthesia was induced by Tiletamine (6 mg kg-1, i.m.; Virbac, Kolding, Denmark), Zolazepam (6 mg kg-1, i.m.; Virbac) and Azaperone i.m. (4 mg kg-1). In addition, Atropine (1.5 mg, i.m.; Mylan, Stockholm, Sweden) was given to prevent excessive salivation. The animals received two peripheral catheters (1.1 mm, Venflon Pro Safety, BD, Helsingborg, Sweden) in auricular veins. Propofol (1-2 mg kg-1, i.v.; Fresenius Kabi, Uppsala, Sweden) was given if needed. The animals were orally intubated in the prone position with a 6-mm endotracheal tube (Covidien, Tullamore, Ireland). Anesthesia was maintained by Propofol (10 mg kg-1 h-1, i.v.) and Petidin (1 ml kg-1 h-1) applied by motorized syringe pumps (Alaris CC, Cardinal Health, Rulle, Switzerland). The depth of anesthesia was intermittently monitored by pain response. No muscle relaxants were given. Ringer`s acetate (10 ml kg-1 h-1, i.v.; Fresenius Kabi) and 10% Glucose with 40 mM sodium and 20 mM potassium (0.5 ml kg-1 h-1, i.v.; Fresenius Kabi) were administered by volume pumps (Alaris GP, CareFusion), to substitute for fluid loss. The pigs were ventilated using volume-control ventilation mode (PV 501, Breas Medical AB, Sweden) to achieve an arterial Pco2 of 5.0-5.3 kPa and Fio2 was adjusted to maintain arterial Po2 at 12-18 kPa. The animals were placed on a thermal mattress and covered with a forced-air warming blanket. At the end of the experiment euthanasia was performed with a rapid i.v. injection of 40 mmol potassium chloride (B. Braun, Danderyd, Sweden), and asystole and circulatory arrest were confirmed with ECG and blood pressure recordings.
Surgical preparation and measurements
A 4-Fr introducer was placed in the right carotid artery by open exploration, for the measurement of systemic blood pressure as well as blood sampling. A midline abdominal incision was performed. A 14-Fr Foley catheter was inserted in the urinary bladder and fixed with a purse-string suture. Rectal perforation was performed with a scissor. The perforation was located 8 cm above the anal verge. The diameter of the hole was 3 cm. The midline incision was sutured at the end of the procedure with a continuous suture.
Protocol
Using a sealed envelope system, the animals were randomized before the operation into two groups: 1. An experimental group with rectal perforation and 2. A sham operated control group. After the operation, there was an intervention free period of one hour. Blood samples for mRNA analysis were taken before the laparotomy, and 4 hours after the rectal perforation (Image1).
mRNA analysis
mRNA analysis was performed by Bioinformatics and Expression Analysis, core facility (BEA) , which is supported by the Faculty Board of research at the Karolinska Institute and the Committee for Research at the Karolinska University Hospital,Stockholm, Sweden. Total RNA was isolated from PAXgene Blood RNA tubes with PAXgene Blood RNA Kit standard protocol on a QIAcube, Qiagen. Total RNA quality was assessed by Agilent Technologies 2200 Tapestation and concentrations were measured by NanoDrop ND-1000. Spectrophotometer. Label protocol: 150ng of total RNA was used to generate amplified sense strand DNA targets using Affymetrix WT Plus Kit followed by fragmentation and biotinylation Hybridization protocol: 2.2ug of ss DNA target was hybridized to Porcine Gene 1.0 ST Arrays in Affymetrix Gene Chip Hybridization Oven 645(26). Hybridization, washing and staining was carried out on Affymetrix GeneChip® Fluidics Station 450, according to the manufacturer’s protocol. The fluorescent intensities were determined with Affymetrix GeneChip Scanner 3000 7G. Protein identification was performed according to gene databases. (www.uniprot.org, www. geneontology.org)
Statistical analysis
Statistical analysis was performed with unpaired T-test with the assumption that the values were normally distributed, in R-interface software system, version 3.6.1. P-values were adjusted for multiple testing using with Benjamini-Hochberg procedure. P < 0.05 considered significant.