Thirty female New Zealand rabbits, three months old and with a median weight of 2.5 kg (range 2.0 to 3.1 kg) were included. Female rabbits were used throughout the study to avoid gender as a potential confounding factor, although gender is not a factor in the healing of small intestinal anastomoses . All rabbits were housed in wire cages with a shelter and plateau, including wood shavings and hay. They were kept at room temperature (18–20°C) with 40-60% humidity and a 12-hour light/dark cycle. All rabbits had access to standard laboratory diet and water as needed, and they were acclimatized for seven days before surgery.
The animals were randomized to treatment with IFX (n = 15) or placebo (n = 15). The treatment group received a single bolus (10 mg/kg) of infliximab (REMICADE, Centocor, Leiden, Holland) intravenously, and the placebo group received the same volume of isotonic saline. The rabbits underwent surgery one week after medication and were sacrificed at the third postoperative day (POD3) based on the results (unpublished) from a pilot study, where we found a rupture in the anastomotic line in 66% of the anastomosis on POD3 and in only 14% of the anastomosis on POD7. Our interpretation was, that the anastomosis was weaker in the POD3 group, which is supported by the fact that at this time the inflammatory reaction in wound healing is high and gradually replaced by the proliferating phase. In addition to this, it is well known that anastomotic leakage most often develop from POD3 to POD5. The exact time course of strength development in the rabbit small intestine is to our knowledge not available in the literature.
All rabbits were sedated with a 0.3 ml/kg mixture of fentanyl citrate 0.315 mg/ml and fluanisone 10 mg/ml (HYPNORM, Vetpharma, Leeds, United Kingdom) administered subcutaneously together with 2 mg/kg of midazolam (DORMICUM, 5 mg/ml; Roche, Basel, Schweiz). In addition 10 mg/kg of propofol (PROPOVET, 10 mg/ml, Abbott Laboratories Ltd, Berkshire, Great Britain) was given intravenously. After sedation, all rabbits were intubated with a size 3.5 endotracheal tube (Rüsch, Kernen, Germany). The animals were anesthetized with 4 vol.% sevoflurane (SEVOFLURANE, Abbott Scandinavia AB, Solna, Sweden) via a MCM 801-ventilator (Dameca, Rødovre, Denmark). For perioperative analgesia, fentanyl (HALDID, 50 μg/ml, Janssen-Cilag, Beerse, Belgium) was administered intravenously at a rate of 5 ml/hour using an infusion pump (Terumo STC-526, New Jersey, USA). The rabbits were monitored by a pulse oximeter (NONIN 8500 V, Nonin Medical, Minnesota, USA).
Under aseptic conditions, a 4 cm midline laparotomy was made and the extremitas appendices vermiformis caeci was identified. Two separate end-to-end anastomoses were made, approximately 25 and 50 cm proximal to the appendix. All anastomoses were made with interrupted inverted single-layer 5–0 non-absorbable sutures (PROLENE, Ethicon, Johnson & Johnson Nordic, Birkerød, Denmark). The musculofascial layer was closed with interrupted 3–0 absorbable sutures (VICRYL, Ethicon, Johnson & Johnson Nordic, Birkerød, Denmark) and the skin with a continuous 4–0 non-absorbable suture intracutaneously (ETHILON, Ethicon, Johnson & Johnson Nordic, Birkerød, Denmark). Prior to skin incision 0.2 ml/kg of a mixture of sulfadoxin 200 mg/ml and trimethoprim 40 mg/ml (DUOPRIM VET, Intervet International B.V, Boxmeer, Holland) was given intravenously and 5 ml of isotonic saline were administrated subcutaneously after the surgery.
At POD3, the rabbits were euthanized with an overdose of 2 ml intravenous pentobarbital (PENTOBARBITAL, 200 mg/ml; KU Life, Copenhagen, Denmark). A relaparotomy was performed and the two anastomoses were identified and carefully freed from adhesions. The anastomoses were resected with a 2 cm margin on each side, and cleaned for fecal contents with saline. The sutures were left in place. The proximal anastomosis was used to test anastomotic breaking strength and the distal anastomosis to histopathological analysis.
The choice of performing the anastomoses in the small intestine was justified by the fact that the majority of intestinal resections in inflammatory bowel disease involve small intestines and/or proximal colon. Another important aspect was that, that the anatomy of the rabbit colon is significant different from humans and anastomosis on/to the colon would necessitate extensive mobilization of the colon with increased risk of postoperative morbidity, which could be a confounding factor.
Anastomotic breaking strength
The proximal anastomosis was mounted, with 20 mm between the clamps and with the anastomosis in the middle, in a testing machine (LF Plus; Lloyds Instruments, Fareham, UK) equipped with an XLC 10 N load cell (Lloyds Instruments, Fareham, UK). The intestine was stretched at a constant deformation rate of 10 mm/min. The anastomotic breaking strength, defined as the minimal strength necessary to rupture the anastomosis, was derived from the load-strain curve calculated by the software (Nexygen, Lloyds Instruments, Fareham, UK). The site of rupture was noted as either; located in the anastomosis or outside the anastomotic line.
Although the specimen was mounted in the machine with precise accuracy, there always was the uncertainty about the tension test machine to apply an equally distractive force to the entire circumference in each specimen. To minimize this possible error, we programmed the test to start recording after the specimen had been subjected to a tension of 0.1 N. By doing this, we had the opportunity to detect signs on distraction, and we could interrupt the test, remount the specimen and restart if necessary.
A sample of the distal anastomosis with the sutures in situ was fixed in 4% formaldehyde. The sample was dehydrated and embedded in paraffin blocks and sliced 3 μm thin perpendicular to the anastomotic line. Staining was made with hematoxylin and eosin. A conventional binocular Leica DMR (Leica Microsystems A/S, Herlev, Denmark) light microscope with objective 40/0.75 was used. The area within two millimetres related to the anastomotic line was examined and the grade of inflammatory infiltration response in the anastomotic line was scored using a four-graded scale, with a 0–3 point value; absent (0 points), slight (1 point), mild (2 points) and intense (3 points). The examiner of the histological specimens was blinded with regard to treatment group to avoid bias.
Due to the lack of a priori information for sample size calculations, we conducted an interim analysis in order to adjust for the final sample size. Results from that interim analysis, based on 6 rabbits treated with IFX, suggested the anastomotic breaking strength on small intestinal anastomosis to be 1.61 N with a standard deviation of 0.3. Assuming a true, clinical significant difference of 20% between the groups with respect to anastomotic breaking strength, 14 rabbits in each group were sufficient to show a statistical significant difference between the groups with a power of 80% at a significance level of 5% (two-sided). Drop-outs were not replaced due to time constraints.
The data was analyzed according to type, i.e., categorical variables were presented as frequencies and corresponding percentages and continuous variables by mean ± standard deviation (SD). The assumption of a normal distribution of data was assessed visually by means of a quantile-quantile plot for the respective variable. Student’s t-test with unequal variances was applied to compare the weight loss between the groups. Mann–Whitney U test (Wilcoxon rank-sum test) was performed to compare the weight, anastomotic breaking strength and number of sutures between the groups. Fisher’s exact test (two-sided) was used to test the site of rupture and to compare the histological parameters between the groups. P-values <0.05 were considered statistically significant without adjustment for multiple testing. The sample size calculation and statistical analysis was performed using Stata/IC 11.0 (StataCorp LP, College Station, Texas, USA).
The project was performed at the central animal facility of the University of Southern Denmark, and a approval (No. 2005/561 - 1066) to perform the project was granted by the Danish Animal inspectorate on April 15th, 2010.