730 hemostasis and tissue sealing starting directly after surgery [10]. Collagen fleeces are mainly animal-derived collagen matrices that have been found to assist in the healing of gastrointestinal wounds [11]. Up to now, most studies using this approach of sealant- or fleece-reinforced anastomoses concluded there was only limited or no significant improvement in anastomotic healing [12–14]. In addition, experimental anastomoses were constructed with up to 20 sutures leading to clinical leakage rates close to zero. In such cases, this results in extremely small interstices which is not comparable with the human situation in which leaks often result from larger interstices. Particularly in these cases, the sealing effect of fibrin glue together with the reinforcement of a collagen fleece could theoretically improve anastomotic healing. We therefore hypothesized that a collagen fleece secured to the anastomosis with fibrin glue could significantly improve anastomotic healing in a rat model with a 4-suture left-sided colonic anastomosis. Material and methods Male Wistar rats, weighing 300 to 350 g were housed and cared for at the Central Animal Facilities of Maastricht University with food and water ad libitum. The experimental protocol complied with the Dutch Animal Experimentation Act and was approved by the Animal Ethics Committee of Maastricht University. After construction of the anastomosis, we assigned rats to one of three treatment groups following randomization with sealed envelopes: no additional intervention (control, n=20), covering the anastomosis with fibrin glue (fibrin glue, n=20), covering the anastomosis with a collagen fleece, kept in place with fibrin glue (collagen fleece, n=21). At either 3 or 7 days follow-up, half of each group was sacrificed (n=10 per group at each time point, except n=11 for 7 days collagen fleece). Surgical procedure Anastomoses were constructed as described earlier [15]. In brief, rats received 0.1 mg/kg of buprenorphine subcutaneously for analgesia. Anesthesia was induced with isoflurane 5% and maintained with isoflurane 2.0– 2.5%. Rats were not fastened prior to surgery. The abdomen was shaved and the skin disinfected with 2% iodine. Following a 4-cm midline incision, a small segment of the left colon, extending from 2 to 1 cm above the peritoneal reflection was resected. An end-to-end anastomosis was constructed with four evenly distributed, interrupted polypropylene 6×0 sutures. In case of applying fibrin glue, Tisseel Duo (Baxter, Vienna, Austria) at 37°C Int J Colorectal Dis (2011) 26:729–735 was sprayed all around the anastomosis using the Easyspray (Baxter) at a pressure of 1.5 bar. In case of applying a collagen fleece, a single layer of Tissufleece (Baxter) extending 6 mm to each side of the anastomosis was wrapped around the anastomosis after applying fibrin glue. Then, the peritoneum and fascia were closed with a running polyglactin 4 × 0 suture. The skin was closed intracutaneously with a running polyglecaprone 4 × 0 suture. On the first day postoperatively, a single shot of 0.1 mg/kg buprenorphine was administered to all rats. Bowel obstruction Bowel obstructions were defined as a colon distended more than two times its normal diameter proximal to the anastomosis and tapering to a normal diameter distal to the anastomosis. Bursting pressure At either 3 or 7 days, rats were anesthetized as described above. The midline laparotomy was opened and the anastomotic region identified without any manipulation of adhering tissue. The colon was transected approximately 3 cm proximal to the anastomosis, followed by insertion of the tube of a manometer (OJB Industries, Groningen, The Netherlands) which was secured with a suture. Distally from the anastomosis, a clamp was placed just above the peritoneal reflection. Subsequently, the abdomen was filled with 0.9% NaCl solution at 37°C until full submersion of the anastomotic region. Then, the air pressure was gradually increased. The bursting pressure was defined as the maximum pressure read out on the manometer before bubbles appeared. Thereafter, rats were sacrificed by opening the diaphragm and cutting the ventricles. The anastomotic region was resected and fixed in 4.0% formaldehyde for 24 h after which the specimens were embedded in paraffin. Histological assessment Slices 4 μm thick were stained with hematoxylin and eosin (H&E) using standard histological techniques. Tissue sections were also stained for collagen using picro-sirius red. For this purpose, sections were deparaffinized in xylene, rehydrated in graded ethanol to distilled water, and subsequently pretreated with 0.2% phosphomolybdic acid for 5 min. Then, sections were stained for 90 min in 0.1% solution of Sirius Red in saturated aqueous picric acid followed by a 2-min wash in 0.01 N HCL. Hereafter, sections were dehydrated in ethanol and mounted in entallan. Pictures of the anastomotic and perianastomotic