Development of a Rat Model for Investigation of Experimental Splinted Uretero-ureterostomy, Ureteral Stenting and Stenosis

Materials and Methods

We used 32 male Sprague-Dawley rats (Charles River Laboratories Research Models and Services, Germany GmbH, Sulzfeld, Germany) with an initial weight between 300 and 320 g in the test series in two groups. In group 1, 16 rats underwent sham surgery without uretero-ureteral anastomosis but with intraoperative dissection and manipulation of the ureter with a blunt instrument (reference group); in group 2, 16 animals underwent surgery with transsection of the left ureter, insertion of a ureteral stent and an end-to-end uretero-ureteral anastomosis by suturing.

For preoperative analgesia, buprenorphine (Temgesic®) in a dose of 0.1 mg/kg body weight was administered s.c. 1 h before starting the procedure. Anesthesia was induced by intramuscular injection of ketamine (dose 100 mg/kg body weight), (Ketanest S 25®) plus xylazine (dose 5 mg/kg body weight) (Rompun®). Approximately 10 min after injection, anesthesia level was sufficient for further preparation and commencement of surgery. All surgical procedures were performed with conventional surgical instruments under sterile conditions.

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Figure 1.

A: Intraoperative image (macro lens 100 mm, 1:2.8) showing the left retroperitoneum, the ureter, the ileolumbal vessels and the lower pole of the left kidney. B: Cut left ureter with inserted 12 mm-stent (Grilamid® L25 Natur; eucatech AG, Rheinfelden, Germany) cranially of the ileolumbal vessels. C: Totally-inserted stent in the left ureter with a cranial fixation suture (10-0 nylon) to avoid a dislocation of the stent. D: Finished end-to-end ureteral anastomosis of the left ureter with single sutures (Ethilon®, black, monofil, non absorbable, CS-44C+Cs44C, USP 10-0/0,30m, Ethicon).

The abdomen was opened by a midline incision with extension from the suprapubic area to the xiphoid process. In the control animals (group 1), the left ureter, with a medium outside diameter of 0.4-0.6 mm, was dissected-free and manipulated with a blunt instrument at ×20-40 magnification under a surgical microscope. Closure of the abdominal wall was performed in two layers with separate running sutures continuously.

In group 2, the left ureter was dissected-free at ×20-40 magnification under a surgical microscope, diagonally transected at the level of the common iliac vessels. A Grilamid® L25 Natur stent with a caliber of 0.40/0.20 mm and 12 mm length (eucatech AG, Rheinfelden, Germany) was inserted into the ureter and fixed with a suture of 10-0 nylon to avoid dislocation. Subsequently, ureteral anastomosis was performed with six single sutures to avoid urine leakage and the stent was fixed by an additional suture cranial to the anastomosis [Ethilon®, black, monofil, non absorbable, CS-44C+CS44C, USP 10-0/ 0,30m, Ethicon (Figure 1)].

Closure of the abdominal wall was performed in the same way as for the control group in two layers with separate running sutures (Marlin® violet, monofil, absorbable, DS24, USP 3-0/0,7m, Catgut). For all animals, blood tests were performed pre- and postoperatively (white blood cells, red blood cells, hemoglobin, platelets and creatinine) (9,10).

For postoperative histology, 5-bromo-2-deoxyuridine (BrdU, B5002-5G, Sigma-Aldrich, Steinheim, Germany) (100 mg/kg body weight BrdU or 5 ml/kg at a concentration of 20 mg/ml) was injected intraperitoneally on days 1 to 8 postoperatively, at the same time every morning.

For surveillance of the animals, a vigilance score (see appendix) was counted and evaluated on every postoperative day. This score included behavior, body weight, eating behavior, mobility, appearance and serum creatinine. Points were scored as 0.0, 0.2 or 0.4 p per criterion to the termination criterion end-point. At ≥1.2 points, additional measures were considered if necessary (11,12).

The animals were sacrificed on day 28 and a histological examination of the left ureter was done (13).

The study had been approved by the Ethics Committee of the University Medical Center of Rostock University and the State Office of Agriculture, Food Safety and Fisheries, Rostock, Germany (AZ 7221.3-1-026/11).

Histology and immunohistochemistry. Histopathological examination of the ureter was analyzed by light microscopy using the urothelium in the area of the anastomosis with respect to its structure and preservation of the top cell layer. In addition, an examination of the kidney on the operated left side was made in comparison to the contralateral kidney. Histological quantification was carried out by the application of BrdU (BrdU, B5002-5G, Sigma-Aldrich, Steinheim, Germany), a thymidine analogue capable of replacing thymidine in DNA synthesis during the cell cycle. By pulse labelling with BrdU, cellular DNA synthesis can be analyzed by detection with antibodies to BrdU. Antibodies against BrdU recognize BrdU in single-stranded DNA, produced by partial denaturation of double-stranded DNA, as well as free BrdU and BrdU bound to a protein carrier (14). With the BrdU immunohistochemical staining, all newly-formed cells during the 8-day BrdU application are stained brown (Figure 2). A primary monoclonal antibody (Clone Bu20a, M 0744, DakoCytomation, Germany, Hamburg) binds to BrdU and thus forms an antibody-antigen complex. With the help of a secondary antibody, an enzyme reaction and a dye, the bound antibodies are visible under microscopy.

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Figure 2.

A: Histological image (HE/BrdU, 10/40) of a cross-section of the ureter with normal pre- and postoperative formation of basal urothelial cells. B: Hypertrophia of the urothelium in the area of the anastomosis. 5-bromo-2-deoxyuridine-staining shows postoperatively the newly-formed urothelial cells with a dark-colored appearance (counterstaining HE/BrdU, 10/10). Persistent cell activation in the area of anastomosis is remarkable with different enlargement of nuclei and/or a shifted nuclear-cytoplasmatic ratio in the basal and apical layer of the urothelium; furthermore, migration of fibroblasts and granulocytes as sign of a local inflammation and wound healing is apparent. C: Ureteral wall with inserted stent in the lumen (cross-section, HE, 10/100) with moderate hypertrophia of the urothelium. D: Anastomosis region of the ureter with visible suture material (0.02 mm) (see arrows) and increased fibrotic and scarring structures (HE, 10/10).

Statistical analysis. 16 animals per group were expected to be sufficient for the statistical evidence targeting hypertrophic fibrosis of the ureter (normal distribution with standard deviation, error αset at 5%, the difference between the true value of null hypothesis and alternative hypothesis being d=1 with standard deviation s=1 (15, 16).