Wire-Calibrated Induction of Segmental Ureteral Strictures in Rats via Midline Laparotomy

Summary

This protocol describes a reproducible rat model for inducing graded segmental ureteral strictures using wire‑calibrated partial ligation via midline laparotomy. A 6‑0 ligature around the ureter and a metal calibration wire generate a defined residual lumen, enabling immediate functional confirmation of obstruction severity.

Abstract

Ureteral strictures remain a major challenge in reconstructive urology and can arise from iatrogenic injury, chronic inflammation, trauma, congenital anomalies, or radiation.​ These lesions frequently lead to progressive obstructive uropathy and loss of renal function. Despite advances in endoscopic and surgical techniques, ureteral strictures remain challenging to address, and their remodeling effects on the renal parenchyma remain the subject of ongoing research, underscoring the need for robust preclinical models. This study establishes a reproducible rat model for controlled induction of various degrees of segmental ureteral strictures using wire‑calibrated partial ligation via midline laparotomy. The protocol enables segmental, non‑transecting constriction of the ureter, intended to approximate fibrotic narrowing at a functional and anatomical level while minimizing evident ischemic damage and maintaining macroscopic tissue viability with ureteral continuity. By providing a reliable experimental platform with low inter‑animal and inter-surgeon variability, this approach supports translational research on biomolecular mechanisms, biomaterials, imaging modalities, and reconstructive techniques for ureteral stricture disease.

Introduction

Ureteral strictures are relatively common and pose a significant challenge in urology. They are reported in up to 4.9% of patients following endoscopic procedures for stone disease^1,2,3, with even higher incidences among high-risk populations, such as kidney transplant recipients, where up to 7% may be affected⁴. Although many strictures are caused by surgical injury or medical interventions, non-iatrogenic causes also comprise an important proportion. These include chronic inflammation, urinary tract infections, physical trauma, congenital abnormalities, and radiation therapy^5,6. Each of these etiologies can ultimately result in urinary obstruction, recurrent infections, pain, stone formation, and - if untreated - irreversible loss of renal function. Affected patients often experience morbidity and reduced quality of life, as shown in both institutional cohorts and systematic reviews^5,7,8. The long‑term burden is substantial; in a multi‑institutional cohort of patients with ureteral strictures after ureteroscopic stone surgery, nephrectomy of the affected side was required in 25% of cases, underscoring the considerable morbidity of this condition⁹.

This clinical burden underscores the need for robust preclinical models that can enable research on associated biomolecular processes and inform treatment optimization. The most common treatment modalities are endoscopic methods, such as dilation or laser incision, as well as stenting, with surgical reconstruction reserved for more complex cases¹⁰. Despite improved early outcomes, recurrence and restenosis remain problematic. Reported recurrence rates after intervention range from 11% to 40%, and many patients require multiple operations or long‑term stenting for durable relief and organ protection^11,12. Treatment success is influenced by stricture site, length, etiology, and details of previous treatments; several recent reviews and large cohort studies underline the need for improved techniques and a deeper mechanistic understanding^4,5,13.

Effective translational research requires experimental models that closely replicate the fibrotic mechanisms, histopathologic features, anatomical context, and upstream renal processes of human ureteral stricture disease. Among these, rodent models have become reliable platforms to study ureteral fibrosis and renal functional loss and to evaluate anti‑fibrotic interventions and reconstructive strategies^{6,14,15,16,17}. The pathophysiologic foundation for these models was laid by Chevalier et al., who reviewed different strategies of ureteral obstruction, focusing on unilateral ureteral obstruction (UUO) as a reproducible model of renal interstitial fibrosis and obstructive nephropathy in rats⁶.

Building on this foundation, contemporary models have refined the level and pattern of obstruction. Vroomen et al. introduced a technically safe approach using irreversible electroporation to induce partial unilateral urinary obstruction with renal scarring¹⁴. Similarly, Chen et al. advanced a reversible UUO model enabling dynamic study of fibrosis regression under variable hemodynamic conditions¹⁵. Nan et al. and Khater et al. further elucidated the molecular cascades driving ureteral fibrosis progression and therapeutic modulation in partial‑obstruction^16,17.

Earlier models that relied on complete ureteral ligation, segmental resection, burying in the psoas muscle, or thermal injury often resulted in uncontrolled, unstandardized degrees of obstruction, extensive ischemia, and necrosis, leading to severe renal parenchymal atrophy rather than graded obstruction. These approaches, as described by Chevalier et al. and Thornhill et al., provided valuable mechanistic insights into obstructive nephropathy, but did not recapitulate the localized fibrotic narrowing characteristic of clinical benign ureteral stricture disease^6,18. There remains a lack of simple, standardized rodent models that generate calibrated, non‑transecting segmental strictures with preserved tissue viability and defined degrees of renal urinary obstruction.

To address this gap, this study establishes a standardized surgical protocol for the controlled induction of partial ureteral strictures in rats. The model is designed to be reproducible, anatomically precise, and versatile for use in preclinical research encompassing reconstructive urology, intraoperative imaging evaluation, and tissue‑engineering applications. By minimizing inter‑operator variability and replicating clinically relevant fibrotic narrowing, this model offers a translational platform to advance mechanistic understanding and accelerate therapeutic development for ureteral stricture disease.

In practical use, the degree of obstruction can be tailored by selecting the appropriate calibration wire diameter to match the research question. Larger diameters (e.g., 0.65 mm) approximate mild partial obstruction with near‑physiological urine flow, whereas smaller diameters (0.30–0.20 mm) model high‑grade obstruction with marked flow reduction. Furthermore, this surgical approach can be applied either as an acute terminal model to study immediate hemodynamic and functional changes or adapted for survival experiments to investigate chronic tissue remodeling, fibrosis progression, and long‑term renal adaptation.

In this model, partial obstruction is achieved by tying a 6‑0 ligature around the ureter together with a metal calibration wire of defined diameter and subsequently removing the wire, leaving a residual lumen that corresponds to the wire size. This design enables graded, geometrically controlled narrowing that can be directly correlated with renal functional urinary outflow.

Protocol

All animal activities described here were conducted in accredited facilities and approved by the institutional animal care and use committee (IACUC) of the Baden-Württemberg Regional Council in Karlsruhe, Germany. Experimental animals were handled in accordance with institutional protocols and in compliance with German legislation governing animal welfare, as well as the guidelines set forth by the European Community Council (2010/63/EU) and the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. Male Sprague Dawley rats (average weight: 400 g) were used following a 7-day acclimatization period in the animal facility under standardized conditions. The reagents and the equipment used are listed in the Table of Materials.

1. Anesthesia and analgesia

1. Induce sedation using 4 vol% isoflurane delivered in 100% oxygen via an induction chamber at a flow rate of 5 L/min.
2. Upon loss of righting reflex, administer a subcutaneous injection of 100 mg/kg ketamine (10% solution; about 0.35 mL) and 4 mg/kg xylazine (1:10 dilution of 2% solution; about 0.75 mL) for dissociative anesthesia, analgesia, and sedation.
3. Confirm anesthetic depth by the absence of response to a firm toe-pinch stimulus using forceps.
4. Apply ophthalmic ointment to both eyes to prevent corneal desiccation.
5. Achieve additional analgesia through a subcutaneous injection of 5 mg/kg carprofen (1:10 dilution of 5% solution; about 0.35 mL).
6. Maintain anesthesia with supplemental 4 vol% isoflurane via a neonatal face mask as needed throughout the procedure.
7. Maintain body temperature at 36.5–37.5 °C using a thermostatically controlled warming pad throughout the procedure.

2. Operating site and instrument preparation

1. Prepare the scrub table with all required instruments, including atraumatic preparation forceps, blunt overholt clamps, preparation scissors, non-resorbable monofilament 6-0 ligatures, metal calibration wires (alloy with electropolished surface and deburred ends; 0.65, 0.55, 0.30, 0.20 mm in diameter; sterilized (autoclaved) prior to use), humidified cotton wool swabs, gauze swabs, 1 mL syringe with integrated 30 G needle, methylene blue (0.5% solution, undiluted), isotonic sodium chloride solution (0.9% NaCl), and capillary tubes.
2. Mount the animal on the rodent surgical exposure apparatus as described in previous reports^19,20.
3. Shave the abdominal access area and perform alternating triple scrubs with 70% ethanol and povidone-iodine or chlorhexidine-based antiseptic swabs in concentric circles from the incision site outward. Repeat three times.
4. Cover the animal with sterile drapes, leaving only the surgical field exposed.
5. Handle volatile anesthetics, sharps, and chemical dyes in accordance with institutional biosafety regulations, using appropriate gas scavenging systems, protective equipment, and approved sharps disposal containers.

3. Surgical access via midline laparotomy

1. Perform a median abdominal cutaneous incision of approximately 3 cm using fine scissors.
2. Incise the linea alba longitudinally in the midline to access the peritoneal cavity.
3. Use preparation hooks and moist compresses to retract the abdominal wall and expose the retroperitoneum.

​4. Identification and mobilization of the left ureter

1. Identify the kidney and retroperitoneal space and gently mobilize the surrounding retroperitoneal fat using blunt dissection with forceps and humidified cotton swabs (Figure 1A).
2. Puncture the renal pelvis through the renal parenchyma with a fine 30 G needle (Figure 1B).
3. Inject 0.01–0.2 mL of undiluted methylene blue to facilitate visual identification of the ureter (Figure 1B.5).
4. Observe progressive dye demarcation to visually track the ureteral course from the renal pelvis towards the bladder (Figure 1B.6–10).
5. Gently separate the surrounding connective and adipose tissue from the dye-demarcated ureter using fine forceps and humidified cotton swabs with a plucking motion (Figure 1C). Avoid direct grasping, compression, or excessive traction of the ureteral wall to prevent ruptures or fistulas. Mobilize the ureter until it can be followed continuously from the renal pelvis toward the vesicoureteral junction (Figure 1D).

Figure 1: Dye-assisted identification and mobilization of the left ureter. The green arrow depicts the renal injection site. The grey arrow depicts the naïve ureter. The blue arrow depicts a dye‑demarcated ureter. The black box depicts a subsequently magnified area. (A) Left retroperitoneal exposure after midline laparotomy. (B) Dye‑assisted ureter demarcation. (C) Blunt dissection mobilizes the ureter from surrounding tissue. (D) Fully exposed left ureter from renal pelvis to vesicoureteral junction.​ (E) Schematic illustration of the described steps, including syringe and methylene blue. Please click here to view a larger version of this figure.

5. Distal calibrated partial ligation of the left ureter using a metal wire

1. Elevate a mobilized segment of the distal ureter using atraumatic forceps without compressing the ureter (Figure 2A.1–2).
2. Pass a 6-0 monofilament suture underneath the ureter using blunt forceps (Figure 2A.3).
3. Place a metal calibration wire of the desired diameter (0.65, 0.55, 0.30, or 0.20 mm) parallel to the ureter within the ligature loop (Figure 2B.1–6).
4. Tighten the ligature gradually using a sliding knot with several loop throws in the same direction until the suture sling adapts tightly to the ureter‑wire complex without loosening, which requires at least 4 throws with slowly bouncing finger movements to allow for suture settling in between throws.
   ​NOTE: The suturing tension should be just below the tensile breaking strength, which is 5–7 N for a 6-0 non-resorbable suture, so 3 N is recommended. This translates to a pulling weight of about 306 g and can either be measured by using a spring scale or is comparable to the digital strength when holding a regular cell phone with two fingers in a pinch grip. The essential aspect is that the suture is completely closed to the ureter‑wire complex and that there are so many loop throws that they do not become loose before the locking throws follow. Due to the fragile nature of the 6-0 sutures, there is no “too tight” as the suture will break before it would be too tight for the ureter tissue to degenerate. Now secure the knot with 3 counterwise locking loop throws to prevent air knots (Figure 2B.7–10).
5. Carefully withdraw the calibration wire in a slow, straight motion to avoid shearing injuries to the ureteral wall, leaving a standardized residual ureteral lumen corresponding to the wire diameter (Figure 2C.1–5). The polished wire surface minimizes friction during removal and reduces the risk of tissue damage.

Figure 2: Distal calibrated partial ligation of the left ureter using a metal wire. (A) Distal ureter isolated and elevated with overholt clamps; 6-0 suture placed underneath.​ (B) Calibration wire positioned parallel to the ureter within the ligature and ligation using a sliding knot. (C) Wire withdrawal, leaving a standardized residual lumen. (D) Schematic illustration of the described steps with the ureter (blue), calibration wire (gray), and ligature (black). Please click here to view a larger version of this figure.

6. Identification and mobilization of the right ureter

1. Repeat the steps in analogy to the contralateral side (Figure 3).

Figure 3: Dye-assisted identification and mobilization of the right ureter. (A) Right retroperitoneal exposure with dye‑assisted ureter demarcation. (B) Blunt dissection mobilizing the ureter from surrounding tissue. (C) Schematic illustration of the described steps. Please click here to view a larger version of this figure.

7. Proximal complete ligation of the right ureter using a sliding knot without wire

1. Elevate a mobilized segment of the proximal ureter using atraumatic forceps without compressing the ureter (Figure 4A.1–3).
2. Pass a 6-0 non-resorbable monofilament suture underneath the ureter using blunt forceps (Figure 4A.4,5).
3. Tighten the ligature gradually using a sliding knot with several loop throws in the same direction until the suture sling tightly ligates the ureter without loosening, as described above. Then, secure with counterwise locking loop throws in order to achieve near-complete luminal closure (Figure 4B.1–4).

Figure 4: Proximal complete ligation of the right ureter using a sliding knot without wire. (A) Proximal ureter isolated and elevated with Overholt clamps; 6-0 suture placed underneath. (B) Complete ligation without wire using a sliding knot. (C) Schematic illustration of the described steps. Please click here to view a larger version of this figure.

8. Functional validation of partial and complete ureteral obstruction

1. For validation of partial obstruction on the left side, inject 0.2 mL undiluted methylene blue transparenchymatously into the renal pelvis using a syringe and observe antegrade filling of the ureter distal to the ligation site (Figure 5A.1–3).
2. Inject 1.0 mL saline into the renal pelvis to progressively clear the dye signal by wash-out and confirm continued ureteral patency (Figure 5A.3). To further increase certainty of incomplete ureteral obstruction, continue injection until the bladder volume capacity is surpassed and visible fluid outflow from the urethra can be observed (Figure 5A.4–5).
3. For validation of complete obstruction on the right side, inject 0.2 mL undiluted methylene blue transparenchymatously into the renal pelvis and confirm absence of distal dye passage (Figure 5B.1,2).
4. Identify proximal ureteral caliber dilation upstream of the ligation site as an indicator of complete outflow block (Figure 5B.3).
5. For additional validation beyond the feasible usability of the animal model, transect both ureters distally and position the open ureteral ends directly adjacent to glass capillaries for urinary collection measurements (Figure 5C).
6. For validation of this model, quantify the urinary production per kidney over a 30-min period under physiological conditions and after partial ligation using calibration wires of 0.65, 0.55, 0.30, and 0.20 mm diameters (Figure 5C.2).

Figure 5: Functional validation of partial and complete ureteral obstruction. (A) Partial obstruction: dye colorization and saline wash-out demonstrate distal ureter filling and urethral outflow, indicating residual patency. (B) Near-complete obstruction: absence of distal dye, with a ballooned proximal ureter upstream of the ligature. (C) Capillary collection of unilateral urinary volume over 30 min per kidney shows a graded reduction of urine output from physiological conditions to complete occlusion. (D) Microscopic measures of (1) ligature diameter, (2) outer ureter diameter, (3) inner ureter diameter, and (4) ureter wall thickness. Asterisks indicate exploratory significance levels. Please click here to view a larger version of this figure.

9. Terminal steps and considerations

1. Close the abdominal wall in multiple layers under sterile conditions as described in previous reports^19,20,21.
2. Following surgery, observe each animal continuously until it regains sternal recumbency and spontaneous mobility. Rehouse animals only after they are fully alert and capable of coordinated movement. Administer postoperative analgesia with subcutaneous carprofen at 5 mg/kg twice daily for 48 h and ensure that veterinary staff conduct daily clinical evaluations to monitor recovery and detect potential complications.
3. Document animal welfare using standardized scoring systems such as the Rat Grimace Scale²² and the Body Condition Score²³. Humane endpoints include a Rat Grimace Score ≥ 6 or a Body Condition Score of 1 despite adequate analgesia. Terminate animals presenting these findings, as well as those exhibiting postoperative wound infection or abdominal wall dehiscence, immediately according to institutional standards of care.
4. For non-survival experiments or for termination after follow-up, euthanize the animal by sharp cardiectomy under deep anesthesia or by cervical dislocation (following institutionally approved protocols). Optionally, resect both kidneys and ureters en bloc for subsequent anatomical inspection, biomolecular analysis, or histopathological imaging.
5. Dispose of animal carcasses, resected tissues, sharps, and remaining chemical solutions according to institutional animal facility and biosafety guidelines.

Results

The calibrated ligation technique produced a diameter‑dependent reduction in urine flow across all experimental groups. Urine flow was assessed in a total of 14 rats using both kidneys, resulting in measurements from 5 organs per partial obstruction level (0.65, 0.55, 0.30, and 0.20 mm diameter). Additionally, 4 animals were included that received Sham treatment on one side (the ureter was mobilized, and a loose, non-constricting air-knot ligature was tied without wire) and complete occlusion on the other (a ligatu...

Discussion

This study establishes a reproducible surgical method for inducing unilateral ureteral strictures in rats through midline laparotomy and calibrated partial ligation, resulting in segmental ureteral narrowing with preserved macroscopic tissue viability that functionally and anatomically resembles benign human ureteral strictures.

Clinically, ureteral stricture disease remains a major contributor to upper tract obstruction and renal morbidity with long-term functional loss, frequently arising af...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
atraumatic preparation forceps	Aesculap	FB395R	DE BAKEY ATRAUMATA atraumatic forceps, straight
blunt overholt clamps	Aesculap	BJ012R	BABY-MIXTER preparation and ligature clamp, bent, 180 mm
cannula	BD (Beckton, Dickinson)	301300	BD Microlance 3 cannula 20 G
capillary tubes	Sigma Aldrich	P1174	Microcapillary tube, calibrated 100 µL
carprofen	cp pharma	115	carprofen 0,5% (5% with 50 mg/mL diluted 1:10) (Carprosol®)
cotton whool swaps	Amazon	ASIN: B0FPDB8ZGS	non-sterile cotton whool swaps (autoclave for survival experiments)
fixation rods	legefirm	‎500343896	tuning forks used as y-shaped metal fixation rods
gauze swaps	Medrull	ASIN: B09164D1JG	sterile gauze swaps
heating pad	Royal Gardineer	IP67	Royal Gardineer Heating Pad Size S, 20 Watt
isoflurane	Piramal Critical Care	PZN / EAN 09714675 / 4150097146757	100% isoflurane 250 mL
isoflurane vaporizer	UNO ROESTVASTSTAAL BV	180000002	isoflurane vaporizer
isotonic sodiumchloride solution	B. Braun	ASIN: B007PZJOQ4	0,9% sodiumchloride solution
ketamine	cp pharma	1202	ketamine 10% (100 mg/mL)
metal calibration wire (0.20 mm diameter)	QUARKZMAN	ASIN: B0DCZBT4LW	Cr20Ni80-Nichrom metal wire (0.20 mm diameter)
metal calibration wire (0.30 mm diameter)	Rayher	4006166052859	Cr20Ni80-Nichrom metal wire (0.30 mm diameter)
metal calibration wire (0.55 mm diameter)	SKYPRO	UPC: 792138793816	Cr20Ni80-Nichrom metal wire (0.55 mm diameter)
metal calibration wire (0.65 mm diameter)	twipp	ASIN: B07WFWNNRM	Cr20Ni80-Nichrom metal wire (0.65 mm diameter)
methylene blue 0,5%	Provepharm	PZN / EAN 10179678 / 4150101796787	METHYLTHIONINIUMCHLORID Proveblue 5 mg/mL in 10 ml vials
non-resorbable monofilament 6-0 ligatures	COVIDIEN	VP-889-X	monofilament surgical suture from non-resorbable poly-propylene; needle can be removed
ophthalmic ointment	Bayer Vital GmbH	1578681	5% dexpanthenol
plastic perfusor tube	M. Schilling GmbH	S702NC150	connecting tube COEX 150 cm
preparation scissors	Aesculap	BC177R	JAMESON preparation scissors, bent, fine model, blunt/blunt, 150 mm (6")
steel plate	Maschinenbau Feld GmbH	C010206	Galvanized sheet plate, 40 x 50 cm, thickness 4.0 mm
syringe with integrated needle	BLPRKOT	BL2509L3yici08	1 ml syringe with integrated 30 G needle
xylazine	cp pharma	1206	xylazine 0,2% (2% with 20 mg/mL diluted 1:10) (Xylavet)