An endoluminal thermal injury rabbit ureteral stricture model and a construction method and application thereof

Abstract

The application discloses an intracavitary thermal injury rabbit ureteral stenosis model and a construction method and application thereof. The construction method comprises the following steps: placing a flexible ureteroscope into a urethra, reversely sending a micro guide wire into a renal pelvis through a target side ureteral opening under direct vision, positioning the guide wire through retrograde ureteral angiography, placing an electrocoagulation guide wire with a bare metal conductive tip at the front end into the guide wire after the angiography agent is discharged, positioning a target section at the upper edge of the fourth lumbar vertebra under X-ray fluoroscopy, connecting an electrosurgical generator to generate electricity in a bipolar coagulation mode to perform intracavitary point coagulation, causing local thermal injury of the ureter from inside to outside, and forming unilateral ureteral stenosis 28 days after the operation. The method is completed through a natural cavity, no body surface incision is needed, and the ureter does not need to be separated, cut or sutured. The collagen of the formed stenosis section is deposited in a centripetal manner, and the proximal ureter of the stenosis section is expanded and the ipsilateral hydronephrosis is accompanied. The model can be used for drug screening for treating ureteral stenosis and evaluation of intracavitary treatment devices for the ureter.

Description

Technical Field

[0001] This invention relates to the construction and application of experimental animal models, specifically to a rabbit ureteral stricture model caused by intraluminal thermal injury, its construction method, and its application. Background Technology

[0002] Ureteral stricture refers to an obstructive lesion of the upper urinary tract caused by narrowing of the ureteral lumen. Its causes include iatrogenic injury, ureteral stones, inflammation, infection, radiation damage, and malignant tumors. Benign ureteral stricture is pathophysiologically characterized by excessive activation of interstitial fibroblasts, abnormal deposition of extracellular matrix, and local scar contracture, leading to centripetal narrowing of the lumen. After stricture formation, urine outflow is obstructed, which can sequentially lead to dilation of the ureter proximal to the stricture, dilation of the renal pelvis and calyces, increased intrarenal pressure, thinning of the renal cortex, and progressive damage to the function of the affected kidney. In severe cases, it can cause irreversible renal parenchymal damage or even loss of function of the affected kidney.

[0003] Iatrogenic injuries account for approximately 75% of all ureteral injuries, surpassing traumatic injuries to become the leading cause. With the widespread adoption of transurethral endoscopy and energy devices, the epidemiological characteristics of ureteral injuries have changed significantly: endoscopic procedures have gradually become the primary cause of benign ureteral strictures, accounting for approximately 58% of all cases. In recent years, with the increasing use of high-power and / or high-frequency lasers in flexible ureteroscopic lithotripsy, the incidence of ureteral stricture after ureteroscopy is approximately 5%, while the incidence can be as high as 34% after endoscopic lithotripsy for impacted ureteral stones. Studies have reported that approximately 5.8% of cases develop ureteral stricture after endoscopic procedures using high-power lasers, with 62.5% of these patients requiring additional surgery. Multicenter studies further show that approximately 25% of patients with ureteral strictures caused by iatrogenic endoscopic thermal injury eventually undergo nephrectomy due to obstructive renal insufficiency. Although ureteral reconstruction and endovascular treatment have a success rate of over 90% in most cases, their efficacy is limited for refractory, recurrent, and multifocal long-segment stenosis, resulting in decreased quality of life, reduced stenosis-free survival, and increased adverse events.

[0004] The localized high temperatures generated during the operation of energy devices can cause damage to the ureteral mucosal barrier, protein denaturation and coagulative necrosis, smooth muscle layer structure destruction, and local microcirculatory disturbances. This leads to inflammatory cell infiltration, fibroblast transformation into myofibroblasts (marked by increased expression of α-smooth muscle actin, i.e., α-SMA), and excessive deposition of extracellular matrix components such as type I collagen and fibronectin, ultimately resulting in scar contracture and luminal narrowing. The severity of thermal injury can be measured by the thermal dose characterized by Cumulative Equivalent Minutes at 43 °C (CEM43); studies have shown that tissues exposed to temperatures above 56 °C can suffer immediate damage within 1 second. Protective measures such as ureteral access sheaths, low-power lasers, temperature-controlled irrigation, and active negative pressure suction can keep the intraoperative intraluminal thermal dose below the injury threshold. However, transient blockage of the access sheath leading to irrigation interruption, improper control of the safe distance between the laser fiber and the mucosa, and excessively long operation time can still cause the local temperature to temporarily exceed the injury threshold, resulting in intraluminal thermal injury. At the cellular level, thermal injury is characterized by heat shock response, protein denaturation, and coagulative necrosis. Its tissue destruction mode and subsequent fibrosis process have significantly different pathological characteristics from simple mechanical cutting, ligation, and suturing injuries, requiring specialized animal models for further study.

[0005] To investigate the mechanisms of fibrosis and stricture formation following ureteral thermal injury, and to evaluate antifibrotic drugs, drug-eluting ureteral stents, dilating balloons, and other endovascular therapeutic devices, it is necessary to establish a stable, reproducible animal model of ureteral stricture that closely resembles the clinical pathogenesis. Existing models mainly suffer from the following three problems: (i) Large animal models are costly and have low throughput. Because pigs have relatively long and large ureters that can accommodate standard clinical endoscopic instruments, current reports on endoscopic ureteral stricture models are mostly focused on large animals such as pigs. While these models have good clinical relevance, they suffer from high cost per animal, long breeding cycles, and complex anesthesia and husbandry management, making it difficult to meet the sample size requirements for early large-scale screening of antifibrotic drugs and batch evaluation of stent products.

[0006] (II) Small animal models are limited by anatomical conditions, requiring open surgical procedures. While small mammals such as rabbits, rats, and mice have low breeding costs and allow for larger sample sizes, their ureteral lumens are small. For example, in rabbits, the inner diameter of the ureter is approximately 1 mm, while the narrowest point of the ureteral wall is only about 0.50 mm. Clinically, the guidewire's outer diameter is typically 0.81 mm, making it impossible to pass through the ureteral opening into the renal pelvis. Furthermore, the bilateral ureteral wall segments form approximately a 90° angle with the endoscope, making it difficult for ordinary rigid instruments to enter smoothly. Due to these limitations, all reported rabbit ureteral stricture models rely on open transabdominal procedures, including: opening the ureter to free it and then performing electrocoagulation or thermal ablation; ureteral ligation; and partial ureteral resection followed by anastomosis.

[0007] (III) Open surgery alters the direction of scar repair and introduces additional trauma. Open surgery requires freeing the ureter, which easily causes full-thickness (outside-in) ureteral damage, leading to urine extravasation, urinary cyst formation, abdominal infection, and adhesions to surrounding tissues. Collagen fibers expand centrifugally around the extravasated area, preventing sufficient centripetal scar contraction on the inner side of the lumen, resulting in unstable stenosis formation and low modeling success rates. Furthermore, the additional trauma caused by open surgery interferes with local inflammation and fibrosis processes, making it difficult for the model to accurately simulate intraluminal thermal damage caused by clinical transurethral endoscopic procedures. Open models also suffer from long surgical times and high animal mortality rates, and standardized ureteral stent placement is difficult to achieve without ureteral transection, making them unsuitable for evaluating endoluminal devices such as drug-eluting stents.

[0008] In summary, there is an urgent need in this field for a method to model ureteral stricture caused by thermal injury in small animals that can be completed through natural orifices without external incisions, without abdominal surgery, with precise control over the location and energy of injury, high modeling success rate, few serious complications, low cost, and retains the condition of ureteral stent placement through natural orifices. Summary of the Invention

[0009] To address the aforementioned problems, this invention provides a rabbit ureteral stricture model caused by intraluminal thermal injury and its construction method. The method involves inserting a flexible ureteroscope via the urethra under anesthesia. Under direct endoscopic visualization, a microguidewire is advanced retrogradely through the target ureteral opening to the renal pelvis. A thin-diameter ureteral catheter is then inserted along the microguidewire, and retrograde pyelography is performed to complete anatomical localization. After aspirating and waiting for the contrast agent to drain, an electrocoagulation guidewire with an exposed metal conductive tip is inserted through the ureteral catheter. Under X-ray fluoroscopy, the target location is determined using vertebral bony landmarks. The conductive end of the electrocoagulation guidewire is connected to an electrosurgical generator, and low-power, short-duration targeted electrocoagulation is performed on the target ureteral segment to create focal intraluminal thermal injury from the inside out. The instruments are then withdrawn, and postoperative management and follow-up evaluation are completed. This method achieves a stable and repeatable unilateral ureteral stricture model without external incisions or laparotomy.

[0010] The technical solution of this invention is: A method for constructing a rabbit ureteral stricture model caused by intraluminal thermal injury includes the following steps: (1) Preparation and anesthesia fixation of experimental animals: Take experimental rabbits, fast them before the operation and give them prophylactic anti-infective drugs, implement inhalation anesthesia, fix the experimental rabbits in a supine position, and complete the perineal skin preparation, disinfection and draping. (2) Endoscope inserted through the urethra: A flexible ureteroscope is inserted into the bladder through the urethra, and the bladder is filled with normal saline. Under direct vision, the orifices of both ureters are identified and the orifice of the target ureter is selected. (3) Retrograde insertion of microguidewire: The microguidewire is delivered to the ureteral opening on the target side through the working channel of the flexible ureteroscope. The bending angle of the front end of the flexible ureteroscope is adjusted so that the direction of the working channel is consistent with the inner segment of the ureteral wall. Under direct vision, the microguidewire is retrogradely inserted into the ureter on the target side and advanced into the renal pelvis. (4) Insertion of ureteral catheter and retrograde angiography: Under fluoroscopic monitoring, the ureteral catheter is inserted retrogradely along the microguidewire, and contrast agent is injected through the ureteral catheter to perform retrograde pyelourethrography to confirm that the device is located in the lumen of the ureter and to determine the predetermined site of injury. (5) Emptying the contrast agent: After the positioning is completed, the residual contrast agent is aspirated through the ureteral catheter and left to stand for 5 to 30 minutes to empty or dilute the contrast agent in the target ureteral segment; (6) Inserting the electrocoagulation guidewire and determining the target location: retain the position of the ureteral catheter, withdraw the microguidewire, insert the electrocoagulation guidewire retrogradely through the ureteral catheter, and adjust the position of the electrocoagulation guidewire under fluoroscopic guidance so that the exposed metal conductive tip at its front end is located at the predetermined injury site; the predetermined injury site is the target ureteral segment corresponding to the level of the upper edge of the fourth lumbar vertebra under fluoroscopic guidance, which is located in the middle and upper part of the ureter and is far away from the ureteropelvic junction and the vesicoureteral junction; (7) Targeted electrocoagulation injury: The conductive end of the electrocoagulation guidewire is connected to an electrosurgical generator to perform targeted electrocoagulation on the ureteral wall at the predetermined injury site to cause focal intraluminal thermal injury of the ureter from the inside out; the power of the electrocoagulation is 5 to 20 W, the single energization time is 0.5 to 5 s, and the total energy delivered to the predetermined injury site is 2.5 to 100 J; (8) Remove instruments and perform postoperative care: After electrocoagulation is completed, stop the output of the electrosurgical generator and disconnect it. Then remove the electrocoagulation guidewire, ureteral catheter and flexible ureteroscope in sequence, and perform postoperative warming and recovery, fluid replacement, analgesia and anti-infection treatment. (9) Follow-up and endpoint assessment: Peripheral blood was collected on postoperative day 1, day 7 and day 28 for hematological and biochemical tests, and imaging and histological assessments were performed to confirm the formation of ureteral stricture, thereby obtaining a rabbit ureteral stricture model of intraluminal thermal injury.

[0011] In one embodiment of the present invention, the experimental animal in step (1) is a female New Zealand white rabbit with a weight of 3.5 to 5.0 kg, preferably 4.0 to 4.5 kg.

[0012] In one embodiment of the present invention, in step (1), the experimental rabbits are fasted for 8 to 16 hours before surgery, preferably 12 hours; and an anti-infective drug is administered via the marginal ear vein 0.5 to 2 hours (preferably 1 hour) before surgery. The anti-infective drug is amoxicillin, and the dosage is 30 to 80 mg / kg, preferably 50 mg / kg.

[0013] In one embodiment of the present invention, the anesthesia in step (1) is induced and maintained by isoflurane inhalation anesthesia. The respiratory rate, heart rate, muscle tone, and reflexes of the experimental animal are continuously monitored during the procedure to maintain an appropriate depth of anesthesia.

[0014] In one embodiment of the present invention, the experimental animals described in step (1) are acclimatized for at least 7 days under conditions of 22-24 °C and 12 h light / 12 h dark cycle before entering the experiment.

[0015] In one embodiment of the present invention, the outer diameter of the flexible ureteroscope in step (2) is 6.5 to 8.5 Fr, and it has a working channel and an adjustable bend front end; preferably 7.0 to 8.0 Fr, and more preferably 7.5 Fr (corresponding to an outer diameter of about 2.5 mm).

[0016] In one embodiment of the present invention, the target side in step (2) can be any side of the ureter, preferably the right side, that is, the right ureter is used as the modeling side, and the left ureter is kept intact to provide functional compensation for the contralateral kidney, thereby reducing the animal mortality rate.

[0017] In one embodiment of the present invention, the outer diameter of the microguidewire in step (3) is 0.40 to 0.50 mm, more preferably 0.46 mm; this outer diameter is smaller than the inner diameter of the narrowest part of the inner wall of the rabbit ureter (about 0.50 mm), so it can pass smoothly through the ureteral opening.

[0018] In one embodiment of the present invention, when resistance is encountered during the advancement of the guidewire in step (3), the forced advancement is stopped, and the guidewire is allowed to travel naturally along the lumen of the ureter by adjusting the bending angle of the endoscope tip and the direction of the guidewire, so as to avoid mechanical damage or false passage formation.

[0019] In one embodiment of the present invention, the outer diameter of the ureteral catheter in step (4) is 0.80 to 0.90 mm, more preferably 0.90 mm; catheters with an outer diameter greater than 0.90 mm are difficult to pass through the rabbit ureteral opening even under the guidance of a microguidewire.

[0020] In one embodiment of the present invention, the contrast agent in step (4) is an iodine-containing contrast agent, preferably a non-ionic iodine-containing contrast agent, and more preferably iodixanol or iohexol.

[0021] In one embodiment of the present invention, in step (5), after aspirating the contrast agent, the mixture is left to stand for 5 to 30 minutes, preferably 10 to 20 minutes, and more preferably 15 minutes, before electrocoagulation is performed.

[0022] In one embodiment of the present invention, the electrocoagulation guidewire in step (6) includes an exposed metal conductive tip at the front end, an exposed metal conductive tail end at the tail end, and an insulating section between the two, wherein the insulating section has an insulating coating; the exposed length of the conductive tip is 0.2 to 2.0 mm, preferably 0.3 to 0.7 mm, and more preferably 0.5 mm; the exposed length of the conductive tail end is 5 to 20 cm, for connection to an electrosurgical generator.

[0023] In one embodiment of the present invention, the electrosurgical generator in step (7) employs a bipolar coagulation mode; the bipolar mode eliminates the need to place a negative electrode plate on the animal's body surface, and the current loop is limited to the vicinity of the conductive tip, resulting in a more localized and controllable range of thermal damage. In another embodiment, the electrosurgical generator may also employ a unipolar coagulation mode or other modes with equivalent thermal damage output effects.

[0024] In one embodiment of the present invention, the electrocoagulation power in step (7) is 8-12 W, the single energizing time is 1-3 s, and the total energy delivered to the predetermined injury site is 8-36 J. More preferably, the electrocoagulation in step (7) is performed in a bipolar coagulation mode, with a power of 10 W, a single energizing time of 2 s, and the total energy delivered to the predetermined injury site is 20 J. In the present invention, the total energy refers to the energy delivered by the electrosurgical generator to the predetermined injury site through the electrocoagulation guidewire, which is numerically equal to the product of the output power and the energizing time; when using intermittent multiple short pulse energizing, the total energy is the sum of the energy delivered in each energizing. When the power is below 5 W or the energizing time is less than 0.5 s, the local temperature of the target segment is difficult to maintain at the threshold of protein denaturation and coagulative necrosis, and the thermal damage is reversible. However, stable luminal stenosis cannot be formed 28 days after the operation. When the power is above 20 W or the energizing time is longer than 5 s, the thermal damage depth exceeds the full thickness of the ureteral wall, which easily leads to perforation, urine extravasation, and urinary cyst formation. The direction of scar repair changes to centrifugal expansion, which reduces the success rate of modeling.

[0025] In one embodiment of the present invention, the fixed-point electrocoagulation in step (7) is carried out in bipolar coagulation mode, and no negative electrode plate is placed on the surface of the experimental rabbit; the fixed-point electrocoagulation in step (7) is a single energization, or an intermittent multiple short pulse energization, with an interval of 5 to 60 s between two adjacent energizations, in order to reduce the risk of excessive thermal damage and perforation; the position of the electrocoagulation guide wire is kept stable during the electrocoagulation process to avoid the movement of the conductive end leading to an expansion of the damage range.

[0026] In one embodiment of the present invention, a sham surgery control group is also included, which receives the same anesthesia, endoscopic insertion, microguidewire insertion, ureteral catheter insertion, retrograde angiography and electrocoagulation guidewire positioning operation as the modeling group, but does not activate the electrosurgical generator output.

[0027] In one embodiment of the present invention, postoperative treatment in step (8) includes one or more of the following: warming and recovery, fluid replacement, analgesia, continuous administration of anti-infective drugs for the first to third days after surgery, daily observation of mental state and food and water intake, observation of urination and monitoring of gross hematuria.

[0028] In one embodiment of the present invention, the follow-up time points in step (9) include peripheral blood collection 1 hour before surgery and on postoperative days 1, 7 and 28. The test items include one or more of the following: white blood cell count, red blood cell count, alanine aminotransferase, aspartate aminotransferase, serum creatinine and serum neutrophil gelatinase-associated lipid transport protein.

[0029] In one embodiment of the present invention, the imaging assessment in step (9) includes one or more of the following: plain radiograph of urinary tract (KUB), retrograde pyelography (RGP), and CT urography (CTU); the histological assessment includes one or more of the following: hematoxylin-eosin (HE) staining, Masson trichrome staining, immunofluorescence, immunohistochemistry, Western blot, and real-time quantitative PCR.

[0030] In one embodiment of the present invention, the criteria for confirming ureteral stricture in step (9) include one or more of the following: narrowing of the target segment ureteral lumen, dilation of the ureter proximal to the stricture, dilation of the renal pelvis and calyces, restricted passage or delayed excretion of contrast agent, decreased enhancement of the renal cortex, decreased thickness of the renal cortex, increased collagen deposition in the target segment, disordered smooth muscle structure, increased expression of α-SMA, and increased expression of type I collagen (COL-1) or fibronectin (Fn).

[0031] The present invention also provides a rabbit ureteral stricture model caused by intraluminal thermal injury, which is constructed by the above-described construction method.

[0032] In one embodiment of the present invention, the model exhibited the following characteristics on postoperative day 28: segmental stenosis of the ureter in the electrocoagulation target segment with a lumen area reduced by more than 50% compared to the sham-operated control; dilation of the ureter proximal to the stenosis, with the diameter of the dilated segment increasing to more than three times that of the sham-operated control; dilation of the ipsilateral renal pelvis and calyces, decreased renal cortical thickness, and delayed contrast agent uptake and excretion; centripetal collagen deposition in the stenotic segment wall and a significant increase in collagen layer thickness; elevated expression of α-SMA and COL-1 in the stenotic segment and the dilated segment proximal to the stenosis; increased α-SMA-positive cells and elevated Fn expression in the ipsilateral renal interstitium, along with renal tubular dilation, epithelial cell flattening, and cast formation.

[0033] The present invention also provides the application of animal models constructed by the above construction method in screening drugs for the prevention and / or treatment of ureteral stricture.

[0034] The present invention also provides a method for screening drugs for the prevention and / or treatment of ureteral stricture, the method comprising: administering the drug to be screened to a rabbit ureteral stricture model with intraluminal thermal injury constructed by the above construction method before, during and / or after modeling, and determining the effectiveness of the drug to be screened in the prevention and / or treatment of ureteral stricture based on changes in the model in imaging, histopathology and expression of fibrosis-related molecules after administration.

[0035] The present invention also provides the application of the animal model constructed by the above construction method in evaluating ureteral intraluminal therapeutic devices, wherein the ureteral intraluminal therapeutic device includes at least one of ureteral stent, drug-eluting ureteral stent, biodegradable ureteral stent, thermo-expandable metal stent and dilating balloon.

[0036] The evaluation was performed after the intraurethral treatment device was inserted through the natural urethral orifice under direct visualization with a flexible ureteroscope along a guidewire.

[0037] Compared with the prior art, the present invention has the following beneficial effects: (1) No incision, minimally invasive, and safe. This invention completes modeling through the natural urethral cavity, eliminating the need for open abdominal incisions, as well as the need to free, cut, or suture the ureter, significantly reducing additional damage caused by open surgery. The measured average operation time was 27.6±6.7 min, significantly shorter than the 81.6±12.6 min of open ureteral electrocoagulation and the 111.4±14.8 min of open ureteral resection and anastomosis (all P<0.001). No animal deaths occurred in the modeling group of this invention, while the mortality rates in the open electrocoagulation group and the open resection and anastomosis group were 30.0% and 40.0%, respectively. No urine extravasation or gross hematuria occurred in the modeling group of this invention, significantly lower than in the open resection and anastomosis group (all P=0.033). The overall complication rate was 10.0%, while it was 60.0% in both open modeling groups.

[0038] (2) Intraluminal thermal injury, with the direction of injury consistent with clinical findings. This invention performs focal thermal injury from inside the ureteral lumen, with the direction of injury from inside to outside, which is closer to the actual process of ureteral thermal injury and delayed stenosis caused by energy devices such as ureteroscopes, lasers or electrocoagulation in clinical practice; the resulting collagen deposits are centripetally distributed, which can effectively cause centripetal narrowing of the lumen.

[0039] (3) Precise positioning and good consistency. This invention significantly improves the accuracy of guidewire placement and electrocoagulation target positioning and the consistency among different animals through four methods: direct endoscopic visualization, retrograde pyelography, vertebral bony anatomical landmark positioning, and guidance by a thin-diameter ureteral catheter. The stenosis position shown by the retrograde angiography after surgery is consistent with the positioning level of the electrocoagulation guidewire during modeling.

[0040] (4) Low energy and low complications. This invention uses low power and short-term fixed-point electrocoagulation, which can form irreversible thermal damage and subsequent fibrotic stenosis with low energy, while significantly reducing the risk of serious complications such as ureteral perforation, urine extravasation, abdominal infection and animal death.

[0041] (5) Complete phenotype and multi-level evaluation. The model established in this invention can stably show manifestations such as ureteral target segment stenosis, ureteral dilation proximal to the stenosis, ipsilateral hydronephrosis, renal cortical thinning, delayed contrast agent excretion, collagen deposition, myofibroblast activation, secondary renal interstitial fibrosis, and renal tubular injury. It can comprehensively reflect the pathological characteristics of ureteral stenosis from multiple levels, including imaging, histopathology, protein, and mRNA.

[0042] (6) Preservation of natural cavities, suitable for evaluation of endovascular devices. The model of this invention preserves the complete natural cavity pathway, and the ureteral double-J stent can be placed along the guidewire under direct vision with a flexible ureteroscope. Moreover, the operation can be repeated on the same animal without the need for laparotomy again. Therefore, it is particularly suitable for the preclinical evaluation of endovascular therapeutic devices such as drug-eluting stents, biodegradable stents, thermo-expandable metal stents and dilating balloons. In contrast, the open electrocoagulation model is difficult to complete standardized stent placement without cutting the ureter.

[0043] (7) Unilateral modeling, good animal welfare, and low cost. This invention adopts unilateral (right-side) modeling, which preserves the compensatory function of the contralateral kidney. The animal's overall condition is stable and can tolerate long-term follow-up, meeting animal welfare requirements. Compared with large animal models such as pigs, the rabbit model has significantly lower procurement, feeding, and experimental costs, which facilitates the expansion of sample size. It is suitable for the study of fibrosis mechanism after ureteral thermal injury and large-scale preclinical screening and evaluation of antifibrotic drugs, drug-eluting ureteral stents, dilating balloons, and other endovascular treatment devices. Attached Figure Description

[0044] The accompanying drawings are provided to further illustrate the invention and form part of the specification. The embodiments shown in the drawings and their descriptions are for explaining the invention and do not constitute a limitation on the scope of protection of the invention. Unless otherwise specified, data in each quantitative graph are expressed as mean ± standard deviation, with n = 5 animals per group, and the experimental unit being a single animal; normality was first determined using the Shapiro-Wilk test, and for those conforming to a normal distribution, an unpaired two-tailed t-test was used; for those not conforming to a normal distribution, the Mann-Whitney U test was used; significance was marked as... P < 0.05 P < 0.01, P < 0.001, P < 0.0001, ns indicates no statistical significance. The sham surgery group represents the sham surgery control group, the modeling group (US) represents the intraluminal thermal injury modeling group of this invention, US-OUE represents the open ureter electrocoagulation group, and US-OUTA represents the open ureter resection and anastomosis group.

[0045] Figure 1This is a schematic diagram of the modeling process of the present invention, wherein: A: Experimental animal preparation and anesthesia fixation; B: Insertion of a flexible ureteroscope (7.5 Fr / 2.5 mm) through the urethra and retrograde insertion of a microguidewire (0.46 mm, i.e. 0.018 inches) through the working channel; C: Insertion of a ureteral catheter (2.6 Fr, outer diameter approximately 0.9 mm) along the microguidewire; D: Retrograde pyelography performed by injecting a non-ionic iodine-containing contrast agent through the ureteral catheter; E: Positioning the exposed metal conductive tip (approximately 0.5 mm long) of the electrocoagulation guidewire at the right ureteral segment corresponding to the upper edge of the fourth lumbar vertebra under X-ray fluoroscopy; F: Performing a single intraluminal fixed-point electrocoagulation with a bipolar coagulation mode of 10 W for 2 s (20 J).

[0046] Figure 2 The diagram shows the key endoscopic procedures during the modeling process. A: Urethral view; B: Bladder and bilateral ureteral openings (white arrow indicates the right ureteral opening, green arrow indicates the left ureteral opening); C: Enlarged view of the right ureteral opening within the wall; D: Microguidewire insertion; E: Ureteral catheter insertion; F: Electrocoagulation guidewire insertion.

[0047] Figure 3 The following is a timeline of X-ray imaging localization, experimental design, and follow-up during the modeling process. A: X-ray imaging localization during the modeling process, from left to right: preoperative urinary tract plain film, localization after microguidewire placement (white arrow indicates microguidewire), localization and local magnification after ureteral catheter placement (red arrow indicates ureteral catheter), retrograde pyelography (green arrow indicates the visualized right renal pelvis, ureter, and bladder), and X-ray guided electrocoagulation guidewire localization and electrocoagulation, with local magnification (blue arrow indicates the fourth lumbar vertebra, yellow arrow indicates the exposed conductive end of the electrocoagulation guidewire); B: Experimental design and follow-up timeline, showing the processing and testing on 1 day before modeling, the day of modeling, 1 day after modeling, 7 days after modeling, and 28 days after modeling.

[0048] Figure 4 The images show the gross specimens of the urinary system and the measurements of the anteroposterior, transverse, and superior-inferior diameters of the affected kidney on postoperative day 28. A: Gross specimen image of the urinary system; left: sham surgery group; right: modeling group; bottom row: affected kidney after dissection; background grid squares with side length 10 mm. B: Anteroposterior diameter of the affected kidney (mm); C: Transverse diameter of the affected kidney (mm); D: Superior-inferior diameter of the affected kidney (mm).

[0049] Figure 5The graph shows the dynamic changes in peripheral blood routine and blood biochemistry before surgery and on days 1, 7 and 28 after surgery. A: White blood cell count (WBC), B: Red blood cell count (RBC), C: Alanine aminotransferase (ALT), D: Aspartate aminotransferase (AST), E: Serum creatinine (CREA), F: Neutrophil gelatinase-associated lipocalin (NGAL). The horizontal axis represents the number of days after surgery (0 represents preoperative days).

[0050] Figure 6 This image shows a comparison of plain radiographs and retrograde pyelography of the urinary tract on postoperative day 0 and day 28. The left half represents the sham surgery group, and the right half represents the modeling group. Within each group, from left to right, the images show the right renal pelvis (KUB) on day 0, the right ureter (RGP) on day 0, the KUB on day 28, and the RGP on day 28. The blue arrow indicates the right renal pelvis, the green arrow indicates the right ureter, and the red arrow indicates the ureteral stenosis located at the level of the upper margin of the fourth lumbar vertebra.

[0051] Figure 7 This image shows the quantitative results of CT urography (CTU) on postoperative day 28, including the mean CT value of the affected renal cortex, the time to first visualization of the ureter, the mean thickness of the renal cortex, the diameter of the dilated ureteral segment, the head-to-tail diameter of the renal pelvis, and the width of the renal calyx fornix. A: CTU images, top row is transverse, bottom row is coronal, left column is the sham surgery group, right column is the modeling group, scale bar 50 mm. B to G show, respectively, the mean CT value (HU) of the affected renal cortex 1 min after contrast agent injection, the time required for first visualization of the affected ureter (min), the mean thickness of the affected renal cortex (mm), the diameter of the dilated ureteral segment proximal to the stenosis (mm), the head-to-tail diameter of the right renal pelvis (mm), and the width of the renal calyx fornix (mm).

[0052] Figure 8 Image 28 shows HE and Masson staining results and corresponding histological quantitative findings of the ureteral stenosis, proximal dilated ureter, and affected kidney on postoperative day 28. A, B, and C: HE staining (top row) and Masson trichrome staining (bottom row, collagen fibers appear blue) of the ureteral stenosis (scale bar 500 μm), proximal dilated ureter (scale bar 500 μm), and affected kidney (scale bar 5 mm). D to G represent, respectively, the luminal area of ​​the stenosis (mm²), the maximum collagen layer thickness of the stenosis (μm), the average collagen layer thickness of the proximal dilated ureter (mm), and the maximum cross-sectional area of ​​the affected renal pelvis (mm²).

[0053] Figure 9Immunofluorescence staining and quantification results of the ureteral stenosis, the proximal dilated stenosis, and the affected kidney tissue are shown. A and B: Immunofluorescence staining and quantification of α-SMA and type I collagen (COL-1) in the ureteral stenosis; C and D: Corresponding results in the proximal dilated stenosis; E and F: Corresponding results of α-SMA and fibronectin (Fn) in the affected kidney tissue. The immunofluorescence images, from top to bottom, are α-SMA (green), COL-1 or Fn (red), DAPI (blue), and a merged image. The left column represents the sham-operated group, and the right column represents the modeled group. The scale bar is 50 μm.

[0054] Figure 10 Image showing HE staining and renal tubular injury score of the affected kidney tissue. A: HE staining of the affected kidney tissue (high power field), top row is the sham surgery group, bottom row is the modeling group, scale bar 50 μm; blue arrows indicate flattening of renal tubular epithelial cells, green arrows indicate dilation of renal tubular lumen, and red arrows indicate formation of casts within the lumen. B: Renal tubular injury score.

[0055] Figure 11 The figures show the results of Western blot detection of fibrosis-related proteins in the ureteral stricture and the results of qRT-PCR detection of related gene mRNAs. A: Western blot protein bands, from top to bottom: Collagen I, α-SMA, Fibronectin, Vimentin, and the internal control GAPDH. The left half represents the sham-operated group, and the right half represents the modeling group. The figure shows three technically repeated lanes on the same membrane from a representative animal. B: Relative protein expression levels normalized to GAPDH. C: Relative mRNA expression levels of Col1a1, Vim, Acta2, and Fn1 detected by qRT-PCR, with Gapdh as the internal control gene.

[0056] Figure 12 Schematic diagrams and intraoperative photographs for open ureterocoagulation modeling and open ureterotomy anastomosis modeling. A: Open ureterocoagulation (OUE) modeling; the leftmost image shows a self-made puncture electrocoagulation needle and a schematic diagram; the rest are intraoperative photographs. Black arrows indicate the puncture electrocoagulation needle, green arrows indicate the freed right ureter, and white arrows indicate ureteral segments after electrocoagulation thermal injury. B: Open ureterotomy anastomosis (OUTA) modeling; the leftmost image is a schematic diagram, and the rest are intraoperative photographs. Green arrows indicate the right ureter, yellow arrows indicate the bladder, and blue arrows indicate the end-to-end anastomosis of the ureter.

[0057] Figure 13These are imaging and endoscopic views of the double-J ureteral stent inserted through a natural orifice in the model of this invention. A: X-ray fluoroscopic image and magnified view; B: Endoscopic image under a flexible ureteroscope. The red arrow indicates the 3Fr hydrophilic coated double-J ureteral stent, with its proximal end coiled in the right renal pelvis and its distal end coiled in the bladder.

[0058] Figure 14 This image shows a comparative analysis of HE and Masson staining and histological quantification of the ureter and affected kidney on postoperative day 28 using three different modeling methods. A, B, and C represent, respectively, the HE staining (top row), Masson staining (middle row), and Masson staining of the affected renal parenchyma for the intraluminal thermal injury group (US), the open ureteral electrocoagulation group (US-OUE), and the open ureterotomy and anastomosis group (US-OUTA), and their respective sham-operated controls. The scale bar is 500 μm. D to G represent, respectively, the luminal area (mm²) of the injured ureteral segment, the average collagen thickness (μm) of the injured segment, the average anteroposterior diameter (mm) of the affected renal pelvis, and the renal tubular injury score. Each image contains six bars from left to right, representing the three modeling methods and their sham-operated controls.

[0059] Figure 15 Comparative CT images of the urinary tract on postoperative day 28 for the three modeling methods. The top row shows transverse sections, and the bottom row shows coronal sections. From left to right, the images represent the US group, the US-OUE group, and the US-OUTA group. The scale bar is 50 mm. Blue arrows indicate the right kidney, green arrows indicate the right ureter, red arrows indicate cysts communicating with the ureter, and yellow arrows indicate contrast agent leaking into the abdominal cavity. Detailed Implementation

[0060] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. For those skilled in the art, conventional substitutions or equivalent transformations of specific materials, instrument models, anesthesia methods, parameter ranges, and detection methods without departing from the spirit and substance of the present invention should all fall within the scope of protection of the present invention. Where specific experimental steps or conditions are not specified in the following embodiments, they can be performed according to the conventional experimental steps or conditions described in the literature in the field; reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0061] All animal experiments in this invention were conducted in accordance with relevant guidelines and regulations and were approved by the Ethics Committee of Xiangya Third Hospital of Central South University (Approval No. 25150). The experimental methods and results were reported in accordance with the ARRIVE guidelines.

[0062] The main instruments, reagents, and equipment used in the various embodiments of this invention are sourced or have the following catalog numbers: New Zealand white rabbits (Hubei Yizhicheng Biotechnology Co., Ltd., Wuhan); flexible ureteroscope (7.5 Fr, outer diameter 2.5 mm, Hunan Yiweidi Medical Device Co., Ltd., Changsha); microguidewire (outer diameter 0.46 mm); ureteral catheter (outer diameter 0.90 mm); electrocoagulation guidewire (with an exposed metal conductive tip of about 0.5 mm at the front end and an exposed metal conductive tail end at the rear end, the rest being insulated); bipolar electrocoagulation device (EMED); 3 Fr hydrophilic coated double-J ureteral stent; X-ray fluoroscopy equipment; non-ionic iodine-containing contrast agent iodixanol; iohexol injection (Omnipaque 300); isoflurane and small animal inhalation anesthesia device; amoxicillin; fully automated animal blood analyzer (BC-5000Vet, Mindray); fully automated biochemical analyzer (BS-240VET, Mindray); rabbit NGAL enzyme-linked immunosorbent assay kit (Rabbit). Lipocalin-2 / NGAL ELISA Kit (NovusBiologicals); Anti-fibronectin antibody (Abcam, ab2413); Anti-α-smooth muscle actin antibody (Abcam, ab5694); Anti-type I collagen antibody (Abcam, ab34710); Anti-vimentin antibody (Abcam, ab8069); Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Abcam, ab8245); Alexa Fluor 488 / 594 labeled fluorescent secondary antibody (Invitrogen); RIPA lysis buffer (NCM, WB3100); Protease inhibitor (Selleck, B14002); Enhanced chemiluminescence reagent (Thermo Fisher Scientific, 32106); Chemiluminescence imaging system (ChemiDoc, Bio-Rad); Total RNA extraction reagent (TRnaZol RNA Kit, NCM Biotech, Suzhou); Reverse transcription kit (PrimeScript RTReagent Kit, Takara); Real-time quantitative PCR instrument (ABI PRISM). 7500); image analysis software MicroDicomViewer (v3.0); image analysis software ImageJ (v1.54, NIH); statistical analysis software GraphPad Prism (v10.1).

[0063] A total of 75 female New Zealand white rabbits, weighing 4.0–4.5 kg, were used in the various embodiments of this invention. Since ureteral tissue fixed and embedded for histological examination could not be used for protein and RNA extraction, the model characterization experiment was conducted using three independent cohorts. Each cohort contained 5 rabbits in a sham-operated group and 5 rabbits in a modeling group, for a total of 30 rabbits: Cohort 1 was used for serial blood sample collection, imaging evaluation, gross specimen observation, and HE staining, Masson staining, and immunofluorescence detection; Cohort 2 was used for fresh frozen tissue for Western blot analysis; and Cohort 3 was used for RNA extraction and qRT-PCR detection. An additional 45 non-overlapping rabbits were used for comparison of the three modeling methods, with 10 rabbits for each method used for observation of operation time and complications, and 5 sham-operated controls for histological comparison.

[0064] Example 1: Construction of a rabbit ureteral stricture model caused by intraluminal thermal injury I. Experimental Objective This embodiment aims to utilize a flexible ureteroscope, microguidewire, and ureteral catheter to retrogradely enter the ureter through the rabbit urethra and bladder, and perform low-power, short-duration endovascular electrocoagulation on a predetermined ureteral segment under direct endoscopic visualization and X-ray imaging guidance, thereby constructing an endovascular thermal injury rabbit ureteral stenosis model without open surgery.

[0065] II. Laboratory Animals and Grouping Ten healthy female New Zealand white rabbits, weighing 4.0–4.5 kg, were randomly divided into a sham surgery group (Sham group) and a modeling group (US group), with five animals in each group. The animals were acclimatized for 7 days under a temperature of 22–24 °C and a 12-hour light / 12-hour dark cycle, with free access to food and water. All animals underwent general observation before the experiment, excluding those with urinary system abnormalities, infections, significant emaciation, or other conditions unsuitable for surgery. All surgeries were performed by the same urologist.

[0066] III. Experimental Methods 1. Preoperative preparation Rabbits were fasted for 12 hours prior to surgery. One hour before surgery, amoxicillin 50 mg / kg was administered via the marginal ear vein for prophylactic anti-infection treatment. Anesthesia was induced and maintained using isoflurane inhalation. After satisfactory anesthesia, the rabbits were fixed in a supine position, and routine perineal cleaning, disinfection, and draping were performed. During the operation, the rabbits' respiratory rate, heart rate, muscle tone, and reflexes were continuously monitored to maintain an appropriate depth of anesthesia.

[0067] 2. Insertion of a flexible ureteroscope via the urethra A fully lubricated 7.5 Fr (2.5 mm outer diameter) flexible ureteroscope was slowly inserted into the bladder of the experimental rabbit through the urethra. After entering the bladder, a small amount of 0.9% sodium chloride solution was instilled to moderately fill the bladder and maintain a clear endoscopic view. Under direct visualization with the ureteroscope, the orifices of both ureters were identified, and the right ureter was selected as the modeling side. Since the inner segments of both ureteral walls in the rabbit formed an approximately 90° angle with the scope, the bending angle of the flexible ureteroscope tip needed to be adjusted so that the working channel direction was basically consistent with the course of the inner segment of the right ureteral wall, allowing the guidewire to enter the ureteral orifice. The procedures for urethral, ​​bladder, bilateral ureteral orifices, and instrument insertion in the experimental rabbit are as follows: Figure 2 As shown.

[0068] 3. Retrograde insertion of the microguidewire A 0.46 mm outer diameter microguidewire was advanced to the right ureteral orifice through the working channel of a flexible ureteroscope. Under direct endoscopic visualization, the microguidewire was slowly inserted retrogradely into the right ureter and continued proximally into the right renal pelvis. When resistance was encountered during guidewire advancement, forced advancement was stopped. The angle of the ureteroscope tip and the direction of the guidewire were adjusted to allow the guidewire to travel naturally along the ureteral lumen, avoiding mechanical damage to the ureter or false passage formation. After the microguidewire entered the right ureter and renal pelvis, its position was confirmed by X-ray fluoroscopy. The imaging localization results of the microguidewire and subsequent catheters are shown below. Figure 3 As shown in Figure A.

[0069] 4. Insertion of a ureteral catheter and administration of retrograde angiography. With the microguidewire in place, a 0.90 mm outer diameter ureteral catheter was retrogradely inserted into the right ureter along the microguidewire. Preliminary experiments confirmed that catheters with an outer diameter exceeding 0.90 mm, even under microguidewire guidance, were difficult to pass through the rabbit ureteral orifice; this value reflects the maximum dilatation limit of the rabbit ureteral orifice. After the ureteral catheter reached the appropriate position, iodixanol was slowly injected through the catheter to perform retrograde pyelography. The imaging of the right renal pelvis, calyces, and ureter was observed under X-ray fluoroscopy to confirm that the microguidewire and ureteral catheter were located within the ureteral lumen, and the subsequent electrocoagulation location was determined based on the ureteral course and vertebral projection. After retrograde pyelography, residual contrast agent in the renal pelvis and ureter was aspirated through the ureteral catheter, and 15 minutes were allowed for further drainage to reduce the impact of residual conductive fluid on the local electrocoagulation area.

[0070] 5. Insert the electrocoagulation guide wire and determine the electrocoagulation target position. The ureteral catheter was retained, and the microguidewire was withdrawn. Then, an electrocoagulation guidewire with a 0.5 mm exposed conductive metal tip was inserted into the right ureter via the ureteral catheter. Under fluoroscopic guidance, the position of the electrocoagulation guidewire was adjusted so that its exposed conductive metal tip was positioned in the right ureteral segment corresponding to the upper border of the fourth lumbar vertebra, and the positions of the electrocoagulation guidewire and ureteral catheter were kept stable. Choosing the ureteral segment corresponding to the upper border of the fourth lumbar vertebra as the electrocoagulation target site is beneficial because it utilizes the vertebral body as a stable radiographic landmark, thereby improving the consistency of electrocoagulation positions among different animals. Simultaneously, this segment is located in the upper-middle ureter, away from the ureteropelvic junction and the vesicoureterovesical junction, avoiding interference from these physiological stenosis sites on the interpretation of results. The electrocoagulation guidewire positioning process is as follows: Figure 1 and Figure 3 As shown in Figure A.

[0071] 6. Intracavitary fixed-point electrocoagulation The exposed metal conductive end of the electrocoagulation guidewire was connected to the output of the EMED bipolar coagulation electrosurgical device. In the modeling group, bipolar coagulation mode was used, with continuous energization of a predetermined ureteral segment for 2 seconds at a power of 10 W (total energy delivered at a single point: 20 J), performing a single intraluminal targeted electrocoagulation to induce focal thermal injury of the right ureter. Using bipolar mode eliminates the need for a negative electrode plate on the animal's surface, confining the current loop to the vicinity of the conductive tip, resulting in a more localized and controllable thermal injury area. During electrocoagulation, the guidewire position was kept stable to prevent movement of the conductive end and subsequent expansion of the injury area. After electrocoagulation, the electrosurgical device output was stopped and disconnected, followed by the sequential removal of the guidewire, ureteral catheter, and flexible ureteroscope. The sham surgery group received the same anesthesia, ureteroscope placement, microguidewire placement, ureteral catheter placement, retrograde angiography, and guidewire positioning procedures as the modeling group, but the electrosurgical device was not activated, and no thermal injury was inflicted on the ureter.

[0072] 7. Postoperative management After the procedure, the rabbits were placed in a warm environment for resuscitation. Amoxicillin was continued for anti-infection treatment from day 1 to day 3 post-surgery, along with analgesia and necessary fluid support according to animal welfare requirements. The rabbits' mental state, activity level, food and water intake, urination, gross hematuria, and other possible post-operative complications were observed daily, and they continued to be fed until day 28 post-surgery.

[0073] 8. Detection time point The experimental procedure and detection time points in this embodiment are as follows: Figure 3As shown in Figure B. Peripheral blood was collected 1 hour before surgery and on postoperative days 1, 7, and 28 for complete blood count, serum biochemistry, and renal injury-related markers. Urinary tract imaging was performed preoperatively and on postoperative day 28. After the imaging examinations were completed on postoperative day 28, the experimental rabbits were euthanized, and both kidneys, ureters, and bladders were separated. The electrocoagulated damaged segment of the right ureter, the proximal dilated segment of the stenosis, and ipsilateral kidney tissue were collected for gross observation, histopathological examination, and molecular biological analysis.

[0074] IV. Experimental Results After the flexible ureteroscope is inserted into the bladder via the urethra, the orifices of both ureters can be identified under direct vision. By adjusting the bending angle of the tip of the flexible ureteroscope, a microguidewire can be inserted into the right ureteral orifice and travel retrogradely along the ureter to the renal pelvis. The ureteral catheter can be inserted into the right ureter along the microguidewire. After retrograde injection of contrast agent, the course of the right renal pelvis, renal calyces, and ureter can be clearly visualized. Under X-ray fluoroscopy, the upper edge of the fourth lumbar vertebra can be used as a bony landmark to stably position the exposed metal conductive segment of the electrocoagulation guidewire at the predetermined right ureteral segment. Using a bipolar coagulation mode, intraluminal targeted thermal injury can be successfully completed under conditions of 10 W and 2 s (20 J). The entire modeling process does not require laparotomy to expose or cut the ureter, and the average operation time is 27.6 ± 6.7 min. Animals in both the sham-operated group and the modeling group completed the corresponding operations without intraoperative mortality. Some animals in the modeling group experienced transient light bloody urine in the early postoperative period, which subsided within 24 hours and was not considered a gross hematuria complication. All experimental animals were able to continue to be housed until the scheduled observation period.

[0075] V. Conclusions of the Examples The above results indicate that the present invention can retrogradely enter the ureter through the rabbit urethra and bladder, and complete the placement of microguidewire, ureteral catheter, retrograde angiography and focal endovascular electrocoagulation under endoscopic and X-ray guidance; the modeling operation path is feasible, the electrocoagulation position is relatively stable, and additional tissue damage caused by open surgery can be avoided.

[0076] Example 2: Imaging and renal function verification of a rabbit model of ureteral stricture caused by intraluminal thermal injury. I. Experimental Objective To verify whether the model constructed in Example 1 can form stable ureteral stenosis, ureteral dilation proximal to the stenosis, ipsilateral hydronephrosis, and ipsilateral urinary excretion dysfunction, this example evaluates the model's imaging and renal function phenotype through gross urinary system anatomy, retrograde pyelourethrography, CT urinary tract imaging, and peripheral blood testing.

[0077] II. Laboratory Animals and Grouping Using the method described in Example 1, ten female New Zealand white rabbits were randomly divided into a sham-operated group and a modeling group, with five rabbits in each group. The sham-operated group received anesthesia, flexible ureteroscope placement, microguidewire placement, ureteral catheter placement, retrograde pyelography, and guidewire localization via electrocoagulation, but without initiating electrosurgical equipment. The modeling group underwent a single-point electrocoagulation at 10 W for 2 seconds (20 J) in a bipolar coagulation mode at the right ureteral segment corresponding to the upper border of the fourth lumbar vertebra, as described in Example 1. Both groups of animals continued to be fed until day 28 post-surgery.

[0078] III. Experimental Methods 1. Peripheral blood and serum marker detection Peripheral blood (2 mL) was collected via the marginal ear vein 1 hour before surgery and on postoperative days 1, 7, and 28. A portion of the blood was placed in anticoagulant tubes containing EDTA, and white blood cell and red blood cell counts were determined using an automated animal blood analyzer. The remaining blood was centrifuged to separate serum, and serum creatinine, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels were measured using an automated biochemical analyzer. Serum NGAL levels were also measured using enzyme-linked immunosorbent assay (ELISA) according to the kit instructions. All samples were tested in duplicate. Results are shown below. Figure 5 .

[0079] 2. Preoperative imaging examinations Twenty-four hours prior to modeling, all experimental rabbits underwent CT urinary tract imaging (2 mL / kg of iohexol injected via the marginal ear vein). Plain, enhanced, and excretion-phase images were acquired to rule out preoperative urinary system structural abnormalities and to record baseline parameters. On the day of modeling, a supine plain urinary tract radiograph was first performed to confirm that the contrast agent had been emptied. Then, retrograde pyelography was performed as described in Example 1 to confirm the course of the right ureter and the location of the electrocoagulation target segment.

[0080] 3. Imaging examinations on postoperative day 28 On postoperative day 28, both the sham surgery group and the modeling group underwent plain urinary tract radiography, retrograde pyelography, and CT urinary tract imaging again. During retrograde pyelography, following the method described in Example 1, a flexible ureteroscope was inserted into the bladder via the urethra. Under direct endoscopic visualization, a microguidewire and ureteral catheter were inserted into the right ureter. Contrast agent was slowly injected through the ureteral catheter, and the passage of the contrast agent through the electrocoagulation target segment, the width of the ureteral lumen in the target segment, the dilation of the ureter proximal to the stenosis, and the morphology of the renal pelvis and calyces were observed. The results of retrograde pyelography are shown below. Figure 6 .

[0081] 4. Evaluation of CT urinary tract imaging On postoperative day 28, after an intravenous injection of iohexol 2 mL / kg via the marginal ear vein, continuous CT scans were performed. Enhanced and excretory images of the renal cortex were acquired 1 minute after contrast agent injection. The following parameters were measured using MicroDicom Viewer: mean CT value of the ipsilateral renal cortex 1 minute after contrast agent injection, time required for first visualization of the ipsilateral ureter, mean thickness of the ipsilateral renal cortex, diameter of the dilated ureter proximal to the stenosis, head-to-tail diameter of the right renal pelvis, and width of the renal calyx fornix. All parameters were measured by the same researcher using uniform window width and level and anatomical landmarks. Results are shown below. Figure 7 A~G.

[0082] 5. Gross observation of the urinary system On postoperative day 28, after imaging examinations, the experimental rabbits were euthanized, and both kidneys, ureters, and bladders were completely dissected. The degree of stenosis in the electrocoagulation target segment of the right ureter, the degree of ureteral dilation proximal to the stenosis, and the degree of hydronephrosis on the ipsilateral side were observed. The anteroposterior, transverse, and superior-inferior diameters of the right kidney were measured. Gross specimens of the urinary system from the sham-operated group and the modeling group are shown below. Figure 4 A, the results of the kidney diameter measurements are shown below. Figure 4 B~D.

[0083] 6. Statistical Analysis Continuous variables are expressed as mean ± standard deviation. Normality was first assessed using the Shapiro-Wilk test; if normality was found, an unpaired two-tailed t-test was performed; otherwise, the Mann-Whitney U test was used. For blood parameters collected repeatedly from the same batch of animals at four time points, two-way repeated measures ANOVA was used, with Šídák's method used to correct for comparisons at each time point. The experimental unit was a single animal, and technical replicates were averaged first. P < 0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism.

[0084] IV. Experimental Results 1. The modeling group developed ureteral stenosis, proximal ureteral dilatation, and ipsilateral hydronephrosis. Gross observation of the urinary system on postoperative day 28 showed that the right ureter in the sham surgery group had a natural course, with no obvious luminal stenosis, ureteral dilation, or hydronephrosis. Compared with the sham surgery group, the modeling group showed localized ureteral stenosis at the electrocoagulation target segment corresponding to the upper edge of the fourth lumbar vertebra, with significant dilation of the ureter proximal to the stenotic segment, accompanied by dilation of the ipsilateral renal pelvis and calyces. The affected kidney in the modeling group was larger, with its anteroposterior diameter, transverse diameter, and superior-inferior diameter all significantly larger than those in the sham surgery group, and the differences were statistically significant (all P < 0.0001, see below). Figure 4 A and Figure 4 (B~D). The above results indicate that targeted thermal injury within the ureteral lumen can lead to stable unilateral upper urinary tract obstruction by postoperative day 28.

[0085] 2. Retrograde angiography confirmed luminal stenosis in the electrocoagulation target segment. Retrograde pyelography showed that the right ureteral lumen was relatively patent in both groups of animals preoperatively, allowing contrast agent to pass smoothly into the renal pelvis. On postoperative day 28, no significant luminal narrowing was observed in the right ureter in the sham-operated group, and the morphology of the renal pelvis and calyces was normal. In the modeling group, significant segmental luminal stenosis was observed at the predetermined electrocoagulation location, with dilation of the ureter proximal to the stenosis, accompanied by dilation of the right renal pelvis and calyces. The location of the stenosis was essentially consistent with the positioning of the electrocoagulation guidewire at the upper edge of the fourth lumbar vertebra during modeling (see [link to modeling]). Figure 6 This result demonstrates that the present invention can utilize bony anatomical landmarks to relatively stably locate the site of thermal injury and form a ureteral stricture at that location.

[0086] 3. CT urography confirmed obstruction on the affected side and delayed contrast agent excretion. CT urography showed that on postoperative day 28, the right renal pelvis and calyces in the modeling group were significantly dilated, the outer diameter of the ureter proximal to the stenosis increased, and the renal cortex on the affected side was thinned (see...). Figure 7 A). The mean thickness of the renal cortex on the affected side was 4.3±1.7 mm in the modeling group and 11.7±2.0 mm in the sham-operated group. The modeling group showed a reduction of approximately 63% compared to the sham-operated group, and the difference was statistically significant (P<0.001, see A). Figure 7 D). The diameter of the ureter proximal to the stenosis in the modeling group was approximately 4.1 times that in the sham-operated group (P < 0.0001, see D). Figure 7 E), indicating that ureteral stenosis obstructs urine outflow and causes significant mechanical dilation proximal to the stenosis. Compared with the sham surgery group, the modeling group showed a significant increase in the right renal pelvis head-to-tail diameter (P < 0.01) and the width of the renal calyx fornix region (P < 0.0001), further confirming the presence of obstructive hydronephrosis on the affected side (see [link to modeling group]). Figure 7 F, G).

[0087] 4. Impaired contrast agent uptake and excretion function of the affected kidney. One minute after contrast agent injection, the mean CT enhancement value of the right renal cortex in the modeling group was significantly lower than that in the sham surgery group (P < 0.0001), suggesting a decreased uptake and early enhancement capacity of the affected kidney. Serial scans showed that the time required for the first contrast agent appearance in the right ureter in the modeling group was significantly longer than that in the sham surgery group (P < 0.01), indicating that ureteral obstruction delayed contrast agent excretion on the affected side (see...). Figure 7 (B, C). These results indicate that, in addition to causing anatomical stenosis of the ureter, the model of this invention can also induce unilateral urinary excretion dysfunction similar to clinical obstructive hydronephrosis.

[0088] 5. Peripheral blood parameters reflect early inflammation and persistent kidney damage. Peripheral blood tests showed that the white blood cell count in the modeling group was higher than that in the sham-operated group on postoperative days 1 and 7, but returned to near baseline levels by postoperative day 28, suggesting that intraluminal thermal injury can cause a transient inflammatory response. Serum creatinine in the modeling group showed a transient increase in the early postoperative period, but due to compensation by the contralateral kidney, there was no significant difference between the two groups on postoperative day 28. Unlike the changes in serum creatinine, serum NGAL in the modeling group remained elevated postoperatively, suggesting that even if overall serum creatinine recovered after compensation by the contralateral kidney, persistent damage remained in the affected kidney. There were no significant differences in red blood cell count, alanine aminotransferase, and aspartate aminotransferase between the two groups at any of the detection time points (all P > 0.05), indicating that the modeling method of this invention did not cause significant blood loss or liver function damage (see...). Figure 5 ).

[0089] V. Conclusions of the Examples The results of this embodiment demonstrate that, following targeted thermal injury of the rabbit ureter using the method described in Example 1, significant ureteral stenosis can be formed at the predetermined electrocoagulation site on postoperative day 28, further leading to ureteral dilation proximal to the stenosis, ipsilateral hydronephrosis, renal cortical thinning, and delayed contrast agent excretion. The gross anatomy, retrograde ureterography, CT urography, and renal injury indicators corroborate each other, indicating that the model constructed in this invention can simultaneously simulate the anatomical obstruction of ureteral stenosis and secondary ipsilateral renal dysfunction.

[0090] Example 3: Histopathological and fibrotic verification of a rabbit model of ureteral stricture caused by intraluminal thermal injury I. Experimental Objective To verify whether the model constructed in Example 1 has the characteristics of scarring stenosis such as ureteral wall structure destruction, collagen deposition, myofibroblast activation and extracellular matrix remodeling, this example uses gross dissection, hematoxylin-eosin staining, Masson trichrome staining, immunofluorescence, Western blot and qRT-PCR to detect the ureteral stenosis segment, the dilated segment proximal to the stenosis and the affected kidney tissue.

[0091] II. Laboratory Animals and Grouping The sham-operated group and the modeling group were established using the method described in Example 1, with 5 rabbits in each group. Since the fixed and embedded tissues could no longer be used for protein and RNA extraction, the histological examination, Western blot examination, and qRT-PCR examination in this example were performed on 3 independent cohorts, with each cohort containing 5 rabbits in the sham-operated group and 5 rabbits in the modeling group. The experimental rabbits were euthanized after imaging examinations were completed on the 28th day after surgery, and the right kidney, ureter, and bladder were completely separated. Based on the intraoperative electrocoagulation location, the results of postoperative retrograde pyelography, and gross anatomical findings, the following tissues were collected: (1) the ureteral stenosis corresponding to the electrocoagulation location; (2) the dilated segment proximal to the ureteral stenosis; and (3) the ipsilateral kidney tissue. The tissues used for histopathology and immunofluorescence detection were fixed in fixative; the tissues used for Western blot and qRT-PCR detection were flash-frozen in liquid nitrogen and stored at −80 °C.

[0092] III. Experimental Methods 1. Pathological examination of ureter and kidney tissue The ureteral stenosis, the proximal dilated segment of the stenosis, and the affected kidney tissue were fixed overnight in 4% paraformaldehyde at 4 °C. After dehydration, clearing, and paraffin embedding, 4 μm serial sections were prepared. Hematoxylin-eosin and Masson's trichrome staining were performed to observe mucosal continuity, lumen area, smooth muscle layer arrangement, inflammatory cell infiltration, renal tubular structure, and collagen deposition. ImageJ was used to quantify the lumen area of ​​the stenosis, the maximum collagen thickness of the stenosis, the average collagen thickness of the dilated segment, and the maximum cross-sectional area of ​​the affected renal pelvis. Stained images are shown below. Figure 8 A through C, quantitative results are shown in […]. Figure 8 D~G.

[0093] 2. Renal tubular injury score Under high magnification, pathological changes in the affected kidney tissue, including flattening of renal tubular epithelial cells, dilation of renal tubules, and formation of intraluminal casts, were observed. A semi-quantitative score was assigned based on the proportion of renal tubules with pathological changes. Multiple fields of view were randomly selected by the evaluators for scoring, and the average renal tubular injury score for each animal was calculated. Results of hematoxylin-eosin staining of kidney tissue and renal tubular injury scoring are shown below. Figure 10 .

[0094] 3. Immunofluorescence detection After dewaxing and hydration, paraffin sections were subjected to heat-induced antigen retrieval in pH 6.0 citrate buffer. After blocking with 5% bovine serum albumin, α-smooth muscle actin antibody, type I collagen antibody, and fibronectin antibody (all 1:200) were added, and incubated overnight at 4°C. Following washing, Alexa Fluor 488 or 594-labeled fluorescent secondary antibody (1:500) was added, and incubation was performed at room temperature in the dark for 1 hour. Cell nuclei were counterstained with DAPI, and a species-matched IgG control was provided. After image acquisition using a fluorescence microscope, the mean fluorescence intensity after background correction was measured using ImageJ within a normalized 1 mm² area. Five non-overlapping fields of view were randomly selected from each animal, and the mean was calculated. Results are shown below. Figure 9 A~F.

[0095] 4. Western blot detection Frozen ureteral stricture tissue was collected, and proteins were extracted using RIPA lysis buffer containing protease and phosphatase inhibitors. The concentration was determined by the BCA method. Equal volumes of protein were transferred to polyvinylidene fluoride membranes after SDS-polyacrylamide gel electrophoresis. After blocking, primary antibodies against type I collagen, α-smooth muscle actin, fibronectin, and vimentin were added, and the membranes were incubated overnight at 4 °C. Horseradish peroxidase-labeled secondary antibody was added, followed by chemiluminescence imaging with enhanced contrast agents. Lysate from each animal was repeatedly loaded onto the same membrane in three lanes, with each membrane containing both one sham-operated group and one modeling group animal. The relative expression level of the target protein was calculated using GAPDH as an internal control, and the mean of the three technical replicates was taken as the value for that animal. Results are shown below. Figure 11 A, B.

[0096] 5. qRT-PCR detection Frozen ureteral stricture tissue was collected, and RNA was extracted using total RNA extraction reagent. RNA concentration and purity were then measured. The qualified RNA was reverse transcribed into cDNA, followed by real-time quantitative PCR. Genes detected included Col1a1 (encoding type I collagen), Vim (encoding vimentin), Acta2 (encoding α-smooth muscle actin), and Fn1 (encoding fibronectin), with Gapdh used as an internal control gene. Each sample was tested in duplicate, and the mean Ct value was calculated. A 2-1 PCR was performed. -ΔΔCt The relative expression levels of each target gene were calculated using a method. Relevant mRNA detection results are shown below. Figure 11 C.

[0097] 6. Statistical Analysis The statistical methods used are the same as in Example 2.

[0098] IV. Experimental Results 1. Thermal injury to the ureter leads to luminal narrowing and centripetal collagen deposition. Hematoxylin-eosin staining results showed that in the sham-operated group, the ureteral mucosa was continuous, the lumen morphology was regular, the smooth muscle bundles were relatively neatly arranged, and the structure of each layer of the ureteral wall was clear. In the modeling group, the electrocoagulation-damaged segment of the ureter showed focal inflammatory cell infiltration, discontinuous mucosal structure, and significant narrowing of the lumen; the smooth muscle layer of the ureter was disordered and the continuity was interrupted in some areas, with collagen fibers interspersed between the smooth muscle bundles and replacing part of the normal muscle layer structure (see...). Figure 8 (A-C). Quantitative results showed that the luminal area of ​​the ureteral stenosis in the modeling group decreased from 0.215 mm² in the sham surgery group to 0.088 mm², a reduction of 59.1%, which was statistically significant (P < 0.001, see A-C). Figure 8 D). Masson trichrome staining results showed that the sham-operated group had fewer collagen fibers in the ureteral wall, and the arrangement was relatively regular; the modeling group showed a large number of dense collagen fiber deposits in the stenotic segment of the ureteral wall, and the maximum thickness of the collagen layer was significantly increased (P < 0.0001, see D). Figure 8 E). The above results indicate that intraluminal targeted thermal injury can cause damage to the ureteral wall structure, centripetal collagen deposition, and scar contraction, ultimately leading to luminal stenosis.

[0099] 2. Fibrotic remodeling also occurs in the proximal dilated segment of the stenosis. In addition to the localized stenosis, significant collagen deposition was also observed in the proximal dilated ureteral segment of the modeling group. Unlike the irregular collagen fiber deposition in the stenotic segment, the collagen layer in the proximal dilated segment showed a more regular, ring-like thickening, forming a dense fibrous sheath-like structure surrounding the ureteral intrinsic muscle layer. The average collagen layer thickness in the proximal dilated segment of the modeling group reached 1.40 ± 0.20 mm, significantly higher than that in the sham surgery group (P < 0.0001, see...). Figure 8 F). Immunofluorescence results showed that the expression of α-smooth muscle actin (P < 0.0001) and type I collagen (P < 0.001) was significantly increased in the narrow segment of the modeling group (see F). Figure 9 A, B); α-smooth muscle actin was also significantly elevated in the proximal dilated segment of the narrowing (P < 0.01), while the increase in type I collagen was relatively smaller (P < 0.05) (see A, B). Figure 9 (C, D) Elevated α-smooth muscle actin expression suggests the transformation of local fibroblasts into myofibroblasts or myofibroblast activation; elevated type I collagen expression indicates increased extracellular matrix synthesis and deposition. These results indicate that after ureteral stricture, fibrotic remodeling is not limited to the electrocoagulation injury site but can also affect the dilated ureteral segment proximal to the stricture; thickening of fibrosis in this segment may affect peristaltic wave conduction and urine drainage, thereby exacerbating increased intrarenal pressure and impaired renal function on the affected side.

[0100] 3. Ureteral stricture leads to secondary hydronephrosis and renal interstitial fibrosis. Histopathological results of the affected kidney tissue showed that the renal pelvis was significantly dilated in the modeling group, and the renal parenchyma was compressed to varying degrees. The maximum cross-sectional area of ​​the affected renal pelvis in the modeling group was 80.6 mm², while that in the sham-operated group was 29.6 mm², and the difference was statistically significant (P < 0.0001, see below). Figure 8 G). Masson trichrome staining showed a significant increase in collagen deposition in the renal cortex and medulla of the affected side in the modeling group, suggesting that persistent ureteral obstruction further led to renal interstitial fibrosis (see G). Figure 8 (A-C). Immunofluorescence results showed that, compared with the sham-operated group, the modeling group had an increased number of α-smooth muscle actin-positive cells and elevated fibronectin expression in the glomeruli and tubulointerstitium of the affected side (both P < 0.0001), indicating increased activation of renal interstitial fibroblasts and increased extracellular matrix deposition (see [link to study]). Figure 9 E, F).

[0101] 4. Ureteral obstruction leads to damage to the renal tubular structure. Hematoxylin-eosin staining results showed that the renal tubular structure was relatively intact and the renal tubular epithelial cells were regularly arranged in the sham-operated group. In the modeling group, obstructive renal injury manifestations such as flattened renal tubular epithelial cells, tubular lumen dilation, and intraluminal cast formation were observed in the affected kidney tissue. The renal tubular injury score in the modeling group was significantly higher than that in the sham-operated group (P < 0.0001, see...). Figure 10 This result corroborates the imaging findings in Example 2, such as thinning of the renal cortex on the affected side, hydronephrosis, and delayed excretion of contrast agent.

[0102] 5. Increased expression of fibrosis-related proteins and mRNA Western blot results showed that, compared with the sham surgery group, the expression levels of type I collagen, α-smooth muscle actin, fibronectin, and vimentin in the ureteral stricture tissue of the modeling group were significantly increased (see...). Figure 11 A, B). qRT-PCR results showed that the mRNA expression levels of Col1a1, Vim, Acta2, and Fn1 in the ureteral stricture tissue of the modeling group were significantly higher than those in the sham-operated group (see A, B). Figure 11 C). The protein and mRNA detection results were consistent with the histopathological and immunofluorescence results, further demonstrating that the model established in this invention has obvious characteristics of myofibroblast activation and extracellular matrix deposition.

[0103] V. Conclusions of the Examples The results of this embodiment demonstrate that, after targeted thermal injury to the ureteral lumen using the method described in Example 1, the ureteral stenosis in experimental rabbits resulted in a reduced lumen area, disruption of the smooth muscle layer structure, increased centripetal collagen deposition, and elevated expression of fibrosis-related molecules. Simultaneously, varying degrees of fibrotic remodeling and obstructive damage were observed in the proximal dilated ureter and the affected kidney tissue. These pathological, immunofluorescence, protein, and mRNA detection results corroborate each other, indicating that the model constructed in this invention can stably simulate the main pathological features of scar formation, luminal stenosis, and secondary kidney injury following clinical ureteral thermal injury.

[0104] Example 4: Comparison of the efficacy and safety of the model of the present invention and the open ureteral stricture model I. Experimental Objective To evaluate the modeling effect, safety, and applicability of intraluminal instruments of the transurethral endoscopic ureteral thermal injury model of this invention compared with the open ureteral electrocoagulation model and the open ureteral resection and anastomosis model, this embodiment uses three methods to construct rabbit ureteral injury models and compares the operation time, ureteral stricture formation, postoperative complications, animal mortality, and feasibility of ureteral stent placement.

[0105] II. Laboratory Animals and Grouping Forty-five healthy female New Zealand white rabbits, weighing 4.0–4.5 kg, were randomly divided into three groups. Three different modeling methods were used to construct models for each group. Ten rabbits were used for each modeling method to observe surgical time and complications. Five sham-operated controls were included in each group for histological comparison. (1) Intraluminal thermal injury group, i.e. US group: The model was constructed using the transurethral endoscopic ureteral fixed-point electrocoagulation method described in Example 1; (2) Open ureter electrocoagulation group, i.e. US-OUE group: The right ureter was exposed by open surgery, and the ureteral wall was punctured by electrocoagulation needle to cause thermal damage. (3) Open ureterotomy and anastomosis group, namely US-OUTA group: the right ureter is exposed and cut through open surgery, followed by end-to-end anastomosis.

[0106] All three groups of experimental rabbits received the same preoperative fasting, anesthesia, anti-infection treatment, and postoperative care, and were followed up until day 28 postoperatively.

[0107] III. Experimental Methods 1. Intracavitary thermal injury group The intracavitary thermal injury model was constructed according to the method described in Example 1. A 7.5 Fr flexible ureteroscope was inserted into the bladder via the urethra. Under direct endoscopic visualization, a microguidewire and ureteral catheter were retrogradely inserted through the right ureteral orifice. After completing retrograde pyelography, the electrocoagulation guidewire was positioned at the right ureteral segment corresponding to the upper edge of the fourth lumbar vertebra. A bipolar coagulation mode was used, with continuous electrocautery at 10 W for 2 seconds (20 J) to perform a single intracavitary targeted thermal injury. The entire modeling process did not involve open surgery, nor was the ureter cut or freed.

[0108] 2. Open ureteral electrocoagulation group Open ureteral electrocoagulation procedure, such as Figure 12 As shown in Figure A. After anesthesia, the rabbits were placed in a supine position. Routine disinfection and draping were performed, and a 5 cm incision was made in the midline of the abdomen to enter the abdominal cavity layer by layer. The greater omentum and right colon were retracted to expose the right retroperitoneal region and the right ureter was freed. A self-made electrocoagulation needle was used to induce thermal injury to the ureter. The metal core of the electrocoagulation needle was approximately 0.5 mm in diameter, and all parts except the distal end were insulated. The distal end was connected to a bipolar electrocoagulation device. At the level of the fourth lumbar vertebra, the electrocoagulation needle was transversely inserted through the wall of the right ureter and into the ureteral lumen. A single point electrocoagulation was performed using parameters of 10 W and 2 seconds (20 J). After electrocoagulation, the needle was removed, and local bleeding and urine extravasation were observed. The abdominal cavity was then closed layer by layer. The sham surgery control underwent the same laparotomy and freeing procedures but without electrocoagulation.

[0109] 3. Open ureterotomy and anastomosis group Open ureterotomy and anastomosis procedure, such as Figure 12 As shown in Figure B. After anesthesia, rabbits were placed in a supine position. A 5 cm incision was made in the midline of the abdomen. Upon entering the abdominal cavity, the greater omentum and right colon were retracted to expose and free the right ureter. Using the level of the fourth lumbar vertebra as the operating center, microvascular clamps were used to temporarily occlude the ureter approximately 1 cm proximal and 1 cm distal to the ureter. The right ureter was transversely severed between the two clamps, followed by interrupted end-to-end anastomosis using 5-0 absorbable sutures. After anastomosis, the microvascular clamps were removed, and the patency of the anastomosis and any significant urine extravasation were observed. Once confirmed, the abdominal cavity was closed layer by layer. The sham control group did not undergo transection or anastomosis.

[0110] 4. Feasibility evaluation of ureteral stent placement The compatibility of three modeling methods with ureteral double-J stent placement was evaluated. In the endovascular thermal injury group, a microguidewire was retrogradely inserted into the right renal pelvis under direct visualization with a flexible ureteroscope, followed by placement of a 3Fr hydrophilic-coated double-J ureteral stent along the guidewire, with the proximal end of the stent coiled in the right renal pelvis and the distal end coiled in the bladder. In the open ureterotomy and anastomosis group, a double-J ureteral stent was placed along the guidewire after ureterotomy, and ureteral end-to-end anastomosis was performed after stent placement. In the open ureterocoagulation group, it was difficult to directly complete standardized double-J stent placement via an open surgical approach without ureterotomy. Results of ureteral stent placement and imaging localization are shown below. Figure 13 .

[0111] 5. Evaluation of Modeling Results On postoperative day 28, CT urinary tract imaging was performed on the three groups of experimental rabbits to observe the following indicators: (1) whether ureteral stenosis was formed at the site of injury; (2) whether ureteral dilation was observed proximal to the stenosis; (3) whether hydronephrosis was observed on the affected side; and (4) whether extravasation of urine, ascites, or periureteral cysts were observed. After completing the imaging examination, the experimental rabbits were euthanized, and the ureteral injury segment and the affected kidney tissue were collected. Hematoxylin-eosin staining and Masson trichrome staining were performed, and the lumen area of ​​the ureteral injury segment, the average thickness of the collagen layer of the injury segment, the anteroposterior diameter of the renal pelvis on the affected side, and the renal tubular injury score were quantitatively analyzed. The histopathological results of the three modeling methods are shown in the figure. Figure 14 A through C, quantitative results are shown in […]. Figure 14 D-G, CT urinary tract imaging results are shown in... Figure 15 .

[0112] 6. Surgical time and evaluation of complications The total surgical time from the start of the procedure to the end of the surgery was recorded for three groups of experimental rabbits, and the following complications were observed within 28 days after the operation: (1) animal death; (2) incision infection; (3) urine extravasation; (4) abdominal hematoma; (5) gross hematuria; (6) intestinal obstruction; (7) abdominal infection. When one or more of the above complications occurred in the same animal, it was counted as the total complication.

[0113] 7. Statistical Analysis Continuous variables are expressed as mean ± standard deviation. Unpaired t-tests are used for comparisons between two groups within the same modeling method (Mann-Whitney U test is used for groups not conforming to a normal distribution). Comparisons between the three modeling methods are performed using one-way ANOVA and Tukey's post-hoc test, with eta-squared reported as the effect size. Categorical variables are expressed as quantity and percentage, and Fisher's exact test is used for comparisons. P < 0.05 is considered statistically significant.

[0114] IV. Experimental Results 1. Three modeling methods yielded different collagen deposition patterns and pathological outcomes. In the intraluminal thermal injury group, significant ureteral luminal stenosis was formed at the predetermined electrocoagulation site. Collagen fibers in the stenotic segment were deposited centripetally along the ureteral wall, accompanied by ureteral dilation proximal to the stenosis and hydronephrosis on the affected side. Both the open ureteral electrocoagulation group and the open ureterotomy and anastomosis group showed varying degrees of urine extravasation, forming large cysts around the ureteral injury area. Numerous dense but unevenly distributed collagen fibers were visible around the cysts. Unlike the centripetal collagen deposition in the intraluminal thermal injury group, the collagen fibers in the open modeling group mainly extended centrifugally around the urine extravasation area. Due to the extension of scar tissue to the periphery of the ureter, sufficient scar contraction did not form on the inner side of the ureteral lumen, and the degree of luminal stenosis was not significant (see...). Figure 14 (A~C).

[0115] Quantitative results showed that only the average thickness of the collagen layer in the damaged segment of the endoluminal thermal injury group was significantly thicker than that of its sham-operated control, with an increase of 3.25 times (P < 0.0001); there were no statistically significant differences between the open ureteral electrocoagulation group (P = 0.083) and the open ureterotomy and anastomosis group (P = 0.054) and their respective sham-operated controls. Comparison among the three modeling methods showed that collagen deposition in the endoluminal thermal injury group was significantly higher than that in the two open modeling groups (one-way ANOVA P < 0.0001, eta² = 0.94; Tukey post-hoc test showed P < 0.0001 for both open groups), while there was no statistically significant difference between the two open modeling groups (P = 0.712). Similarly, only the intracavitary thermal injury group showed a significant decrease in the luminal area of ​​the injured segment (P < 0.001, eta² = 0.63), a significant increase in the anteroposterior diameter of the affected renal pelvis (P < 0.001, eta² = 0.73), and a significant increase in the renal tubular injury score (P < 0.0001, eta² = 0.95); the above indicators in both open modeling groups were not significantly different from their respective sham-operated controls (see...). Figure 14 D~G).

[0116] 2. Imaging results showed that the open model was prone to urine extravasation. CT urography revealed segmental ureteral stenosis at the predetermined location in the intraluminal thermal injury group, accompanied by ureteral dilation proximal to the stenosis and hydronephrosis on the affected side. In the open ureteral electrocoagulation group, a cyst communicating with the ureter was observed, suggesting full-thickness ureteral wall injury and urine extravasation caused by puncture electrocoagulation. In the open ureterotomy and anastomosis group, contrast agent entered the peritoneal cavity, indicating urine leakage at the ureteral anastomosis site. Compared to the intraluminal thermal injury group, neither of the two open modeling groups developed significant obstructive hydronephrosis (see...). Figure 15The above results indicate that open-procedure-induced full-thickness ureteral injury and urine extravasation can alter the direction of normal scar repair and reduce the stability of ureteral stenosis formation.

[0117] 3. The method of this invention significantly shortens the operation time. The average operation time was 27.6 ± 6.7 min in the endovascular thermal injury group, 81.6 ± 12.6 min in the open ureteral electrocoagulation group, and 111.4 ± 14.8 min in the open ureteral resection and anastomosis group. Compared with the endovascular thermal injury group, the operation time in both open modeling groups was significantly prolonged (P < 0.001), and the overall difference among the three groups was also statistically significant (P < 0.001). There were no significant differences among the three groups in baseline parameters such as body weight and the anteroposterior diameter, transverse diameter, and superior-inferior diameter of the affected kidney (see Table 1).

[0118] 4. The method of this invention reduces postoperative mortality and serious complications. No animal deaths occurred in the intraluminal thermal injury group, while the mortality rate was 30.0% in the open ureterocoagulation group and 40.0% in the open ureterotomy and anastomosis group. The overall complication rate was 10.0% in the intraluminal thermal injury group and 60.0% in both open modeling groups (P = 0.057, reported as a trend). No urinary extravasation or gross hematuria occurred in the intraluminal thermal injury group; the urinary extravasation rate was 40.0% and the gross hematuria rate was 30.0% in the open ureterocoagulation group; and both urinary extravasation and gross hematuria rates were 50.0% in the open ureterotomy and anastomosis group. Compared with the intraluminal thermal injury group, the incidence of urinary extravasation and gross hematuria was significantly higher in the open ureterotomy and anastomosis group (P = 0.033 for both). In the intracavitary thermal injury group, only one rabbit developed abdominal infection and hematoma, which was controlled after anti-infection treatment. The main causes of death in the two open modeling groups were urinary peritonitis and abdominal infection caused by persistent urinary extravasation. A detailed comparison of baseline characteristics and postoperative complications among the three groups is shown in Table 1.

[0119] 5. The model of this invention is applicable to the evaluation of ureteral stents. In the intraluminal thermal injury group, a microguidewire was retrogradely inserted into the renal pelvis under direct visualization with a flexible ureteroscope, and a 3Fr hydrophilic-coated double-J ureteral stent was placed along the guidewire. Imaging examination showed that the proximal end of the stent was coiled in the right renal pelvis, and the distal end was coiled in the bladder; the stent was well positioned (see [link to relevant documentation]). Figure 13The open ureterotomy and anastomosis group can also place a stent after ureterotomy, but it requires open surgery and ureterotomy; the open ureterocoagulation group is difficult to perform standardized stent placement through an open route without ureterotomy. Therefore, the model of this invention can not only simulate ureteral stricture caused by clinical intraluminal thermal injury, but also retain the operating conditions for ureteral stent placement through natural orifices, and is suitable for the evaluation of drug-eluting stents, biodegradable stents and other endoluminal therapeutic devices.

[0120] Table 1. Baseline characteristics and postoperative complications of three ureteral injury modeling methods

[0121] Note: The US group refers to the ureteral intraluminal thermal injury group under transurethral endoscopy of this invention; the US-OUE group refers to the open ureteral electrocoagulation group; and the US-OUTA group refers to the open ureteral resection and anastomosis group. The three diameters of the affected kidney were measured based on CT urography. P a The p-value is the comparison between the US-OUE group and the US group; P b The p-value is the comparison between the US-OUTA group and the US group; P c For comparisons among three groups (one-way ANOVA), categorical variables are not applicable (NA).

[0122] V. Conclusions of the Examples The results of this embodiment demonstrate that, compared to open ureteral electrocoagulation and open ureterotomy and anastomosis methods, the transurethral endoscopic intraluminal thermal injury method of this invention can create more stable ureteral stenosis at a predetermined location, while significantly shortening the operation time and reducing urine extravasation, abdominal infection, and animal mortality. Furthermore, the model of this invention can complete ureteral stent placement through natural orifices, making it more suitable for preclinical research on the formation mechanism of ureteral stenosis, antifibrotic drugs, and novel ureteral stents.

[0123] Example 5: Application of the model of the present invention in the evaluation of antifibrotic drugs and ureteral intraluminal therapeutic devices I. Experimental Objective This embodiment illustrates how to use the intraluminal thermal injury rabbit ureteral stricture model constructed in Example 1 to evaluate candidate drugs for the prevention and / or treatment of ureteral stricture and intraluminal ureteral treatment devices.

[0124] II. Screening methods for anti-fibrotic drug candidates The model was constructed according to the method described in Example 1. Animals were randomly divided into a sham-operated group, a model control group (administered with solvent), and a drug treatment group (administered with different doses of the selected drug), with no fewer than 5 animals in each group. Drug administration could begin before modeling (prophylactic administration), during modeling, or after modeling (therapeutic administration). The route of administration could be intravenous injection, intraperitoneal injection, gavage, or local delivery via the ureteral lumen. The administration cycle covered the critical time window for postoperative fibrosis formation (usually 0–28 days postoperatively). On postoperative day 28, the degree of improvement of the following indicators was evaluated according to the methods described in Examples 2 and 3: (1) Imaging indicators, including target segment lumen width, degree of dilation of the proximal ureter of the stenosis, thickness of the renal cortex on the affected side, renal pelvis diameter, enhanced CT value of the renal cortex, and time to first visualization of the ureter; (2) Histological indicators, including lumen area of ​​the stenotic segment, collagen layer thickness, maximum cross-sectional area of ​​the renal pelvis, and renal tubular injury score; (3) Molecular indicators, including protein expression of α-SMA, type I collagen, fibronectin, and vimentin, and mRNA expression of Acta2, Col1a1, Fn1, and Vim; (4) Systemic safety indicators. If the drug treatment group significantly improved the above indicators compared with the model control group, the candidate drug was deemed effective.

[0125] III. Evaluation Methods for Endourological Devices for Ureteral The model was constructed according to the method described in Example 1. At predetermined time points during or after modeling, a flexible ureteroscope was inserted into the bladder via the urethra. Under direct vision, a microguidewire was retrogradely advanced into the right renal pelvis, and the endoureteral therapeutic device to be evaluated (including but not limited to a standard double-J stent, drug-eluting stent, biodegradable stent, thermo-expandable metal stent, and dilating balloon) was inserted along the guidewire. The position was confirmed by X-ray fluoroscopy after insertion. At predetermined follow-up time points, the device position, patency, displacement, and crusting were evaluated using plain urinary tract radiographs, retrograde pyelography, and CT urography. Histological evaluation was performed at the endpoint, comparing the differences in luminal area, collagen layer thickness, epithelial hyperplasia, and renal injury indicators between the device-implanted group and the non-implanted group to determine the device's effectiveness, safety, and degradation / release characteristics. Because the model of this invention completely preserves the natural cavitary pathway, the above evaluations can be repeated on the same animal without requiring further laparotomy.

Claims

1. A method for constructing a rabbit ureteral stricture model caused by intraluminal thermal injury, characterized in that, Includes the following steps: (1) Preparation and anesthesia fixation of experimental animals: Take experimental rabbits, fast them before the operation and give them prophylactic anti-infective drugs, implement inhalation anesthesia, fix the experimental rabbits in a supine position, and complete the perineal skin preparation, disinfection and draping. (2) Endoscope inserted through the urethra: A flexible ureteroscope is inserted into the bladder through the urethra, and the bladder is filled with normal saline. Under direct vision, the orifices of both ureters are identified and the orifice of the target ureter is selected. (3) Retrograde insertion of microguidewire: The microguidewire is delivered to the ureteral opening on the target side through the working channel of the flexible ureteroscope. The bending angle of the front end of the flexible ureteroscope is adjusted so that the direction of the working channel is consistent with the inner segment of the ureteral wall. Under direct vision, the microguidewire is retrogradely inserted into the ureter on the target side and advanced into the renal pelvis. (4) Insertion of ureteral catheter and retrograde angiography: Under fluoroscopic monitoring, the ureteral catheter is inserted retrogradely along the microguidewire, and contrast agent is injected through the ureteral catheter to perform retrograde pyelourethrography to confirm that the device is located in the lumen of the ureter and to determine the predetermined site of injury. (5) Emptying the contrast agent: After the positioning is completed, the residual contrast agent is aspirated through the ureteral catheter and left to stand for 5 to 30 minutes to empty or dilute the contrast agent in the target ureteral segment; (6) Inserting the electrocoagulation guidewire and determining the target location: retain the position of the ureteral catheter, withdraw the microguidewire, insert the electrocoagulation guidewire retrogradely through the ureteral catheter, and adjust the position of the electrocoagulation guidewire under fluoroscopic guidance so that the exposed metal conductive tip at its front end is located at the predetermined injury site; the predetermined injury site is the target ureteral segment corresponding to the level of the upper edge of the fourth lumbar vertebra under fluoroscopic guidance, which is located in the middle and upper part of the ureter and is far away from the ureteropelvic junction and the vesicoureteral junction; (7) Targeted electrocoagulation injury: The conductive end of the electrocoagulation guidewire is connected to the electrosurgical generator to perform targeted electrocoagulation on the ureteral wall at the predetermined injury site to cause focal intraluminal thermal injury of the ureter from the inside to the outside; the power of the electrocoagulation is 5 to 20 W, the single energization time is 0.5 to 5 s, and the total energy delivered to the predetermined injury site is 2.5 to 100 J; (8) Remove instruments and perform postoperative care: After electrocoagulation is completed, stop the output of the electrosurgical generator and disconnect it. Then remove the electrocoagulation guidewire, ureteral catheter and flexible ureteroscope in sequence, and perform postoperative warming and recovery, fluid replacement, analgesia and anti-infection treatment. (9) Follow-up and endpoint assessment: Peripheral blood was collected on postoperative day 1, day 7 and day 28 for hematological and biochemical tests, and imaging and histological assessments were performed to confirm the formation of ureteral stricture, thereby obtaining a rabbit ureteral stricture model of intraluminal thermal injury.

2. The method for constructing a rabbit ureteral stricture model caused by intraluminal thermal injury according to claim 1, characterized in that: The experimental rabbits were female New Zealand white rabbits, weighing 3.5–5.0 kg. In step (1), the experimental rabbits were fasted for 8–16 hours before the operation and were given an anti-infective drug via the marginal ear vein 0.5–2 hours before the operation. The anti-infective drug was amoxicillin, and the dosage was 30–80 mg / kg. The anesthesia was induced and maintained by isoflurane inhalation anesthesia.

3. The method for constructing a rabbit ureteral stricture model caused by intraluminal thermal injury according to claim 1, characterized in that: The flexible ureteroscope in step (2) has an outer diameter of 6.5 to 8.5 Fr and has a working channel and an adjustable bend front end; the microguidewire in step (3) has an outer diameter of 0.40 to 0.50 mm; the ureteral catheter in step (4) has an outer diameter of 0.80 to 0.90 mm.

4. The method for constructing a rabbit ureteral stricture model caused by intraluminal thermal injury according to claim 1, characterized in that: The contrast agent mentioned in step (4) is a non-ionic iodine-containing contrast agent; in step (5), it is left to stand for 10 to 20 minutes.

5. The method for constructing a rabbit ureteral stricture model caused by intraluminal thermal injury according to claim 1, characterized in that: The electrocoagulation wire in step (6) includes an exposed metal conductive tip at the front end, an exposed metal conductive tail end at the tail end, and an insulating section between the two. The insulating section has an insulating coating. The exposed length of the conductive tip is 0.2 to 2.0 mm, and the exposed length of the conductive tail end is 5 to 20 cm.

6. The method for constructing a rabbit ureteral stricture model caused by intraluminal thermal injury according to claim 1, characterized in that: The electrocoagulation power in step (7) is 8-12 W, the single energizing time is 1-3 s, and the total energy delivered to the predetermined damage site is 8-36 J.

7. The method for constructing a rabbit ureteral stricture model caused by intraluminal thermal injury according to claim 6, characterized in that: The electrocoagulation in step (7) is carried out in bipolar coagulation mode with a power of 10 W, a single energizing time of 2 s, and a total energy delivered to the predetermined damage site of 20 J.

8. The method for constructing a rabbit ureteral stricture model caused by intraluminal thermal injury according to claim 1, characterized in that: The fixed-point electrocoagulation described in step (7) is carried out in bipolar coagulation mode, and no negative electrode plate is placed on the surface of the experimental rabbit; the fixed-point electrocoagulation described in step (7) is a single energization, or an intermittent multiple short pulse energization, with an interval of 5 to 60 seconds between two adjacent energizations.

9. A rabbit ureteral stricture model caused by intraluminal thermal injury, characterized in that: The model is constructed using the method described in any one of claims 1 to 8.

10. The use of the intraluminal thermal injury rabbit ureteral stricture model of claim 9 in screening drugs for the prevention and / or treatment of ureteral stricture.

11. The application of the endoluminal thermal injury rabbit ureteral stenosis model according to claim 9 in evaluating endoluminal ureteral therapeutic devices, characterized in that: The ureteral intraluminal treatment device includes at least one of the following: ureteral stent, drug-eluting ureteral stent, biodegradable ureteral stent, thermo-expandable metal stent, and dilation balloon.

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