Holmium:YAG Laser-Assisted Induction of Bladder Neck Contracture in a Rat Model

Summary

This protocol demonstrates transvesical Ho:YAG laser ablation to create a reproducible rat model of bladder neck fibrosis for studying thermal injury-induced fibrotic remodeling and therapeutic interventions.

Abstract

Bladder neck contracture (BNC) is a fibrotic narrowing of the bladder outlet and a challenging complication after transurethral resection of the prostate (TURP). Existing animal models often rely on extravesical ligation or chemical cauterization, which do not adequately reproduce the thermal injury-driven fibrosis that develops after endoscopic surgery and may show limited reproducibility. Here, we describe a rat model of bladder neck fibrosis induced by transvesical Holmium:YAG laser (Ho:YAG laser) ablation of the bladder neck. After a small lower midline laparotomy and cystotomy, a 200-µm Ho:YAG laser fiber is introduced into the bladder lumen under direct visualization. Laser energy is delivered in contact mode at 1.0 J and 10 Hz for 5 s per point at the 3 and 9 o’clock positions of the bladder neck, corresponding to 50 J per point and 100 J per animal. Four weeks later, treated animals develop gross bladder enlargement, bladder neck narrowing, bladder wall thickening, and histological evidence of fibrotic remodeling, including increased collagen deposition and increased collagen type I and III expression. This protocol provides a practical model that mimics localized thermal injury-induced scarring at the bladder outlet. Because female rats are used and functional urodynamic testing was not performed in the current study, the model is best suited for studying bladder neck fibrosis and structural remodeling rather than fully reproducing male post-prostatic-surgery BNC. The model may be useful for studying the fibrotic mechanisms of BNC and for preclinical evaluation of anti-fibrotic or reconstructive interventions.

Introduction

Bladder neck contracture (BNC) is a fibrotic narrowing of the bladder neck that commonly occurs after transurethral procedures, particularly TURP^1,2,3. Its pathological basis is excessive scar formation and collagen deposition after tissue injury and abnormal wound healing^4,5,6. Because BNC can lead to persistent voiding dysfunction, recurrent interventions, and substantial morbidity, a reproducible experimental model is needed to investigate its pathogenesis and treatment⁷.

Several animal models have been used to study bladder outlet obstruction or peri-urethral fibrosis, including extravesical ligation of the proximal urethra⁸ and other open surgical approaches⁹. Although these models can induce urinary obstruction and detrusor hypertrophy, they do not specifically reproduce the localized thermal injury that is thought to initiate postoperative BNC after endoscopic surgery. In addition, the anatomical target and degree of injury are often difficult to standardize, which can increase inter-animal variability.

An ideal BNC-related model should meet three criteria: it should target the anatomical bladder neck, reproduce controlled thermal injury similar to that produced by endoscopic energy devices, and reliably generate fibrotic narrowing with acceptable surgical complexity. To address these needs, we developed a transvesical Ho:YAG laser-based protocol in female rats. Female animals were selected to facilitate surgical exposure and to isolate bladder neck-specific thermal injury and fibrotic remodeling without interference from prostatic tissue. Using direct intravesical visualization, this method enables accurate delivery of laser energy to the bladder neck mucosa and submucosa and produces reproducible fibrotic narrowing of the bladder outlet.

Protocol

All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the Experimental Animal Welfare Ethics Committee of Beijing Institute of Traditional Chinese Medicine (Approval No. BJTCM-R-2025-03-02). Animals were housed under standard conditions with ad libitum access to food and water. In the current protocol, a total of 16 rats were used, including 8 rats in the no-surgery control group and 8 rats in the Ho:YAG laser-treated group. Two animals in the Ho:YAG laser-treated group died postoperatively. No additional animals were excluded because of perforation, severe bleeding, or failed model induction.

1. Preoperative preparation

1. Prepare pharmaceutical-grade meloxicam for perioperative analgesia (Figure 1A), the small-animal inhalation anesthesia system (Figure 1B), the Ho:YAG laser system with a 200-µm laser fiber (Figure 1C), sterile microsurgical instruments (Figure 1D), 8-0 absorbable braided sutures (coated polyglycolic acid, PGA) for cystotomy closure, 4-0 absorbable braided polyglactin 910 sutures for skin closure, sterile saline, sterile drapes, sterile cotton swabs, and sterile lubricant (Figure 1E).
2. Induce anesthesia with 3%–4% isoflurane in oxygen in an induction chamber, and maintain anesthesia with 1.5%–2.5% isoflurane through a nose cone during surgery.
3. Adjust the concentration according to respiratory rate and the approved institutional animal protocol. Confirm adequate anesthetic depth by the absence of the toe pinch reflex before incision and reassess anesthetic depth throughout the procedure.
4. Clip the fur widely from the lower abdomen so that the drape covers any remaining fur and prevents contact between instruments and unclipped hair.
5. Disinfect the surgical field with alternating povidone-iodine and 70% isopropyl alcohol scrubs; perform three applications of each solution before draping.
6. Place the animal in the supine position on a heating pad set at 37 °C and maintain sterile conditions throughout the procedure.
7. If bladder filling or intraoperative identification of the bladder neck is required, lubricate a small sterile urethral catheter or cannula with sterile lubricant, gently insert it through the urethral meatus into the bladder, and slowly instill sterile saline. Avoid forceful insertion or overfilling. Remove or secure the catheter according to the planned surgical exposure.

2. Exposure of the bladder

1. Make a 3–4 cm lower midline skin incision under sterile conditions (Figure 2A).
2. Using fine-tipped forceps and microscissors, divide the subcutaneous tissue and open the linea alba to enter the peritoneal cavity.
3. Gently exteriorize the urinary bladder with atraumatic forceps (Figure 2B).
4. Identify the bladder neck as the junction between the bladder base and the proximal urethra.
5. Place a blunt probe beneath the bladder neck to provide gentle support and stabilize the target area (Figure 2C).

3. Transvesical Ho:YAG laser ablation

1. Distend the bladder with sterile saline until the bladder wall becomes moderately tense. Avoid overdistension, which may obscure the anatomical landmarks or increase tension at the cystotomy site.
2. Create a 0.5 cm longitudinal cystotomy at the anterior bladder dome using fine microscissors (Figure 2D).
3. Introduce the prepared 200-µm Ho:YAG laser fiber through the cystotomy into the bladder lumen.
4. Advance the fiber under direct visualization to the bladder neck.
5. Gently place the fiber tip in direct contact with the bladder neck mucosa. Maintain contact mode without visible indentation of the tissue; ensure the tip-to-tissue distance is approximately 0 mm throughout energy delivery.
6. Deliver laser energy at 1.0 J and 10 Hz for 5 s at the 9 o’clock position (Figure 3A) and repeat at the 3 o’clock position (Figure 3B). This setting delivers 50 J per point and 100 J per animal, with power fixed at 10 W across animals.
   NOTE: A successful ablation is indicated by uniform mucosal whitening and formation of a shallow mucosal-to-submucosal crater without carbonization.
7. Keep the fiber stable during the 5 s application and avoid pushing the fiber into the wall. Avoid deep crater formation or full-thickness perforation. Do not direct the fiber toward the ureteral orifices or distal urethra.
8. Remove the fiber and irrigate the bladder lumen with sterile saline to remove debris and confirm hemostasis.
9. Close the cystotomy with 2–3 interrupted 8-0 absorbable braided sutures (coated PGA) in a watertight fashion.
10. Refill the bladder with 0.5–1.0 mL sterile saline to confirm the absence of leakage from the closure line (Figure 3C).
11. Close the abdominal wall and skin in layers (Figure 3D).

4. Postoperative care

1. Keep the rat on a heating pad until it has fully recovered from anesthesia.
2. Administer meloxicam at 1 mg/kg subcutaneously for postoperative analgesia and continue once daily for 1–3 days, or according to the approved animal protocol.
3. Monitor the animal daily for at least 7 days for signs of pain, distress, hematuria, urinary retention, wound dehiscence, or infection.
4. Provide hydration support and softened food as needed during the early recovery period.

5. Evaluation of bladder neck contracture

1. Allow the animals to recover for 4 weeks after surgery to permit fibrotic remodeling at the bladder neck.
2. At the experimental endpoint, euthanize the animals using sodium pentobarbital overdose (150 mg/kg, intraperitoneally) or another method approved by the institutional animal protocol, and confirm death before tissue harvest. Harvest the bladder, bladder neck, and proximal urethra en bloc.
3. Open the specimen longitudinally and photograph the bladder neck to document gross narrowing.
4. Fix the tissue in 4% paraformaldehyde at 4 °C for 24 h, dehydrate through graded ethanol, clear in xylene, embed in paraffin, and obtain serial 4-µm cross-sections through the bladder neck region.
5. Stain sections with hematoxylin and eosin, Masson’s trichrome, and picrosirius red according to standard histological protocols or the manufacturer’s instructions to evaluate lumen narrowing, bladder neck wall thickening, and collagen deposition.
6. For immunohistochemistry, deparaffinize sections, perform antigen retrieval, block endogenous peroxidase and nonspecific binding, incubate sections with antibodies against collagen type I and collagen type III, apply the appropriate secondary antibody, develop with chromogen, and counterstain with hematoxylin.
   NOTE: The antibodies are diluted according to the manufacturer’s recommendations.
7. Quantify lumen area and collagen-positive area using ImageJ/Fiji or equivalent image analysis software.
   1. Calibrate the pixel-to-micrometer scale using the microscope scale bar, manually delineate the bladder neck lumen and tissue region of interest, and measure lumen area.
   2. For Masson’s trichrome staining, apply a consistent color threshold to identify blue collagen-positive pixels within the region of interest.
   3. Calculate the collagen-positive area fraction as collagen-positive area divided by total tissue area.
   4. Perform measurements on comparable sections and fields from each animal, and analyze images in a blinded manner.
      NOTE: Successful model induction is supported by gross bladder outlet narrowing together with histological evidence of concentric fibrosis and increased collagen deposition at the lesion site. The operational success rate was 6/8 in the Ho:YAG laser-treated group, based on the development of the expected gross and histological phenotype.

Results

Four weeks after surgery, rats treated with Ho:YAG laser showed reproducible gross and histological changes consistent with bladder neck fibrotic narrowing. In this study, the no-surgery control group included 8 rats, and the Ho:YAG laser-treated group included 8 rats. 6/8 treated animals developed the expected phenotype; attrition consisted of 2 postoperative deaths in the Ho:YAG laser-treated group, with no additional exclusions. Grossly, control bladders showed normal size and a smooth external appearance (

Discussion

In this study, we established a rat model of bladder neck fibrotic narrowing using transvesical Ho:YAG laser ablation. The main strength of this method is that it produces controlled thermal injury at the anatomical bladder neck, followed by progressive fibrotic remodeling. This differs from traditional extravesical ligation models, which mainly generate obstruction through external compression rather than intrinsic scar formation¹⁰. Because postoperative BNC in clinical practice is closely linked...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Absorbable braided suture (coated PGA), 8-0	Shanghai Pudong Jinhuan Medical Products Co., Ltd., Shanghai, China	R831
Anti-collagen I antibody, mouse monoclonal	Proteintech Group, Inc., Wuhan, China	66761-1-Ig
Anti-collagen III antibody, mouse monoclonal	Proteintech Group, Inc., Wuhan, China	68320-1-Ig
Coated Vicryl Plus suture (polyglactin 910), 4-0	Ethicon, Inc. (Johnson & Johnson)	VCP773D	SH 26 mm ½-circle taper needle; violet braided; absorbable; Lot: RLMCJX
Hematoxylin and eosin (HE) staining kit	Servicebio, Wuhan, China	2310005
Holmium-YAG Laser System (LITHO)	Quanta System S.p.A, Italy	https://www.quantasystem.com/laser/litho/	1 J, 10 Hz, 10 W, 200 μm fiber
HRP-conjugated goat anti-mouse IgG secondary antibody	Boster, Wuhan, China	BA1050
Isoflurane	RWD	2025010601
Masson's trichrome staining kit	Zhuhai Baso Biotechnology Co., Ltd., Zhuhai, Guangdong, China	BA4079B
Meloxicam Injection	Qilu Animal Health, Shandong, China	E135H003
Paraformaldehyde (4%)	Servicebio, Wuhan, China	2510H0070148
Picrosirius red staining kit	Nanjing Senbeijia Biological Technology Co., Ltd., Nanjing, Jiangsu, China	20250228
Safety intravenous catheter, 24 G	Guangdong Lily Medical Technology Co., Ltd., Foshan, Guangdong, China	AD4-240827
Small-animal inhalation anesthesia system	RWD Life Science, Shenzhen, China	https://www.rwdstco.com/inhalation-anesthesia-solutions/	Induce with 3%–4% isoflurane in oxygen