Urethral Stricture Induction Followed by Buccal Mucosa Graft Urethroplasty in a Rat Model

Summary

In the present protocol, a urethral stricture induction was developed in Wistar rats, followed by urethral reconstruction with a buccal mucosa graft. A retrograde urethrogram and laser Doppler assessment were performed, validating urethral reconstruction (after stricture formation) and graft placement.

Abstract

Urethral reconstruction is an important area of expertise for urologists. The buccal mucosa is considered the best option when urethral grafting is necessary, although in some cases, it is inappropriate or needs to be optimized to repair a given stricture. Therefore, developing innovative procedures and evaluating their putative success in experimental models is crucial to fit the clinical need. With this goal, this study describes a protocol in which urethral stricture was induced by electrocautery in Wistar rats. Urethral reconstruction was performed 1 week later with a buccal mucosa graft, harvested from the lower lip, and placed in a ventral onlay fashion. A retrograde urethrogram showed a significant improvement in urethral diameter after urethroplasty compared to the respective value after stricture induction. Additionally, the graft placement was assessed by blood perfusion analysis using laser Doppler. As expected, a dark blue area corresponded to the non-vascularized buccal mucosa graft. This procedure can successfully simulate the normal pathophysiological process of urethral injury and tissue modulation, as well as urethral reconstruction using a buccal mucosa graft in a reproducible manner, and serve as the basis for future research based on tissue engineering or urethral grafts.

Introduction

Urethral reconstruction is a major challenge for urological surgeons in the management of urethral injury in the setting of strictures, trauma, or congenital defects¹. With curative intent, urethroplasty is the treatment of choice for most patients, with long (>2 cm) and anterior urethral defects requiring some form of substitution urethroplasty². Many tissues have been used as urethral substitutes, including full or split-thickness skin grafts of genital or extragenital areas, bladder wall mucosa, or the widespread buccal mucosa². Buccal mucosa grafts have several advantages, such as coming from ....

Protocol

All animal procedures were performed according to Directive 2010/63/EU. The procedures were approved by the institutional Animal Welfare Body, licensed by DGAV, the Portuguese competent authority for animal protection (license number 0421/000/000/2021). Male Wistar Han IGS (Crl:WI(Han) rats (400-500 g) at 12-14 weeks of age were used for the present study. The animals were obtained from a commercial source (see Table of Materials).

1. Preparation of solutions

1. Anesthetic solution
   1. Fill a 3 mL syringe with 2.3 mL of medetomidine (1 mg/mL; 0.715 mg/kg body weight) and transfer to a 15 mL....

Results

A total of 12 male Wistar rats, weighing 400-500 g and 12-14 weeks old, were used for urethral stricture induction. A retrograde urethrogram (RUG1) was performed 1 week later⁸, confirming the success of the technique. The urethral diameter was measured in millimeters at the level of the stricture induction. After this, a urethroplasty with a buccal mucosa graft was performed in the ventral face of the rat urethra. The same rats were submitted to a second retrograde urethrogram (RUG2) 14 days after.......

Discussion

Urethroplasty with buccal mucosa grafts is a major cornerstone in urethral reconstruction. However, innovative procedures should be developed to optimize those already described and establish new ones, such as tissue-engineered materials and biological grafts, to reduce complications and morbidity. Several procedures have been published to establish preclinical models and define surgical techniques. Souza et al.¹ conducted a study including 12 New Zealand rabbits. A ventral longitudinal skin incis.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Atipamezole	OrionPharma		ANTISEDAN (atipamezole) is indicated for the reversal of the sedative and analgesic effects.
Buprenorphine	RichterPharma		Buprenorphine is a derivative of the opioid alkaloid thebaine that is a more potent (25-40 times) and longer lasting analgesic than morphine.
Carl Zeiss Opmi-1 FC Surgical Stereo Microscope	Carl Zeiss Microscopy, Germany		OPMI 1 FC from ZEISS symbolizes quality, precision and reliability. The manual system is easy to use and delivers high fidelity images with the legendary ZEISS optics.
Carprofen	Zoetis		Carprofen is a non-steroidal anti-inflammatory drug (NSAID) of the propionic acid class.
Catheter 22 G. x 1''	B Braun	Introcan Safety	IV Catheter of fluorinated ethylene propylene (FEP) with firmer construction for arterial access.
Enrofloxacin	Bayer		Enrofloxacin is a fluoroquinolone antibiotic.
Fentanyl	Braun	3644960	Fentanyl is a powerful synthetic opioid analgesic.
Flumazenil	Fresenius Kabi		Benzodiazepine antagonist is used for the complete or partial reversal of the sedative effects caused by benzodiazepines.
High temperature cautery	Fiab	F7244	Disposable cautery, sterile, high temperature (1200 °C), 28 mm fine tip.
Instillagel gel	Farco-pharma		Cellulose-based lubricant with local anaesthetic and disinfecting properties.
Iopromide	Bayer		Non-ionic injectable contrast medium, with iodine.
Laser Doppler imaging system (perfusion imager moorLDI2-HIR and dedicated software)	MoorLDI-V6.0, Moor Instruments Ltd, Axminster, UK	5710	The angiogenesis models uses Laser Doppler imaging to assess blood perfusion in the hind limbs, one of which is ligated surgically. Dedicated measurement and comparison software allows the definition of regions of interest for blood flow assessment on the ischaemic versus non-ischaemic limb to establish a "reperfusion ratio" which can be assessed as often as needed and over a number of days on the same subject.
Lubrithal Eye Gel	Dechra		Eye gel used in animals for prevention of dry eyes during anaesthesia. Lubricating and moisturising action on cornea and conjunctiva.
Male Wistar Han IGS (Crl:WI(Han) rats	Charles River Laboratories, Spain		Twelve to fourteen-week-old
Metedomidin	Virbac	037/01/07RFVPT	Medetomidine is a synthetic drug used as surgical anesthetic.
Midazolam	Labesfal		Benzodiazepine medication is used for anesthesia and procedural sedation.
Monoplan Angiography System	Philips Medical Systems	Azurion 7 M20	A stationary diagnostic fluoroscopic x-ray system specifically designed to optimize the capability of users to visually and quantitatively evaluate the anatomy and function of blood vessels of the heart, brain and other organs, as well as the lymphatic system.
Mosquito forceps	Henry Schein	102-4346	Hartman-Mosquito Hemostatic Forcep Curved 3-1/2" Stainless Steel
Needle Holder	Henry Schein	100-2570	Needle holder Mayo-Hegar, stainless steel, 14 cm
Ophtalmic Needle Holder	Asico	AE-6143	Needle holder barranquer most delicate without lock
Pentobarbital Sodium	Ecuphar		Pentobarbital Sodium is the sodium salt of pentobarbital used for euthanasia.
Pointed Forceps	Aesculap	BD335R	Microforceps, 0.30 mm tip
Polysorb 6.0	Medtronic (Covidien)	UL-101	Coated Synthetic Absorbable Suture aimed to reduce the inflammatory reaction in tissues, followed by gradual encapsulation of the suture by fibrous connective tissue.
Providone-Iodine	Mylan		Povidone-iodine 10% is an antiseptic drug, used as a disinfectant before and after surgery.
Scalpel Blade nº11	B Braun	BB511	Carbon steel, sterile
Spring Scissor	Henry Schein	600-4826	Surgical scissors 31 castroviejo
Surgical Forceps	Aesculap	BD33R	Microforceps, 0.20mm tip
Surgical Scissor	Aesculap	MA873R	Micro Iris Scissor, curved shrap tips
SurgiPro 7.0	Medtronic (Coviden)	VP-702-X	Non-Absorbable Monofilament Polypropylene Suture indicated for use in general soft tissue ligation.