Name: urethral stricture induction followed by buccal mucosa graft urethroplasty in a rat model
Uploaded: 2023-04-28T13:00:00.000+00:00
Duration: 5 min 9 s
Description: Watch this Scientific Journal Video about Urethral Stricture Induction Followed by Buccal Mucosa Graft Urethroplasty in a Rat Model at JoVE.com

We thank Jo&#227;o Leit&#227;o, head of the Radiology department of Centro Hospitalar Universit&#225;rio Lisboa Norte, and Catia Fernandes, technician of the Radiology department of Centro Hospitalar Universit&#225;rio Lisboa Norte, for their cooperation in the realization of urethrograms.

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
<ol>
	<li>Anesthetic solution
	<ol>
		<li>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 centrifuge tube.</li>
		<li>Fill a 3 mL syringe with 1.55 mL of fentanyl (0.05 mg/mL; 0.02 mg/kg body weight) and transfer to the same 15 mL centrifuge tube.</li>
		<li>Fill a 10 mL syringe with 6.15 mL of midazolam (5 mg/mL; 9.5 mg/kg body weight) and transfer to the same 15 mL centrifuge tube, thus making a 10 mL anesthetic solution.</li>
		<li>Label the tube and store it at 4 &#176;C in the dark. 
		NOTE: This anesthetic combination (medetomidine, fentanyl, and midazolam) provides an up to 3 h anesthetic surgical window. The administration of atipamezole and flumazenil may revert the effect.</li>
	</ol>
	</li>
	<li>Anti-sedative solution
	<ol>
		<li>Fill a 1 mL syringe with 0.7 mL of atipamezole (5 mg/mL; 3.72 mg/kg body weight) and transfer to a 15 mL centrifuge tube.</li>
		<li>Fill a 10 mL syringe with 9.3 mL of flumazenil (0.1 mg/mL; 1.56 mg/kg body weight) and transfer to the same 15 mL centrifuge tube, thus making a 10 mL anti-sedative solution.</li>
		<li>Label the tube and store it at 4 &#176;C in the dark.</li>
	</ol>
	</li>
	<li>Postoperative analgesia solutions
	<ol>
		<li>Fill a 1 mL syringe with 0.5 mL of carprofen (50 mg/mL; 5 mg/kg body weight) and transfer to a 15 mL centrifuge tube.</li>
		<li>Add 9.5 mL of NaCl solution (0.9%), obtaining a 10 mL carprofen solution.</li>
		<li>Fill a 1 mL syringe with 1 mL of buprenorphine (0.3 mg/mL; 0.01-0.05 mg/kg body weight) and transfer to a 15 mL centrifuge tube.</li>
		<li>Add 9 mL of NaCl solution (0.9%), obtaining a 10 mL buprenorphine solution.</li>
		<li>Label the tubes and store them at 4 &#176;C in the dark.</li>
	</ol>
	</li>
	<li>Perioperative antibiotic
	<ol>
		<li>Fill a 2.5 mL syringe with 2.5 mL of enrofloxacin (25 mg/mL; 10 mg/kg body weight) and transfer to a 15 mL centrifuge tube.</li>
		<li>Add 7.5 mL of NaCl solution (0.9%), obtaining a 10 mL enrofloxacin solution.</li>
		<li>Label the tube and store it at 4 &#176;C in the dark. 
		&#8203;NOTE: The details of all the reagents to prepare the above solutions are listed in the Table of Materials.</li>
	</ol>
	</li>
</ol>
2. Surgical induction of urethral stricture
NOTE: The surgical procedures were performed using a stereo microscope (10x) (see Table of Materials).
<ol>
	<li>Sterilize all surgical tools before use: scalpel blade (number 11), pointed forceps, spring scissors, surgical forceps, ophthalmic needle holder, surgical scissors, and a needle holder. Use sterile cotton balls to clean the surgical field.</li>
	<li>Load the syringe with the anesthetic solution before holding the rat.</li>
	<li>Restrain the animal using a tube or a towel and elevate the tail to expose the abdomen.</li>
	<li>Keep the animal restrained and perform an intraperitoneal injection of the anesthetic solution (prepared in step 1.1).</li>
	<li>Test the rat&#39;s pedal withdrawal reflexes to assess anesthesia.</li>
	<li>Apply protective eye gel in both eyes of the animal. Perform a subcutaneous injection of the antibiotic solution (prepared in step 1.1), at 10 mg/kg of body weight.</li>
	<li>Place the rat in the dorsal decubitus position on a heated pad and use a dissection microscope at 10x or 20x magnification to perform the surgical procedure.</li>
	<li>Clean the penis and surrounding abdominal skin with povidone-iodine (100 mg/mL).</li>
	<li>Manually retract the foreskin and place a superficial stay suture (7.0 suture; see Table of Materials) in the dorsal aspect of the penile glans to apply traction to the penis, leaving the needle holder in place to keep the penis retracted.</li>
	<li>Place a 22 G venous catheter into the urethra for catheterization using a lubricant gel.</li>
	<li>Using a surgical scalpel blade (number 11), perform a 1 cm longitudinal ventral incision in the penile skin.</li>
	<li>Using the pointed forceps and the spring scissors, dissect the penile tissue layers until exposing the urethra at the mid-shaft level.</li>
	<li>With an electrocautery device (see Table of Materials), apply a current with 10 W, for 1 s, in the lateral aspects of the urethra (in one location on each side), ventrally at the level of the midshaft of the penis.</li>
	<li>Close the incision with a 6.0 absorbable running suture (see Table of Materials).</li>
	<li>Remove the urethral catheter. Remove the penile traction suture.</li>
	<li>Perform the subcutaneous injection of analgesia: Carprofen at 5 mg/kg of body weight, and buprenorphine at 0.03 mg/kg of body weight.</li>
	<li>Load the syringe with the anti-sedative solution (prepared in step 1.2) and, with the animal in ventral decubitus, tent the loose skin to administer a subcutaneous injection of the anti-sedative solution.</li>
</ol>
3. Surgical procedure of urethroplasty with a buccal mucosa graft
NOTE: The surgical procedures were performed using a stereo microscope (10x) (see Table of Materials).
<ol>
	<li>Sterilize all surgical tools needed for this intervention: pointed forceps, spring scissors, ophthalmic needle holder, surgical scissors, needle holder, three mosquito forceps, and a scalpel blade (number 11). Use small sponges to clean the surgical field.</li>
	<li>Administer the anesthetic, the antibiotic, the restrain and place the animal as described previously (steps 2.2-2.8).</li>
	<li>Clean the buccal mucosa of the lower lip, the penis, and the surrounding abdominal skin with povidone-iodine (100 mg/mL).</li>
	<li>Place three stay sutures (7.0 sutures) in the lower lip, at both sides and in the middle, and leave a mosquito in each one to retract the lower lip and expose the inner mucosa.</li>
	<li>Using spring scissors and pointed forceps, harvest a 4 mm diameter graft of the lower lip inner buccal mucosa, and place it in a small recipient with sterile saline (0.9% NaCl).</li>
	<li>Apply compression in the donor area with a sponge for hemostasis.</li>
	<li>Remove the previously placed stay sutures of the lower lip.</li>
	<li>Expose the penis as described previously (step 2.9).</li>
	<li>Place a 22 G venous catheter into the urethra for catheterization using a lubricant gel.</li>
	<li>Using spring scissors, perform a circumferential, sub-coronal incision and deglove the penis to the base.</li>
	<li>Using the pointed forceps and the spring scissors, dissect the remaining layers and expose the urethra.</li>
	<li>Using a surgical scalpel blade (number 11) and spring scissors, perform a longitudinal ventral incision, starting 3 mm distal to the coronal sulcus in an extension of 4 mm, spatulating the urethra at the level of the previously inducted stricture (step 2).</li>
	<li>Place two stay sutures of 7.0 material, one at each side of the spatulation, and leave a mosquito in each one to retract the urethra.</li>
	<li>Place two 7.0 non-absorbable sutures, one at each end of the spatulation.</li>
	<li>Place the buccal mucosa graft in a ventral onlay fashion with the mucosa side facing the urethral lumen.</li>
	<li>Pass one of the sutures through the graft end and perform a half ellipse with a running suture.</li>
	<li>Repeat step 3.15 and step 3.16 with the other suture on the other side of the graft.</li>
	<li>Remove the urethral catheter. Reposition the penile skin.</li>
	<li>Close the circumferential, sub-coronal incision with a 6.0 absorbable interrupted suture.</li>
	<li>Remove the penile traction suture.</li>
	<li>Administer the analgesia followed by the anti-sedative solution, as described in steps 2.16-2.17.</li>
</ol>
4. Postoperative monitoring
<ol>
	<li>Observe the rats three to four times every hour, confirming their recovery from anesthesia. Monitor respiration and assess pedal and eye reflexes.</li>
	<li>Subcutaneously inject analgesia every 12 h for 48 h. 
	NOTE: Carprofen was administered at 1 mL/500 mg of body weight, and buprenorphine was administered at 0.5 mL/500 mg of body weight (see Table of Materials).</li>
	<li>Provide soft food for 48 h after each procedure and water ad libitum.</li>
	<li>Monitor the rats daily following surgery and register their health status and surgery site appearance. Evaluated signs include facial expression, vocalization, activity state, any sign of pain, food, and drink ingestion, voiding, and bleeding.</li>
</ol>
5. Blood perfusion evaluation
NOTE: Blood flow is measured immediately before stricture induction, immediately before urethroplasty, and immediately after urethroplasty.
<ol>
	<li>Perform laser Doppler perfusion imaging.
	<ol>
		<li>Anesthetize the rats using the anesthetic solution (step 1.1).</li>
		<li>Put the rat supine on a 37 &#176;C heating pad with penile traction, as described in step 2.9.</li>
		<li>Start the laser Doppler perfusion imager (see Table of Materials) to obtain data. Pre-set the area of interest to be read by the laser beam.</li>
		<li>Apply the anti-sedative solution (step 1.2) to revert the anesthesia.</li>
		<li>Using the image analysis software, draw the region of interest (ROI) around the area of the penis and register the flux values over time.</li>
	</ol>
	</li>
</ol>
6. Radiographic evaluation
NOTE: Stricture induction confirmation and stricture resolution after urethroplasty are confirmed with a retrograde urethrogram. This evaluation is performed 1 week after stricture induction (before urethroplasty) and 2 weeks after urethroplasty.
<ol>
	<li>Perform a retrograde urethrogram using a monoplan angiography system (see Table of Materials).
	<ol>
		<li>Anesthetize the rats using the anesthetic solution.</li>
		<li>Place the animal on the angiography mattress in a right oblique decubitus, with penile traction, as described in step 2.9.</li>
		<li>Focus the cone beam on the animal&#39;s pelvic area, including the penis.</li>
		<li>Place a 22 G venous catheter advanced 2 mm into the distal urethra.</li>
		<li>Start instillating 1 mL of iodine radiographic contrast material (1:1 ratio of 623 mg/mL iopromide solution and 0.9% NaCl) into the urethra.</li>
		<li>Simultaneously, perform plain radiography to identify the radiographic contrast fulfilling the urethral lumen and assess the urethral diameter.</li>
		<li>On completion of imaging, revert the anesthesia with the anti-sedative solution.</li>
	</ol>
	</li>
</ol>
7. Euthanasia
NOTE: Euthanasia is performed 3 weeks after urethroplasty (4 weeks after stricture induction), immediately after the last perfusion evaluation.
<ol>
	<li>Fill a 2.5 mL syringe with 2 mL/kg pentobarbital sodium (400 mg/mL).</li>
	<li>Perform an intraperitoneal injection of the solution to euthanize the animal.</li>
</ol>

<ol>
	<li>Souza, G. F., Calado, A. A., Delcelo, R., Ortiz, V., Macedo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathological+evaluation+of+urethroplasty+with+dorsal+buccal+mucosa:+an+experimental+study+in+rabbits.">Histopathological evaluation of urethroplasty with dorsal buccal mucosa: an experimental study in rabbits.</a> International Brazilian Journal of Urology. 34 (3), 345-354 (2008).</li><li>Andrich, D. E., Mundy, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urethral+strictures+and+their+surgical+treatment.">Urethral strictures and their surgical treatment.</a> BJU International. 86 (5), 571-580 (2000).</li><li>Bhargava, S., Chapple, C. R., Bullock, A. J., Layton, C., MacNeil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+buccal+mucosa+for+substitution+urethroplasty.">Tissue-engineered buccal mucosa for substitution urethroplasty.</a> BJU International. 93 (6), 807-811 (2004).</li><li>Fu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substitution+urethroplasty+for+anterior+urethral+stricture+repair:+comparison+between+lingual+mucosa+graft+and+pedicled+skin+flap.">Substitution urethroplasty for anterior urethral stricture repair: comparison between lingual mucosa graft and pedicled skin flap.</a> Scandinavian Journal of Urology. 51 (6), 479-483 (2017).</li><li>Blain, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+grafts+in+the+rat+model:+an+anatomic+study.">Vascular grafts in the rat model: an anatomic study.</a> Microsurgery. 21 (3), 80-83 (2001).</li><li>Schmauss, D., Weinzierl, A., Schmauss, V., Harder, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+rodent+flap+models+in+experimental+surgery.">Common rodent flap models in experimental surgery.</a> European Surgical Research. 59 (3-4), 255-264 (2018).</li><li>You, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rat+orthotopic+renal+transplantation+model+for+renal+allograft+rejection.">A rat orthotopic renal transplantation model for renal allograft rejection.</a> Journal of Visualized Experiments. (180), e63464 (2022).</li><li>Park, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balloon-expandable+biodegradable+stents+versus+self-expandable+metallic+stents:+a+comparison+study+of+stent-induced+tissue+hyperplasia+in+the+rat+urethra.">Balloon-expandable biodegradable stents versus self-expandable metallic stents: a comparison study of stent-induced tissue hyperplasia in the rat urethra.</a> Cardiovascular and Interventional Radiology. 42 (9), 1343-1351 (2019).</li><li>Mart&#237;n-Cano, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+and+immunohistochemical+changes+in+the+rat+oral+mucosa+used+as+an+autologous+urethral+graft.">Histological and immunohistochemical changes in the rat oral mucosa used as an autologous urethral graft.</a> Journal of Pediatric Surgery. 48 (7), 1557-1564 (2013).</li><li>Hofer, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+primary+urethral+wound+healing+in+the+rat.">Analysis of primary urethral wound healing in the rat.</a> Urology. 84 (1), e1-7 (2014).</li><li>Tavukcu, H. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effect+of+platelet-rich+plasma+on+urethral+injury+model+of+male+rats.">Protective effect of platelet-rich plasma on urethral injury model of male rats.</a> Neurourology and Urodynamics. 37 (4), 1286-1293 (2018).</li></ol>

The authors have nothing to disclose.

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...

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 (&gt;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 a wet and hairless environment, being easy to harvest, being resistant to infection, a thick epithelium, a reduced likelihood of pseudo-diverticulum formation, and a thin lamina, allowing early imbibition and inosculation3. In contrast with flaps, grafts have no blood supply, depending on the recipient's vascular bed to survive4.
Animal models of grafts or flaps have been used extensively to develop or refine surgical techniques, study and understand tissue physiology, underlying mechanisms, and causes of failure, and evaluate innovative treatment strategies5,6. Although bigger animals facilitate technical execution, rodents, namely rats and mice, are easier to handle and maintain, resistant to diseases, more cost-effective, and, importantly, with the tools to investigate molecular mechanisms, crucial to test innovative therapies5,6. Several models of flaps and grafts have been described in rats using different tissues, namely skin, bone, muscle6, vessels5, and even solid organs7. However, there is scarce investigation in murine models into grafts for urethral reconstruction or tissue engineering. 
Nevertheless, advances in translational science depend on animal models that mimic disease. So far, the local pathophysiology environment has not been addressed, since urethral reconstruction is performed immediately after its stricture. Herein, this study aims to perform a urethral reconstruction using a buccal mucosa graft in a local pathophysiology environment. With this objective, the urethral stricture was induced 1 week before its reconstruction. This experimental model, performed in rats, allows to test innovative therapies and investigate their molecular mechanisms and clinical advantages in the future.

<table><tbody><tr><td>Atipamezole</td><td>OrionPharma</td><td></td><td>ANTISEDAN (atipamezole) is indicated for the reversal of the sedative and analgesic effects.</td></tr><tr><td>Buprenorphine</td><td>RichterPharma</td><td></td><td>Buprenorphine is a derivative of the opioid alkaloid thebaine that is a more potent (25-40 times) and longer lasting analgesic than morphine.</td></tr><tr><td>Carl Zeiss Opmi-1 FC Surgical Stereo Microscope</td><td>Carl Zeiss Microscopy, Germany</td><td></td><td>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.</td></tr><tr><td>Carprofen</td><td>Zoetis</td><td></td><td>Carprofen is a non-steroidal anti-inflammatory drug (NSAID) of the propionic acid class.</td></tr><tr><td>Catheter 22 G. x 1&#039;&#039;</td><td>B Braun</td><td>Introcan Safety</td><td>IV Catheter of fluorinated ethylene propylene (FEP) with firmer construction for&lt;br/&gt; arterial access.</td></tr><tr><td>Enrofloxacin</td><td>Bayer</td><td></td><td>Enrofloxacin is a fluoroquinolone antibiotic.</td></tr><tr><td>Fentanyl</td><td>Braun</td><td>3644960</td><td>Fentanyl is a powerful synthetic opioid analgesic.</td></tr><tr><td>Flumazenil</td><td>Fresenius Kabi</td><td></td><td>Benzodiazepine antagonist is used for the complete or partial reversal of the sedative effects caused by benzodiazepines.</td></tr><tr><td>High temperature cautery</td><td>Fiab</td><td>F7244</td><td>Disposable cautery, sterile, high temperature (1200 &amp;deg;C), 28 mm fine tip.</td></tr><tr><td>Instillagel gel</td><td>Farco-pharma</td><td></td><td>Cellulose-based lubricant with local anaesthetic and disinfecting properties.</td></tr><tr><td>Iopromide</td><td>Bayer</td><td></td><td>Non-ionic injectable contrast medium, with iodine.</td></tr><tr><td>Laser Doppler imaging system (perfusion imager moorLDI2-HIR and dedicated software)</td><td>MoorLDI-V6.0, Moor Instruments Ltd, Axminster, UK</td><td>5710</td><td>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 &quot;reperfusion ratio&quot; which can be assessed as often as needed and over a number of days on the same subject.</td></tr><tr><td>Lubrithal Eye Gel</td><td>Dechra</td><td></td><td>Eye gel used in animals for prevention of dry eyes during anaesthesia. Lubricating and moisturising action on cornea and conjunctiva.</td></tr><tr><td>Male Wistar Han IGS (Crl:WI(Han) rats</td><td>Charles River Laboratories, Spain</td><td></td><td>Twelve to fourteen-week-old</td></tr><tr><td>Metedomidin</td><td>Virbac</td><td>037/01/07RFVPT</td><td>Medetomidine is a synthetic drug used as surgical anesthetic.</td></tr><tr><td>Midazolam</td><td>Labesfal</td><td></td><td>Benzodiazepine medication is used for anesthesia and procedural sedation.</td></tr><tr><td>Monoplan Angiography System</td><td>Philips Medical Systems</td><td>Azurion 7 M20</td><td>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.</td></tr><tr><td>Mosquito forceps</td><td>Henry Schein</td><td>102-4346</td><td>Hartman-Mosquito Hemostatic Forcep Curved 3-1/2&quot; Stainless Steel</td></tr><tr><td>Needle Holder</td><td>Henry Schein</td><td>100-2570</td><td>Needle holder Mayo-Hegar, stainless steel, 14 cm</td></tr><tr><td>Ophtalmic Needle Holder</td><td>Asico</td><td>AE-6143</td><td>Needle holder barranquer most delicate without lock</td></tr><tr><td>Pentobarbital Sodium</td><td>Ecuphar</td><td></td><td>Pentobarbital Sodium is the sodium salt of pentobarbital used for euthanasia.</td></tr><tr><td>Pointed Forceps</td><td>Aesculap</td><td>BD335R</td><td>Microforceps, 0.30 mm tip</td></tr><tr><td>Polysorb 6.0</td><td>Medtronic (Covidien)</td><td>UL-101</td><td>Coated Synthetic Absorbable Suture aimed to reduce the inflammatory reaction in tissues, followed by gradual encapsulation of the suture by fibrous connective tissue.</td></tr><tr><td>Providone-Iodine</td><td>Mylan</td><td></td><td>Povidone-iodine 10% is an antiseptic drug, used as a disinfectant before and after surgery.</td></tr><tr><td>Scalpel Blade n&amp;ordm;11</td><td>B Braun</td><td>BB511</td><td>Carbon steel, sterile</td></tr><tr><td>Spring Scissor</td><td>Henry Schein</td><td>600-4826</td><td>Surgical scissors 31 castroviejo</td></tr><tr><td>Surgical Forceps</td><td>Aesculap</td><td>BD33R</td><td>Microforceps, 0.20mm tip</td></tr><tr><td>Surgical Scissor</td><td>Aesculap</td><td>MA873R</td><td>Micro Iris Scissor, curved shrap tips</td></tr><tr><td>SurgiPro 7.0</td><td>Medtronic (Coviden)</td><td>VP-702-X</td><td>Non-Absorbable Monofilament Polypropylene Suture indicated for use in general soft tissue ligation.</td></tr></tbody></table>

urethral stricture induction followed by buccal mucosa graft urethroplasty in a rat model

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.

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...

Watch this Scientific Journal Video about Urethral Stricture Induction Followed by Buccal Mucosa Graft Urethroplasty in a Rat Model at JoVE.com

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

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.

Urethral reconstruction is an important area of expertise for urologists. The buccal mucosa is considered the best option when urethral grafting is ...

urethral-stricture-induction-followed-buccal-mucosa-graft

Research

JoVE Journal

Medicine

862 Views.  Faculdade de Medicina da Universidade de Lisboa. 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.

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

Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and expels it with a precisely orchestrated contraction. A number of congenital and acquired urological anomalies including posterior urethral valves, benign prostatic hyperplasia, and neurogenic bladder secondary to spina bifida/spinal cord injury can result in pathologic tissue remodeling leading to impaired compliance and reduced capacity1. Functional or anatomical obstruction of the urinary tract is frequently associated with these conditions, and can lead to urinary incontinence and kidney damage from increased storage and voiding pressures2. Surgical implantation of gastrointestinal segments to expand organ capacity and reduce intravesical pressures represents the primary surgical treatment option for these disorders when medical management fails3. However, this approach is hampered by the limitation of available donor tissue, and is associated with significant complications including chronic urinary tract infection, metabolic perturbation, urinary stone formation, and secondary malignancy4,5.
Current research in bladder tissue engineering is heavily focused on identifying biomaterial configurations which can support regeneration of tissues at defect sites. Conventional 3-D scaffolds derived from natural and synthetic polymers such as small intestinal submucosa and poly-glycolic acid have shown some short-term success in supporting urothelial and smooth muscle regeneration as well as facilitating increased organ storage capacity in both animal models and in the clinic6,7. However, deficiencies in scaffold mechanical integrity and biocompatibility often result in deleterious fibrosis8, graft contracture9, and calcification10, thus increasing the risk of implant failure and need for secondary surgical procedures. In addition, restoration of normal voiding characteristics utilizing standard biomaterial constructs for augmentation cystoplasty has yet to be achieved, and therefore research and development of novel matrices which can fulfill this role is needed.
In order to successfully develop and evaluate optimal biomaterials for clinical bladder augmentation, efficacy research must first be performed in standardized animal models using detailed surgical methods and functional outcome assessments. We have previously reported the use of a bladder augmentation model in mice to determine the potential of silk fibroin-based scaffolds to mediate tissue regeneration and functional voiding characteristics.11,12 Cystometric analyses of this model have shown that variations in structural and mechanical implant properties can influence the resulting urodynamic features of the tissue engineered bladders11,12. Positive correlations between the degree of matrix-mediated tissue regeneration determined histologically and functional compliance and capacity evaluated by cystometry were demonstrated in this model11,12. These results therefore suggest that functional evaluations of biomaterial configurations in rodent bladder augmentation systems may be a useful format for assessing scaffold properties and establishing in vivo feasibility prior to large animal studies and clinical deployment. In the current study, we will present various surgical stages of bladder augmentation in both mice and rats using silk scaffolds and demonstrate techniques for awake and anesthetized cystometry.

Surgical stages of bladder augmentation are described using 3-D scaffolds in murine and rat models. To test the efficacy of biomaterial configurations for use in bladder augmentation, techniques for both awake and anesthetized cystometry are presented.

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and ...

Bioengineering

A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive nephropathy induced by unilateral ureteric obstruction (UUO), which can either be irreversible (UUO) or reversible (R-UUO). In the irreversible UUO model, the ureter may be obstructed for variable periods of time in order to induce increasingly severe renal inflammation and interstitial fibrotic scarring. In the reversible R-UUO model the ureter is obstructed to induce hydronephrosis, tubular dilation and inflammation. After a suitable period of time the ureteric obstruction is then surgically reversed by anastomosis of the severed previously obstructed ureter to the bladder in order to allow complete decompression of the kidney and restoration of urinary flow to the bladder. The irreversible UUO model has been used to investigate various aspects of renal inflammation and scarring including the pathogenesis of disease and the testing of potential anti-inflammatory or anti-fibrotic therapies. The more challenging model of R-UUO has been used by some investigators and does offer significant research potential as it allows the study of inflammatory and immune processes and tissue remodeling in an injured and scarred kidney following the removal of the injurious stimulus. As a result, the R-UUO model offers investigators the opportunity to explore the resolution of kidney inflammation together with key aspects of tissue repair. These experimental models are of relevance to human disease as patients often present with obstruction of the renal tract that requires decompression and are commonly left with significant residual kidney impairment that has no current treatment options and may lead to eventual end stage kidney failure.

The murine model of irreversible unilateral ureteric obstruction (UUO) is presented together with the model of reversible UUO in which the ureteric obstruction is relieved by anastomosis of the severed ureter into the bladder. These models enable the study of renal inflammation and scarring as well as tissue remodeling.

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive ...

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal model to investigate bladder remodeling in pBOO are female rodents undergoing partial obstruction at the proximal urethra. Variability in the degree of obstruction and animal mortality are major concerns with proximal obstruction. Furthermore, dissecting around the proximal urethra and bladder neck jeopardizes bladder innervation.
We developed a nerve-sparing mid-urethral obstruction (NeMO) model for pBOO avoiding the disadvantages of the traditional model. We approached the urethra just inferior to the pubic symphysis, which obviated the need for laparotomy as well as for dissection in this area; also, the striated urethral sphincter remained untouched. We performed NeMO in female Sprague-Dawley rats (12 obstructions, 6 sham animals) as well as in female C57/bl6 mice (20 obstructions, 18 sham animals). After two weeks, we evaluated bladder function, bladder mass, and body mass.
We had no mortalities among obstructed- or sham-operated female rats; as described for the traditional proximal pBOO-method, we tied the suture around the proximal urethra and a temporarily placed 0.9 mm metal rod. NeMO induced an 85% increase in bladder mass after two weeks, average residual urine volume was 0.4 mL in partially obstructed rats while only 0.03 mL in sham animals. In mice, we tested 3 sizes of cannulas that we placed along the urethra when tying the suture. We found that using a 27-gauge cannula resulted in over 50% animal mortality; placing the 25-gauge cannula did not yield the desired response in increasing bladder mass; utilizing a 26-gauge cannula yielded favorable results with minimal animal mortality (1/8) yet a significant 2-fold increase in bladder mass.

Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents

Traditional modeling of partial bladder outlet obstruction in rodents is fraught with animal mortality. A denervation injury from dissection around the proximal urethra and bladder neck is also of major concern. We developed and evaluated a safe and reliable mid-urethral obstruction model, avoiding the shortcomings of the traditional model.

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal ...

Vessel-sparing Excision and Primary Anastomosis

Urethroplasty is considered to be the standard treatment for urethral strictures since it provides excellent long-term success rates. For isolated short bulbar or posterior urethral strictures, urethroplasty by excision and primary anastomosis (EPA) is recommended. As EPA only requires the excision of the narrowed segment and the surrounding spongiofibrosis, a full-thickness transection of the corpus spongiosum, as performed in the traditional transecting EPA (tEPA), is usually unnecessary. Jordan et al. introduced the idea of a vessel-sparing approach in 2007, aiming to reduce surgical trauma, especially to the dual arterial blood supply of the urethra, and, thus, potentially reducing the risk of postoperative erectile dysfunction or glans ischemia. This approach could also be beneficial for subsequent urethral interventions such as redo urethroplasty using a free graft, in which a well-vascularized graft bed is imperative. Nevertheless, these potential benefits are only assumptions as prospective studies comparing the functional outcome of both techniques with validated questionnaires are currently lacking. Moreover, vessel-sparing EPA (vsEPA) should at least be able to provide similar surgical outcomes as tEPA. The aim of this paper is to give an elaborate, step-by-step overview of how to manage patients with isolated short bulbar or posterior urethral strictures with vsEPA. The main objective of this manuscript is to outline the surgical technique and to report the representative surgical outcome. A total of 117 patients were managed according to the described protocol. The analysis was performed on the entire patient cohort and on the bulbar (n = 91) and posterior (n = 26) vsEPA group separately. Success rates were 93.4% and 88.5% for the bulbar and posterior vsEPA, respectively. To conclude, vsEPA, as outlined in the protocol, provides excellent success rates with low complication rates for isolated short bulbar and posterior urethral strictures.

Here, we present an elaborate and efficient protocol to treat isolated short bulbar or posterior urethral strictures with vessel-sparing excision and primary anastomosis.

Urethroplasty is considered to be the standard treatment for urethral strictures since it provides excellent long-term success rates. For isolated short ...

Accessing Collagen Scaffold for Single-Stage Urinary Diversion Surgery in Pigs

Surgical Model for Single-Staged Tissue-Engineered Urothelial Tubes in Minipigs

Reconstructive surgeries are often challenged by a lack of grafting tissue. In the treatment of urogenital malformations, the conventional solution has been harvesting gastrointestinal tissue for non-orthotopic reconstruction due to its abundance to reestablish normal function in the patient. The clinical outcomes after rearranging native tissues within the body are often associated with significant morbidity; thus, tissue engineering holds specific potential within this field of surgery. Despite substantial advances, tissue-engineered scaffolds have not yet been established as a valid surgical treatment alternative, mainly due to the costly and complex requirements of materials, production, and implantation. In this protocol, we present a simple and accessible collagen-based tubular scaffold embedded with autologous organ-specific tissue particles, designed as a conduit for urinary diversion. The scaffold is constructed during the primary surgical procedure, comprises commonly available surgical materials, and requires conventional surgical skills. Secondly, the protocol describes an animal model designed to evaluate the short-term in vivo outcomes post-implantation, with the possibility of additional variations to the procedure. This publication aims to demonstrate the procedure step-by-step, with special attention to the use of autologous tissue and a tubular form.

Tissue-engineered implants for reconstructive surgery rarely progress beyond preclinical trials due to laborious ex vivo culturing, which includes complex and expensive scaffold components. Here, we present a single-staged procedure designed for urinary diversion with an accessible collagen-based tubular scaffold containing autologous micrografts.

Reconstructive surgeries are often challenged by a lack of grafting tissue. In the treatment of urogenital malformations, the conventional solution has ...

Development of a Uterosacral Ligament Suspension Rat Model

Pelvic organ prolapse (POP) is a common pelvic floor disorder (PFD) with the potential to significantly impact a woman's quality of life. Approximately 10%-20% of women undergo pelvic floor repair surgery to treat prolapse in the United States. PFD cases result in an overall $26.3 billion annual cost in the United States alone. This multifactorial condition has a negative impact on the quality of life and yet the treatment options have only dwindled in the recent past. One common surgical option is uterosacral ligament suspension (USLS), which is typically performed by affixing the vaginal vault to the uterosacral ligament in the pelvis. This repair has a lower incidence of complications compared to those with mesh augmentation, but is notable for a relatively high failure rate of up to 40%. Considering the lack of standard animal models to study pelvic floor dysfunction, there is an urgent clinical need for innovation in this field with a focus on developing cost-effective and accessible animal models. In this manuscript, we describe a rat model of USLS involving a complete hysterectomy followed by fixation of the remaining vaginal vault to the uterosacral ligament. The goal of this model is to mimic the procedure performed on women to be able to use the model to then investigate reparative strategies that improve the mechanical integrity of the ligament attachment. Importantly, we also describe the development of an in situ tensile testing procedure to characterize interface integrity at chosen time points following surgical intervention. Overall, this model will be a useful tool for future studies that investigate treatment options for POP repair via USLS.

Pelvic organ prolapse affects millions of women worldwide and yet some common surgical interventions have failure rates as high as 40%. The lack of standard animal models to investigate this condition impedes progress. We propose the following protocol as a model for uterosacral ligament suspension and in vivo tensile testing.

Pelvic organ prolapse (POP) is a common pelvic floor disorder (PFD) with the potential to significantly impact a woman's quality of life. Approximately ...

Efficient Rat Model for Urethral Stricture: Electric Knife Method

Iatrogenic Injury Recapitulated: Electroexcision Technique for Urethral Stricture Modeling in Rats

Urethral stricture (US) is a common clinical condition in urology, characterized by high prevalence and morbidity across all ages. Current treatments for US, such as urethral dilatation and internal urethrotomy, fail to fully resolve the condition and are associated with high rates of recurrence and complications. 
Additionally, the pathogenesis of US is not well understood. To explore the pathogenesis of US and develop new therapeutic strategies, it is crucial to establish a standardized rat model that accurately reflects the clinical manifestations. This study outlines a straightforward and repeatable method for inducing US in rats using a high-frequency electric knife. The method involves making a longitudinal incision with the electric knife set to a unipolar mixed cutting mode at 4 W, which inflicts significant urethral damage. Histopathological analysis shows thickening of the urothelium, inflammatory infiltration, and disorganized collagen fibers. This model effectively replicates iatrogenic injury through electroexcision in the rat urethra. In summary, this study successfully establishes a new, efficient, and stable rat model of US that closely mimics the clinical scenario, providing a valuable tool for further research into the mechanisms and novel treatments for US.

In this study, we present a novel, efficient, and stable rat model of urethral stricture created through electroexcision of the rat urethra, which effectively simulates iatrogenic injury observed in clinical settings.

Urethral stricture (US) is a common clinical condition in urology, characterized by high prevalence and morbidity across all ages. Current treatments for ...

Urethroplasty with Pedicled Tunica Vaginalis for the Treatment of Long-segment Anterior Urethral Stricture Caused by Lichen Sclerosus of Glans Penis

Urethroplasty for the management of long-segment urethral strictures associated with lichen sclerosus presents considerable clinical challenges. Oral mucosal grafts are commonly employed but are vulnerable to posttransplantation infection and recurrent stricture formation. Furthermore, the necessity for anesthesia and oral graft harvesting restricts their application in primary healthcare settings.
The single layer of flattened epithelium of the tunica vaginalis can serve as a potential alternative to oral mucosa. Animal experiments have demonstrated that the tunica vaginalis can readily form a tight connection with the multilayered urothelium of the urethra. Utilizing the tunica vaginalis as a scaffold for urethral re-epithelialization may help reduce the risk of recurrence of urethral stricture after surgery. Over a 19 year period, pedicled tunica vaginalis urethroplasty has been used for successfully treating 86 cases. 
The surgical procedure involves dorsally incising the urethral stricture segment, then covering it with a pedicled tunica vaginalis patch followed by suturing. Postoperatively, the pedicled tunica vaginalis graft exhibits good vascularization and take rate, facilitating urethral re-epithelialization. The surgical procedure is conducted in a sterile environment to mitigate the potential for infectious complications. Moreover, the operation can be executed under spinal anesthesia, which facilitates its implementation in primary healthcare settings.

This article introduces a surgical method using a pedicled tunica vaginalis to treat long-segment urethral stricture resulting from lichen sclerosus. The tunica vaginalis is an excellent substitute for the urethra, leading to quick postoperative recovery for patients.

Urethroplasty for the management of long-segment urethral strictures associated with lichen sclerosus presents considerable clinical challenges. Oral ...

An Immature Murine Model of Reversible Unilateral Ureteral Obstruction

Unilateral ureteral obstruction (UUO) is a common cause of chronic kidney disease (CKD), leading to the progression of renal interstitial fibrosis and ultimately resulting in irreversible kidney damage. The alleviation of UUO is crucial. Several animal models of reversible unilateral ureteral obstruction (RUUO) have been established in the literature, enabling the observation of structural changes and functional damage while also simulating physiological and pathophysiological changes following the relief of ureteral obstruction. In this study, a reversible obstruction model was established in the unilateral murine ureter using a silicone tube. Significant renal damage was observed prior to obstruction relief, with partial recovery noted afterward. Unlike UUO, this model prevents progressive hydronephrosis, leading to distinct pathological outcomes. This simple surgical procedure demonstrates a high success rate and holds promise as a classical model for investigating reversible obstructive nephropathy and potential treatments for renal interstitial fibrosis. Furthermore, it provides a practical platform for studying the mechanisms of recovery from obstructive nephropathy, renal cell regeneration, and tissue remodeling.

The present protocol describes a step-by-step, reproducible model of unilateral ureteral obstruction.

Unilateral ureteral obstruction (UUO) is a common cause of chronic kidney disease (CKD), leading to the progression of renal interstitial fibrosis and ...

Unilateral Ureteral Obstruction Murine Model: A Surgical Procedure to Obstruct the Urinary Flow from a Kidney in Mouse Models

Source: Yanai, K. et al. <a href="https://www.jove.com/video/61383" target="_blank">Quantitative real-time PCR evaluation of microRNA expressions in mouse kidney with unilateral ureteral obstruction.</a> J. Vis. Exp. (2020)
This video demonstrates the procedure to generate a unilateral ureteral obstruction mouse model. The procedure involves cutting one of the ureters of the mouse to obstruct urine flow.

Source: Yanai, K. et al. Quantitative real-time PCR evaluation of microRNA expressions in mouse kidney with unilateral ureteral obstruction. J. Vis. Exp. ...