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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#39;&#39;</td>
			<td>B Braun</td>
			<td>Introcan Safety</td>
			<td>IV Catheter of fluorinated ethylene propylene (FEP) with firmer construction for 
			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 &#176;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 &#34;reperfusion ratio&#34; 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&#34; 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&#186;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

824 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

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 months. It is critical to have suitable animal models to mimic the hemodynamic spread of the metastatic cells in the clinical scenario. Here, we are introducing the use of small animal ultrasound imaging to guide an accurate injection of brain tropical breast cancer cells into the left ventricle of athymic nude mice. Longitudinal MRI is used to assessing intracranial initiation and growth of brain metastases. Ultrasound-guided intracardiac injection ensures not only an accurate injection and hereby a higher successful rate but also significantly decreased mortality rate, as compared to our previous manual procedure. In vivo high resolution MRI allows the visualization of hyperintense multifocal lesions, as small as 310 &#181;m in diameter on T2-weighted images at 3 weeks post injection. Follow-up MRI reveals intracranial tumor growth and increased number of metastases that distribute throughout the whole brain.

A breast cancer brain metastasis mouse model is established with ultrasound imaging-guided intracardiac injection of MDA-MB231/Br-GFP cells. Development of multifocal intracranial metastases has been monitored longitudinally using high-resolution 9.4 T MRI.

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans ...

A Rat Model of Ventricular Fibrillation and Resuscitation by Conventional Closed-chest Technique

A rat model of electrically-induced ventricular fibrillation followed by cardiac resuscitation using a closed chest technique that incorporates the basic components of cardiopulmonary resuscitation in humans is herein described. The model was developed in 1988 and has been used in approximately 70 peer-reviewed publications examining a myriad of resuscitation aspects including its physiology and pathophysiology, determinants of resuscitability, pharmacologic interventions, and even the effects of cell therapies. The model featured in this presentation includes: (1) vascular catheterization to measure aortic and right atrial pressures, to measure cardiac output by thermodilution, and to electrically induce ventricular fibrillation; and (2) tracheal intubation for positive pressure ventilation with oxygen enriched gas and assessment of the end-tidal CO2. A typical sequence of intervention entails: (1) electrical induction of ventricular fibrillation, (2) chest compression using a mechanical piston device concomitantly with positive pressure ventilation delivering oxygen-enriched gas, (3) electrical shocks to terminate ventricular fibrillation and reestablish cardiac activity, (4) assessment of post-resuscitation hemodynamic and metabolic function, and (5) assessment of survival and recovery of organ function. A robust inventory of measurements is available that includes &#8211; but is not limited to &#8211; hemodynamic, metabolic, and tissue measurements. The model has been highly effective in developing new resuscitation concepts and examining novel therapeutic interventions before their testing in larger and translationally more relevant animal models of cardiac arrest and resuscitation.

This article describes a rat model of electrically-induced ventricular fibrillation and resuscitation by chest compression, ventilation, and delivery of electrical shocks that simulates an episode of sudden cardiac arrest and conventional cardiopulmonary resuscitation. The model enables gathering insights on the pathophysiology of cardiac arrest and exploration of new resuscitation strategies.

A rat model of electrically-induced ventricular fibrillation followed by cardiac resuscitation using a closed chest technique that incorporates the basic ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

Acupuncture in a Rat Model of Asthma

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes methods for the fabrication of the high platform, establishes a rat model of asthma and measures changes in respiratory function using a noninvasive and real-time whole-body plethysmography (WBP) system.

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes ...

Retinal Pathophysiological Evaluation in a Rat Model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal detachment, breakdown of the blood-retinal barrier (BRB), and retinal angiogenesis. An in vivo rat model is a valuable experimental tool to examine the changes in the structure and function of the retina. We propose three different experimental techniques in the rat model to identify morphological changes of retinal cells, retinal vasculature, and compromised BRB. Retinal histology is used to study the morphology of various retinal cells. Also, quantitative measurement is performed by retinal cell count and thickness measurement of different retinal layers. A BRB breakdown assay is used to determine the leakage of extraocular proteins from the plasma to vitreous tissue due to the breakdown of BRB. Fluorescence angiography is used to study angiogenesis and leakage of blood vessels by visualizing retinal vasculature using FITC-dextran dye.

Diabetic retinopathy is one of the leading causes of blindness. Histology, blood-retinal barrier breakdown assay, and fluorescence angiography are valuable techniques to understand the pathophysiology of the retina, which could further enhance the efficient drug screening against diabetic retinopathy.

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal ...

Establishment and Evaluation of a Porcine Vein Graft Disease Model

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the investigation of disease mechanisms and the development of therapeutic strategies. 
To perform the surgery, we enter the cardiac chamber through the third intercostal space and carefully dissect the internal mammary vein and immerse it in normal saline. The right main coronary artery is then treated for ischemia. The target vessel is incised, a shunt plug is placed, and the distal end of the graft vein is anastomosed. The ascending aorta is partially blocked, and the proximal end of the graft vein is anastomosed after perforation. The graft vein is checked for patency, and the proximal right coronary artery is ligated. 
CABG surgery is performed in minipigs to harvest the left internal mammary vein for its use as a vascular graft. Serum biochemical tests are used to evaluate the physiological status of the animals after surgery. Ultrasound examination shows that the proximal, middle, and distal end of the graft vessel are unobstructed. In the surgical model, turbulent blood flow in the graft is observed upon histological examination after the CABG surgery, and venous graft stenosis associated with intimal hyperplasia is observed in the graft. The study here provides detailed surgical procedures for the establishment of a repeatable CABG-induced VGD model.

In this protocol, novel pig vein bypass grafting was performed through a small incision in the left chest wall without cardiopulmonary bypass. A postoperative pathology study was done, which showed intimal thickening.