The work was funded by a CHIR operating grant (DJB).

The following experimental protocol was approved by the institution&#39;s animal care committee.
1. NeMO in Female Rats
<ol>
	<li>Preparation
	<ol>
		<li>Place a female Sprague-Dawley rat weighing around 200 g under 3% isoflurane anesthesia, weigh the animal, and shave and disinfect the prepubic area 3 times using an iodine surgical scrub.</li>
		<li>On a heating pad, tape hind limbs and tail down and catheterize the bladder with a 20-gauge angiocatheter, which is also gently taped down to prevent sliding.</li>
	</ol>
	</li>
	<li>Dissection
	<ol>
		<li>Use a scalpel to perform an 8 mm longitudinal skin incision from the palpable pubic symphysis towards the urethral meatus. Lift off the skin on either side to facilitate later wound closure.</li>
		<li>Dissect bluntly to identify the stented urethra by longitudinal spreading with fine scissors. Place fine self-retaining skin hooks to enhance exposure of the urethra.</li>
		<li>Perform some gentle longitudinal spreading using curved scissors dorsal to the urethra to develop the plane between urethra and vagina. Then, bluntly pass a 4-0 silk suture behind the urethra and prepare the double throw of a surgeon&#39;s knot.</li>
	</ol>
	</li>
	<li>Partial urethral obstruction
	<ol>
		<li>Remove the angiocatheter, place a 0.9 mm metal rod parallel to the urethra, and then tighten the knot around urethra and metal rod, so that the latter still slides out easily thereafter.</li>
		<li>Secure the knot with 3 more throws using gentle tension before removing the rod. Cut the suture ends about 3 mm long, or 4 mm if later release of obstruction is part of the experiment.</li>
	</ol>
	</li>
	<li>Wound closure
	<ol>
		<li>Approximate the paraurethral glands using a buried single stitch of absorbable braided suture 4-0.</li>
		<li>Close the skin with a buried horizontal mattress stitch using the same absorbable suture. Note:&#160;While it may seem unconventional to use an absorbable suture for a skin-penetrating stitch, we did not encounter complications such as wound infections, and that suture removal can be avoided. &#160;</li>
	</ol>
	</li>
	<li>After care
	<ol>
		<li>Inject buprenorphine 0.1 mg/kg subcutaneously before placing the rat in a recovery cage.</li>
		<li>Offer some soft recovery diet for the first 24 h to the single housed rat. After this period, house animals again in the same pairs as before the procedure provided they are doing well and belong to the same treatment group.</li>
		<li>Check the rats daily for bladder size and general well-being; record their weight at least weekly.</li>
	</ol>
	</li>
</ol>
2. NeMO in Female Mice
<ol>
	<li>Preparation
	<ol>
		<li>Place a female C57bl/6 mouse weighing around 18 g and proceed as detailed in step 1.1.1.</li>
		<li>Proceed as detailed in step 1.1.2, except use a 24-gauge angiocatheter.</li>
	</ol>
	</li>
	<li>Dissection
	<ol>
		<li>Use a scalpel to perform a 6 mm longitudinal skin incision from the palpable pubic symphysis towards the urethral meatus.</li>
		<li>Dissect bluntly to identify the stented urethra by longitudinal spreading with fine scissors. Lift off the neuro-vascular bundle running ventrally on the urethra by analogous blunt dissection.</li>
		<li>Perform some gentle longitudinal spreading using curved scissors dorsal to the urethra to develop the plane between urethra and vagina. Then, bluntly pass a 5-0 non-absorbable braided suture behind the urethra and prepare the double throw of a surgeon&#39;s knot.</li>
	</ol>
	</li>
	<li>Partial urethral obstruction
	<ol>
		<li>Connect the angiocatheter with a 1 mL syringe and inject 0.1 mL of normal saline into the bladder.</li>
		<li>Place a 26-gauge cannula parallel to the urethra. Gently tighten the knot around urethra and cannula while pulling back the angiocath.</li>
		<li>Secure the knot with 3 more throws using gentle tension before removing the cannula.</li>
		<li>Test for urine appearing at the meatus upon gentle pressure on the animal&#39;s bladder; loosen the tie if unable to express urine. Otherwise, cut the suture ends about 3 mm long.</li>
	</ol>
	</li>
	<li>Wound closure
	<ol>
		<li>Close the skin with a buried horizontal mattress stitch using a 5-0 braided absorbable suture.</li>
	</ol>
	</li>
	<li>After care
	<ol>
		<li>Inject 0.1 mg/kg buprenorphine subcutaneously before placing the mouse in a recovery cage.</li>
		<li>Offer some soft recovery diet for the first 48 h to the single housed mouse. After this period, house animals again in the same groups as before the procedure provided they are doing well and belong to the same treatment group. 
		NOTE: Mice with a palpable distended bladder 24 h after the procedure that do not void when handled are over-obstructed and will die within the next 1 - 2 days unless taken back under anesthesia for removal of the obstructing ligature.</li>
		<li>Check the mice daily for bladder size and general well-being; record their weight at least weekly.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Andersson, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Storage+and+voiding+symptoms:+pathophysiologic+aspects.">Storage and voiding symptoms: pathophysiologic aspects.</a> Urology. 62 (5), 3-10 (2003).</li><li>Boyle, P., 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=The+prevalence+of+lower+urinary+tract+symptoms+in+men+and+women+in+four+centres.+The+UrEpik+study.">The prevalence of lower urinary tract symptoms in men and women in four centres. The UrEpik study.</a> BJU Int. 92 (4), 409-414 (2003).</li><li>Coyne, K. S., Wein, A., Nicholson, S., Kvasz, M., Chen, C. I., Milsom, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Economic+burden+of+urgency+urinary+incontinence+in+the+United+States:+a+systematic+review.">Economic burden of urgency urinary incontinence in the United States: a systematic review.</a> J Manag Care Pharm. 20 (2), 130-140 (2014).</li><li>Buttyan, R., Chen, M. W., Levin, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+bladder+outlet+obstruction+and+molecular+insights+into+the+basis+for+the+development+of+bladder+dysfunction.">Animal models of bladder outlet obstruction and molecular insights into the basis for the development of bladder dysfunction.</a> Eur. Urol. 32 (Suppl 1), 32-39 (1997).</li><li>Mattiasson, A., Uvelius, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+contractile+properties+in+hypertrophic+rat+urinary+bladder.">Changes in contractile properties in hypertrophic rat urinary bladder.</a> J Urol. 128 (6), 1340-1342 (1982).</li><li>Chang, S., 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=Alteration+of+the+PKC-mediated+signaling+pathway+for+smooth+muscle+contraction+in+obstruction-induced+hypertrophy+of+the+urinary+bladder.">Alteration of the PKC-mediated signaling pathway for smooth muscle contraction in obstruction-induced hypertrophy of the urinary bladder.</a> Lab Invest. 89 (7), 823-832 (2009).</li><li>Moore, J. A., Brading, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+porcine+model+of+bladder+outlet+obstruction+incorporating+radio-telemetered+cystometry.">A porcine model of bladder outlet obstruction incorporating radio-telemetered cystometry.</a> BJU Int. 100 (5), 1192-1193 (2007).</li><li>Beamon, C. R., Mazar, C., Salkini, M. W., Phull, H. S., Comiter, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+sildenafil+citrate+on+bladder+outlet+obstruction:+a+mouse+model.">The effect of sildenafil citrate on bladder outlet obstruction: a mouse model.</a> BJU Inte. 104 (2), 252-256 (2009).</li><li>Pandita, R. K., Fujiwara, M., Alm, P., Andersson, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystometric+evaluation+of+bladder+function+in+non-anesthetized+mice+with+and+without+bladder+outlet+obstruction.">Cystometric evaluation of bladder function in non-anesthetized mice with and without bladder outlet obstruction.</a> J Urol. 164 (4), 1385-1389 (2000).</li><li>Kang, Y. J., 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=Early+Sequential+Changes+in+Bladder+Function+after+Partial+Bladder+Outlet+Obstruction+in+Awake+Sprague-Dawley+Rats:+Focus+on+the+Decompensated+Bladder.">Early Sequential Changes in Bladder Function after Partial Bladder Outlet Obstruction in Awake Sprague-Dawley Rats: Focus on the Decompensated Bladder.</a> Korean J Urol. 52 (12), 835-837 (2011).</li><li>Schroeder, A., 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+an+oral+endothelin+converting+enzyme+inhibitor+on+rat+detrusor+function+after+outlet+obstruction.">Protective effect of an oral endothelin converting enzyme inhibitor on rat detrusor function after outlet obstruction.</a> J Urol. 172 (3), 1171-1174 (2004).</li><li>Sidler, M., Aitken, K., Jiang, J., Bijos, D., B&#228;gli, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="sometimes+a+sham+is+just+a+sham"+-+why+the+rodent+model+of+partial+bladder+outlet+obstruction+needs+adjustment.">"sometimes a sham is just a sham" - why the rodent model of partial bladder outlet obstruction needs adjustment.</a> J Urol. 195 (4), e379 (2016).</li><li>Jin, L. 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=Persistent+detrusor+overactivity+in+rats+after+relief+of+partial+urethral+obstruction.">Persistent detrusor overactivity in rats after relief of partial urethral obstruction.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 301 (4), R896-R904 (2011).</li></ol>

The authors have nothing to disclose.

Animal mortality is one of the major concerns of the traditional model of pBOO at the proximal urethra, at 15% or more in many reports10,11,13. NeMO appears to have minimal mortality when applied in female rats. Proximal urethral obstruction in mice is technically more challenging than in rats and thus even more prone to complications. Using a 26-gauge cannula as a placeholder for our mid-urethral approach, we h...

Partial bladder outlet obstruction (pBOO) has a high prevalence and can result in severe bladder dysfunction1; the spectrum ranges from congenital malformations such as posterior urethral valves or hypospadias, over acquired urethral strictures to benign prostatic hyperplasia. The latter affecting more than 30% of men sixty years and older2. Great patient burden and immense health care costs associated with pBOO warrant the considerable research effort put into studying bladder remodeling in response to increased outflow resistance3. From 2006 until 2015, over 220 PubMed indexed articles were published concerning the effect of pBOO on the urinary bladder.
Although animal models for pBOO have been devised in several species4, such as the rat5, the rabbit6, pig7, and mouse8,9, arguably the most commonly used animal model however are female rats undergoing partial obstruction of the proximal urethra; access to the animals&#39; abdomen, exteriorizing of the bladder, and dissection around the bladder neck are inevitable with this traditional proximal urethral obstruction technique. Variability in the degree of obstruction and animal mortality are only some of the concerns associated with this procedure10,11; we showed that in sham operated animals, dissection around the proximal urethra leads to physiologic changes that correlate with loss of nerve fibers at the bladder neck12. This finding indicates, that the most commonly used pBOO animal model, which involves accessing the proximal urethra in female rodents, leads to a denervation injury with associated structural and functional changes of the bladder, affecting sham and obstructed animals. Therefore, an alternate approach avoiding denervation injury was needed. Our lab developed and evaluated a Nerve-sparing Mid-urethral Obstruction (NeMO) approach, effective in inducing expected obstruction-associated changes in the bladder such as increase in organ mass and residual urine, whilst sham-operated animals were indistinguishable from unoperated control animals. Also, the striated urethral sphincter remained untouched as it lies proximal to the level of dissection. Furthermore, variability in obstruction-induced increase in bladder mass was significantly lower than in traditional proximal urethral obstruction and animal mortality was zero.
We also successfully applied NeMO in female mice with a less than 10% mortality in obstructed animals, while all pBOO models for mice described to date were associated with a mortality around 50%. Studying bladder remodeling in the context of pBOO in mice will benefit from applicability of the whole spectrum of transgenic modifications.
Dissecting around the mid-urethra in female rodents does not induce the undesirable and confounding structural or functional changes in the urinary bladder observed in the traditional proximal obstruction model. Nevertheless, inducing a partial obstruction at the mid-urethral level still induces bladder hypertrophy and increased residual urine, as expected from an animal model for pBOO. Importantly, performing NeMO in mice opens investigation of bladder remodeling in pBOO to transgenic methods, which are virtually unavailable in larger rodents.

<table><tbody><tr><td>Female rat&amp;nbsp;</td><td>Charles-River Canada</td><td>Sprague Dawley Rat (Crl:SD); Strain Code: 400</td><td>about 200 g</td></tr><tr><td>Isoflorane vaporizer</td><td>Benson medical industries inc.</td><td>TEC III Isoflurane; product code: 53-T3ISO&amp;nbsp;</td><td></td></tr><tr><td>Isoflorane</td><td>Fresenius Kabi Animal Health</td><td>Isoflorane; product code CP0406V2</td><td>&amp;nbsp;250 mL bottle</td></tr><tr><td>Disposable scalpel</td><td>Ted Pella, Inc.</td><td>#15 Sterile, Stainless Steel Scalpels; catalogue number 549-9-15S</td><td></td></tr><tr><td>Microscopy forceps</td><td>Ted Pella, Inc.</td><td>Aesculap Microscopy Forceps, Finepoint, 115mm; product number 5443</td><td></td></tr><tr><td>Adson forceps</td><td>Ted Pella, Inc.</td><td>Aesculap Adson Dissection Forceps, Curved, 120mm; product number 5002-9</td><td></td></tr><tr><td>Curved surgical scissors</td><td>Ted Pella, Inc.</td><td>Surgical Scissors, Curved, S/S, 105mm; catalogue number 1376</td><td></td></tr><tr><td>0.9 mm inox metal rod</td><td>goodfellow.com</td><td>Stainless Steel - AISI 316L Wire Diameter:0.9mm; order code 911-684-65</td><td></td></tr><tr><td>Skin hooks - self made from inox steel</td><td>goodfellow.com</td><td>Stainless Steel - AISI 316L Wire Diameter:0.9mm; order code 911-684-65</td><td></td></tr><tr><td>4-0 Silk suture</td><td>esutures.com</td><td>4-0 Sofsilk black 98&quot; strand; product number LS640</td><td></td></tr><tr><td>5-0 Vicry suture</td><td>esutures.com</td><td>5-0 Vicryl undyed 27&quot; RB-1 taper; product number J213H</td><td></td></tr><tr><td>Needle holder</td><td>Medsupplier.com</td><td>Integra Miltex Carb-N-Sert Baumgartner Needle Holder - 5 1/2&quot;; product code SKU NEE4484-8-40TC&lt;br/&gt; SKU NEE4484-8-40TC&lt;br/&gt; SKU NEE4484-8-40TC&amp;nbsp;</td><td></td></tr><tr><td>Temgesic (Buprenorphine)</td><td>first veterinary supply</td><td>Buprenex 0.3MG/ml inj amps; item #075701</td><td></td></tr><tr><td>1 mL syringe with 27 G needle</td><td>Fisher Scientific</td><td>BD&amp;nbsp;Tuberculin Syringe, BD 309623</td><td></td></tr><tr><td>20 G Angiocath</td><td>Fisher Scientific</td><td>Andwin Scientific&amp;nbsp;ANGIOCATH 20 GA; Catalog No. NC9561906</td><td></td></tr><tr><td>Recovery diet</td><td>clearh2o</td><td>DietGel 31M; 72-08-5022</td><td></td></tr><tr><td>C57Bl/6 female mice</td><td>Charles-River Canada</td><td>C57BL/6 Mouse (C57BL/6NCrI); Strain Code: 027</td><td>about 18 g</td></tr><tr><td>24 G angiocath</td><td>Fisher Scientific</td><td>Hanna Pharmaceutical Supply Co., Inc.&amp;nbsp;IV CATH ANGIOCATH 24GX3/4IN; Catalog No. NC9814340</td><td></td></tr><tr><td>5-0 Ethibond</td><td>esutures.com</td><td>5-0 Ethibond green 30&quot; RB-1 taper; product number X550H</td><td></td></tr><tr><td>26 G syringe needle</td><td>Sigma-Aldrich</td><td>BD Precisionglide syringe needle, Gauge 26; z192392</td><td></td></tr><tr><td>Normal Saline</td><td>Baxter</td><td>0.9% Sodium Chloride Injection, USP, 100 mL MINI-BAG; product code 2B0043</td><td></td></tr></tbody></table>

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 Rats
Mortality
We performed a mid-urethral obstruction on over 40 female rats so far and had no mortalities.
Bladder mass
Average relative bladder mass (bladder-t...

Watch this Scientific Journal Video about Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents at JoVE.com

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.

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 ...

nerve-sparing-mid-urethral-obstruction-nemo-in-female-small-rodents

Research

JoVE Journal

Medicine

9.6K Views.  The Hospital for Sick Children. 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.

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

Transurethral Induction of Mouse Urinary Tract Infection

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on Luria-Bertani (LB) agar at 37&deg;C in ambient air. UPEC strains grow as yellowish-white translucent colonies on LB agar. Following confirmation of appropriate colony morphology, single colonies are then picked to be cultured in broth. LB broth can be used for most uropathogenic bacterial strains. Two serial, overnight LB broth cultures can be employed to enhance expression of type I pili, a well-defined virulence factor for uropathogenic bacteria. Broth cultures are diluted to the desired concentration in phosphate buffered saline (PBS). Eight to 12 week old female mice are placed under isoflurane anesthesia and transurethrally inoculated with bacteria using polyethylene tubing-covered 30 gauge syringes. Typical inocula, which must be empirically determined for each bacterial/mouse strain combination, are 106 to 108 cfu per mouse in 10 to 50 microliters of PBS. After the desired infection period (one day to several weeks), urine samples and the bladder and both kidneys are harvested. Each organ is minced, placed in PBS, and homogenized in a Blue Bullet homogenizer. Urine and tissue homogenates are serially diluted in PBS and cultured on appropriate agar. The following day, colony forming units are counted.

This video will demonstrate methods to transurethrally induce mouse urinary tract infections and quantify the extent of resulting infections.

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on ...

Immunology and Infection

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
	After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
	Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice

Urinary tract infections (UTI) are extremely common worldwide, incurring significant morbidity and healthcare-associated expenses. Small animal models, which accurately reflect disease establishment and progression, permit dissection of host-pathogen interactions and generation of immunity to infection. In mice, intravesical instillation of uropathogenic E. coli, the causative agent in more than 85% of community acquired UTI, recapitulates many of the stages of infection observed in humans. Until recently, however, UTI could only be modeled in female animals. This limitation has hindered the study of sex-related differences in UTI, as well as other bladder pathologies, such as cancer. Here, we describe a method to instill male mice that allows direct comparison between female and male animals and provide a detailed protocol to assess bladder tissue by flow cytometry as a means to better understand host responses to infection. Together, these approaches will aid in the identification of host factors that contribute to sex biases observed in UTI and other bladder-associated diseases.

The established model of transurethral catheterization of mice allows the study of bladder pathologies, including urinary tract infection, but can only be performed in females. A new model of male transurethral instillation, presented here, will enable research in an area marked by strong clinical and epidemiological differences between the sexes.

Urinary tract infections (UTI) are extremely common worldwide, incurring significant morbidity and healthcare-associated expenses. Small animal models, ...

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 ...

In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents

ATP, released from the urothelium in response to bladder distension, is thought to play a significant sensory role in the control of micturition. Therefore, accurate measurement of urothelial ATP release in a physiological setting is an important first step in studying the mechanisms that control purinergic signaling in the urinary bladder. Existing techniques to study mechanically evoked urothelial ATP release utilize cultured cells plated on flexible supports or bladder tissue pinned into Ussing chambers; however, each of these techniques does not fully emulate conditions in the intact bladder. Therefore, an experimental setup was developed to directly measure ATP concentrations in the lumen of the rodent urinary bladder.
In this setup, the bladders of anesthetized rodents are perfused through catheters in both the dome of the bladder and via the external urethral orifice. Pressure in the bladder is increased by capping the urethral catheter while perfusing sterile fluid into the bladder through the dome. Measurement of intravesical pressure is achieved using a pressure transducer attached to the bladder dome catheter, akin to the setup used for cystometry. Once the desired pressure is reached, the urethral catheter's cap is removed, and fluid collected for ATP quantification by luciferin-luciferase assay. Through this experimental setup, the mechanisms controlling both mechanical and chemical stimulation of urothelial ATP release can be interrogated by including various agonists or antagonists into the perfusate or by comparing results between wildtype and genetically modified animals.

In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents

This protocol describes the procedure for measuring ATP concentrations in the lumen of the bladder in an anesthetized rodent.

ATP, released from the urothelium in response to bladder distension, is thought to play a significant sensory role in the control of micturition. ...

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 ...

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.

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 ...

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 ...

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.

Mysi model jednostronnej niedrożności moczowodu: zabieg chirurgiczny mający na celu zablokowanie przepływu moczu z nerki w modelach mysich

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

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

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A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction

In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents

Development of a Uterosacral Ligament Suspension Rat Model

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

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

An Immature Murine Model of Reversible Unilateral Ureteral Obstruction

Mysi model jednostronnej niedrożności moczowodu: zabieg chirurgiczny mający na celu zablokowanie przepływu moczu z nerki w modelach mysich