Name: nerve-sparing mid-urethral obstruction (nemo) in female small rodents
Uploaded: 2017-04-25T13:00:00
Description: Watch this Scientific Journal Video about Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents at JoVE.com

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 gentl...

<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>

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.

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	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Female rat&#160;</td>
			<td style="width:123px;">Charles-River Canada</td>
			<td style="width:559px;">Sprague Dawley Rat (Crl:SD); Strain Code: 400</td>
			<td style="width:167px;">about 200g</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Isoflorane vaporizer</td>
			<td style="width:123px;">Benson medical industries inc.</td>
			<td style="width:559px;">TEC III Isoflurane; product code: 53-T3ISO&#160;</td>
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			<td height="40" style="height:40px;width:216px;">Isoflorane</td>
			<td style="width:123px;">Fresenius Kabi Animal Health</td>
			<td style="width:559px;">Isoflorane; product code CP0406V2</td>
			<td style="width:167px;">&#160;250ml bottle</td>
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			<td height="40" style="height:40px;width:216px;">Disposable scalpell</td>
			<td style="width:123px;">Ted Pella, Inc.</td>
			<td style="width:559px;">#15 Sterile, Stainless Steel Scalpels; catalogue number 549-9-15S</td>
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			<td style="width:559px;">Aesculap Adson Dissection Forceps, Curved, 120mm; product number 5002-9</td>
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			<td height="40" style="height:40px;width:216px;">Curved surgical scissors</td>
			<td style="width:123px;">Ted Pella, Inc.</td>
			<td style="width:559px;">Surgical Scissors, Curved, S/S, 105mm; catalogue number 1376</td>
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			<td height="40" style="height:40px;width:216px;">0.9mm inox metal rod</td>
			<td style="width:123px;">goodfellow.com</td>
			<td style="width:559px;">Stainless Steel - AISI 316L Wire Diameter:0.9mm; order code 911-684-65</td>
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			<td height="40" style="height:40px;width:216px;">Skin hooks - self made from inox steel</td>
			<td style="width:123px;">goodfellow.com</td>
			<td style="width:559px;">Stainless Steel - AISI 316L Wire Diameter:0.9mm; order code 911-684-65</td>
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			<td height="40" style="height:40px;width:216px;">4-0 Silk suture</td>
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			<td style="width:559px;">4-0 Sofsilk black 98&#34; strand; product number LS640</td>
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			<td height="40" style="height:40px;width:216px;">Needle holder</td>
			<td style="width:123px;">Medsupplier.com</td>
			<td style="width:559px;">Integra Miltex Carb-N-Sert Baumgartner Needle Holder - 5 1/2&#34;; product code SKU NEE4484-8-40TC 
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			SKU NEE4484-8-40TC&#160;</td>
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			<td height="40" style="height:40px;width:216px;">Temgesic (Buprenorphine)</td>
			<td style="width:123px;">first veterinary supply</td>
			<td style="width:559px;">Buprenex 0.3MG/ml inj amps; item #075701</td>
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			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:559px;">BD&#160;Tuberculin Syringe, BD 309623</td>
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			<td height="40" style="height:40px;width:216px;">20G Angiocath</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:559px;">Andwin Scientific&#160;ANGIOCATH 20 GA; Catalog No. NC9561906</td>
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			<td height="40" style="height:40px;width:216px;">Recovery diet</td>
			<td style="width:123px;">clearh2o</td>
			<td>DietGel 31M; 72-08-5022</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">C57Bl/6 female mice</td>
			<td style="width:123px;">Charles-River Canada</td>
			<td style="width:559px;">C57BL/6 Mouse (C57BL/6NCrI); Strain Code: 027</td>
			<td style="width:167px;">about 18g</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">24G angiocath</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:559px;">Hanna Pharmaceutical Supply Co., Inc.&#160;IV CATH ANGIOCATH 24GX3/4IN; Catalog No. NC9814340</td>
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			<td style="width:123px;">esutures.com</td>
			<td style="width:559px;">5-0 Ethibond green 30&#34; RB-1 taper; product number X550H</td>
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			<td height="42" style="height:42px;width:216px;">26G syringe needle</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:559px;">BD Precisionglide syringe needle, Gauge 26; z192392</td>
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			<td height="42" style="height:42px;width:216px;">Normal Saline</td>
			<td style="width:123px;">Baxter</td>
			<td style="width:559px;">0.9% Sodium Chloride Injection, USP, 100 mL MINI-BAG; product code 2B0043</td>
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</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 (Video) | JoVE

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 ...

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