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

Summary

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.

Abstract

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.

Introduction

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 mouse^8,9, arguably the most commonly used animal model however are female rats undergoing partial obstruction of the proximal urethra; access to the animals' 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 procedure^10,11; 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.

Protocol

The following experimental protocol was approved by the institution's animal care committee.

1. NeMO in Female Rats

1. Preparation
   1. 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.
   2. 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.
2. Dissection
   1. 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.
   2. 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.
   3. 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's knot.
3. Partial urethral obstruction
   1. 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.
   2. 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.
4. Wound closure
   1. Approximate the paraurethral glands using a buried single stitch of absorbable braided suture 4-0.
   2. Close the skin with a buried horizontal mattress stitch using the same absorbable suture. Note: 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.  
5. After care
   1. Inject buprenorphine 0.1 mg/kg subcutaneously before placing the rat in a recovery cage.
   2. 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.
   3. Check the rats daily for bladder size and general well-being; record their weight at least weekly.

2. NeMO in Female Mice

1. Preparation
   1. Place a female C57bl/6 mouse weighing around 18 g and proceed as detailed in step 1.1.1.
   2. Proceed as detailed in step 1.1.2, except use a 24-gauge angiocatheter.
2. Dissection
   1. Use a scalpel to perform a 6 mm longitudinal skin incision from the palpable pubic symphysis towards the urethral meatus.
   2. 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.
   3. 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's knot.
3. Partial urethral obstruction
   1. Connect the angiocatheter with a 1 mL syringe and inject 0.1 mL of normal saline into the bladder.
   2. Place a 26-gauge cannula parallel to the urethra. Gently tighten the knot around urethra and cannula while pulling back the angiocath.
   3. Secure the knot with 3 more throws using gentle tension before removing the cannula.
   4. Test for urine appearing at the meatus upon gentle pressure on the animal's bladder; loosen the tie if unable to express urine. Otherwise, cut the suture ends about 3 mm long.
4. Wound closure
   1. Close the skin with a buried horizontal mattress stitch using a 5-0 braided absorbable suture.
5. After care
   1. Inject 0.1 mg/kg buprenorphine subcutaneously before placing the mouse in a recovery cage.
   2. 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.
   3. Check the mice daily for bladder size and general well-being; record their weight at least weekly.

Results

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...

Discussion

Animal mortality is one of the major concerns of the traditional model of pBOO at the proximal urethra, at 15% or more in many reports^10,11,13. 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...

Materials

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