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 publ....

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 in....

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

List of materials used in this article

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