The authors do not have any acknowledgements.

All the procedures with animals, including the method and duration of anesthesia, as well as postoperative care, were discussed with a veterinarian and approved by the Institutional Animal Care and Use Committee (IACUC) at the affiliated institution. In this manuscript, all procedures were performed with 8 to 15-week-old C57BL/6 male mice and the protocol follows the animal care guidelines approved by the IACUC at the University of Colorado.
1. Preparation of Tubing
N...

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J., Bushman, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+bacterial+infection+and+inflammation+incite+reactive+hyperplasia+in+a+mouse+model+of+chronic+prostatitis.">Chronic bacterial infection and inflammation incite reactive hyperplasia in a mouse model of chronic prostatitis.</a> Prostate. 67 (1), 14-21 (2007).</li><li>Boehm, B. J., Colopy, S. A., Jerde, T. J., Loftus, C. J., Bushman, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+bacterial+inflammation+of+the+mouse+prostate.">Acute bacterial inflammation of the mouse prostate.</a> Prostate. 72 (3), 307-317 (2012).</li><li>Bjorling, D. E., Wang, Z. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+and+host+responses+to+Escherichia+coli+urinary+tract+infection+in+genetically+distinct+mouse+strains.">Time course and host responses to Escherichia coli urinary tract infection in genetically distinct mouse strains.</a> Infect Immun. 66 (6), 2798-2802 (1998).</li><li>Dinis, P., Charrua, A., Avelino, A., Cruz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravesical+resiniferatoxin+decreases+spinal+c-fos+expression+and+increases+bladder+volume+to+reflex+micturition+in+rats+with+chronic+inflamed+urinary+bladders.">Intravesical resiniferatoxin decreases spinal c-fos expression and increases bladder volume to reflex micturition in rats with chronic inflamed urinary bladders.</a> BJU Int. 94 (1), 153-157 (2004).</li><li>Cayan, 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=Botulinum+toxin+type+A+may+improve+bladder+function+in+a+rat+chemical+cystitis+model.">Botulinum toxin type A may improve bladder function in a rat chemical cystitis model.</a> Urol Res. 30 (6), 399-404 (2003).</li><li>Jerde, T. J., Bjorling, D. E., Steinberg, H., Warner, T., Saban, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+mouse+bladder+inflammatory+response+to+E.+coli+lipopolysaccharide.">Determination of mouse bladder inflammatory response to E. coli lipopolysaccharide.</a> Urol Res. 28 (4), 269-273 (2000).</li><li>Saban, M. R., 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=LPS-sensory+peptide+communication+in+experimental+cystitis.">LPS-sensory peptide communication in experimental cystitis.</a> Am J Physiol Renal Physiol. 282 (2), F202-F210 (2002).</li><li>Schilling, J. D., Mulvey, M. A., Vincent, C. D., Lorenz, R. G., Hultgren, S. 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The authors have nothing to disclose.

This manuscript describes in detail a method of transurethral instillation in adult male mouse. Differential primary seeding area can be reached by using PE tubing of different diameters. PE-50 is recommended for successfully reaching the urinary bladder lumen, while a larger diameter PE-10 is used to reach the AP and SV (Figure 3A-B). In addition to the choice of size of PE tubing and target organ for inoculation, other factors, such as the volume of instilled inoculum, als...

The transurethral instillation approach has been used in previous studies as one of the methods to create rodent models of bladder pathologies1,2,3,4 and can be used to evaluate the effectiveness of locally delivered treatments in mice. Although animal models cannot fully recapitulate human pathologies, identification of the underlying mechanisms in animal studies provides a better understanding of the human LUT disorders such as interstitial cystitis/bladder pain syndrome, neurogenic cystitis, autoimmune cystitis, and prostatic inflammation5.
The transurethral instillation procedure performed on an adult male mouse is more technically challenging than intravesical instillation in an adult female mouse6. The naturally curved anatomy of the male urethra along with its small diameter make it technically difficult to accomplish the transurethral insertion of a catheter. Therefore, detailed instructions for the transurethral induction of mouse urinary tract infection6 and LUT inflammation7,8,9,10,11,12,13,14,15 via bladder-inserted catheter were previously outlined for female mice only. This manuscript aims to provide a step-by-step description of the technique for transurethral instillation of substances in male mice including the video clips, images, and illustrations. Transurethral instillation procedures in an adult male mouse can be performed with variable inoculates similar to that previously described in the female mouse7 and merged with additional techniques such as electromyogram recordings of visceromotor responses (VMR)16.

<table>
	<tbody>
		<tr>
			<td>BD INTRAMEDIC Polyethylene Tubing/ PE 10</td>
			<td>BD Medical</td>
			<td>63019-004</td>
			<td></td>
		</tr>
		<tr>
			<td>BD INTRAMEDIC Polyethylene Tubing/ PE 50</td>
			<td>BD Medical</td>
			<td>63019-048</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Insyte-N Autoguard/ Angiocath</td>
			<td>BD Medical</td>
			<td>311411</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (EtOH)</td>
			<td>Fisher Scientific</td>
			<td>BP2818500</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Needle Only 22 Gauge (G) 1 inch</td>
			<td>Becton Dickson</td>
			<td>205155</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Needle Only 27 Gauge (G) 1/2 inch</td>
			<td>Becton Dickson</td>
			<td>305129</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer stub, 22 G X 0.5 in / Blunt Needle</td>
			<td>Instech</td>
			<td>LS22</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer stub, 27 G X 0.5 in / Blunt Needle</td>
			<td>Instech</td>
			<td>LS27</td>
			<td></td>
		</tr>
		<tr>
			<td>6&#34; Long x 2&#34; Wide x 1&#34; Thick, Aluminum Oxide Sharpening Stone / Oil Stone</td>
			<td>Norton</td>
			<td>40204034</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Lubricant Sterile Bacteriostatic Folilpac</td>
			<td>Surgilube</td>
			<td>281020543</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Fluriso</td>
			<td>13985-528-60</td>
			<td></td>
		</tr>
		<tr>
			<td>SomnoSuite Low-Flow Anesthesia System</td>
			<td>Kent Scientific</td>
			<td>SS-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Forceps, Curved</td>
			<td>vwr</td>
			<td>82027-392</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 1 ml Syringe</td>
			<td>BD Medical</td>
			<td>309626</td>
			<td></td>
		</tr>
		<tr>
			<td>India Ink Solution., 0.2% in PBS buffer</td>
			<td>Alfa Aesar</td>
			<td>J61007</td>
			<td></td>
		</tr>
		<tr>
			<td>8~12 weeks old male C57B/6 Mice</td>
			<td>Jackson Lab</td>
			<td>#000664</td>
			<td></td>
		</tr>
	</tbody>
</table>

transurethral instillation procedure in adult male mouse

Transurethral instillation can be used to deliver different solutions with active ingredients (e.g., drugs, chemicals, bacteria, and viruses) locally into the urinary bladder to either induce animal models of bladder pathologies or evaluate the effectiveness of intravesical treatments. Most rodent models of lower urinary tract (LUT) pathologies are induced in female mice due to ease of intravesical instillation of the substances via the female urethra. However, due to anatomical differences between the female and male LUT, transurethral instillation in a male mouse has been deemed a very challenging procedure, and it has not been previously described. In this manuscript, we provide a detailed description of how to prepare polyethylene (PE) tubing for subsequent insertion into the urethra of a male mouse. In addition, we discuss the ideal types of PE tubing to be used depending on the desired site of inoculation. Furthermore, we describe point by point how to prepare an animal for a successful transurethral instillation to avoid injury to the urethra and ensure the delivery of the solution to the desired location. The procedure is started by retracting the prepuce and the glans to expose the opening of the urethral meatus. Next, the glans are grasped by blunt non-crushing forceps to stabilize the penis and the PE tubing. The PE tubing is first inserted into the urethral meatus parallel to the animal body, then its angle is adjusted by tilting the catheter to maneuver it to follow the natural curvature of the urethra. This technique can be used to induced murine models of bladder pathologies and/or evaluate the effectiveness of intravesical treatments in male mice.

Transurethral instillation of blue dye via PE 50 tubing resulted in instillation of the dye into the urinary bladder (Figure 4A), and via PE 10 tubing in colorization of the SV and AP (Figure 4B), respectively. To evaluate whether the performed transurethral instillation causes an instant reflux of the inoculum to the kidneys or not, the kidney and ureter were observed followed by instillation of 100 &#181;L of the blue dye and f...

Watch this Scientific Journal Video about Transurethral Instillation Procedure in Adult Male Mouse at JoVE.com

Transurethral Instillation Procedure in Adult Male Mouse

Transurethral instillation is a challenging procedure and has not been well described in the literature. The aim of this manuscript is to describe a technique for transurethral insertion of a catheter for intravesical delivery of liquids with active substances into the urinary bladder and/or prostate in adult male mice.

Transurethral instillation can be used to deliver different solutions with active ingredients (e.g., drugs, chemicals, bacteria, and viruses) locally into ...

The overall goal of this procedure is to use transurethral catheter insertion for the intravesical delivery of active substances into the urinary bladder and/or prostate of adult male mice. This method can help answer key questions in the field of bladder pathology about how to study the underlying mechanisms involved in lower urinary tract disease in males. The main advantages of this technique are that it can be used to induce animal models of bladder pathology and to evaluate the effectiveness of intravesicular treatment.Transurethra instillation in male is challenging due to the natural curvature of the urethra, the thinness of the urethral wall, and the need for repetitive active substance instillation. Before beginning the procedure, select the PE tubing size based on the target organ and the age of the animal, and cut an approximately 2.5-inch long piece from the tube. Dull the sharp edge of a compatibly sized needle on a commercially available oil stone, and carefully introduce the needle into the lumen of one end of the tubing.Then, immerse the needle and tubing in 70%ethanol, flushing both pieces with ethanol to confirm the absence of leakage. Next, using gentle pressure, massage the lower abdomen of the mouse to induce voiding of the bladder. Then, place the anesthetized animal on a warm heating blanket.After placing the nose cone, confirm a lack of response to toe pinch, and retract the prepuce. Using the thumb and index finger of one hand, apply pressure to the pubic region and pull out the glans penis with the thumb and index finger of the other hand. Remove the tubing and catheter from the ethanol.Rinse the tubing and needle in sterile saline and load the needle onto a 1-mL syringe containing the inoculum of interest. Lubricate the tip of the catheter and the glans with sterile surgical lubricant. Gently grasp the tip of the penis with dull forceps and apply gentle pressure to the glans to open the urethral meatus.Using a tripod finger grip, carefully insert the PE tubing into the meatus, adjusting the angle of the needle to follow the curvature of the urethra as necessary. When the tubing has reached the appropriate organ, depress the plunger to instill the full experimental volume of the inoculum. In these representative images, PE-52 tubing was used to facilitate the transurethral instillation of a blue dye into the mouse urinary bladder, and PE-10 tubing was used to colorize the seminal vesicles and the anterior prostate of a second animal.However, histological evaluation of the ureters or kidneys is highly recommended to confirm the absence of indirect contamination of the ureters or kidneys from transurethral instillations. Once mastered, this technique can be performed within 10 to 15 minutes if it is performed properly. After watching this video, you should have a good understanding of how to perform transurethra catheterization in an adult male mouse.

transurethral-instillation-procedure-in-adult-male-mouse

Research

JoVE Journal

Immunology and Infection

15.1K vistas.  University of Colorado School of Medicine. The overall goal of this procedure is to use transurethral catheter insertion for the intravesical delivery of active substances into the urinary bladder and/or prostate of adult male mice. This method can help answer key questions in the field of bladder pathology about how to study the underlying mechanisms involved in lower urinary tract disease in males. The main advantages of this technique are that it can be used to induce animal models of bladder pathology and to evaluate the effective...