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 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.
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.
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
NOTE: To instill the inoculum, a needle attached to the polyethylene (PE) tubing (Figure 1A) or a small gauge (<24 G) angiocatheter can be used (Figure 1G). The use of an angiocatheter requires no preparation other than sterilization with 70% ethanol (EtOH). The inoculum is the substance that is instilled/injected/deposited into the urinary tract. This can be a solution containing bacteria, virus, or a chemical. This is prepared ahead of time and per the research protocol. For example, if the study aims to evaluate the effects of E. coli infection on the lower urinary tract, the researcher will need to prepare the inoculum containing bacteria, which can be inoculated/instilled in the lower urinary tract.
2. Catheterization Procedure
3. In Vivo Imaging of the Lower Urinary Tract and Kidneys
NOTE: The purpose of in vivo imaging in this manuscript is: (1) to confirm the spatial localization of the inoculum; and (2) to assess the incidence of reflux of the inoculum to the kidneys. For this purpose, either an anesthetized or euthanized mouse can be used.
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 µL of the blue dye and fluorescent dye (Figure 4A-B). None of the instillation procedure using two different sizes of PE tubing and two different dyes resulted in colorization of the ureters or kidneys.
Figure 1: 22 G needle preparation for insertion into polyethylene (PE) tubing 50. The sharp needle (A) is grinded against an oil stone (B) to dull the sharp edges (C). Once the needle is dull, the needle is inserted into the lumen of the PE tubing (D) and advanced over the needle with fine forceps (E). The tubing is then cut to approximately 1.5 inches (F). 24 G angiocath (G) can also be used. Please click here to view a larger version of this figure.
Figure 2: Male mouse prepuce covers the glans and urethral meatus (A), with slight traction of prepuce the glans can be exposed (B), and with slight traction of the glans (C), the entire prepuce can be retracted (D), thus allowing for easy access to the urethral meatus and urethra. Please click here to view a larger version of this figure.
Figure 3: Illustrations of lower urinary tract organs; urinary bladder, prostate, and seminal vesicle (SV) showing transurethral instillation using PE 50 tubing (A) and PE 10 tubing (B) for primary seeding of the urinary bladder and SV/anterior prostate, respectively. Please click here to view a larger version of this figure.
Figure 4: Images of upper and lower urinary tract organs; kidneys, urinary bladder, prostate, and seminal vesicle (SV) after transurethral instillation using PE 50 tubing (A) and PE 10 tubing (B). Please click here to view a larger version of this figure.
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, also play a role in the end location and distribution of the solution. Our experiments demonstrate that transurethral instillation of 100 µL of blue dye using either PE-10 or PE-50 does not trigger the instant reflux of the dye to the kidneys. However, the reflux of urine to the kidneys may happen during the post-instillation period and may result in kidney infection, as was previously shown in adult female mice8. Therefore, histological evaluation of the kidneys is highly recommended to confirm the absence of indirect contamination of the kidneys from transurethral instillations.
There are several important key steps to perform a successful transurethral instillation of the inoculum. First, the PE tubing should be of a sufficient length — at least 2.5 inches. This would allow for additional adjustments of the tubing length, if necessary, during the tubing preparation process, or following the transurethral instillation procedure. Multiple attempts and failures during step 9 of this protocol can cause a blockage of the inserted end of the PE tubing by body fluids. In this situation, the tip of the PE tubing should be cut out to have a clear tip for subsequent attempts. Once the tip of the PE tubing is cut, the surgical lubricant should be re-applied. Second, a proper grip of either the syringe or PE tubing itself does increase the success rate. Third, a sufficient length of the PE tubing should be inserted and the prepuce, glans penis, and penile body should be rigidly lined up without hand grip during the process of catheterization. Otherwise, backflow of the inoculum may happen when the syringe plunger is pushed.
Better outcomes for the transurethral instillation procedure can be a result of practice accompanied by avoiding damage to the urethra and target organ during the procedure. Performance outcomes can be validated by using colored dyes or fluorescent dyes as the procedure to confirm primary localization of the inoculum and any signs of reflux to the kidneys.
The authors have nothing to disclose.
The authors do not have any acknowledgements.
BD INTRAMEDIC Polyethylene Tubing/ PE 10 | BD Medical | 63019-004 | |
BD INTRAMEDIC Polyethylene Tubing/ PE 50 | BD Medical | 63019-048 | |
BD Insyte-N Autoguard/ Angiocath | BD Medical | 311411 | |
Ethanol (EtOH) | Fisher Scientific | BP2818500 | |
BD Needle Only 22 Gauge (G) 1 inch | Becton Dickson | 205155 | |
BD Needle Only 27 Gauge (G) 1/2 inch | Becton Dickson | 305129 | |
Luer stub, 22 G X 0.5 in / Blunt Needle | Instech | LS22 | |
Luer stub, 27 G X 0.5 in / Blunt Needle | Instech | LS27 | |
6" Long x 2" Wide x 1" Thick, Aluminum Oxide Sharpening Stone / Oil Stone | Norton | 40204034 | |
Surgical Lubricant Sterile Bacteriostatic Folilpac | Surgilube | 281020543 | |
Isoflurane | Fluriso | 13985-528-60 | |
SomnoSuite Low-Flow Anesthesia System | Kent Scientific | SS-01 | |
Dissecting Forceps, Curved | vwr | 82027-392 | |
BD 1 ml Syringe | BD Medical | 309626 | |
India Ink Solution., 0.2% in PBS buffer | Alfa Aesar | J61007 | |
8~12 weeks old male C57B/6 Mice | Jackson Lab | #000664 |