Ultrasonography of the Adult Male Urinary Tract for Urinary Functional Testing

Summary

We describe the use of high frequency ultrasound with contrast imaging as a method to measure bladder volume, bladder wall thickness, urine velocity, void volume, void duration, and urethral diameter. This strategy can be used to assess voiding dysfunction and treatment efficacy in various mouse models of lower urinary tract dysfunction (LUTD).

Abstract

The incidence of clinical benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) is increasing due to the aging population, resulting in a significant economic and quality of life burden. Transgenic and other mouse models have been developed to recreate various aspects of this multifactorial disease; however, methods to accurately quantitate urinary dysfunction and the effectiveness of new therapeutic options are lacking. Here, we describe a method that can be used to measure bladder volume and detrusor wall thickness, urinary velocity, void volume and void duration, and urethral diameter. This would allow for the evaluation of disease progression and treatment efficacy over time. Mice were anesthetized with isoflurane, and the bladder was visualized by ultrasound. For non-contrast imaging, a 3D image was taken of the bladder to calculate volume and evaluate shape; the bladder wall thickness was measured from this image. For contrast-enhanced imaging, a catheter was placed through the dome of the bladder using a 27-gauge needle connected to a syringe by PE50 tubing. A bolus of 0.5 mL of contrast was infused into the bladder until a urination event occurred. Urethral diameter was determined at the point of the Doppler velocity sample window during the first voiding event. Velocity was measured for each subsequent event yielding a flow rate. In conclusion, high frequency ultrasound proved to be an effective method for assessing bladder and urethral measurements during urinary function in mice. This technique may be useful in the assessment of novel therapies for BPH/LUTS in an experimental setting.

Introduction

Benign prostatic hyperplasia (BPH) is a disease that develops in men as they age and affects nearly 90% of men over 80 years of age^1,2. Although the development of BPH is generally associated with aging, other factors including obesity and metabolic syndrome can lead to BPH in relatively younger men^3,4. Many men with BPH develop lower urinary tract symptoms (LUTS) that significantly decrease their quality of life, and some experience complications that may include bleeding, infection, bladder outlet obstruction (BOO), bladder stones, and renal failure. The cost of treatment for BPH exceeds $4 billion annually^5,6,7. Diagnosis of LUTS caused by BPH generally relies on the use of the AUA symptom index (AUASI) score, uroflowmetry, and assessment of prostate size⁸. The etiology of BPH/LUTS is complex and multifactorial, and disease development and progression has been associated with prostatic hyperplasia (prostate proliferation), smooth muscle contractility, and fibrosis. Current treatments include the use of α-adrenergic blockers to regulate smooth muscle tone within the bladder and prostate to alleviate LUTS and/or 5α-reductase inhibitors to decrease androgen metabolism and decrease prostate size. Better disease models, murine and other, to allow the accurate study of the effects of varied causative and therapeutic factors in this disease process over time is highly desirable⁹.

Rodent models have been extensively used to study urodynamics; however, most studies are focused on female micturition and disease¹⁰. In order to fully examine all aspects of male LUTS, rodent models have been developed and used to study different aspects of BPH including changes in cellular proliferation, smooth muscle function, collagen deposition, and inflammation^11,12,13,14. However, rodent and human prostate anatomy differ. While the human prostate is compact and encased by a condensed fibromuscular layer, the rodent prostate is lobular; and these differences complicate direct comparisons of disease progression and treatment efficacy. Additionally, LUTS are difficult to assess in mice, since it is not possible to directly measure bother. Instead, current methods for studying disease correlate histological features with physiological features (i.e., bladder volume and wall thickness with uroflowmetry, void spot assays, and cystometry endpoint data) that compare the level of urinary dysfunction between BPH model and control animals^{12,15,16,17,18}. Physiological features are frequently evaluated as post-mortem necropsy endpoints, and there is an inability within the same animal to observe BOO across time. Recently, we have identified a subdivision of the pelvic urethra (the prostatic urethra) where exogenous hormone implants cause a narrowing based on post-mortem necropsy assessments¹². Current methods do not allow for the direct, in vivo assessment of urethral narrowing during voiding.

Ultrasound is a non-invasive diagnostic and evaluation technique that has successfully been used in other disease models. It is used to quantify organ volume and assess vascular flow^19,20,21. Ultrasound is also used to visualize and guide microinjections, allowing for targeted injections of stem cells or other drugs, and to evaluate systolic and diastolic cardiac function.

This protocol describes the use of high frequency ultrasound to evaluate lower urinary tract anatomy and assess urinary physiology in anesthetized mice. We describe the use of ultrasound for measuring bladder volume and wall thickness. We also describe the use of contrast-enhanced ultrasound to measure urine velocity, urine volume, void duration, and urethra diameter. The use of ultrasound provides a more comprehensive understanding of the lower urinary tract in vivo, determines how disease alters normal voiding function, and gives us the tools to better evaluate the effectiveness of new therapeutic options. Currently, the non-contrast imaging protocol is non-terminal, while the current contrast-enhanced imaging protocol is a terminal procedure.

Protocol

Procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Wisconsin – Madison.

1. Animal preparation

1. Place a 24-month-old, C57Bl6/J male mouse in a pre-charged chamber with 3-5% isoflurane until the righting reflex is lost and the breathing rate slows.
2. If necessary, use clippers to shave the abdominal hair from the animal for surgery and/or imaging. Remove all remaining hair using a depilatory cream. 
3. Place the mouse in a supine position in a nose cone with 2% isoflurane on a heated platform to maintain anesthesia. Confirm the depth of anesthesia by loss of motion from the animal in response to a pedal-withdrawal reflex (Figure 1B).

2. Ultrasound set-up

1. Connect a MV707 probe with center frequency of 30 MHz to the active-port, with the application preset to "abdominal imaging" (Figure 1A).
2. Position the ultrasound probe parallel to the long axis of the bladder (Figure 1C).
3. Long and short axis images of the bladder, the prostate, and the urethra are made in B-Mode (Figure 1D).
4. Use the "xy" micro-manipulators to move the mouse.

3. Non-contrast imaging protocol

1. Measure the bladder wall thickness using the linear distance measurement tool and tracing the outside edge to inside edge of the bladder wall b-mode post acquisition.
2. Measure the bladder 3D volume with the volumetric tool on the 3D mode acquisition by tracing the inside of the bladder walls to create a contour. Multiple contours are then generated through the thickness of the bladder to calculate volume.

4. Microbubble contrast resuspension/activation

1. Activate the contrast agent (e.g., DEFINITY) by shaking in the vortex mixer for 45 s to encapsulate the microbubbles in solution. This step is critical for optimum contrast enhancement.

5. Catheter insertion

1. With the mouse anesthetized and taped to the heated platform, expose the bladder with a midline incision using straight sharp/blunt scissors through the skin and abdominal wall.
2. Insert a 27-gauge needle connected to a syringe by flexible polyethylene tubing (PE 50) into the bladder. Prefill the tubing with saline to ensure no air bubbles are injected into the bladder.

6. Contrast-enhanced imaging protocol

1. To confirm needle placement, instill 10 µL of saline into the bladder while being observed via ultrasound.
2. Replace the saline syringe with a syringe containing contrast to improve visualization of urethral walls and voiding events, because the urethra is normally collapsed. Once a complete long axis view of the urethra is obtained and an image saved, rotate the probe 90° to obtain a short axis view and an M-Mode image.
3. Instill a bolus of microbubbles at 0.5 mL per 3 s into the bladder until a urination event occurs.
4. During the first voiding event, measure the urethral diameter at the point of the Doppler velocity sample window using the linear distance tool and measuring edge to edge.
5. With the urethra properly located, angle the probe in relation to the urethra to become more parallel to the urine flow.
6. Instill a second bolus of microbubbles into the bladder, and measure the event velocity using the velocity time integral (VTI) tool.
7. After data collection, euthanize the mouse with cervical dislocation.

7. Data Calculation and Analysis

1. Select the VTI tool to measure velocity by tracing the recorded images.
2. Measure the diameter of the urethra from the B-mode or M-mode image using the leading edge to leading edge convention.
3. Calculate the cross-sectional area (CSA) using the following formula (CSA ) using image measurements obtained above.
4. Calculate the void volume using the CSA of the urethra and multiplying that by the area under the Doppler trace (velocity time integral)(CSA x VTI = volume).
5. Calculate the actual voided urine volume assuming one gram per cubic centimeter density.

Results

Ultrasound can be used with or without contrast enhancement depending on the experimental design and endpoint measurement. Mice are anesthetized with isoflurane and shaved and all traces of hair removed with a depilatory cream. Anesthetized animals are placed on a heated platform with the ultrasound probe positioned along the long axis of the bladder (Figure 1).

Figure 2 shows representative ultrasound images of a mouse bladder acquir...

Discussion

Current techniques for evaluating the lower urinary tract of rodents are limited by their ability to directly correlate changes in voiding physiology with changes in prostatic histology consequent to disease progression. Void spot assays and uroflowmetry can be used to assess spontaneous urination events in rodents, and these techniques can be used to evaluate changes over a period of time^15,16,17. However, for both techniques, ...

Materials

Name	Company	Catalog Number	Comments
21mm Clear Tubing	Supera Anesthesia Innov	301-150
27 gauge needle	BD	Z192376
4 port Manifold	Supera Anesthesia Innov	RES536
DEFINITY	Lantheus Medical Imaging	DE4
F/AIR Canister	Supera Anesthesia Innov	80120
Graefe forceps (Serrated, Straight)	F.S.T.	11050-10
Inlet/Outlet Fittings	Supera Anesthesia Innov	VAP203/4
Isoflurane	Midwest Vet Supply	193.33161.3
Isoflurane Vaporizer	Supera Anesthesia Innov	VAP3000
MV707 probe	Fujifilm VisualSonics Inc
Oxygen Flowmeter	Supera Anesthesia Innov	OXY660
Polyethylene 50 tubing	BD	427516
Pressure Reg/Gauge	Supera Anesthesia Innov	OXY508
Rebreathing Circuits	Supera Anesthesia Innov	CIR529
Small Mice Nose Cone	Supera Anesthesia Inov	ACC526
Sterile saline	Midwest Vet Supply	193.74504.3	NaCl 0.9%, Injectable
Straight Sharp/Blunt Scissors	Fine Scientific Tools (F.S.T)	14054-13
Syringe	BD	309646	5mL
Vevo 770	Fujifilm VisualSonics Inc
VIALMIX	Lantheus Medical Imaging	VMIX