This protocol uses high-frequency ultrasonography to evaluate the changes in the bladder and lower urinary tract in mice. This technique allows a longitudinal examination of the urinary tract in vivo for anatomical and physiological measurements and is complementary to other techniques for measuring urinary flow. The ability to assess in vivo voiding changes in rodent models that recapitulate aspects of human urological diseases can provide insight into disease progression and treatment efficacy.
Begin by connecting a probe with a center frequency of 30 megahertz to the active port of the ultrasound system and presetting the application to abdominal imaging. For optimum contrast enhancement, shake an appropriate contrast agent in a vortex mixer for 45 seconds to encapsulate the microbubbles in solution. After confirming a lack of response to toe pinch, place an anesthetized 24-week-old C57 black 6 male mouse in the supine position on a heated platform and shave the hair from the abdomen.
Remove any remaining hair with depilatory cream and position the ultrasound probe parallel to the long axis of the bladder. Then using the XY manipulator to move the mouse as necessary, obtain long and short axis images of the bladder. Before imaging, make a midline incision through the skin and abdominal wall to expose the bladder and pre-fill a piece of flexible polyethylene tubing with saline.
Then connect a syringe equipped with a 27 gauge needle to the piece of tubing and insert the needle into the bladder. To confirm correct placement of the needle, instill 10 microliters of saline into the bladder. Replace the saline syringe with a syringe containing the vortexed contrast agent and collect another B-mode image of the catheterized bladder.
To facilitate measurement of the urethra diameter, instill a 0.5 milliliter bolus of microbubbles over a three-second period into the bladder until a urination event occurs. To facilitate measurement of the velocity, correct the Doppler sample window angle until it is parallel to the urine flow and instill a second bolus of microbubbles into the bladder. Then collect a 3D image of the full bladder.
When all of the images have been obtained, trace the outside to inside edges of the bladder wall and use the linear distance measurement tool to measure the bladder. Use the volumetric tool to trace the inside of the bladder walls to create a contour. Multiple contours can then be generated through the thickness of the bladder to calculate the volume of the bladder in the 3D acquisition mode.
For the first voiding event, use the linear distance tool to measure the urethral diameter edge to edge at the point of the Doppler velocity sample window. For the first voiding event, use the velocity time integral tool to measure the urethral velocity. Here, representative ultrasound images of a mouse bladder acquired without contrast agent are shown.
The bladder wall is echogenic and the bladder wall thickness can be measured using a software measurement package. A bladder size and shape can be rendered in 3D to determine bladder volume. Here, a representative image of a microbubble filled echogenic bladder is shown.
After low frequency ultrasonic burst, the bubbles are destroyed and the bladder becomes transiently echogenic before the bubbles reform confirming the structure as the bladder. During a urination event, the urethral lumen diameter can be acquired during urination along with the flow velocity of the urine passing through that region of the urethra. Using contrast imaging, diameter measurements can be made throughout the entire visible length of the urethra.
From these measurements, further calculations can be made to assess the urinary flow and bladder compliance. Activating the contrast agent correctly is essential to the success of this protocol. We can reconstruct rodent urinary tracts with MRI and microcomputed tomography to confirm the ultrasound flowmetry as well as introduce new complex models of urologic flow dynamics and 3D reconstruction.
We have begun using this technique to quantitatively examine models of lower urinary tract disease, the effects of aging, and the efficacy of therapeutic treatments on urinary function.
