Doppler Ultrasonography for Live Imaging and Quantification of Ovarian Vascular Function in Mice

This research aims to characterize urine preovulatory ovarian blood flow using a non-invasive in vivo method. The field lacks non-invasive methods to study ovarian vascular remodeling in real-time without disrupting normal physiology in mice. To begin, switch on the main power located on the back of the imaging system.

After the system boots, toggle the computer standby switch on the left side of the cart to wake the monitor and computer. Connect the MS700 transducer to the active transducer port on the imaging unit located at the rightmost port. Align the locking pin on the transducer connector with the notch in the port.

Push the connector in fully and turn the locking lever to the vertical position to secure it. Launch the analysis software by clicking on the software icon. Click New to create a new study and choose the appropriate name from the dropdown menu.

In the Study Information window, enter the study name, series name. Application package is ovary and measurement package is vascular. Once the program opens and the transducer is automatically detected, select the ovary application package.

Next, apply a small amount of electrode cream to each of the electrodes to ensure proper contact for heart rate monitoring. Once the animal is fully sedated, redirect gas flow by opening the stopcock to the nose cone on the imaging platform, and closing the stopcock to the induction chamber. Gently transfer the mouse onto the heated imaging platform.

Place the mouse in the prone position with the dorsal side facing upward and secure the nose cone over its nose to maintain anesthesia. Tape each foot in the electrode cream onto the corresponding electrode pad for physiological monitoring. Apply ophthalmic ointment to the eyes to prevent corneal drying during anesthesia.

Confirm adequate anesthesia depth using the toe pinch reflex method. Monitor the animal's vital signs, such as rate of breathing and heartbeat, movements indicating discomfort, and the platform temperature every five minutes. Next, shave the lower right quadrant of the back using an electric shaver from the right hind limb to the midline, staying lateral to the spine.

Apply a thin layer of body hair remover cream. After a minute, wipe off the cream thoroughly with damp gauze. Then, apply a generous and even layer of ultrasound gel to the shaved region.

Position the transducer probe in transverse mode relative to the mouse on the mechanical stand with the orientation notch facing to the left of the operator. Secure the probe in place using the clamp. Press the B-Mode key on the control panel to activate the standard grayscale imaging window.

Ensure the mouse and platform are flat and that the transducer is perpendicular to the surface. Then, pull the skin to the left and position the transducer near the upper shaved region, closer to the midline of the back. Lower the transducer gently onto the shaved area coated with ultrasound gel.

Align the left edge of the probe with the spine and the right edge over the right flank. To identify the ovary, first identify the kidney, a large light gray ovoid structure with cortex and medulla. Push the imaging platform slightly toward the mouse's head to reveal the ovary and periovarian adipose tissue.

Press the Cine Store button to capture the B-Mode video. Now, press the key for Power Doppler Mode on the control panel to visualize blood vessels with high sensitivity to low velocity flow. Adjust the Doppler gain to 32 to 55 decibels, sensitivity to 5, dynamic range to 15 DR, and velocity to one kilohertz.

Move the platform gently back and forth to get the best view of the ovarian vasculature and identify the ovarian artery entering the ovarian hilum, medullary vessels in the central medulla, and cortical vessels around developing follicles. Press Cine Store to save the displayed video of the ovary and vasculature in Power Doppler Mode. To distinguish arterial from venous flow, press Color Doppler Mode.

Red shows flow toward the transducer and blue shows flow away from it. The vessel with the higher average velocity is the ovarian artery. Press the Pulsed Wave PW Doppler button to bring up the sample datelines.

Press it again to activate the Doppler Spectral Waveform. After positioning the sample gate in the center of the vessel lumen, adjust the insonation angle to be less than or equal to 60 degrees and parallel to flow using the angle knob. For the ovarian artery, place the gate just before it enters the ovary.

For the medullary vessel, place it at the central bifurcation. For cortical vessels, place it at the base of a growing follicle. Press Cine Store to save both the ultrasound image and spectral wave form.

When imaging is complete, wipe off ultrasound gel from the back and gently remove the mouse from the imaging platform. And place the mouse on a heating pad until fully awake. Power Doppler Imaging revealed a visible increase in profusion within the medullary vessels at one hour post-human chorionic gonadotropin injection, followed by a return to baseline at eight hours and further decrease by 12 hours post-injection.

Color Doppler Imaging enabled clear differentiation between the ovarian artery and ovarian vein and improved visualization of flow direction within ovarian vessels. Representative PW Doppler waveforms displayed distinct cycles with measurable peak systolic and end diastolic velocities in the ovarian artery of a hormonally responsive mouse, whereas a non-responsive mouse exhibited low signal traces with no detectable waveform. At one hour post-human chorionic gonadotropin injection, blood flow velocity increased in the ovarian artery, medullary vessels, and cortical vessels in mice that responded to stimulation, but no such increase was observed in the nonresponsive mouse.

In nonresponsive mice, the velocity in cortical vessels dropped below the detection threshold at selected time points indicated by assigned placeholder values. Using Doppler ultrasonography, we determined that ovarian blood flow velocity increases rapidly after DLH surge in mice. Doppler ultrasonography allows a repeated, noninvasive assessment of ovarian hemodynamics during the preovulatory period, which other methods cannot achieve.

After characterizing normal preovulatory hemodynamics, future studies can now use this protocol to examine blood flow disruptions in models of ovarian disorders like PCOS.