Ultrasound-based Pulse Wave Velocity Evaluation in Mice

Arterial stiffness represents a key factor in cardiovascular disease and pulse wave velocity (PWV) can be considered as a surrogate index for arterial stiffness. This protocol describes an image processing algorithm for calculating PWV in mice based on ultrasound image processing that is applicable at different arterial sites.

The overall goal of this procedure is to provide an image processing algorithm for assessing arterial stiffness in mice based on the ultrasound imaging of different arterial sites. Arterial stiffness is associated with cardiovascular disease and is increasing used as therapeutic target due to its prognostic value. After confirming the appropriate levels of sedation by toe pinch, place the animal on a 40 degree Celsius temperature controlled board in the supine position and moisten the animals eyes with ointment.

Coat the four limbs of the animal with conductive paste and tape the limbs onto the ECG electrodes embedded in the board. Measure the body temperature with a rectal probe lubricated with petroleum jelly and confirm that all of the physiological measurements are correctly displayed. Next, use depilatory cream to chemically remove the hair from the abdomen and coat the exposed skin with acoustic coupling gel.

Place a 13 to 24 mega hertz ultrasound probe in the mechanical arm and fix the probe parallel to the animal. Adjust the probe so that the desired longitudinal images of the abdominal aorta located in the focal zone can be obtained and select high frame rate ECG gate at acquisition with a frame rate acquisition of 700 frames per second. Start the acquisition, then using the same scan projection select pulse wave doppler and move the sample volume to the center of the vessel.

Obtain images with the cineloop of at least three seconds, maintaining a small an angle correction as possible. After all of the images have been acquired, remove the animal from the board with monitoring until full recovery. For post processing analysis of the ultrasound data, first export the B mode and post wave doppler images as dicom files.

After saving the files, convert the post wave doppler dicom files to tif images Next using the dedicated graphical user interface, initialize the contours by drawing a line close to the far wall of the vessel and double click close to the near wall. A line parallel to the drawn line will automatically appear. Click analyze to apply the algorithm on a single frame.

If the edges have been correctly identified click go"to apply the algorithm to the whole cineloop. Then click record"and save the corresponding mat file containing the instantaneous diameter values related to a single cardiac cycle. For the diameter velocity loop implementation, open the dedicated graphical user interface.

To begin the pulse wave doppler image processing, click velocity"generating a single beat mean velocity curve. After identifying the post wave doppler trace, select white line"to locate the line corresponding to a velocity value equal to zero. Then, in the calibration panel, click velocity"to draw lines with lengths that correspond to the calibration factor.

Repeat the operation for the time calibration clicking time. Using the region of interest physio button, manually select a region of interest containing the physiological signals, then click region of interest signal to manually select a region of interest containing the post wave doppler trace. Select analyze"to check if the envelope is identified.

If the result is not satisfactory, enter the new value in the velocity threshold editable text field and analyze the envelope again. Next, click elaboration"and check for errors in our peaks detection of the ECG signal. Change the minimum peaks distance value, and then click update"for a correct identification of our peaks.

Click choose beats, select all of the beats that are uncorrupted by noise or located in the inspiration phase. Press the return button on the keyboard to re-sample selected beats and to obtain a single beat velocity wave form, then select the mean velocity check box to achieve a single beat mean velocity signal and click okay. Now click diameter"and press the interpolate button on the keyboard to obtain a single beat diameter signal with the same sample frequency as the single beat velocity signal.

Click okay, then select the second derivative approach as the alignment method and click update. The two curves will be automatically time aligned using the second derivative method. The natural logarithm of the single beat diameter values are automatically plotted against the single beat mean velocity values.

And the corresponding pulse wave value is assessed. In this image, edge detection and contour tracking techniques were applied to B mode images acquired with a high frame rate, ECG gated modality to provide the diameter wave form. Identification of the post wave doppler signal envelope and the average of data from different cardiac cycle leads to the single beat mean velocity curve assessment.

Single beat diameter and mean velocity wave forms are interpolated in both the frequency and time domain and then time aligned. A diameter velocity loop is obtained by plotting the natural logarithm diameter values against the mean velocity measurements, allowing assessment of the pulse wave velocity value. This is possible by calculating the slope of the linear part of the loop, which is known to correspond to the early systolic phase as it happens in humans using a similar approach.

to assess local arterial stiffness, noninvasively.