Pulsed Wave Doppler Assessment of Diastolic Dysfunction in the ZSF-1 Rat Model of Pulmonary Hypertension Due to Left Heart Disease

Our research focuses on heart failure with preserved ejection fraction and the significant sex differences the disease manifests. Diastolic dysfunction is a hallmark of HFpEF with indices that can be assessed using Doppler sonography without 2D echocardiography, which is expensive and requires sophisticated equipment. To begin, turn on the tablet connected to the ECG tracing pad.

Select RS Monitor from the available applications. Then turn on the ECG pad by pressing the round button in front. Tap settings in the right lower corner of the user interface.

Select presets from the menu of experimental settings. Tap the dropdown menu under saved presets, then choose Rat CV and click OK in the bottom right corner. Next, clean the ECG pad with 70%ethyl alcohol to remove any ECG gel residue.

Obtain highly conductive electrode gel for the electrocardiogram and water-soluble hypoallergenic ultrasound gel for the Doppler. Place the anesthetized animal supine on the non-invasive ECG pad with the nose positioned in the appropriately-sized nose cone with a vaporizer output set to 1.5 to 2%Using a cotton-tipped applicator, apply a small amount of electrode gel to the dorsal surface of the four paws and to the plantar side of the hind paws. Secure the paws with tape on the electrode contact surface of the ECG pad, ensuring sufficient contact between the gel and the pad.

Then use a gentle depilatory cream to remove the chest and abdominal hair. Wipe off any residual cream to avoid skin irritation. Ensure that the isoelectric baseline and the P, QRS, and T-wave forms are clearly displayed on the computer monitor.

Create a new folder identifying the animal and the data of interest. Turn on the pulse Doppler transceiver device using the power switches on the front and the back of the large blue box. Then turn on the Doppler signal processor.

Next, open the Doppler signal processing workstation software. Click on Setup and select System Setup. Select the 20 megahertz probe frequency for the experiment.

Plug in the 20 megahertz probe when investigating a young rat or mouse. Use a 10 megahertz probe for older, larger, or obese rats. If both probes are connected, use the toggle switch on the front of the transceiver module to switch between frequencies as needed.

Ensure that the handheld probe transducer is connected to channel A or B receptacle on the transceiver module. Select the corresponding channel to operate the handheld probe. Set the direction switch to TWD to display flow toward the probe above the zero flow baseline.

Then adjust the range to 2.5 to 3.5 millimeters for mice or four to six millimeters for rats. Set filter to 15 to smooth the phasic velocity output to a damped signal. Press go in the top left corner of the user interface.

In the pop-up window, enter the relevant acquisition information, including animal type, gender, date of birth, age on acquisition date, and weight. Select the desired pre-created folder from the directory dropdown menu and click OK.Adjust the ECG baseline by dragging the axis scale bar up or down and until the R-waves are clearly visible. To perform Doppler measurements, first apply a small amount of ultrasound gel to the substernal and epigastric region of the anesthetized animal's chest and abdomen.

Place the Doppler probe midline under the diaphragm just below the sternum with the head pointing cephalad toward the left ear of the animal. Slowly scan for blood flow by moving the probe in small circular motions with increasing radius. Maintain gentle pressure of the probe against the skin under the diaphragm at all times.

If mitral flow is not detected, adjust the range dial in increments of 0.5 millimeters until the transmitral wave form is identified. Ensure that the Doppler wave form and the electrocardiogram are displayed simultaneously on the screen. Create snapshots using the foot pedal once the correct flow is identified and repeat until optimal data quality is recorded.

Stop data acquisition by clicking stop when satisfied with the data quality. Click on File and select Open from the dropdown menu. Choose the highest quality data file from the pre-created folder for analysis.

Press on Analysis and then Analysis Mode. Choose step two, general setup and set data type to Doppler. Then set signal to mitral inflow.

Select the appropriate animal model and choose all measurement names under default measurement names. Select step five, R-peak editor from the analysis control dropdown menu. Enable display beat and R-peak markers and click auto-calculate.

Then choose step six, beat editor from the analysis control dropdown menu. Enable display beat and R-peak markers and click select all beats. Next, choose step seven, marker editor.

Click display analysis markers and choose the desired parameter under current marker selected. Ensure the given parameters are available for the completeness of data. Select step eight, measurement results.

Click copy results to clipboard to copy the data into an Excel file and save it. Remove the tape from the animal's paws after turning off the vaporizer. Then remove the animal from the electrode pad.

Clean off any remaining depilatory cream and electrode gel from the animal. Then transfer the animal back into its cage with free access to water and food for recovery. For the most accurate assessment of diastolic function, antegrade and retrograde flow patterns related to the opening and closing of the mitral and aortic valve need to be visualized within the same cardiac cycle over multiple cycles.

Multiple indices were determined from the biphasic transmitral flow pattern, including the early filling peak, the atrial contraction peak, the ratio between these two peaks, and the deceleration time of the early filling peak. The isovolumic relaxation time, or IVRT was a good indicator of diastolic dysfunction in rodents. IVRT increased during aging in ZS F1 rat model of pulmonary hypertension left heart disease.

This change could be detected as early as 16 weeks of age. 24-week-old obese male rats showed higher IVRT compared to earlier time points in lean controls. This protocol allows the investigator to study established echocardiographic indices of HFpEF, including the E/A ratio, IVRT, or E-wave deceleration time.

This technique lacks 2D visualization of the heart, and some markers of diastolic function are load-dependent, a condition that is difficult to control for. This is a low-cost technique that reliably detects disease progression over time in cardiopulmonary diseases that manifest diastolic dysfunction.