The overall goal of this procedure is to non-evasively assess vascular endothelial function in living rats by measuring flow-mediated dilation of the brachial and superficial femoral artery. The methods described provide assessment of arterial and endothelial function without animal instrumentation or sacrifice, thereby providing investigators the ability to make multiple measurements across time during an intervention in a single rat. Generally individuals new to this protocol will struggle because ultrasound imaging is difficult, but even moreso in rats due to the anatomical size of the arteries.
After anesthetizing the animal, place it in a supine position on the examination table and apply ophthalmic ointment. Restrain each upper limb and each lower limb to the table with surgical tape. Now, use depilatory cream on the right upper limb to expose the skin.
On the same limb, position a 10 millimeter standard vascular occlusion cuff. Keep the occluder off the platform or the limb may move and ruin the imaging when the occluder is put to use. Next, set the ultrasound machine to B-mode.
Then, apply a small amount of ultrasound gel over the brachial artery. Manually align an ultra-high frequency linear array transducer, attached to a stereotactic holder, with the upper limb. The brachial artery should be visible at about three to five millimeters deep.
To confirm that the artery and not the vein is being imaged, switch to PW-mode. The artery has a pulsatile blood flow as opposed to the adjacent vein, which has continuous blood flow. Prepare the animal as before, only now elevate the right lower limb.
Then, apply and remove depilatory cream and take note of the femoral vein, which is visible to the naked eye. In this preparation, put the occlusion cuff proximal to the right ankle without resting the cuff on the platform. Then, set the ultrasound machine to B-mode.
Apply a small amount of ultrasound gel proximal to the occlusion cuff, and manually align an ultra-high frequency linear array transducer, attached to a stereotactic holder, with the femoral vein. The superficial femoral artery should be visible at about one to two millimeters deep. Use the PW-mode to confirm that it is not the femoral vein.
The artery has a pulsatile blood flow as opposed to the adjacent vein, which has continuous blood flow. To optimize the B-mode image, first ensure that a horizontal, longitudinal image of the vessel is observed. Then, make slight adjustments to the ultrasound probe placement to maximize the view of the artery.
Other standard optimization strategies can also be used. After optimizing the image, turn on the ECG gating option so only images captured during the R-wave are displayed. Then, ensure that only one diameter frame is collected during each diastolic portion of each cardiac cycle.
Without ECG gating, the combination of a high heart rate in rats and a high frame rate to capture the diastolic diameter only allows for 10-to 20-second clips. The number of clips and the amount of data increases the analysis burden substantially. Now, set the clip recording length to the maximum number of frames per clip, or at least 10 to 20 frames per clip higher than the rat's heart rate in a minute.
Then, record 60 seconds of baseline data in B-mode. Next, switch to PW-mode. Then, place the cursor at the middle of the lumen.
Because the sample gates will be placed automatically in reference to the cursor, adjust the width using the ultrasound keyboard. Next, adjust the insonation angle to not more than 60 degrees by altering the doppler beam angle and then making fine adjustments with the ultrasound keyboard. For more adjustment, tilt the probe to a more optimum angle.
Now, in PW-mode, with the insonation angle correctly set, set the clip recording length to 10 seconds and record 10 seconds of velocity data. To begin, inflate the vascular occluder using an air-filled, 10 milliliter syringe. Then, fold the tube on itself and seal the air in with a binder clip.
In PW-mode, confirm the cuff occlusion. There should be an obvious reduction in blood velocity. Then, switch to B-mode and set the clip length to 60 seconds.
Record data until the occlusion has been in place for four minutes and 45 seconds. Note the heart rate at the beginning and end of each recording, and the total time of each recording. At four minutes and 45 seconds, switch to PW-mode and record for five seconds.
Then, release the cuff and record for five seconds before and after the cuff release. When switching between ultrasound modes during cuff release, the image may shift, requiring immediate adjustment to the ultrasound probe. Practice performing this step should improve your efficiency and confidence at this step.
Now, switch back to B-mode and record data in 60-second clips for three minutes. After starting each clip, take note of the heart rate and the time. After the test, remove the animal from the examination table and monitor it until it has regained sufficient consciousness to maintain sternal recumbency.
The described procedure was used to study FMD in brachial and superficial femoral arteries of eight Lister rats. The analysis showed that there is a similar vasodilatory response between the brachial and superficial femoral arteries when expressed as a percent change from baseline. The absolute change from baseline was significantly higher in the brachial artery, which was probably due to significant size differences between the vessels.
Despite this, there is a strong linear relationship between brachial and superficial femoral artery FMD when expressed as a percent or absolute change from baseline. Other cardiovascular variables were also measured during the baseline, occlusion and hyperemic phases. After normalizing to shear stimulus, vasodilation to peak shear rate was higher in the brachial artery than the superficial femoral artery.
This was also true for FMD, expressed as an absolute change from baseline after being normalized to peak shear rate. However, there was a strong linear relationship in percent and absolute FMD normalized to peak shear rate. Once mastered, this procedure can be performed in 20 to 30 minutes.
When performing this technique, it's important to keep track of time and maintain the same image site. We have adapted this technique from human studies to be performed in rats. After its development in humans, this technique paved the way for researchers in cardiovascular physiology to explore the relation between arterial and endothelial function and its relation to clinical cardiovascular disease.
