Name: ultrasound assessment of flow-mediated dilation of the brachial and superficial femoral arteries in rats
Uploaded: 2016-11-03T13:00:00.000+00:00
Duration: 9 min 40 s
Description: Watch this Scientific Journal Video about Ultrasound Assessment of Flow-Mediated Dilation of the Brachial and Superficial Femoral Arteries in Rats at JoVE.com

All animal imaging was performed at the Small Animal Imaging Core Facility, University of Utah.
This study was funded in part by grants from the National Institutes of Health (R21 AG043952, R01 AG040297, K01 AG046326, K02 AG045339, and R01 DK100505).

All animal procedures conformed to the Guide for the Care and Use of Laboratory Animals14 and were approved by the University of Utah and Salt Lake City Veterans Affairs Medical Center Animal Care and Use.
1. Animal Preparation
<ol>
	<li>Place animal in anesthesia induction chamber containing 3% isoflurane in 100% oxygen. Leave the animal in the induction chamber until it is unresponsive to external stimuli.</li>
	<li>Remove the animal from the induction chamber and place it on a heated examination table equipped with electrocardiogram (ECG) electrodes. Maintain anesthesia at 3% isoflurane in 100% oxygen. Brachial and superficial femoral artery FMD cannot be performed simultaneously. Therefore, preparation instructions for each measurement are listed below.</li>
</ol>
2. Brachial Artery Preparation
<ol>
	<li>Position the animal supine and restrain the left upper limb and each lower limb of the animal to the examination table with surgical tape.</li>
	<li>Restrain the right upper limb of the animal, so that the lower portion of the upper limb is slightly elevated (~0.2-0.5 cm) above the platform.</li>
	<li>Apply depilatory agent (e.g., Nair) to the animal&#39;s right upper limb to remove hair.</li>
	<li>Position an occlusion cuff (10 mm lumen diameter standard vascular occluder) on the right upper limb distal to the elbow. Do not rest the occluder on the platform, as inflation/deflation will move the limb and disturb ultrasound imagery.</li>
	<li>Set the ultrasound machine to B-mode using the ultrasound keyboard.</li>
	<li>Apply a small amount of ultrasound gel to the upper limb of the animal, proximal to the occlusion cuff.</li>
	<li>Manually align an ultra-high frequency linear array transducer attached to a stereotactic holder with the upper limb. The brachial artery should be visible 2-3 mm deep.</li>
	<li>To confirm that the brachial artery, not the brachial vein, is being imaged, switch to PW-mode using the ultrasound keyboard. The artery will have pulsatile blood flow as opposed to the adjacent vein which will have continuous blood flow.</li>
</ol>
3. Superficial Femoral Artery Preparation
<ol>
	<li>Position the animal supine and restrain the upper limbs and the left lower limb to the examination table with surgical tape.</li>
	<li>Restrain the right lower limb of the animal to an elevated position (~0.5-1 cm) above the platform using a pad (e.g., folded paper towels).</li>
	<li>Apply depilatory agent (e.g., Nair) to the animal&#39;s right lower limb to remove hair. After hair removal the femoral vein should be clearly visible at the upper inner thigh.</li>
	<li>Position an occlusion cuff (10 mm lumen diameter standard vascular occluder) proximal to the right ankle. Do not rest the occluder on the platform, as inflation/deflation will move the lower limb and disturb ultrasound imagery.</li>
	<li>Set the ultrasound machine to B-mode.</li>
	<li>Apply a small amount of ultrasound gel to the lower limb of the animal, proximal to the occlusion cuff.</li>
	<li>Manually align an ultra-high frequency linear array transducer attached to a stereotactic holder with the femoral vein, which is visible through the skin. The superficial femoral artery should be visible &#60;1 mm deep.</li>
	<li>To confirm that the superficial femoral artery, not the femoral vein, is being imaged, switch to PW-mode. The artery will have pulsatile blood flow as opposed to the adjacent vein which will have continuous blood flow.</li>
</ol>
4. Baseline Phase
<ol>
	<li>Optimize the B-mode image, similar to how it would be done in humans15. Ensure that a horizontal, longitudinal image of the vessel with intima-media visualized in both walls is observed. Optimize the image by slightly adjusting the ultrasound probe placement to ensure that as much of the artery as possible is viewable in the capture window.
	<ol>
		<li>Alternatively, adjust ultrasound settings to obtain a better image by changing the brightness/contrast, focal zones, frequency, dynamic range, and line density. There are other ways to optimize the ultrasound image, but detailed description of those are beyond the scope of this protocol.</li>
	</ol>
	</li>
	<li>After optimization of artery imagery, turn on ECG-gating to display only images captured during the R-wave to ensure that only one diameter frame is collected are during each diastolic portion of a cardiac cycle. 
	NOTE: ECG-gating is available on the ultrasound machine used in this protocol by selecting ECG-gating under the physiological settings option, however, this feature may not be available on all ultrasound machines. ECG-gating should be turned on after the image is optimized, as it is difficult to obtain an image at lower frame rates (i.e., once per R-wave). Without ECG-gating, the combination of a high heart rate in rats and requirement of a high frame rate to capture the diastolic portion of the cardiac cycle only allows for ~10-20 second clips. The cumbersome size and amount of data in each clip increases the analysis burden substantially.</li>
	<li>Record 60 seconds of baseline data using B-mode. 
	NOTE: The ultrasound machine is always recording, however, not all images are stored on the ultrasound machine, as there is a limit on the number of frames that can be recorded in an ultrasound clip. The clip length (i.e., number of frames) can be adjusted in the settings. It is suggested to set for maximum number of frames per clip. When the recording is at the end of a clip (i.e., maximum number of frames reached), the recording continues, but the clip rolls forward capturing the most recent frames. In this case, earlier frames that were captured outside of the maximum frame limit are subsequently deleted. While these intricacies in recording differ between machines, adjustment of recording length may be necessary.</li>
	<li>Switch to PW-mode. Place the cursor in the middle of the lumen. Sample gates will be placed automatically in reference to the cursor, but can be adjusted for width using the ultrasound keyboard. Maintain an insonation angle of &#8804;60&#176;.
	<ol>
		<li>Adjust the insonation angle by altering the Doppler beam angle. Make fine adjustments to the angle by using the ultrasound keyboard. If neither of these provide an angle suitable for measurement, manually adjust the ultrasound probe by tilting the artery to a more optimum angle. If any adjustment of the ultrasound angle is performed, recapture B-mode images.</li>
	</ol>
	</li>
	<li>Record 10 seconds of velocity data.</li>
</ol>
5. Occlusion Phase
<ol>
	<li>Inflate the vascular occluder using an air-filled 10 ml syringe. To keep air pressure constant in the vascular occluder, fold the tube on itself and place a binder clip on the folded tube.</li>
	<li>Switch to PW-mode to confirm cuff occlusion, as evidenced by a large reduction in blood velocity.</li>
	<li>Switch to B-mode and record data in 60 second clips, until 4:45 min of occlusion.</li>
	<li>Switch to PW-mode. Keep a record of heart rate and the time of each ultrasound clip for analysis.</li>
</ol>
6. Hyperemic Phase
<ol>
	<li>Release the cuff while recording in PW-mode by removing the binder clip from the folded tube. Record 5 seconds prior to and 5 seconds after cuff release.</li>
	<li>Switch to B-mode and record data in 60 second clips until 3 minutes after occlusion. Keep a record of heart rate and the time of each ultrasound clip for analysis.</li>
	<li>After completion of FMD remove the animal from the examination table and monitor until it has regained sufficient consciousness to maintain sternal recumbency.</li>
</ol>
7. Analysis
<ol>
	<li>For analysis, export ultrasound as DICOM files to an offline computer equipped with edge-detection software, which allows for unbiased determination of artery diameter at each frame. Analysis is possible on the ultrasound machine, however, it not recommended, as it is extremely time intensive and subject to investigator bias.</li>
	<li>Analyze artery diameter data in 60 second segments during baseline and occlusion phase, and in 10 second segments during the hyperemic phase.</li>
	<li>Analyze blood velocity data using the flow analysis capabilities of the automated edge-detection software. Determine average blood velocity by measuring 5 or more consecutive waveforms of uniform appearance during baseline and occlusion phases. Determine average blood velocity during reactive hyperemia for blood velocities immediately after cuff release. The waveform with highest blood velocity is considered peak blood velocity.</li>
</ol>

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In the present study, a non-invasive measurement of FMD was demonstrated in the brachial and superficial femoral arteries of rats. Similar to humans6, following a 5 min occlusion period, there was a rapid increase in blood velocity (i.e., reactive hyperemia) thereby increasing shear rate on the arterial wall which resulted in the subsequent vasodilation of the artery. FMD was observed in both the brachial and superficial femoral arteries. Additionally, there was a strong relationship in FMD between arteries. Although peak shear rate was higher in the superficial femoral artery, FMD normalized to peak shear rate maintained a strong relationship between arteries. Together, these results indicate that non-invasive measurement of FMD can be successfully performed in rats.
Although measurement of FMD was performed on two different arteries, the magnitude of FMD was similar when expressed as a percent change from baseline. However, when expressed as an absolute change from baseline, FMD was higher in the brachial artery, as that artery had a ~25% larger resting diameter than the superficial femoral artery. These results are similar to those of human studies in which FMD was measured in the brachial and superficial femoral arteries in the same subjects17,18. Despite the differences in arterial diameter, the relationship of FMD, expressed as a percent or absolute change from baseline, was extremely strong between arteries.
Flow-mediated dilation has been proposed to represent a functional bioassay for endothelium-derived NO bioavailability in humans6, as vasodilation occurs via NO release from the endothelium in response to increased shear rate1,3. Thus, a higher FMD normalized to peak shear rate represents an endothelium that has increased sensitivity a given increase in shear rate. After normalizing FMD to peak shear rate, FMD was higher in the brachial artery, regardless of whether it is expressed as a percent or absolute change from baseline. Despite differences in magnitude of FMD normalized to peak shear rate between brachial and superficial femoral arteries, there was a strong linear relationship in percent and absolute FMD normalized to peak shear rate between arteries.
In the present study, the non-invasive measurement of FMD is described in the brachial and superficial femoral arteries with cuff occlusion distal to the ultrasound probe. This cuff placement was selected for several reasons, 1.) in humans, this is most commonly used method for measuring FMD, 2.) the contribution of NO to FMD has been shown to be greater when artery diameter measurement is proximal to occlusion site9, and 3.) there was difficulty in maintaining the ultrasound image after cuff inflation when the measurement site was distal to occlusion. Although this procedure represents a non-invasive measurement of FMD, others have performed FMD in living rats using a surgical occlusion of the common iliac artery with ultrasound measurement distal to occlusion in the femoral artery19. The FMD response using the protocol first described by Heiss et al. was inhibited by infusion of eNOS inhibitors. Indeed, this procedure has been used to demonstrate that pharmacological increases in intracellular NO improves FMD in two rat models of endothelial dysfunction and hypertension20, and exposure to secondhand smoke results in an impairment of FMD in rats21,22. These studies demonstrate the contribution of NO to FMD in rats and establish the relation of FMD to cardiovascular health. However, as this technique is invasive, it might limit the ability to measure FMD longitudinally in the same rats over a period of weeks to years. Using a similar method to the present study, two recent studies have performed non-invasive measurement of FMD in the hind limb of mice23,24, but there were several technical variations between studies (i.e., measurement time course and placement of ultrasound probe and occlusion cuff). Due to discrepancies between these studies and difficulty in replicating these results, the non-invasive measurement of FMD in rats was attempted instead, as rats are commonly used in translational research, but have a larger body and blood vessel size than mice. While measurement of FMD in the femoral artery of rodents is not generally novel, no study has performed measurement of FMD in the brachial artery of any living rodents. The strong relation of FMD between limbs in the present study may illustrate the systemic nature of endothelial function, but also provides a method to non-invasively measure FMD in animals that have disrupted blood flow in the hind limbs (e.g., femoral arteriovenous fistula).
The optimization and maintenance of a high quality ultrasound images are critical skills required for this procedure and requires extensive practice. For example, for measurement of FMD in humans, it is suggested that at least 100 supervised scans are performed before scanning independently5. At times, the image may shift during cuff occlusion and require slight adjustments of the ultrasound probe. A critical step in this protocol is switching between B-mode and PW-mode at specific time points. Simultaneous B-mode and PW-mode imaging are not possible on the ultrasound machine used in this protocol. Therefore, it is required to quickly switch between ultrasound modes to capture velocity and diameter measurements during specific time segments. Having a written out protocol and practice performing the protocol will greatly improve the efficiency of switching between ultrasound modes. Considering the time-sensitive nature of ultrasound recordings during this protocol, errors will occur, so be prepared to note down any protocol abnormalities, such as forgetting to capture an ultrasound clip. Missing an ultrasound clip during the occlusion phase is not critical, however, if a recording is lost during the reactive hyperemia phase it is suggested to perform the procedure again after at least 30 min have passed25.
As with any study there are limitations to the experimental protocol. In this study, anesthesia was administered to rats under 100% oxygen, thus, measurements of FMD might also reflect vasoreactivity to hyperoxia. Other forms of anesthesia, such as sodium pentobarbital could be used to create a more representative blood gas profile to humans and eliminate this concern. Blood pressure was not monitored at any point of this protocol. Although blood pressure does not change in response to acute cuff occlusion in humans, it is unknown if any transient change in blood pressure would occur in rats. Additionally, compressed air was used to fill the vascular occluder, however, filling with water may have resulted in a firmer occlusion of blood flow as water it is not as compressible as air. Lastly, non-invasive measurement of FMD under conditions in which eNOS is inhibited (i.e., L-NMMA infusion) have not been performed. Thus, the contribution of NO to FMD, as it is performed in this protocol, has not been determined.
In conclusion, this article has demonstrated a protocol for the non-invasive measurement of FMD in the brachial and superficial femoral arteries of rats. In conjunction with the recent shift in focus to translational research, the assessment of FMD in rats may provide a valuable tool for translating results in humans to rats, as well as provide the ability to assess endothelial function at several time points in longitudinal studies of rats receiving different treatments. Indeed, reductions in FMD are accompanied by aortic arterial stiffening have been observed following kidney damage in a rat model of chronic kidney failure (unpublished findings), which demonstrates the application of non-invasive FMD as a marker of vascular function in longitudinal animal studies. Future studies to investigate mechanisms of FMD in rats are warranted and would provide further insight into non-invasive FMD measurement in humans.

The vascular endothelium is a cellular monolayer that lines the lumen of arteries and is an important regulator of vascular function. There are numerous molecules released from the endothelium that result in modulation of blood vessel diameter. Among these molecules, nitric oxide (NO), appears to be the primary vasodilatory molecule released from the vascular endothelium in response to stimulation (e.g., insulin, acetylcholine, or changes in shear stress)1. In the vascular endothelium, NO is produced by the enzyme endothelial NO synthase (eNOS) and is subsequently released from endothelial cells2. NO diffuses to the vascular smooth muscle where it causes relaxation and increased vessel diameter3.
Endothelial dysfunction can be assessed non-invasively in humans using the flow-mediated dilation (FMD) technique4,5. FMD has been proposed to represent a functional bioassay for endothelium-derived NO bioavailability in humans, and is typically assessed at the brachial or superficial femoral artery in response to reactive hyperemia following a ~5 min limb occlusion6. Reactive hyperemia increases laminar shear forces that are transduced to the endothelial cell7, signaling a release of NO8. Although in recent years, the proportion of vasodilation initiated by NO release has been debated9,10, FMD is indicative of endothelium-dependent dilation and has consistently been shown to predict cardiovascular events11-13.
To date, thousands of studies have employed the FMD technique for non-invasive measurement of endothelial function in humans. Considering the recent shift in focus to translational research, guidelines for the non-invasive measurement of FMD in rodents would be extremely valuable. Keeping with a translational approach, this protocol was established for measurement of FMD in brachial and superficial femoral arteries of rats, as those sites are most commonly measured in humans. This protocol results in a robust and repeatable FMD response in rats, however, measurement of FMD in rats is technically demanding and may be difficult for other investigators to replicate without video demonstration. Therefore, the following article will demonstrate a method for the non-invasive measurement of FMD in the brachial and superficial femoral arteries of rats.

<table><tbody><tr><td>Vevo 2100 High Resolution Micro-Ultrasound Imaging System</td><td>VisualSonics, Toronto, ON, CAN</td><td></td><td></td></tr><tr><td>MicroScan Ultra-High Frequency Linear Array Transducer - MS-700 30-70 MHz</td><td>VisualSonics, Toronto, ON, CAN</td><td></td><td></td></tr><tr><td>Vevo Imaging Station</td><td>VisualSonics, Toronto, ON, CAN</td><td></td><td></td></tr><tr><td>Thermasonic gel warmer</td><td>Parker Laboratories, Fairfield, NJ, USA</td><td>82-03</td><td>Optional</td></tr><tr><td>Signacreme electrode cream</td><td>Parker Laboratories, Fairfield, NJ, USA</td><td>17-05</td><td></td></tr><tr><td>Transpore surgical tape</td><td>3M, Maplewood, MN, USA</td><td>1527-1</td><td></td></tr><tr><td>Depilatory cream (&lt;i&gt;e.g.&lt;/i&gt;, Nair)</td><td>General supply</td><td></td><td></td></tr><tr><td>Cotton swabs</td><td>General supply</td><td></td><td></td></tr><tr><td>Ultrasound gel</td><td>General supply</td><td></td><td></td></tr><tr><td>Standard vascular occluder, 10 mm lumen diameter</td><td>Harvard Apparatus, Holliston, MA, USA</td><td>62-0115</td><td></td></tr><tr><td>10 ml syringe with Luer-Lok tip</td><td>General Supply</td><td></td><td>Used for occlusion cuff apparatus</td></tr><tr><td>Paperclip</td><td>General Supply</td><td></td><td>Used for occlusion cuff apparatus</td></tr><tr><td>Hypodermic needle &amp;ndash; 18 gauge&amp;nbsp;</td><td>General Supply</td><td></td><td>Used for occlusion cuff apparatus</td></tr><tr><td>Medium binder clip</td><td>General Supply</td><td></td><td>Used for occlusion cuff apparatus</td></tr></tbody></table>

ultrasound assessment of flow-mediated dilation of the brachial and superficial femoral arteries in rats

Arterial vasodilation to increases in wall shear rate is indicative of vascular endothelial function. In humans, the non-invasive measurement of endothelial function can be achieved by employing the flow-mediated dilation technique, typically performed in the brachial or superficial femoral artery. Briefly, a blood pressure cuff placed distal to an ultrasound probe is inflated to a suprasystolic pressure, which results in limb ischemia. After 5 min of occlusion the cuff is deflated, resulting in reactive hyperemia and increases in wall shear rate that signal vasodilatory molecules to be released from the endothelium eliciting vasodilation. Despite the thousands of studies performing flow-mediated dilation in humans, surprisingly, no studies have performed this technique non-invasively in living rats. Considering the recent shift in focus to translational research, the establishment of guidelines for non-invasive measurement of flow-mediated dilation in rats and other rodents would be extremely valuable. In the following article, a protocol is presented for the non-invasive measurement of flow-mediated dilation in brachial and superficial femoral arteries of rats, as those sites are most commonly measured in humans.

Flow-mediated dilation was performed on the brachial and superficial femoral artery of 8 Wistar rats. Positioning of a rat is shown in Figure 1.
Representative ultrasound images of the superficial femoral artery are shown in Figure 2.
<img alt="Figure 1" src="/files/ftp_upload/54762/54762fig1.jpg" /> 
Figure 1. Rat and ultrasound positioning. 
Positioning of rat for measurement of brachial (A) and superficial femoral (B) artery FMD. Positioning of ultrasound probe and occlusion cuff for measurement of brachial (C) and superficial femoral (D) artery FMD. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54762/54762fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54762/54762fig2.jpg" /> 
Figure 2. Representative ultrasound images. 
Ultrasound image of the superficial femoral artery using B-mode imaging for determination of diameter (A). Blood velocity was determined using PW-mode. Shown in the figure are blood velocity at baseline (B), the reduction in blood velocity during the occlusion phase (C), and the rapid increase in blood velocity at cuff release during the hyperemic phase (D). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54762/54762fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
As shown in Figure 3, there is a similar vasodilatory response between the brachial and superficial femoral arteries. When expressed as a percent of baseline, FMD was similar between arteries, however when expressed as an absolute change from baseline, FMD was significantly higher in the brachial artery (P&#60;0.05). This difference is likely due to a larger vessel diameter in the brachial artery than in the superficial femoral artery (498&#177;28 vs. 397&#177;11 &#181;m, P&#60;0.05). Similar to measurement of FMD in humans16, intersession coefficient of variation for brachial and superficial femoral FMD were 9&#177;1 and 10&#177;4%, respectively. Despite differences in vessel size, there was a strong linear relationship between brachial and superficial femoral artery FMD when expressed as a percent or absolute change from baseline.
<img alt="Figure 3" src="/files/ftp_upload/54762/54762fig3.jpg" /> 
Figure 3. Flow-mediated dilation in rats. 
Vasodilation of the brachial and superficial femoralartery following a 5 min period of limb ischemia expressed as percent (A) and absolute (B) change from baseline. FMD was similar between arteries when expressed as a percent of baseline (C). However, when expressed as an absolute change from baseline (D), FMD was significantly higher in the brachial artery. Whether expressed as a percent (E) or absolute (F) change from baseline, there was a strong relationship for FMD between brachial and superficial femoral arteries. * P&#60;0.05 vs. Brachial Artery. Values are mean &#177; SEM. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54762/54762fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
As shown in Table 1, cardiovascular variables were measured during baseline, occlusion, and hyperemic phases. Artery diameter was determined using the intima-to-intima or media-to-media distance during the diastolic portion of the cardiac cycle. Artery diameter was measured for 60 seconds at baseline and continuously in ~60 second intervals through the occlusion and hyperemic phases. Blood velocity was determined using the mean arterial velocity of blood in the lumen during one cardiac cycle. Blood flow was calculated as per the equation: blood flow (&#181;l/min) = (blood velocity (&#181;m/sec) &#183; &#960; &#183; [vessel diameter (&#181;m)/2]2 &#183; 60). Shear rate was calculated according to the equation: shear rate (s-1) = blood velocity &#183; 8/vessel diameter. FMD was calculated according the equation: FMD = (peak vessel diameter - baseline vessel diameter)/baseline vessel diameter.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Brachial Artery</td>
			<td>Superficial Femoral Artery</td>
		</tr>
		<tr>
			<td>Baseline Phase</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160; Heart Rate, bpm</td>
			<td>367&#177;12</td>
			<td>368&#177;16</td>
		</tr>
		<tr>
			<td>&#160; Diameter, &#181;m</td>
			<td>498&#177;28</td>
			<td>397&#177;11*</td>
		</tr>
		<tr>
			<td>&#160; Blood Velocity, &#181;m/sec</td>
			<td>85&#177;8</td>
			<td>76&#177;11</td>
		</tr>
		<tr>
			<td>&#160; Blood Flow, &#181;l/min</td>
			<td>1,027&#177;147</td>
			<td>568&#177;90*</td>
		</tr>
		<tr>
			<td>&#160; Shear Rate, s-1</td>
			<td>1.4&#177;0.1</td>
			<td>1.5&#177;0.2</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Occlusion Phase</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160; Heart Rate, bpm</td>
			<td>362&#177;12</td>
			<td>359&#177;14</td>
		</tr>
		<tr>
			<td>&#160; Diameter, &#181;m</td>
			<td>499&#177;32</td>
			<td>390&#177;11*&#8224;</td>
		</tr>
		<tr>
			<td>&#160; Blood Velocity, &#181;m/sec</td>
			<td>63&#177;9&#8224;</td>
			<td>38&#177;8&#8224;</td>
		</tr>
		<tr>
			<td>&#160; Blood Flow, &#181;l/min</td>
			<td>722&#177;122&#8224;</td>
			<td>272&#177;62*&#8224;</td>
		</tr>
		<tr>
			<td>&#160; Shear Rate, s-1</td>
			<td>1.0&#177;0.2&#8224;</td>
			<td>0.8&#177;0.2&#8224;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hyperemic Phase</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160; Heart Rate, bpm</td>
			<td>363&#177;12</td>
			<td>357&#177;12</td>
		</tr>
		<tr>
			<td>&#160; Peak Diameter, &#181;m</td>
			<td>586&#177;22&#8224;&#8225;</td>
			<td>457&#177;15*&#8224;&#8225;</td>
		</tr>
		<tr>
			<td>&#160; Peak Blood Velocity, &#181;m/sec</td>
			<td>149&#177;11&#8224;&#8225;</td>
			<td>205&#177;12*&#8224;&#8225;</td>
		</tr>
		<tr>
			<td>&#160; Peak Blood Flow, &#181;l/min</td>
			<td>1,778&#177;229&#8224;&#8225;</td>
			<td>1,495&#177;127&#8224;&#8225;</td>
		</tr>
		<tr>
			<td>&#160; Peak Shear Rate, s-1</td>
			<td>2.5&#177;0.3&#8224;&#8225;</td>
			<td>3.7&#177;0.2*&#8224;&#8225;</td>
		</tr>
		<tr>
			<td>* P&#60;0.05 vs. Brachial Artery.&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#8224; P&#60;0.05 vs. Baseline Phase.&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#8225; P&#60;0.05 vs. Occlusion Phase.&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Values are mean &#177; SEM.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1. Cardiovascular variables through each phase of the protocol.
There were no changes in heart rate throughout either protocol, as well as between artery measurements (P&#62;0.05). The diameter of the brachial artery was significantly larger than the superficial femoral artery (P&#60;0.05). During the occlusion phase, there was a significant reduction in blood velocity, blood flow, and shear rate compared to baseline in both arteries (P&#60;0.05). Following cuff release, peak blood velocity, blood flow, and shear rate were all significantly higher than baseline or occlusion phases in both arteries (P&#60;0.05). Similar to measurement of reactive hyperemia in humans16, intersession coefficient of variation for brachial and superficial femoral reactive hyperemia were 24&#177;9 and 19&#177;5%, respectively. There were differences in blood velocity, blood flow, and shear rate between arteries, owing in large part to the difference in artery diameter.
After normalizing to peak shear rate, vasodilation was higher in the brachial artery (Figure 4). This was also true for FMD expressed as an absolute change from baseline. However, despite differences in magnitude, there was a strong linear relationship in percent and absolute FMD normalized to peak shear rate between the brachial and superficial femoral artery.
<img alt="Figure 4" src="/files/ftp_upload/54762/54762fig4.jpg" /> 
Figure 4. Flow-mediated dilation normalized to peak shear rate in rats. 
After normalizing to peak shear rate, FMD expressedas a percent (A) or absolute change (B) from baseline was higher in the brachial artery compared to the superficial femoral artery. 
Despite differences between arteries in FMD normalized to peak shear rate, there was a strong relationship between brachial and superficial femoral arteries when FMD was expressed as a percent (C) or absolute change (D) from baseline. * P&#60;0.05 vs. Brachial Artery. Values are mean &#177; SEM. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54762/54762fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Ultrasound Assessment of Flow-Mediated Dilation of the Brachial and Superficial Femoral Arteries in Rats at JoVE.com

Ultrasound Assessment of Flow-Mediated Dilation of the Brachial and Superficial Femoral Arteries in Rats

Non-invasive assessment of endothelial function in humans can be determined by the flow-mediated dilation technique. Although thousands of studies have used this technique, no study has performed this technique non-invasively in rats. The following article describes non-invasive measurement of flow-mediated dilation in the brachial and superficial femoral arteries of rats.

Arterial vasodilation to increases in wall shear rate is indicative of vascular endothelial function. In humans, the non-invasive measurement of ...

ultrasound-assessment-flow-mediated-dilation-brachial-superficial

Nanyang Technological University

Research

JoVE Journal

Medicine

11.6K Views.  University of Utah.  Niet-invasieve bepaling van de endotheelfunctie bij de mens kan worden bepaald door de flow gemedieerde dilatatie techniek. Hoewel duizenden studies deze techniek gebruikt, heeft geen enkele studie deze techniek uitgevoerd op niet-invasieve bij ratten. Het volgende artikel wordt niet-invasieve meting van de flow gemedieerde dilatatie in de brachiale en oppervlakkige femorale slagaders van ratten. 

Video: Ultrasound Assessment of Flow-Mediated Dilation of the Brachial and Superficial Femoral Arteries in Rats

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

Ultrasound Assessment of Endothelial-Dependent Flow-Mediated Vasodilation of the Brachial Artery in Clinical Research

The vascular endothelium is a monolayer of cells that cover the interior of blood vessels and provide both structural and functional roles. The endothelium acts as a barrier, preventing leukocyte adhesion and aggregation, as well as controlling permeability to plasma components. Functionally, the endothelium affects vessel tone.
Endothelial dysfunction is an imbalance between the chemical species which regulate vessel tone, thombroresistance, cellular proliferation and mitosis. It is the first step in atherosclerosis and is associated with coronary artery disease, peripheral artery disease, heart failure, hypertension, and hyperlipidemia.
The first demonstration of endothelial dysfunction involved direct infusion of acetylcholine and quantitative coronary angiography. Acetylcholine binds to muscarinic receptors on the endothelial cell surface, leading to an increase of intracellular calcium and increased nitric oxide (NO) production. In subjects with an intact endothelium, vasodilation was observed while subjects with endothelial damage experienced paradoxical vasoconstriction.
There exists a non-invasive, in vivo method for measuring endothelial function in peripheral arteries using high-resolution B-mode ultrasound. The endothelial function of peripheral arteries is closely related to coronary artery function. This technique measures the percent diameter change in the brachial artery during a period of reactive hyperemia following limb ischemia.
This technique, known as endothelium-dependent, flow-mediated vasodilation (FMD) has value in clinical research settings. However, a number of physiological and technical issues can affect the accuracy of the results and appropriate guidelines for the technique have been published. Despite the guidelines, FMD remains heavily operator dependent and presents a steep learning curve. This article presents a standardized method for measuring FMD in the brachial artery on the upper arm and offers suggestions to reduce intra-operator variability.

Endothelial dysfunction is associated with numerous disease states and is predictive of adverse cardiovascular events in humans. Flow-mediated vasodilation (FMD) is a non-invasive ultrasound method of evaluating endothelial function. Methodological choices and operator experience may affect results. A systematic approach to FMD in human studies is discussed here.

The vascular endothelium is a monolayer of cells that cover the interior of blood vessels and provide both structural and functional roles. The ...

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol.
This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We describe a technique for measuring aortic stiffness from its pressure-diameter relationship in vivo in mice. Aortic diameter is recorded by ultrasound and aortic pressure is measured invasively with a solid-state pressure catheter. Blood pressure is changed incrementally and the resulting diameter is measured.

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound ...

A Methodological Approach to Non-invasive Assessments of Vascular Function and Morphology

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis. Damage to the endothelium may predispose the vessel to atherosclerosis and increase the risk for cardiovascular disease. Assessments of peripheral endothelial function are good indicators of early abnormalities in the vascular wall and correlate well with assessments of coronary endothelial function. The present manuscript details the important methodological steps necessary for the assessment of microvascular endothelial function using laser Doppler imaging with iontophoresis, large vessel endothelial function using flow-mediated dilatation, and carotid atherosclerosis using carotid artery ultrasound. A discussion on the methodological considerations for each of the techniques is also presented, and recommendations are made for future research.

The present article describes the methodological considerations for several non-invasive assessments of vascular function and morphology that are commonly used in medical research to assess different stages of atherosclerosis.

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis.  Damage to the endothelium may ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Ultrasound Assessment of Endothelial Function: A Technical Guideline of the Flow-mediated Dilation Test

Cardiovascular disease is the primary cause of mortality and a major cause of disability worldwide. The dysfunction of the vascular endothelium is a pathological condition characterized mainly by a disruption in the balance between vasodilator and vasoconstrictor substances and is proposed to play an important role in the development of atherosclerotic cardiovascular disease. Therefore, a precise evaluation of endothelial function in humans represents an important tool that could help better understand the etiology of multiple cardio-centric pathologies.
Over the past twenty-five years, many methodological approaches have been developed to provide an assessment of endothelial function in humans. Introduced in 1989, the FMD test incorporates a forearm occlusion and subsequent reactive hyperemia that promotes nitric oxide production and vasodilation of the brachial artery. The FMD test is now the most widely utilized, non-invasive, ultrasonic assessment of endothelial function in humans and has been associated with future cardiovascular events.
Although the FMD test could have clinical utility, it is a physiological assessment that has inherited several confounding factors that need to be considered. This article describes a standardized protocol for determining FMD including the recommended methodology to help minimize the physiological and technical issues and improve the precision and reproducibility of the assessment.

The flow mediated dilation (FMD) test is the most commonly utilized, non-invasive, ultrasound assessment of endothelial function in humans. Although the FMD test has been related with the prediction of future cardiovascular disease and events, it is a physiological assessment with many inherent confounding factors that need to be considered.

Cardiovascular disease is the primary cause of mortality and a major cause of disability worldwide. The dysfunction of the vascular endothelium is a ...

Ultrasound-based Pulse Wave Velocity Evaluation in Mice

Arterial stiffness can be evaluated by calculating pulse wave velocity (PWV), i.e., the speed with which the pulse wave travels in a conduit vessel. This parameter is being increasingly investigated in small rodent models in which it is used for assessing alterations in vascular function related to particular genotypes/treatments or for characterizing cardiovascular disease progression. This protocol describes an image processing algorithm which leads to non-invasive arterial PWV measurement in mice using ultrasound (US) images only. The proposed technique has been used to assess abdominal aorta PWV in mice and evaluate its age-associated changes.
Abdominal aorta US scans are obtained from mice under gaseous anesthesia using a specific US device equipped with high-frequency US probes. B-mode and Pulse-Wave Doppler (PW-Doppler) images are analyzed in order to obtain diameter and mean velocity instantaneous values, respectively. For this purpose, edge detection and contour tracking techniques are employed. The single-beat mean diameter and velocity waveforms are time aligned and combined in order to achieve the diameter-velocity (lnD-V) loop. PWV values are obtained from the slope of the linear part of the loop, which corresponds to the early systolic phase.
With the present approach, anatomical and functional information about the mouse abdominal aorta can be non-invasively achieved. Requiring the processing of US images only, it may represent a useful tool for the non-invasive characterization of different arterial sites in the mouse in terms of elastic properties. The application of the present technique can be easily extended to other vascular districts, such as the carotid artery, thus providing the possibility to obtain a multi-site arterial stiffness assessment.

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.

Arterial stiffness can be evaluated by calculating pulse wave velocity (PWV), i.e., the speed with which the pulse wave travels in a conduit vessel. This ...

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

Measurement of Pulse Propagation Velocity, Distensibility and Strain in an Abdominal Aortic Aneurysm Mouse Model

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 times of its original size. Clinical and experimental studies have shown that small aneurysms may rupture, while a subpopulation of large aneurysms may remain stable. Thus, in addition to the measurement of intraluminal diameter of the aorta, knowledge of structural traits of the vessel wall may provide important information to assess the stability of the AAA. Aortic stiffening has recently emerged as a reliable tool to determine early changes in the vascular wall. Pulse propagation velocity (PPV) along with the distensibility and radial strain are highly useful ultrasound-based methods relevant for assessing aortic stiffness. The primary purpose of this protocol is to provide a comprehensive technique for the use of ultrasound imaging system to acquire images and analyze the structural and functional properties of the aorta as determined by MILD, PPV, distensibility and radial strain.

This manuscript describes a detailed protocol for using high frequency ultrasound imaging to measure luminal diameter, pulse propagation velocity, distensibility and radial strain on a mouse model of abdominal aortic aneurysm.

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 ...

Immunology and Infection

Using Arterial Stiffness and Endothelial Function for Cardiovascular Assessment

Pulse-Wave Velocity, Flow-Mediated Dilation, and Carotid Intima-Media Thickness to Assess Cardiovascular Risk in Population with Metabolic Syndrome

Pulse-wave velocity (PWV), flow-mediated dilation (FMD), and carotid intima-media thickness (CIMT) are established methods used in research and clinical settings to assess arterial stiffness, endothelial function, and subclinical atherogenesis. These measurements may reflect vascular disease and atherosclerotic progression, which are major causes of adverse cardiovascular events. These methods are particularly valuable in determining cardiovascular dysfunction among populations with different risk factors, such as diabetes mellitus, hypertension, and other metabolic dysfunction-related conditions. They provide a non-invasive and reliable source of information that complements clinical practice. Early detection, risk assessment, and therapeutic decisions regarding cardiovascular disease can be achieved, ultimately contributing to improved patient outcomes. Traditional tools for evaluating cardiovascular disease do not reveal whether metabolic syndrome affects early subclinical cardiovascular disease in patients with obesity. Recent research has highlighted the importance of including arterial stiffness and endothelial function in a comprehensive cardiovascular assessment. Therefore, the aim of the present study is to describe methods that provide information on early subclinical vascular aging, endothelial dysfunction, and atherogenic disease, enabling vascular-targeted risk stratification among populations with obesity and different metabolic profiles.

Author Spotlight: Assessing the Cardiovascular Profile of Patients with Metabolic Syndrome

Endothelial/vascular aging and atherogenesis are key mechanisms that promote the development of cardiovascular diseases. The present protocol describes methods to evaluate arterial stiffness, endothelial dysfunction, and atherogenesis in patients with related risk factors, which are highly valuable in the cardiovascular research field.

Pulse-wave velocity (PWV), flow-mediated dilation (FMD), and carotid intima-media thickness (CIMT) are established methods used in research and clinical ...

Ultrasound Assessment of Flow-Mediated Dilation of the Brachial and Superficial Femoral Arteries in Rats

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