Name: ultrasound assessment of flow-mediated dilation of the brachial and superficial femoral arteries in rats
Uploaded: 2016-11-03T13:00:00
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 ...

<ol>
	<li>Smits, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+release+of+nitric+oxide+contributes+to+the+vasodilator+effect+of+adenosine+in+humans.">Endothelial release of nitric oxide contributes to the vasodilator effect of adenosine in humans.</a> Circulation. 92, 2135-2141 (1995).</li><li>Forstermann, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+synthase+isozymes.+Characterization,+purification,+molecular+cloning,+and+functions.">Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions.</a> Hypertension. 23, 1121-1131 (1994).</li><li>Gardiner, S. M., Compton, A. M., Bennett, T., Palmer, R. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+ultrasound+assessment+of+endothelial-dependent+flow-mediated+vasodilation+of+the+brachial+artery:+a+report+of+the+International+Brachial+Artery+Reactivity+Task+Force.">Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery Reactivity Task Force.</a> J Am Coll Cardiol. 39, 257-265 (2002).</li><li>Celermajer, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+detection+of+endothelial+dysfunction+in+children+and+adults+at+risk+of+atherosclerosis.">Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis.</a> Lancet. 340, 1111-1115 (1992).</li><li>Niebauer, J., Cooke, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+effects+of+exercise:+role+of+endothelial+shear+stress.">Cardiovascular effects of exercise: role of endothelial shear stress.</a> J Am Coll Cardiol. 28, 1652-1660 (1996).</li><li>Sessa, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=eNOS+at+a+glance.">eNOS at a glance.</a> J Cell Sci. 117, 2427-2429 (2004).</li><li>Wray, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+brachial+artery+flow-mediated+vasodilation+provide+a+bioassay+for+NO?.">Does brachial artery flow-mediated vasodilation provide a bioassay for NO?.</a> Hypertension. 62, 345-351 (2013).</li><li>Green, D. J., Dawson, E. A., Groenewoud, H. M., Jones, H., Thijssen, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+flow-mediated+dilation+nitric+oxide+mediated?+A+meta-analysis.">Is flow-mediated dilation nitric oxide mediated? A meta-analysis.</a> Hypertension. 63, 376-382 (2014).</li><li>Green, D. J., Jones, H., Thijssen, D., Cable, N. T., Atkinson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-mediated+dilation+and+cardiovascular+event+prediction:+does+nitric+oxide+matter?.">Flow-mediated dilation and cardiovascular event prediction: does nitric oxide matter?.</a> Hypertension. 57, 363-369 (2011).</li><li>Brevetti, G., Silvestro, A., Schiano, V., Chiariello, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+dysfunction+and+cardiovascular+risk+prediction+in+peripheral+arterial+disease:+additive+value+of+flow-mediated+dilation+to+ankle-brachial+pressure+index.">Endothelial dysfunction and cardiovascular risk prediction in peripheral arterial disease: additive value of flow-mediated dilation to ankle-brachial pressure index.</a> Circulation. , 2093-2098 (2003).</li><li>Gokce, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predictive+value+of+noninvasively+determined+endothelial+dysfunction+for+long-term+cardiovascular+events+in+patients+with+peripheral+vascular+disease.">Predictive value of noninvasively determined endothelial dysfunction for long-term cardiovascular events in patients with peripheral vascular disease.</a> J Am Coll Cardiol. 41, 1769-1775 (2003).</li><li>National Research Council (U.S.). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide for the care and use of laboratory animals. , (2011).</li><li>Alley, H., Owens, C. D., Gasper, W. J., Grenon, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+assessment+of+endothelial-dependent+flow-mediated+vasodilation+of+the+brachial+artery+in+clinical+research.">Ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery in clinical research.</a> J Vis Exp. , e52070 (2014).</li><li>Ghiadoni, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+flow-mediated+dilation+reproducibility:+a+nationwide+multicenter+study.">Assessment of flow-mediated dilation reproducibility: a nationwide multicenter study.</a> J Hypertension. 30, 1399-1405 (2012).</li><li>Thijssen, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+conduit+artery+function+in+humans:+impact+of+arterial+size.">Heterogeneity in conduit artery function in humans: impact of arterial size.</a> Am J Physiol Heart Circ. 295, H1927-H1934 (2008).</li><li>Green, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+isn't+flow-mediated+dilation+enhanced+in+athletes?.">Why isn't flow-mediated dilation enhanced in athletes?.</a> Med Sci Sports. 45, 75-82 (2013).</li><li>Heiss, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+measurement+of+flow-mediated+vasodilation+in+living+rats+using+high-resolution+ultrasound.">In vivo measurement of flow-mediated vasodilation in living rats using high-resolution ultrasound.</a> Am J Physiol Heart Circ. 294, H1086-H1093 (2008).</li><li>Chen, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+inhibition+of+S-nitrosoglutathione+reductase+improves+endothelial+vasodilatory+function+in+rats+in+vivo.">Pharmacological inhibition of S-nitrosoglutathione reductase improves endothelial vasodilatory function in rats in vivo.</a> J Appl Physiol. 114, 752-760 (2013).</li><li>Pinnamaneni, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+exposure+to+secondhand+smoke+reversibly+impairs+endothelial+vasodilatory+function.">Brief exposure to secondhand smoke reversibly impairs endothelial vasodilatory function.</a> Nicotine Tob Res. 16, 584-590 (2014).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairment+of+Endothelial+Function+by+Little+Cigar+Secondhand+Smoke.">Impairment of Endothelial Function by Little Cigar Secondhand Smoke.</a> Tob Regul Sci. 2, 56-63 (2016).</li><li>Schuler, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+endothelium-dependent+vasodilation+in+mice--brief+report.">Measurement of endothelium-dependent vasodilation in mice--brief report.</a> Arterioscler Thromb Vasc Biol. 34, 2651-2657 (2014).</li><li>Erkens, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left+ventricular+diastolic+dysfunction+in+Nrf2+knock+out+mice+is+associated+with+cardiac+hypertrophy,+decreased+expression+of+SERCA2a,+and+preserved+endothelial+function.">Left ventricular diastolic dysfunction in Nrf2 knock out mice is associated with cardiac hypertrophy, decreased expression of SERCA2a, and preserved endothelial function.</a> Free Radic Biol Med. 89, 906-917 (2015).</li><li>Harris, S. 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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. A...

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 border="0" cellpadding="0" cellspacing="0" width="710">
	<tbody>
		<tr height="58">
			<td height="58" style="height:58px;width:203px;">Vevo 2100 High Resolution Micro-Ultrasound Imaging System</td>
			<td style="width:169px;">VisualSonics, Toronto, ON, CAN</td>
			<td style="width:169px;"></td>
			<td style="width:169px;"></td>
		</tr>
		<tr height="77">
			<td height="77" style="height:77px;width:203px;">MicroScan Ultra-High Frequency Linear Array Transducer - MS-700 30-70 MHz</td>
			<td style="width:169px;">VisualSonics, Toronto, ON, CAN</td>
			<td style="width:169px;"></td>
			<td style="width:169px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Vevo Imaging Station</td>
			<td style="width:169px;">VisualSonics, Toronto, ON, CAN</td>
			<td style="width:169px;"></td>
			<td style="width:169px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Thermasonic gel warmer</td>
			<td style="width:169px;">Parker Laboratories, Fairfield, NJ, USA</td>
			<td style="width:169px;">82-03</td>
			<td style="width:169px;">Optional</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Signacreme electrode cream</td>
			<td style="width:169px;">Parker Laboratories, Fairfield, NJ, USA</td>
			<td style="width:169px;">17-05</td>
			<td style="width:169px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Transpore surgical tape</td>
			<td style="width:169px;">3M, Maplewood, MN, USA</td>
			<td style="width:169px;">1527-1</td>
			<td style="width:169px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Depilatory cream (e.g., Nair)</td>
			<td style="width:169px;">General supply</td>
			<td style="width:169px;"></td>
			<td style="width:169px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Cotton swabs</td>
			<td style="width:169px;">General supply</td>
			<td style="width:169px;"></td>
			<td style="width:169px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Ultrasound gel</td>
			<td style="width:169px;">General supply</td>
			<td style="width:169px;"></td>
			<td style="width:169px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Standard vascular occluder, 10 mm lumen diameter</td>
			<td style="width:169px;">Harvard Apparatus, Holliston, MA, USA</td>
			<td style="width:169px;">62-0115</td>
			<td style="width:169px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">10 ml syringe with Luer-Lok tip</td>
			<td style="width:169px;">General Supply</td>
			<td style="width:169px;"></td>
			<td style="width:169px;">Used for occlusion cuff apparatus</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Paperclip</td>
			<td style="width:169px;">General Supply</td>
			<td style="width:169px;"></td>
			<td style="width:169px;">Used for occlusion cuff apparatus</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Hypodermic needle &#8211; 18 gauge&#160;</td>
			<td style="width:169px;">General Supply</td>
			<td style="width:169px;"></td>
			<td style="width:169px;">Used for occlusion cuff apparatus</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Medium binder clip</td>
			<td style="width:169px;">General Supply</td>
			<td style="width:169px;"></td>
			<td style="width:169px;">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. <s...

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 ...

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.

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Medicine

A Novel Application of Musculoskeletal Ultrasound Imaging

Ultrasound is an attractive modality for imaging muscle and tendon motion during dynamic tasks and can provide a complementary methodological approach for biomechanical studies in a clinical or laboratory setting. Towards this goal, methods for quantification of muscle kinematics from ultrasound imagery are being developed based on image processing. The temporal resolution of these methods is typically not sufficient for highly dynamic tasks, such as drop-landing. We propose a new approach that utilizes a Doppler method for quantifying muscle kinematics. We have developed a novel vector tissue Doppler imaging (vTDI) technique that can be used to measure musculoskeletal contraction velocity, strain and strain rate with sub-millisecond temporal resolution during dynamic activities using ultrasound. The goal of this preliminary study was to investigate the repeatability and potential applicability of the vTDI technique in measuring musculoskeletal velocities during a drop-landing task, in healthy subjects. The vTDI measurements can be performed concurrently with other biomechanical techniques, such as 3D motion capture for joint kinematics and kinetics, electromyography for timing of muscle activation and force plates for ground reaction force. Integration of these complementary techniques could lead to a better understanding of dynamic muscle function and dysfunction underlying the pathogenesis and pathophysiology of musculoskeletal disorders.

We describe a new ultrasound-based vector tissue Doppler imaging technique to measure muscle contraction velocity, strain and strain rate with sub-millisecond temporal resolution during dynamic activities. This approach provides complementary measurements of dynamic muscle function and could lead to a better understanding of mechanisms underlying musculoskeletal disorders.

Ultrasound is an attractive modality for imaging muscle and tendon  motion during dynamic tasks and can provide a complementary methodological  approach ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

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 ...

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 ...

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 ...

A Pleural Effusion Model in Rats by Intratracheal Instillation of Polyacrylate/Nanosilica

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study these pulmonary diseases. Previous pleural effusion models paid more attention to biological factors rather than nanoparticles in the environment. Here, we introduce a model to make pleural effusion in rats by intratracheal instillation of polyacrylate/nanosilica, and a method of nanoparticle isolation in the pleural effusion. By intratracheal instillation of polyacrylate/nanosilica with concentrations of 3.125, 6.25 and 12.5 mg/kg&#8729;mL, the pleural effusion in rats presented on day 3, peaked at days 7-10 in 6.25 and 12.5 mg/kg&#8729;mL groups, then slowly decreased and disappeared on day 14. When the concentration of polyacrylate/nanosilica increased, the pleural effusion is produced more and faster. This pleural fluid was detected by ultrasound examination or CT chest scanning and confirmed by dissection of rats. Silica nanoparticles were observed in the rats' pleural effusion by transmission electron microscope. These results showed that the exposure to polyacrylate/nanosilica leads to the induction of pleural effusion, which was consistent with our previous report in humans. Additionally, this model is beneficial for the further study of nanotoxicology and the pleural effusion diseases.

Here, we present a protocol to construct a pleural effusion model in rats by intratracheal instillation of polyacrylate/nanosilica.

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study ...

Brachial Artery Catheterization in Swine

The video describes in detail the catheterization of the distal brachial artery in swine. This technique enables researchers to measure arterial blood pressure continuously and collect arterial blood samples to assess arterial blood gas measurements. Arterial blood pressures and arterial blood gases are important physiological parameters to monitor during experimental procedures. In swine, four common methods of arterial catheterization have been described, including catheterization of the carotid, femoral, auricular, and medial saphenous arteries. Each of these techniques have advantages, such as ease of access for the auricular artery, and disadvantages that include deep tissue dissection for carotid artery catheterization. The described alternative method of arterial catheterization in swine, the catheterization of the distal aspect of the brachial artery, is a rapid procedure that requires relatively minimal tissue dissection and provides information that is in line with data collected from other arterial catheterization sites. The procedure uses a medial approach along an oblique plane of the lower brachium, positioned between the olecranon and the flexor aspect of the elbow joint, and this approach allows researchers the major advantage of unimpeded freedom for procedures that involve the caudoventral, caudodorsal back, or hind limbs of the pig. Due to the location of the upper forelimb of the catheterized vessel and potential challenges of effective homeostasis following catheter removal from the artery, this technique may be limited to non-recovery procedures.

The video describes in detail the catheterization of the distal brachial artery in swine. This procedure accurately measures arterial blood pressure and is a simple and fast method to collect samples for arterial blood gas measurements.

The video describes in detail the catheterization of the distal brachial artery in swine. This technique enables researchers to measure arterial blood ...

Echocardiographic Assessment of Cardiac Anatomy and Function in Adult Rats

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional imaging to study the cardiac anatomy of the left ventricle and M-mode echo to assess its dimensions. However, this could limit a comprehensive study. Herein, we describe a protocol that allows an assessment of the heart chamber size, left ventricular function (systolic and diastolic) and valvular function. A conventional medical ultrasound machine was used in this protocol and different echo views were obtained through left parasternal, apical and suprasternal windows. In the left parasternal window, the long and short axis were acquired to analyze left chamber dimensions, right ventricle and pulmonary artery dimensions, and mitral, pulmonary and aortic valve function. The apical window allows the measurement of heart chamber dimensions and evaluation of systolic and diastolic parameters. It also allows Doppler assessment with detection and quantification of heart valve disturbances (regurgitation or stenosis). Different segments and walls of the left ventricle are visualized throughout all views. Finally, the ascending aorta, aortic arch, and descending aorta can be imaged through the suprasternal window. A combination of ultrasound imaging, Doppler flow and tissue Doppler assessment have been obtained to study cardiac morphology and function. This represents an important contribution to improve the assessment of cardiac function in adult rats with impact for research using these animal models.

A non-invasive protocol for transthoracic echocardiography assessment of cardiac anatomy and function for adult rats is presented in the current study. The heart valves, all four cardiac chambers and the ascending aorta, aortic arch and descending aorta are studied in detail.

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional ...

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart methods.&#160;

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...