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 ...

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 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 ...

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 ...

Providing Visual Biofeedback Using Brightness Mode Ultrasound During a Golf Swing

Using ultrasound biofeedback in conjunction with verbal cueing can increase muscle thickness more than verbal cueing alone and may augment traditional rehabilitation techniques in an athletic, physically active population. Brightness mode (B-mode) ultrasound can be applied using frame-by-frame analysis synchronized with video to understand muscle thickness changes during these dynamic tasks. Visual biofeedback with ultrasound has been established in static positions for the muscles of the lateral abdominal wall. However, by securing the transducer to the abdomen using an elastic belt and foam block, biofeedback can be applied during more specific tasks prevalent in lifetime sports, such as golf. To analyze muscle activity during a golf swing, muscle thickness changes can be compared. The thickness must increase throughout the task, indicating that the muscle is more active. This methodology allows clinicians to immediately replay ultrasound videos for patients as a visual tool to instruct proper activity of the muscles of interest. For example, ultrasound can be used to target the external and internal obliques, which play an important role in swinging a golf club or any other rotational sport or activity. This methodology aims to increase oblique muscle thickness during the golf swing. Additionally, the timing of muscle contraction can be targeted by instructing the patient to contract the abdominal muscles at specific time points, such as the beginning of the downswing, with the goal of improving muscle firing patterns during tasks.

Brightness mode ultrasound can be used to provide visual biofeedback of the muscles of the lateral abdominal wall during a golf swing. Post-swing visual and verbal instruction can increase the muscle activation and timing of the external and internal obliques.

Using ultrasound biofeedback in conjunction with verbal cueing can increase muscle thickness more than verbal cueing alone and may augment traditional ...

Muscle Function Obtained with Motion Mode Ultrasound and Surface Electromyography during Core Endurance Exercise

Motion mode (M-mode) ultrasound allows researchers and clinicians to measure the change of muscle thickness across time. Muscle thickness can be measured between fascial borders at a given time point during an exercise. This selected time point produces a one-dimensional image resulting in real-time, live observation of anatomy. Ultrasound used during functional movement can be referred to as dynamic ultrasound; this is feasible and reliable with the use of a linear transducer, elastic belt, and foam block to secure consistent transducer placement. The lateral abdominal wall is commonly investigated using ultrasound due to the overlapping nature of the muscles. Surface electromyography (sEMG) can complement M-mode ultrasound imaging because it measures the electrical representation of muscle activation. There is minimal evidence using M-mode ultrasound and sEMG simultaneously during core exercise. Exercises that challenge the core musculature involve both isometric holds (e.g., side plank), as well as oscillatory extremity movements (e.g., dead bug). In this study, both instruments will be used simultaneously to measure core muscle function during exercise. Ultrasound measurements will be obtained using a linear transducer and ultrasound unit, and sEMG measurements will be acquired from a wireless sEMG system. To make comparisons between participants and exercises, normalization methods using static, exercise starting positions for both instruments will be used. An activation ratio will be used for ultrasound and calculated by dividing the contracted thickness (thickness during a time point of exercise) by the rested (starting position) thickness. Muscle thickness will be measured in centimeters from the superior inferior fascial border to inferior superior fascial border. These methods aim to offer an innovative and practical measurement of muscle function with M-mode ultrasound and sEMG during core endurance exercises.

This protocol uses motion mode ultrasound and surface electromyography simultaneously to measure muscle function of the core. Muscle thickness and activation of the local stabilizers (e.g., transverse abdominis, internal oblique) and global movers (e.g., external oblique) is achievable during specific time points of the side plank and dead bug exercises.

Motion mode (M-mode) ultrasound allows researchers and clinicians to measure the change of muscle thickness across time. Muscle thickness can be measured ...

Orthopedic Robot-Assisted Femoral Neck System in the Treatment of Femoral Neck Fracture

Cannulated screw fixation is the main therapy for femoral neck fractures, especially in young patients. The traditional surgical procedure uses C-arm fluoroscopy to place the screw freehand and requires several guide wire adjustments, which increases the operation time and radiation exposure. Repeated drilling can also cause damage to the blood supply and bone quality of the femoral neck, which can be followed by complications such as screw loosening, nonunion, and femoral head necrosis. In order to make fixation more precise and reduce the incidence of complications, our team applied robot-assisted orthopedic surgery for screw placement using the femoral neck system to modify the traditional procedure. This protocol introduces how to import a patient's X-ray information into the system, how to perform screw path planning in software, and how the robotic arm assists in screw placement. Using this method, the surgeons can place the screw successfully the first time, improve the accuracy of the procedure, and avoid radiation exposure. The whole protocol includes the diagnosis of femoral neck fracture; the collection of intraoperative X-ray images; screw path planning in the software; precise placement of the screw under the assistance of the robotic arm by the surgeon; and verification of the implant placement.

This article introduces a method of robot-assisted orthopedic surgery for screw placement during the treatment of femoral neck fracture using the femoral neck system, which allows for more accurate screw placement, improved surgical efficiency, and fewer complications.

Cannulated screw fixation is the main therapy for femoral neck fractures, especially in young patients. The traditional surgical procedure uses C-arm ...

Jugular Vein and Femoral Artery Cannulation with Hemorrhagic Shock Induction and Resuscitation in Rats

To begin, insert a lubricated rectal probe to control the temperature of the anesthetized rat during surgery. After locating the jugular region in the lower right neck region of the anesthetized rat above the clavicle, use DeBakey Atraumatic Forceps to gently grasp the skin and make a precise incision with fine sharp scissors or a scalpel. Gently slash the tissue with standard pattern forceps.Locate and gently liberate the jugular vein from its muscular compartment. Place a 4-0 silk suture thread to securely ligate the distal side of the jugular vein. Use the suture with the needle holders to tension the jugular vein.Place a 4-0 suture on the proximal side of the jugular vein and prepare a surgeon knot without tightening it. Using Vannas micro dissecting scissors, make a small incision in the jugular vein. After carefully grasping the vein wall with small forceps, insert the PE50 catheter in the jugular vein.Slightly advance the catheter, and to confirm its correct placement in the vein, draw a small amount of blood and re-inject it. Tighten the previously prepared knot to secure the catheter. Apply a drop of lidocaine to the incised area.Cover the incision with a moist sterile gauze pad. Use DeBakey Atraumatic Forceps to grasp the skin of the Scarpa triangle on the left leg, and make an incision with fine sharp scissors or a scalpel. Gently dilacerate tissue with standard pattern forceps.Locate the femoral triad and place one forceps under the triad. Then with a second standard pattern forceps, carefully separate the artery from the nerve and femoral vein. Place a 4-0 suture to ligate the femoral artery distally and take the suture with the needle holders to put the femoral artery in tension.Now place a 4-0 suture on the proximal side of the femoral artery and prepare a surgeon knot without closing it. After clamping the artery, use Vannas micro dissecting scissors to make a transverse incision in the femoral artery. Grasp the artery wall with standard pattern forceps and cannulate the femoral artery with the catheter holding it with forceps.Gently unclamp the artery and ensure that the blood is not flowing back into the catheter. If the signal is good, advance the catheter slightly into the artery and tighten the previously prepared surgeon knot. Administer heparin into the rat, and place a moistened sterile gauze pad over the incised area.After 10 minutes of stabilization, record the basal hemodynamic values for five minutes. To induce hemorrhagic shock, use a one milliliter syringe to draw five milliliters of blood from the femoral artery over five minutes. Prepare a mix of 50%lactated Ringer solution and 50%collected blood at room temperature.Place a two milliliter syringe filled with the mix at the end of the jugular vein cannula. For hemorrhagic shock maintenance, when mean arterial pressure exceeds 38 millimeters of mercury, withdraw 200 microliters of blood from the femoral artery. If pressure drops below 32 millimeters of mercury, inject 200 microliters of the 50%blood, 50%lactated Ringer's mixture through the jugular vein.After sterile preparation of the tail with a gauze pad, use the tip of the needle to draw a blood drop from the end of the tail to measure peripheral blood lactate. Finally, resuscitate the rat with a lactated Ringer solution using a 20 milliliter syringe through the jugular catheter with a syringe pump.