From the Vascular Integrated Physiology and Experimental Therapeutics (VIPERx) Laboratory, this work was supported by funds from the Department of Surgery, University of California, San Francisco and the Northern California Institute for Research and Education. The project described was supported by Award Number KL2RR024130 from the National Center for Research Resources. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health.

The following procedure, developed as part of an investigator-initiated study, was reviewed and approved by the University of California, San Francisco (UCSF) Committee on Human Research (CHR) and all participants gave informed consent.
1. Equipment
<ol>
	<li>Use an EKG gated image capture system to record and analyze the FMD. Connect a Philips HD11 ultrasound to a desktop PC.</li>
	<li>Connect a video signal from the ultrasound with a special frame-grabber card on the PC.</li>
	<li>Relay an audio signal from the ultrasound to an EKG gating module which amplifies the signal. Carry the amplified signal to the PC, to allow the image capture software to identify and record images at a consistent point in the cardiac cycle. Generate the signal from the sharp deflection of the R-wave in the EKG.</li>
	<li>Use a 5-12 MHz linear array transducer to optimize resolution at the depth of the brachial artery.</li>
</ol>
2. Subject Preparation
<ol>
	<li>Ensure that participants fast and avoid exercise for 8 hr&#160;before the exam as well as avoid caffeine or nicotine for at least four fours. Ensure that participants avoid medications affecting vascular tone or cardiac output for four half-lives. 
	NOTE: Diet, medications, and time of day may affect results.</li>
	<li>Conduct the exam in a quiet, darkened room at 21 &#176;C. When conducting longitudinal studies, hold repeat exams at the same time of day.</li>
</ol>
3. Baseline Measurements
<ol>
	<li>Ask the subject to lie supine on an exam table. Attach a 3-lead EKG in a standard position. Address any orthopedic issues to ensure the subject will be comfortable and refrain from movement during the exam.</li>
	<li>Allow the subject to rest for 10 min before the start of the exam. After 5 min rest, measure the subject&#39;s blood pressure by an oscillometric, non-invasive blood-pressure monitor.
	<ol>
		<li>Apply a 5 cm tourniquet cuff in either a proximal or distal position to demonstrate the upper arm technique.</li>
		<li>Extend the subject&#8217;s arm laterally and maintain at the level of the heart.</li>
		<li>Depending upon the operator&#39;s preference, use a table and pillow to constrain the subject&#8217;s arm.</li>
		<li>Place the arm of the operator in a position that resists fatigue and provides support for the wrist. Try to minimize extension of the wrist and keep the forearm in the anatomic neutral position.</li>
	</ol>
	</li>
	<li>Conduct a cross-sectional scan of the brachial artery, beginning at the insertion of the bicep and proceeding proximally. Use color flow imaging to verify the brachial artery and to locate collateral vessels that may serve as landmarks.</li>
	<li>When a suitable position is found, rotate the probe 90&#176; so that the proximal edge appears on the left of the ultrasound screen. Maintain position on the artery using substantial practice and a delicate touch. Verify the orientation by pushing the tissue near the distal edge. Mark the subject&#8217;s skin along the distal edge of the probe.</li>
	<li>Align the focus setting of the probe with the deep or &#8220;far&#8221; wall of the brachial artery to improve lateral resolution of the image. Vary probe settings on the axial resolution with a higher frequency improving axial resolution.</li>
	<li>Adjust the angle of the probe to optimize contrast resolution of both the near and the far walls. Small changes to the angle can result in improved contrast. Estimate the angle with a simple protractor if serial exams are conducted on the subject.</li>
	<li>To ensure quality measurements, ensure that the vessel is horizontal and aligned with the longitudinal axis. Make small changes in pressure (heeling one edge of the probe) to help align the artery. Overall, keep pressure light to help prevent operator fatigue.</li>
	<li>When optimized, ensure that the &#8220;Double Lines of Pignoli&#8221; can be seen in both walls, corresponding to the intima-media thickness. Use gain adjustments to reduce echo in the vessel lumen. Allow at least 2 cm of intima-mediate thickness (IMT) on both sides for accurate diameter measurements.</li>
</ol>
4. Baseline Measurements
<ol>
	<li>Record the baseline velocity using 2D Doppler mode. Place the sample gates in the middle of the lumen and maintain an insonation angle of 60&#176;. Collect 60 sec of data.</li>
</ol>
5. Occlusion Phase
<ol>
	<li>Inflate the cuff to 50 mm Hg above the subject&#8217;s systolic blood pressure. Using a 5 cm tourniquet cuff will overestimate the systolic pressure. Use the 2D Doppler mode to verify occlusion.</li>
	<li>Use a timer to track the duration of occlusion as many blood pressure cuffs will slowly lose pressure over 5 min. Use 2D Doppler mode to verify complete occlusion.</li>
	<li>After 4:30 of occlusion, place the 2D-Doppler gate slightly superficial to the longitudinal axis of the artery. Adjust the vertical scale to account for velocities 2-3x higher than baseline.</li>
	<li>Adjust the settings on the image capture software for 3:10 of recording.</li>
	<li>Begin recording 10 sec before cuff release to capture the time of cuff release, an important parameter when measuring time to peak diameter during data analysis.</li>
</ol>
6. Hyperemia
<ol>
	<li>Release the cuff. As the artery may shift superficially after cuff release, make small changes to the probe&#39;s position while listening for amplification of the sound to help compensate for the shift. Reposition the Doppler sample gate and insonation angle if the artery shifts.</li>
	<li>After 30 sec of velocity recording, switch the ultrasound to B-Mode.</li>
	<li>Since it is common for the probe to slide proximally during an exam, use vessel landmarks or the marking on the subjects&#39; skin to verify probe position. This phase of the exam is critical for obtaining accurate results.</li>
	<li>Adjust the probe position or angle to optimize the IMT on both walls as small changes can substantially improve the image. Record the diameter for 3 min.</li>
	<li>If repeat measurements are planned, use the marking on the subject&#39;s skin to record distance from the antecubital fossa. Ask the subject to bend their arm 90&#176; and mark the crease. Measure from this line to the line made earlier.</li>
</ol>

The authors have nothing to disclose.

Endothelial dysfunction is an imbalance in the chemical mediators affecting vessel tone and an early step in the development of atherosclerosis. Measuring the reactivity of an artery is a way to assess the state of these chemical pathways. Both direct and indirect methods of assessing reactivity exist for the different vascular beds, ranging from direct infusion of an endothelium agonist in the coronary circulation to non-invasive, pulse waveform analysis in the index finger38.
Brachial artery FMD is an established technique for indirectly assessing endothelial function by high-frequency ultrasound. There are advantages to using FMD in clinical trials. First, the technique is non-invasive and easy to understand, which eases barriers to recruitment. Furthermore, use of invasive techniques, such as coronary angiography in asymptomatic patients raises ethical questions. Next, FMD requires a minimum amount of subject preparation compared to invasive techniques and the entire exam may be completed in a short amount of time. So long as the occlusion duration is kept within guidelines and subjects screened appropriately, FMD presents little concern for safety. The relative ease of an FMD study makes serial exams in longitudinal studies feasible, but its use in testing treatment effects is controversial. Likewise, according to current clinical guidelines, FMD is not appropriate for characterizing an individual&#8217;s risk for cardiovascular events or making clinical decisions39. The development of analysis software allows for quick analysis, blinding, and repeat analysis. Finally, technique is well established and a number of studies have been published using the technique, allowing for comparison of results40.
There are, however, challenges to the successful use of FMD in a trial. First, the technique presents a long learning curve. Current guidelines suggest a new technician complete 100 scans under an experienced operator before working independently. Next, the cost of equipment may be prohibitive for smaller laboratories. Subject preparation is important as factors, such as smoking, medications, post-prandial state, hyperglycemia, time-of-day, ambient temperature, and recent exercise can affect the magnitude of the response19,29. This requires careful instruction to the participant and rigorous adherence to study protocols. In older individuals, changes to vessel distensibility may diminish the predictive value of FMD19.
The protocol, as described above, measures %FMD at 60 sec post-occlusion. Studies have suggested peak FMD may occur outside of this window41,42. This protocol allows for capture of peak FMD by continuous recording for 3 min post-deflation. It should be noted this requires extensive experience and leads to longer analysis time. Likewise, the choice of upper-arm occlusion is controversial. In a study that compared different occlusion positions with similar shear stress stimuli, greater vasodilation was seen with the upper arm technique, suggesting some component of the dilation is not mediated by NO20. In a meta-analysis of studies using FMD, Bots et al. report a wide range of %FMD with the majority of the studies (81.2%) used the upper arm technique43. After adjusting for age, sex, presence of CHD, and diabetes, the lower arm technique was found to decrease %FMD (mean difference 2.47%, CI 0.55-4.39). While the location of the ischemic trigger (upper vs. lower arm) was found to be important, the location of the measurement (antecubital fossa vs. above antecubital fossa) was not significantly related to mean FMD. The difference in signal strength may be related to the size of the ischemic bed. Current guidelines support the use of either the upper arm or lower arm technique, suggesting laboratories adopt a consistent method across trials.
There are a number of critical steps in FMD. First, subject preparation is paramount as a number of factors such as medications, diet, nicotine, and exercise may affect the participant&#8217;s response. Sympathetic activation reduces FMD, so take appropriate steps to minimize distraction or discomfort for the participant44. Next, selecting an appropriate index segment of the brachial artery will improve the accuracy of the test. Distinct intima lines are important for measuring the change in diameter after occlusion. The change in diameter is small, typically 5-10% of a 5 mm artery or 50-100 &#956;m, and the diameter of the artery varies longitudinally. Operator fatigue is common and the ultrasound probe may slide during the exam to a section of the vessel with a different diameter. It is important to have visually distinct landmarks such as collateral vessels or areas of medial calcification to verify the index segment. Use of a stereotactic clamp to hold the probe or simply marking the subject&#8217;s skin can help maintain the index segment. Likewise, steps to prevent the subject from unintentionally moving their arm, such as shoulder and forearm constraining pillows, are recommended.
If holding the probe by hand, care should be taken to prevent operator fatigue or repetitive use injury. We suggest arranging the participant and any equipment so that the operator&#8217;s forearm is in the anatomic neutral position. Minimize the force required to hold the probe by wrapping it in foam tape or use a vertical post to clamp the cable, reducing the force required to hold the probe in a static position for 10 min.
Measuring velocity and diameter in the hyperemia presents technical challenges. This protocol calls for measuring 30 sec of Doppler spectral waveforms to record velocity then switching to B-mode imaging for diameter measurements. The peak velocity will occur between 5-10 sec after cuff release and the vessel may shift position during this time. If the probe is off the longitudinal axis or the insonation angle is &#62;60&#176;, the magnitude of the waveforms will be inaccurate. Positioning the sample gate slightly above the longitudinal axis of the vessel will help account for any shifting. When switching from Doppler to B-mode, it is important to maintain a position over the index segment. It is likely the artery has shifted and there is only a short time-window (25 sec) to optimize the image for the hyperemia diameter measurement. Often, only small changes to probe pressure, longitudinal alignment and angle are necessary to produce a quality image of the index segment. If the vessel is completely lost, a quick transverse scan up the artery will allow identification of the index segment, the most echogenic portion of the intima, and the correct angle of approach. Then, rotating the probe to a longitudinal view will return to a suitable view of the index segment.
Simple steps can improve the quality and consistency of FMD studies. The UCSF Vascular Integrated Physiology and Experimental Therapeutics (VIPERx) lab uses a quality control protocol when conducting FMD studies. First, participants are given standard instructions and pre-visit phone calls to ensure they avoid medications and behavior that affect the study. Next, a standard set of flow-sheets are used by study staff to ensure the exam is conducted the same way for each participant and critical steps are not overlooked. Finally, a post-exam six-point grading system is used to rate each study. Important factors such as the alignment of the vessel relative to the probe, the presence of anatomical landmarks, and the extent of intima lines are included to ensure these critical steps are met.

The human vascular endothelium provides structural and functional roles within the body. In histological sections, the endothelium appears small, comprising a thin layer of cells 1-2 microns thick sitting atop a layer of smooth muscle cells (the media) and a thick layer of connective tissue (the adventitia). Taken as a whole, the endothelium provides a wide area for the exchange of information between the blood and vascular smooth muscle tissue. By one estimate, a cross sectional area of 700 m2 and a mass of 1,000-1,500 grams in a 70 kg man, is comparable in mass to the liver1. A healthy endothelium allows for mechanical to chemical signal transduction to maintain homeostasis of the blood vessel. Endothelial dysfunction is an imbalance of these mediators and the first step in vascular disease, present prior to histological evidence of atherosclerosis. A non-invasive, in vivo method for quantifying the vasodilatory function of human artery exists. This method, endothelium-dependent, flow-mediated vasodilation (FMD) is widely used in clinical trials.
The endothelium acts as a structural component of the vasculature and manufactures components of the extracellular matrix such as glycosaminoglycans and fibronectin2. Long term changes in blood flow and acute injury to the artery may lead to structural changes. Functionally, the vascular endothelial cells participate in regulation of vessel tone, inflammatory processes, antithrombosis, and anticoagulation. Endothelial cells affect vasoconstriction through endothelin while vasodilation is mediated by nitric oxide (NO), prostacyclin, and endothelial derived hyperpolarizing factor (EDHF)3-6.
Endothelial dysfunction is an impairment of any of these mediators and the first step in atherosclerosis. Not surprisingly, as a mechanism of disease, it is associated with a number of clinically important conditions such as coronary artery disease, hypertension and diabetes mellitus7-11. Importantly, endothelial dysfunction can be observed in individuals without diagnosed cardiovascular disease and is predictive of future cardiovascular events7,12,13. One measure of endothelial dysfunction, in combination with the Framingham score, can provide additional prognostic information above either measure alone14.
Measures of endothelial dysfunction may involve the direct infusion of a pharmacological agent. Intercoronary infusion of acetylcholine, for example, combined with quantitative angiography demonstrates vasodilation in subjects with an intact endothelium. However, individuals with endothelial damage experience paradoxical vasoconstriction.15 In peripheral arteries, infusion of a pharmacological agent with measurement of flow by gauge-strain plethysmography is possible16.
Agents that directly affect the endothelium and elicit a chemical signal are termed endothelium-dependent vasodilators. Acetylcholine, for example, acts on muscarinic receptors on endothelial cells, leading to increased intra-cellular calcium concentration, activation of nitric oxide synthase and vasodilation. Agents that affect vasodilation without involvement of the endothelium are called endothelium-independent agents. Nitroglycerin, for example, activates soluble guanyl cyclase and cyclic guanosine-3&#8217;,-5&#8217;-monophasphate (cGMP) which mediates vasodilation in the vessel wall through protein kinases regulating intracellular calcium concentrations17.
There is a non-invasive, in vivo method for quantifying endothelial dysfunction introduced by Celermajer and associates called &#8220;flow-mediated, endothelium-dependent vasodilation&#8221; (FMD)18. Briefly, changes to arterial blood flow open shear stress sensitive ion channels in the endothelium. The signal is tranduced via a second-messenger cascade and activates endothelial nitric oxide synthase (eNOS), generating NO. This species diffuses across the cell membrane to neighboring smooth muscle cells (SMC). Within the SMC, the signal is transduced, lowering intracellular calcium concentration and affecting vasorelaxation19. The diameter of the artery lumen increases, leading to an increase in blood flow consistent with the Hagen-Poiseullie equation. The effect of FMD may be abolished with administration of an NO synthase inhibitor such as mono-methylarginine (L-NMMA)20.
Celermajer et al.&#8217;s innovative work has allowed the use of high resolution B-mode ultrasound to assess the change in artery diameter during the reactive hyperemia that follows ischemia. In this technique, a human subject rests supine and the diameter of the brachial artery is measured in a longitudinal plane. A blood-pressure cuff is used to produce ischemia in the limb. Following release of the blood pressure cuff the diameter of the artery is measured again. The rapid change in shear stress is the stimulus for NO mediated vasodilation. A simple equation describes the change in the diameter relative to the baseline diameter (Equation 1). A full discussion of the parameters of this equation, hyperemia and baseline diameter, can be found in the Protocol and Results sections.

In multiple studies, percent FMD has been found to predict cardiovascular events in patient with established cardiovascular disease21-24. A correlation between brachial artery percent FMD and coronary artery FMD was established by Anderson et al., demonstrating a link between peripheral measurements and the more clinically-relevant ischemic changes to the heart25. FMD does not demonstrate the maximum vasodilation of the vessel. To evaluate this, FMD can be followed by endothelium-dependent, nitroglycerin-mediated vasodilation of the same vessel.
There are technical issues affecting the measurement of percent FMD. Since the introduction of the technique, several studies showed a high degree of within-subject and inter-operator variability26. It has been shown that physiological factors such as cigarette smoking, antihypertensive medications, time of day, and fasting state affect percent FMD. Likewise, technical choices such as the position of the cuff relative to the site of measurement and duration of occlusion have been shown to affect the measurement27,28. Guidelines have been published that describe the current consensus and allow for standardization of technique between laboratories19,29.
Despite the evolving consensus on technique, flow-mediated vasodilation remains heavily operator dependent with a long learning curve. Corretti, for example, recommends the sonographer complete 100 scans under the supervision of an experienced investigator before operating independently. To maintain a level of adequate expertise, it is recommended the technician complete 100 scans annually. For investigators with a small sample population and limited resources, the learning curve presents a barrier to entry. This article will demonstrate a method for flow-mediated vasodilation of the brachial artery in the upper arm and offer technical suggestions to reduce intra-operator variability.

<table><tbody><tr><td>Philips HD 11XE ultrasound</td><td>Philips Healthcare</td><td></td><td></td></tr><tr><td>5-12 MHz linear array transducer</td><td>Philips Healthcare</td><td>L12-5</td><td></td></tr><tr><td>Ultrasound gel</td><td>Parker Laboratories</td><td></td><td></td></tr><tr><td>Vascular Research Tools v.5.0</td><td>Medical Imaging Applications, LLC</td><td></td><td></td></tr><tr><td>MIA Gating module</td><td>Medical Imaging Applications, LLC</td><td></td><td></td></tr><tr><td>Windows XP</td><td>Microsoft, Inc</td><td></td><td></td></tr><tr><td>Hand-held aneroid manometer</td><td>Welch Allyn</td><td>DS66</td><td></td></tr></tbody></table>

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.

The key variables of flow-mediated vasodilation are shown in Table 1.
<table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Variable</td>
			<td>Description</td>
		</tr>
		<tr>
			<td>Mean Velocity (cm/sec)</td>
			<td>The mean arterial velocity of blood in the middle 50% of the lumen during one cardiac cycle estimated from Doppler spectral waveforms, proportional to blood flow and inversely proportional to cross-sectional area (see Figure 1).</td>
		</tr>
		<tr>
			<td>Diameter (mm)</td>
			<td>The intima-intima distance as measured from a longitudinal view along the vessel axis (see Figure 2). This is measured at Baseline and during reactive hyperemia.</td>
		</tr>
		<tr>
			<td>Flow (ml/min)</td>
			<td>The bulk flow of fluid in the circulation, derived mathematically from mean velocity and diameter (see Equation 2).</td>
		</tr>
		<tr>
			<td>Shear Stress (dynes/cm2)</td>
			<td>The frictional force exerted by circulating blood on the intima surface, proportional to velocity and inversely proportional to diameter, derived from mean velocity and diameter (see Equation 3).</td>
		</tr>
		<tr>
			<td>% FMD</td>
			<td>The change in arterial diameter after occlusion in response to hyperemia, over the baseline diameter (see Equation 1).</td>
		</tr>
	</tbody>
</table>
Table 1. The key variables of flow-mediated vasodilation.
This FMD protocol will provide sufficient data to measure % flow-mediated vasodilation, flow, and shear stress. Recording 60 sec of baseline data will help account for normal, physiological variation in heart rate and respiration. Analysis software will calculate diameter during the baseline and hyperemia phases. Some software packages can measure the average velocity (m/sec) during a point in the cardiac cycle by integrating the area under the velocity spectral waveform and dividing by time to arrive at a time-averaged velocity. Diameter and Velocity will allow an investigator to calculate the following variables.
Equation 1. 
% FMD is defined as: <img alt="Equation 1" fo:content-width="2in" src="/files/ftp_upload/52070/52070eq1.jpg" />.
Equation 2. 
Mean Flow in ml/min is defined as: <img alt="Equation 2" fo:content-width="1.5in" src="/files/ftp_upload/52070/52070eq2.jpg" />.
Equation 3. 
Shear Stress is defined as: <img alt="Equation 3" fo:content-width="1in" src="/files/ftp_upload/52070/52070eq3.jpg" /> where Tw is shear stress in dynes/cm2, Q is mean volumetric flow, and &#181;, the viscosity of blood, is assumed to be 0.035 poise.
Example data from studies conducted at the UCSF Vascular Integrated Physiology and Experimental Therapeutics lab (VIPERx) is given in Table 2 and Table 3. Briefly, the example cohort is a randomly chosen subset of participants in the cross-sectional arm of the Omega-PAD trial (NCT01310270)30. All participants were patients referred to the outpatient vascular surgery clinic of the San Francisco Veterans Affairs Medical Center for evaluation of peripheral artery disease (PAD). PAD diagnosis was based on current guidelines of an ankle-brachial index &#60;0.9. Patients with incompressible arteries (ABI&#62;1.4) were excluded. Inclusion in the &#8220;No PAD&#8221; group was based on ABI &#62; 0.9 and the absence of PAD, CAD, and CVD. Statistical analysis was done by t-test for continuous variables or chi-square test for categorical variables.
The example cohort is almost entirely male with a mean age is 68 &#177; 9 years and Caucasian, 74%. As a whole, participants carry a number of cardiovascular risk factors including: hypertension (84%), hyperlipidemia (78%), smoking history (86%), and obesity (mean BMI is 30 &#177; 6). Overall, 16% of participants carry a diagnosis of coronary artery disease (CAD) and 40% a diagnosis of diabetes mellitus.
The prevalence of hypertension was higher in the PAD group than the non-PAD group (96% vs. 72%, p = 0.02) as was CAD (32% vs. 0%, p &#60; 0.001) and diabetes (56% vs. 24%, p = 0.02). The PAD group had greater abdominal adiposity but not to the level of significance (waist-hip ratio of 1.04 vs. 1.00, p = 0.065). Likewise, the PAD group had worse low density lipoprotein (LDL) than the non-PAD group (68 vs. 101 mg/dl, p &#60; 0.001) but better total cholesterol (142 vs. 174 mg/dl, p = 0.002). Both groups were appropriately managed with medications, demonstrating widespread use of statins and antihypertensive medications. The PAD group shows a higher level of aspirin (84% vs. 48%, p = 0.007) and beta-blockers (60% vs. 28%, p = 0.023), consistent with their co-morbidities.
Table 3 demonstrates example flow-mediated vasodilation data from the two groups. The baseline characteristics are similar for each group showing similar diameter, velocity, and flow. The PAD group, however, demonstrate worse flow-mediated vasodilation than the non-PAD group (6.8% vs. 9.1%, p = 0.021). Results for both group fall within the expected range for individuals with cardiovascular risk factors (&#60;10%). A review of several studies suggest a %FMD of 6-10% in healthy adults and a %FMD of 0-5% in CAD populations using lower arm occlusion31-36. Values above 10% were observed in young, healthy adults using the upper arm technique37. The %FMD for each group has a wide standard deviation, presenting an opportunity to further segment the cohort based on %FMD.
Figures 1-4 demonstrate example images collected during the phases of FMD. Figure 1 shows a Doppler Spectral waveform obtained at Baseline. The arrows indicate the extent of one cardiac cycle which forms the basis for calculating mean arterial velocity. The protocol calls for averaging the results of multiple cycles collected during 60 sec. In the example cohort, the mean baseline velocity for all participants was 17 &#177; 6 cm/sec. No significant difference between the PAD and No PAD cohort was seen.
Figure 2 shows an example B-mode image of the Baseline vessel diameter. The arrows indicate the location where the intima-intima distance, the basis for the lumen diameter, was measured. In the example cohort, the mean baseline diameter for all participants was 4.20 &#177; 0.57 mm. No significant difference between the PAD and No PAD groups was seen.
Figure 3 shows an example Doppler Spectral waveform obtained immediately after cuff release in the Reactive Hyperemia phase. The yellow arrow indicates the moment of cuff release. Waveforms obtained in the first 5 sec after cuff are used to calculate the Reactive Hyperemia Velocity. For all participants, the mean Reactive Hyperemia Velocity was 74 &#177; 26 cm/s. No significant difference was seen between the PAD and No PAD groups.
Figure 4 shows example B-mode image obtained 60 sec after cuff release during the Reactive Hyperemia phase. Like the Baseline diameter, the intima-intima distance is used to calculate Reactive Hyperemia Diameter. For all participants, the mean Reactive Hyperemia diameter was 4.53 &#177; 0.59 mm. The difference between PAD and No PAD subgroups was approached, but did not meet, significance (p = 0.08). The difference between Baseline and Reactive Hyperemia diameter forms the basis of the numerator in the %FMD variable.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Characteristics</td>
			<td>All patients 
			(n = 50)</td>
			<td>PAD 
			(n = 25)</td>
			<td>No PAD 
			(n = 25)</td>
			<td>P-value</td>
		</tr>
		<tr>
			<td>Age, Mean (SD), y</td>
			<td>68 &#177; 9</td>
			<td>68 &#177; 6</td>
			<td>68 &#177; 11</td>
			<td>0.89</td>
		</tr>
		<tr>
			<td>Male Sex (%)</td>
			<td>98</td>
			<td>100</td>
			<td>96</td>
			<td>0.31</td>
		</tr>
		<tr>
			<td>Caucasian (%)</td>
			<td>74</td>
			<td>84</td>
			<td>64</td>
			<td>0.37</td>
		</tr>
		<tr>
			<td>BMI</td>
			<td>30 &#177; 6</td>
			<td>29 &#177; 7</td>
			<td>30 &#177; 4</td>
			<td>0.73</td>
		</tr>
		<tr>
			<td>Waist-hip ratio (%)</td>
			<td>1.02 &#177; 0.06</td>
			<td>1.04 &#177; 0.06</td>
			<td>1.00 &#177; 0.05</td>
			<td>0.07</td>
		</tr>
		<tr>
			<td>Systolic Blood Pressure (mm Hg)</td>
			<td>136 &#177; 19</td>
			<td>139 &#177; 22</td>
			<td>134 &#177; 15</td>
			<td>0.33</td>
		</tr>
		<tr>
			<td>Diastolic Blood Pressure (mm Hg)</td>
			<td>79 &#177; 10</td>
			<td>78 &#177; 11</td>
			<td>80 &#177; 10</td>
			<td>0.47</td>
		</tr>
		<tr>
			<td>Index ABI</td>
			<td>0.93 &#177; 0.27</td>
			<td>0.72 &#177; 0.16</td>
			<td>1.14 &#177; 0.16</td>
			<td>&#60;0.001</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>Comorbidities</td>
		</tr>
		<tr>
			<td>Hypertension (%)</td>
			<td>84</td>
			<td>96</td>
			<td>72</td>
			<td>0.02</td>
		</tr>
		<tr>
			<td>Hyperlipidemia (%)</td>
			<td>78</td>
			<td>88</td>
			<td>68</td>
			<td>0.09</td>
		</tr>
		<tr>
			<td>Hx of CAD (%)</td>
			<td>16</td>
			<td>32</td>
			<td>0</td>
			<td>0.00</td>
		</tr>
		<tr>
			<td>Diabetes Mellitus (%)</td>
			<td>40</td>
			<td>56</td>
			<td>24</td>
			<td>0.02</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>Medications</td>
		</tr>
		<tr>
			<td>Aspirin (%)</td>
			<td>66</td>
			<td>84</td>
			<td>48</td>
			<td>0.01</td>
		</tr>
		<tr>
			<td>Ace-inhibitor (%)</td>
			<td>48</td>
			<td>52</td>
			<td>44</td>
			<td>0.57</td>
		</tr>
		<tr>
			<td>&#946;-Blocker (%)</td>
			<td>44</td>
			<td>60</td>
			<td>28</td>
			<td>0.02</td>
		</tr>
		<tr>
			<td>Statin (%)</td>
			<td>66</td>
			<td>68</td>
			<td>64</td>
			<td>0.77</td>
		</tr>
		<tr>
			<td>Insulin (%)</td>
			<td>30</td>
			<td>14</td>
			<td>6</td>
			<td>0.39</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>PAD Risk Factors</td>
		</tr>
		<tr>
			<td>History of smoking (%)</td>
			<td>86</td>
			<td>92</td>
			<td>79</td>
			<td>0.24</td>
		</tr>
		<tr>
			<td>Total Cholesterol (mg/dl)</td>
			<td>158 &#177; 38</td>
			<td>142 &#177; 31</td>
			<td>174 &#177; 37</td>
			<td>0.00</td>
		</tr>
		<tr>
			<td>LDL (mg/dl)</td>
			<td>85 &#177; 32</td>
			<td>68 &#177; 27</td>
			<td>101 &#177; 29</td>
			<td>&#60;0.001</td>
		</tr>
		<tr>
			<td>HDL (mg/dl)</td>
			<td>44 &#177; 11</td>
			<td>43 &#177; 11</td>
			<td>46 &#177; 10</td>
			<td>0.30</td>
		</tr>
		<tr>
			<td>Triglycerides (mg/dl)</td>
			<td>153 &#177; 119</td>
			<td>165 &#177; 125</td>
			<td>141 &#177; 115</td>
			<td>0.49</td>
		</tr>
		<tr>
			<td>Hemoglobin A1C (%)</td>
			<td>6.3 &#177; 1.5</td>
			<td>6.5 &#177; 1.5</td>
			<td>6.1 &#177; 1.6</td>
			<td>0.38</td>
		</tr>
		<tr>
			<td>Serum creatinine (mg/dl)</td>
			<td>1.11 &#177; 0.84</td>
			<td>1.28 &#177; 1.15</td>
			<td>0.95 &#177; 0.22</td>
			<td>0.17</td>
		</tr>
		<tr>
			<td>eGFR (ml/min)</td>
			<td>80 &#177; 21</td>
			<td>75 &#177; 21</td>
			<td>86 &#177; 21</td>
			<td>0.10</td>
		</tr>
		<tr>
			<td>Albumin (g/dl)</td>
			<td>4.0 &#177; 0.3</td>
			<td>4.0 &#177; 0.3</td>
			<td>4.1 &#177; 0.3</td>
			<td>0.43</td>
		</tr>
	</tbody>
</table>
Table 2. Baseline characteristics of a sample cohort. The following data are a randomly chosen subset of participants in the cross-sectional arm of the Omega-PAD trial (NCT01310270) and cohort . All participants were patients referred to the outpatient vascular surgery clinic of the San Francisco Veterans Affairs Medical Center for evaluation of peripheral artery disease (PAD). PAD diagnosis was based on current guidelines of an ankle-brachial index &#60;0.9. Patients with incompressible arteries (ABI &#62; 1.4) were excluded. Inclusion in the &#8220;No PAD&#8221; group was based on ABI &#62; 0.9 and the absence of PAD, CAD, and CVD.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Characteristics</td>
			<td>All patients 
			(n = 50)</td>
			<td>PAD 
			(n = 25)</td>
			<td>No PAD 
			(n = 25)</td>
			<td>P-value</td>
		</tr>
		<tr>
			<td>Baseline Artery Diameter (SD), mm</td>
			<td>4.20 &#177; 0.57</td>
			<td>4.11 &#177; 0.60</td>
			<td>4.29 &#177; 0.53</td>
			<td>0.27</td>
		</tr>
		<tr>
			<td>Baseline Velocity (SD), cm/sec</td>
			<td>17 &#177; 6</td>
			<td>18 &#177; 6</td>
			<td>16 &#177; 5</td>
			<td>0.13</td>
		</tr>
		<tr>
			<td>Baseline Flow (SD), ml/min</td>
			<td>145 &#177; 68</td>
			<td>151 &#177; 84</td>
			<td>138 &#177; 47</td>
			<td>0.51</td>
		</tr>
		<tr>
			<td>Baseline Shear Stress (SD), dynes/cm2</td>
			<td>12 &#177; 4</td>
			<td>13 &#177; 5</td>
			<td>11 &#177; 3</td>
			<td>0.07</td>
		</tr>
		<tr>
			<td>Reactive Hyperemia Diameter (SD), mm</td>
			<td>4.53 &#177; 0.59</td>
			<td>4.38 &#177; 0.60</td>
			<td>4.68 &#177; 0.55</td>
			<td>0.08</td>
		</tr>
		<tr>
			<td>Reactive Hyperemia Velocity (SD), cm/sec</td>
			<td>74 &#177; 26</td>
			<td>70 &#177; 25</td>
			<td>78 &#177; 27</td>
			<td>0.32</td>
		</tr>
		<tr>
			<td>Reactive Hyperemia Flow (SD), ml/min</td>
			<td>735 &#177; 340</td>
			<td>658 &#177; 327</td>
			<td>812 &#177; 342</td>
			<td>0.11</td>
		</tr>
		<tr>
			<td>Reactive Hyperemia Shear Stress (SD), dynes/cm2</td>
			<td>46 &#177; 18</td>
			<td>46 &#177; 19</td>
			<td>47 &#177; 18</td>
			<td>0.79</td>
		</tr>
		<tr>
			<td>Brachial FMD (%)</td>
			<td>8.0 &#177; 3.7</td>
			<td>6.8 &#177; 3.5</td>
			<td>9.1 &#177; 3.6</td>
			<td>0.02</td>
		</tr>
	</tbody>
</table>
Table 3. Flow-Mediated Vasodilation analysis. As described in the protocol, baseline diameter and velocity are the mean of 60 sec of data. Reactive hyperemia diameter was obtained at 60 sec post-occlusion. Reactive hyperemia velocity was the time-averaged velocity of the first 5 sec of Doppler spectral waveforms obtained after cuff-release.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/52070/52070fig1highres.jpg" width="500" /> 
Figure 1. Baseline velocity measurements. Doppler spectral waveforms of the brachial artery are captured by an image analysis system. A single cardiac cycle is shown between the arrows. Image analysis systems can calculate the mean arterial velocity. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52070/52070fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/52070/52070fig2highres.jpg" width="500" /> 
Figure 2. Baseline diameter measurements. The double-lines of Pignoli, corresponding to intima and media boundaries are visible on both the superficial and deep edges of the brachial artery (yellow arrows). The image shows proper horizontal and vertical alignment. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52070/52070fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/52070/52070fig3highres.jpg" width="500" /> 
Figure 3. Hyperemia velocity measurements. Doppler spectral waveforms immediately after cuff release are visible. The moment of cuff release can be appreciated by the sharp increase in velocity to the left of the image (yellow arrow). The top half of the image shows the positioning of the sample gate prior to cuff release. After cuff release, the artery may shift to a more superficial position. Placing the gate above the axis of the occluded artery helps compensate for vertical shifting of the artery after cuff release. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52070/52070fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/52070/52070fig4highres.jpg" width="500" /> 
Figure 4. Hyperemia diameter measurements. A longitudinal view of the index segment after cuff release is visible. The change in diameter is small and may be quantified by image analysis software. The IMT boundary on the superficial wall is clearly visible along the index segment (yellow arrow). The width of the arrow shafts represents a 10% increase from the baseline diameter. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52070/52070fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Ultrasound Assessment of Endothelial-Dependent Flow-Mediated Vasodilation of the Brachial Artery in Clinical Research at JoVE.com

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

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 ...

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27.2K Views.  University of California, San Francisco. 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.

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

Assessing Endothelial Vasodilator Function with the Endo-PAT 2000

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of the panoply of paracrine factors that it secretes, the endothelium regulates the contractile and proliferative state of the underlying vascular smooth muscle, as well as the interaction of the vessel wall with circulating blood elements. Because of its central role in mediating vessel tone and growth, its position as gateway to circulating immune cells, and its local regulation of hemostasis and coagulation, the the properly functioning endothelium is the key to cardiovascular health. Conversely, the earliest disorder in most vascular diseases is endothelial dysfunction.
In the arterial circulation, the healthy endothelium generally exerts a vasodilator influence on the vascular smooth muscle. There are a number of methods to assess endothelial vasodilator function. The Endo-PAT 2000 is a new device that is used to assess endothelial vasodilator function in a rapid and non-invasive fashion. Unlike the commonly used technique of duplex ultra-sonography to assess flow-mediated vasodilation, it is totally non-operator-dependent, and the equipment is an order of magnitude less expensive. The device records endothelium-mediated changes in the digital pulse waveform known as the PAT ( peripheral Arterial Tone) signal, measured with a pair of novel modified plethysmographic probes situated on the finger index of each hand. Endothelium-mediated changes in the PAT signal are elicited by creating a downstream hyperemic response. Hyperemia is induced by occluding blood flow through the brachial artery for 5 minutes using an inflatable cuff on one hand. The response to reactive hyperemia is calculated automatically by the system. A PAT ratio is created using the post and pre occlusion values. These values are normalized to measurements from the contra-lateral arm, which serves as control for non-endothelial dependent systemic effects. Most notably, this normalization controls for fluctuations in sympathetic nerve outflow that may induce changes in peripheral arterial tone that are superimposed on the hyperemic response.
In this video we demonstrate how to use the Endo-PAT 2000 to perform a clinically relevant assessment of endothelial vasodilator function.

A noninvasive procedure to assess endothelial function is demonstrated using the Endo-PAT 2000.

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of ...

The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel underlying neurobiological mechanisms behind drug action and neurotransmitter-related disorders, such as depression and ADHD. Like most of the imaging methods (PET, SPECT, CT) it represents a progress in the investigation of brain disorders and the related function of neurotransmitter pathways in a non-invasive way with respect of the overall neuronal connectivity. Moreover it also provides the ideal tool for translation to clinical investigations. MRI, while still behind in molecular imaging strategies compared to PET and SPECT, has the great advantage to have a high spatial resolution and no need for the injection of a contrast-agent or radio-labeled molecules, thereby avoiding the repetitive exposure to ionizing radiations. Functional MRI (fMRI) is extensively used in research and clinical setting, where it is generally combined with a psycho-motor task. phMRI is an adaptation of fMRI enabling the investigation of a specific neurotransmitter system, such as serotonin (5-HT), under physiological or pathological conditions following activation via administration of a specific challenging drug.
The aim of the method described here is to assess brain 5-HT function in free-breathing animals. By challenging the 5-HT system while simultaneously acquiring functional MR images over time, the response of the brain to this challenge can be visualized. Several studies in animals have already demonstrated that drug-induced increases in extracellular levels of e.g. 5-HT (releasing agents, selective re-uptake blockers, etc) evoke region-specific changes in blood oxygenation level dependent (BOLD) MRI signals (signal due to a change of the oxygenated/deoxygenated hemoglobin levels occurring during brain activation through an increase of the blood supply to supply the oxygen and glucose to the demanding neurons) providing an index of neurotransmitter function. It has also been shown that these effects can be reversed by treatments that decrease 5-HT availability16,13,18,7. In adult rats, BOLD signal changes following acute SSRI administration have been described in several 5-HT related brain regions, i.e. cortical areas, hippocampus, hypothalamus and thalamus9,16,15. Stimulation of the 5-HT system and its response to this challenge can be thus used as a measure of its function in both animals and humans2,11.

The goal of this technique is to assess serotonin (5-HT) neurotransmitter function in the live and free-breathing animal with pharmacological magnetic resonance imaging (phMRI) and an intravenous challenge with a selective serotonin reuptake inhibitor (SSRI), fluoxetine.

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel ...

Non-invasive Assessment of Microvascular and Endothelial Function

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of cardiovascular disease. Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. Percent capillary recruitment is assessed by dividing the increase in capillary density induced by postocclusive reactive hyperemia (postocclusive reactive hyperemia capillary density minus baseline capillary density), by the maximal capillary density (observed during passive venous occlusion). Percent perfused capillaries represents the proportion of all capillaries present that are perfused (functionally active), and is calculated by dividing postocclusive reactive hyperemia capillary density by the maximal capillary density. Both percent capillary recruitment and percent perfused capillaries reflect the number of functional capillaries. The forearm blood flow (FBF) technique provides accepted non-invasive measures of endothelial function: The ratio FBFmax/FBFbase is computed as an estimate of vasodilation, by dividing the mean of the four FBFmax values by the mean of the four FBFbase values. Forearm vascular resistance at maximal vasodilation (FVRmax) is calculated as the mean arterial pressure (MAP) divided by FBFmax. Both the capillaroscopy and forearm techniques are readily acceptable to patients and can be learned quickly.
The microvascular and endothelial function measures obtained using the methodologies described in this paper may have future utility in clinical patient cardiovascular risk-reduction strategies. As we have published reports demonstrating that microvascular and endothelial dysfunction are found in initial stages of hypertension including prehypertension, microvascular and endothelial function measures may eventually aid in early identification, risk-stratification and prevention of end-stage vascular pathology, with its potentially fatal consequences.

Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of ...

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 ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

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

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

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

Clinical Assessment of Spatiotemporal Gait Parameters in Patients and Older Adults

Spatial and temporal characteristics of human walking are frequently evaluated to identify possible gait impairments, mainly in orthopedic and neurological patients1-4, but also in healthy older adults5,6. The quantitative gait analysis described in this protocol is performed with a recently-introduced photoelectric system (see&#160;Materials table) which has the potential to be used in the clinic because it is portable, easy to set up (no subject preparation is required before a test), and does not require maintenance and sensor calibration. The photoelectric system consists of series of high-density floor-based photoelectric cells with light-emitting and light-receiving diodes that are placed parallel to each other to create a corridor, and are oriented perpendicular to the line of progression7. The system simply detects interruptions in light signal, for instance due to the presence of feet within the recording area. Temporal gait parameters and 1D spatial coordinates of consecutive steps are subsequently calculated to provide common gait parameters such as step length, single limb support and walking velocity8, whose validity against a criterion instrument has recently been demonstrated7,9. The measurement procedures are very straightforward; a single patient can be tested in less than 5 min&#160;and a comprehensive report can be generated in less than 1&#160;min.

This protocol is used to evaluate spatial and temporal gait variables of neurological/orthopedic patients and older persons by means of a recently-introduced floor-based photocell system.

Spatial and temporal characteristics of human walking are frequently evaluated to identify possible gait impairments, mainly in orthopedic and ...

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

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

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

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

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

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

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

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

Ultrasound 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.

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 ...

Using Arterial Stiffness and Endothelial Function for Cardiovascular Assessment

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

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

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

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

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