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.
Brac...

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 border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Philips HD 11XE ultrasound</td>
			<td style="width:71px;">Philips Healthcare</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">5-12 MHz linear array transducer</td>
			<td style="width:71px;">Philips Healthcare</td>
			<td style="width:119px;">L12-5</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ultrasound gel</td>
			<td style="width:71px;">Parker Laboratories</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Vascular Research Tools v.5.0</td>
			<td style="width:71px;">Medical Imaging Applications, LLC</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">MIA Gating module</td>
			<td style="width:71px;">Medical Imaging Applications, LLC</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Desktop PC</td>
			<td style="width:71px;">Dell, Inc</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Windows XP</td>
			<td style="width:71px;">Microsoft, Inc</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">5 cm tourniquet blood pressure cuff</td>
			<td style="width:71px;">Hokanson</td>
			<td style="width:119px;">SC 5</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hand-held aneroid manometer</td>
			<td style="width:71px;">Welch Allyn</td>
			<td style="width:119px;">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 Fi...

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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27.1K 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

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 ...

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 ...

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 ...

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

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

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

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

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

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

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

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

Ultrasound-based Pulse Wave Velocity Evaluation in Mice

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

Arterial stiffness represents a key factor in cardiovascular disease and pulse wave velocity (PWV) can be considered as a surrogate index for arterial stiffness. This protocol describes an image processing algorithm for calculating PWV in mice based on ultrasound image processing that is applicable at different arterial sites.

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

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 ...

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 ...

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 ...

Technique for Salivary Gland Ultrasound Examination of Sjogren&#039;s Syndrome

Ultrasonographic Evaluation of Salivary Glands for Sjogren's Syndrome: Diagnostic and Monitoring Insights

Sjogren's syndrome (SS) is a chronic autoimmune condition commonly affecting the exocrine glands, causing oral or ocular dryness and extraglandular manifestations including arthralgia, cytopenia, and lymphoma. The presence of autoantibodies against SSA/Ro, labial salivary gland biopsy, ocular staining, Schirmer's test, or salivary flow assessment are included in the current classification criteria of SS. However, the availability and invasiveness of these procedures limit their widespread use in clinical settings. Salivary gland ultrasonography (SGUS) is a non-invasive imaging modality for the evaluation of the salivary gland parenchyma and is increasingly utilized to aid diagnosis and monitoring in SS. 
This article presents the protocol of SGUS for image acquisition at the parotid and submandibular glands. The objective is to present a standardized, reproducible, and practical approach to diagnostic SGUS for SS in daily clinical settings. Major salivary glands are scanned in a stepwise approach, beginning at the angle of the mandible for the superficial lobe of the parotid gland, followed by the deep lobe below the ramus of the mandible. Submandibular areas are then scanned for the submandibular glands. The steps in obtaining salivary gland images at each anatomical site are explained in the accompanying video. The echogenicity and echotexture at the thyroid gland are taken as a reference. The homogeneity, the presence and distribution of hypoechoic areas within the glands, and the border of the salivary glands are examined. The sizes and features of intra-/peri-glandular lymph nodes are recorded. The most distinctive sonographic feature in SS is glandular heterogeneity with the presence of hypoechoic/hyperechoic areas within the glands. 
In summary, while SGUS cannot diagnose SS on its own, it can supplement the current classification criteria of SS and guide the clinical decision for salivary gland biopsy to support the diagnosis of SS in patients with sicca syndrome or suspicious systemic features, combined with autoantibody testing.

Author Spotlight: A Focus on Standardized Salivary Gland Ultrasound Protocol in Connective Tissue Disease Research

Salivary gland ultrasonography is a promising tool for the diagnosis and evaluation of disease activity and complications in Sjogren's syndrome (SS). The scanning technique for salivary gland ultrasound examination relevant to SS is described in the article.

Sjogren's syndrome (SS) is a chronic autoimmune condition commonly affecting the exocrine glands, causing oral or ocular dryness and extraglandular ...