Name: oxygenation-sensitive cardiac mri with vasoactive breathing maneuvers for the non-invasive assessment of coronary microvascular dysfunction
Uploaded: 2022-08-17T13:50:00
Description: Watch this Scientific Journal Video about Oxygenation-sensitive Cardiac MRI with Vasoactive Breathing Maneuvers for the Non-invasive Assessment of Coronary Microvascular Dysfunction at JoVE.com

This paper and methodology review was made possible by the entire team of the Courtois CMR Research Group at the McGill University Health Centre. Special thanks to our MRI technologists Maggie Leo and Sylvie Gelineau for the scanning of our participants and feedback on this manuscript.

All MRI scans utilizing OS-CMR with vasoactive breathing maneuvers should be performed in compliance with local institutional guidelines. The protocol outlined below has been used in studies approved by several&#160;institutional human research ethics committee. Written consent was obtained for all the human participant data and results described in this protocol and manuscript.
1. Broad overview
<ol>
	<li>Vary the inclusion and exclusion criteria depending on the study popu...

x`The addition of an OS-CMR acquisition with standardized, vasoactive breathing maneuvers to an already established research or clinical MRI protocol adds little time to the overall scan. With this short addition, information about underlying macro- and microvascular function can be obtained (Figure 2). An important consequence of endothelial dysfunction is the inability of the vasculature to respond to physiologic stimuli, as initially demonstrated through abnormal flow-mediated relaxation ...

This corrects the article <a href="https://dx.doi.org/10.3791/64149">10.3791/64149</a>

An erratum was issued for: <a href="https://www.jove.com/t/64149">Oxygenation-sensitive Cardiac MRI with Vasoactive Breathing Maneuvers for the Non-invasive Assessment of Coronary Microvascular Dysfunction</a>. The Abstract, Protocol, and Representative Results&#160;section were&#160;updated.

Erratum: Oxygenation-sensitive Cardiac MRI with Vasoactive Breathing Maneuvers for the Non-invasive Assessment of Coronary Microvascular Dysfunction

Oxygenation-sensitive cardiac magnetic resonance imaging (OS-CMR) uses the inherent paramagnetic properties of deoxyhemoglobin as an endogenous source of MR contrast1,2,3. Used in combination with standardized vasoactive breathing maneuvers (hyperventilation and apnea) as a potent non-pharmacologic vasomotor stimulus, OS-CMR can monitor changes in myocardial oxygenation as a marker for vascular function, thereby circumventing the need for any extrinsic, intravenous contrast or pharmacologic stress agents 4,5,6.
Breathing maneuvers, including breath-holds and hyperventilation, are highly effective vasoactive measures to alter vasomotion and, because of their safety and simplicity, are ideal for controlled endothelial-dependent vasomotion as part of a diagnostic procedure. Studies have shown an added effectiveness when combining hyperventilation with a subsequent breath-hold4,7, as during such a protocol, the vasoconstriction (through the associated decrease of blood carbon dioxide) is followed by vasodilation (increase of blood carbon dioxide); thus, a healthy vascular system transitions through the entire range from vasoconstriction to vasodilation with a strong increase in myocardial blood flow, which in turn increases myocardial oxygenation and, thus, the observable signal intensity in OS-CMR images. The use of cine images for the acquisition also allows for cardiac phase-resolved results with a better signal-to-noise ratio when compared to adenosine infusion8.
Breathing maneuvers can replace pharmacological stress agents for inducing vasoactive changes that can be used for assessing coronary vascular function. This not only reduces patient risk, logistical efforts, and associated costs but also helps in providing results that are clinically more meaningful. Pharmacologic stress agents such as adenosine trigger an endothelium-dependent response and, thus, reflect endothelial function itself. Such specific assessment of endothelial function so far was only possible by an intracoronary administration of acetylcholine as an endothelial-dependent vasodilator. This procedure, however, is highly invasive2,9 and, therefore, rarely performed.
Lacking access to direct biomarkers, several diagnostic techniques have used surrogate markers such as tissue uptake of an exogenous contrast agent. They are limited by the need for one or two intravenous access lines, contraindications such as severe kidney disease or atrioventricular block, and the need for the physical presence of staff with training in managing potentially severe side effects10,11. The most significant limitation of current imaging of coronary function, however, remains that myocardial perfusion as a surrogate marker does not reflect myocardial tissue oxygenation as the most important downstream consequence of vascular dysfunction2.
OS-CMR with vasoactive breathing maneuvers has been utilized to evaluate vascular function in numerous scenarios, including healthy individuals, macrovascular disease in patients with coronary artery disease (CAD), as well as microvascular dysfunction in patients with obstructive sleep apnea (OSA), ischemia with no obstructive coronary artery stenosis (INOCA), after heart transplantation, and heart failure with preserved ejection fraction (HFpEF)4,7,12,13,14,15,16. In a CAD population, the protocol for the breathing-induced myocardial oxygenation reserve (B-MORE) as derived from OS-CMR was proven to be safe, feasible, and sensitive in identifying an impaired oxygenation response in myocardial territories perfused by a coronary artery with a significant stenosis13.
In microvascular dysfunction, OS-CMR demonstrated a delayed myocardial oxygenation response in patients with obstructive sleep apnea, and a blunted B-MORE was found in patients with HFpEF and following heart transplantation12,14,16. In women with INOCA, the breathing maneuver led to an abnormally heterogeneous myocardial oxygenation response, highlighting the advantage of the high spatial resolution of OS-CMR15. This paper reviews the rationale and methodology for performing OS-CMR with vasoactive breathing maneuvers and discusses its clinical utility in the assessment of vascular pathophysiology in patient populations with microvascular dysfunction, specifically as they relate to endothelial dysfunction.
The physiological context of breathing-enhanced oxygenation-sensitive MRI 
Under normal physiologic conditions, an increase in oxygen demand is matched by an equivalent increase in oxygen supply through increased blood flow, resulting in no change in local deoxyhemoglobin concentration. In contrast, induced vasodilation leads to &#34;excess&#34; inflow of oxygenated blood without a change in oxygen demand. Consequently, more of the tissue hemoglobin is oxygenated, and thus, there is less deoxyhemoglobin, leading to a relative increase in OS-CMR signal intensity4,17. If vascular function is compromised, it cannot properly respond to an altered metabolic demand or stimulus to augment myocardial blood flow.
In the setting of a stimulus to elicit vasomotion, such as paced hyperventilation eliciting vasoconstriction or a long breath-hold eliciting carbon dioxide-mediated vasodilation, impaired vasomotor activity would result in a relative increase in local deoxyhemoglobin concentration compared with other regions, and, subsequently, a reduced change in OS-CMR signal intensity. In the setting of inducible ischemia, impaired vascular function would result in increased local demand not met by a local increase in myocardial blood flow even in the absence of epicardial coronary artery stenosis. In OS-CMR images, the net local increase in deoxyhemoglobin concentration leads to a decrease in local signal intensity2,18,19,20.
Attenuated vascular smooth muscle relaxation in response to endothelium-dependent and -independent vasodilators (including adenosine) has been demonstrated in patients with coronary microvascular dysfunction21,22,23,24,25,26,27. Endothelial-independent dysfunction is thought to be due to structural abnormalities from microvascular hypertrophy or surrounding myocardial pathology. In contrast, endothelial dysfunction results in both inadequate vasoconstriction and impaired (endothelium-dependent) vasorelaxation, typically caused by a loss of nitric oxide bioactivity in the vessel wall21,28. Endothelial dysfunction has been implicated in the pathogenesis of a number of cardiovascular diseases, including hypercholesterolemia, hypertension, diabetes, CAD, obstructive sleep apnea, INOCA, and HF23,24,28,29,30,31,32. In fact, endothelial dysfunction is the earliest manifestation of coronary atherosclerosis33. The imaging of endothelial function has very strong potential, given its role as a significant predictor of adverse cardiovascular events and long-term outcomes, with profound prognostic implications in cardiovascular disease states23,29,30,31,34,35.
In contrast to perfusion imaging, the breathing-induced myocardial oxygenation reserve (B-MORE), defined as the relative increase in myocardial oxygenation during a post-hyperventilation breath-hold allows for visualizing the consequences of such a vasoactive trigger on global or regional oxygenation itself2,36. As an accurate downstream marker of vascular function, B-MORE can, therefore, not only identify vascular dysfunction but also actual inducible ischemia, indicating a more severe local perfusion or oxygenation problem18,19,37. This is achieved through the ability of OS-CMR to visualize the relative decrease in deoxygenated hemoglobin, which is abundant in the capillary system of the myocardium, which itself represents a significant proportion of myocardial tissue24.
OS-CMR sequence 
The magnetic resonance imaging (MRI) sequence used for OS-CMR imaging is a prospectively gated, modified, balanced, steady-state, free precession (bSSFP) sequence acquired in two short-axis slices. This bSSFP sequence is a standard clinical sequence available (and modifiable) on all MRI scanners that perform cardiac MRI, making this technique vendor-agnostic and easily implemented. In a regular bSSFP cine sequence, echo time, repetition time, and flip angle are modified to sensitize the resulting signal intensity to the BOLD effect and, thus, create an oxygenation-sensitive sequence. This approach, a T2-prepared bSSFP readout, has previously been shown to be suitable for acquiring oxygenation-sensitive images with a higher signal-to-noise ratio, higher image quality, and faster scan times when compared to previous gradient echo techniques used for BOLD imaging38. Performing breathing-enhanced OS-CMR with this approach can be applied with very few, mild side effects (Table 1). Of note, more than 90% of participants complete this protocol with sufficiently long breath-hold times4,12,13,16.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>balanced SSFP MRI sequence</td>
			<td>Any</td>
			<td></td>
			<td>To modify to create the OS-CMR sequence</td>
		</tr>
		<tr>
			<td>DICOM/ Imaging Viewer</td>
			<td>Any</td>
			<td></td>
			<td>Best if the viewer has the ability for quantitative measurements (i.e., Area19 prototype software)</td>
		</tr>
		<tr>
			<td>Magnetic Resonance Imaging scanner</td>
			<td>Any</td>
			<td></td>
			<td>3 Tesla or 1.5 Tesla</td>
		</tr>
		<tr>
			<td>Metronome</td>
			<td>Any</td>
			<td></td>
			<td>Set to 30 breaths per minute. To use if manually communicating breathing maneuver instructions to participants.</td>
		</tr>
		<tr>
			<td>Speaker system</td>
			<td>Any</td>
			<td></td>
			<td>To communicate breathing maneuver instrucitons to participants through</td>
		</tr>
		<tr>
			<td>Stopwatch</td>
			<td>Any</td>
			<td></td>
			<td>To use if manually communicating breathing maneuver instructions to participants</td>
		</tr>
	</tbody>
</table>

oxygenation-sensitive cardiac mri with vasoactive breathing maneuvers for the non-invasive assessment of coronary microvascular dysfunction

Oxygenation-sensitive cardiac magnetic resonance imaging (OS-CMR) is a diagnostic technique that uses the inherent paramagnetic properties of deoxyhemoglobin as an endogenous source of tissue contrast. Used in combination with standardized vasoactive breathing maneuvers (hyperventilation and apnea) as a potent non-pharmacologic vasomotor stimulus, OS-CMR can monitor changes in myocardial oxygenation. Quantifying such changes during the cardiac cycle and throughout vasoactive maneuvers can provide markers for coronary macro- and microvascular function and thereby circumvent the need for any extrinsic, intravenous contrast or pharmacologic stress agents.
OS-CMR uses the well-known sensitivity of T2*-weighted images to blood oxygenation. Oxygenation-sensitive images can be acquired on any cardiac MRI scanner using a modified standard clinical steady-state free precession (SSFP) cine sequence, making this technique vendor-agnostic and easily implemented. As a vasoactive breathing maneuver, we apply a 4-min breathing protocol of 120 s of free breathing, 60 s of paced hyperventilation, followed by an expiratory breath-hold of at least 30 s. The regional and global response of myocardial tissue oxygenation to this maneuver can be assessed by tracking the signal intensity change. The change over the initial 30 s of the post-hyperventilation breath-hold, referred to as the breathing-induced myocardial oxygenation reserve (B-MORE) has been studied in healthy people and various pathologies. A detailed protocol for performing oxygen-sensitive CMR scans with vasoactive maneuvers is provided.
As demonstrated in patients with microvascular dysfunction in yet incompletely understood conditions, such as inducible ischemia with no obstructive coronary artery stenosis (INOCA), heart failure with preserved ejection fraction (HFpEF), or microvascular dysfunction after heart transplantation, this approach provides unique, clinically important, and complementary information on coronary vascular function.

Interpreting B-MORE 
In previously published studies utilizing OS-CMR with vasoactive breathing maneuvers, the global or regional B-MORE was calculated by comparing the first end-systolic image of the breath-hold to the end-systolic image closest to 15 s, 30 s, 45 s, etc. of the breath-hold. The end-systolic phase of the cardiac cycle was chosen for several reasons. The end-systolic image is the most consistent phase identified among and between readers: it contains the greatest number of pixels in ...

Watch this Scientific Journal Video about Oxygenation-sensitive Cardiac MRI with Vasoactive Breathing Maneuvers for the Non-invasive Assessment of Coronary Microvascular Dysfunction at JoVE.com

Oxygenation-sensitive Cardiac MRI with Vasoactive Breathing Maneuvers for the Non-invasive Assessment of Coronary Microvascular Dysfunction

The assessment of microvascular function by oxygenation-sensitive cardiac magnetic resonance imaging in combination with vasoactive breathing maneuvers is unique in its ability to assess rapid dynamic changes in myocardial oxygenation in vivo and, thus, may serve as a critically important diagnostic technique for coronary vascular function.

Oxygenation-sensitive cardiac magnetic resonance imaging (OS-CMR) is a diagnostic technique that uses the inherent paramagnetic properties of ...

With oxygenation-sensitive MRI, we can track myocardial oxygenation over time, and using breathing maneuvers, we can interfere with the physiology and therefore use this test for assessing microvascular function and overall coronary vascular function. Our technique utilizes an endogenous contrast agent in deoxyhemoglobin and vasoactive breathing maneuvers instead of a pharmacological stress agent to induce changes that we can then use to assess coronary vascular function. The needle and stress-free method allows us to assess vascular function in various diseases, and especially in diseases like heart failure with preserved ejection fraction and other forms of non-coronary ischemia.This will allow for a diagnostic approach that will provide important clinical information for imaging people and for assessing the success of therapeutic interventions. When beginners are using this technique, they should make sure to practice the breathing maneuver with their participants outside of the scanner. As well, when performing the actual acquisition, the MRI technologist should watch the live-scanning window and the respiratory trace to make sure that the participants are accurately and adequately performing the hyperventilation and breath hold.And now, Maggie Leo, our chief technologist here at the McGill University Health Center actually lead you through the procedure. To begin, ensure that every participant passes the MRI safety and compatibility questionnaire of the local institution, which should indicate questions on past medical and surgical history. Identify the presence of an implant, device, or metallic foreign body inside or at the surgical site of the participant.If applicable, obtain a pregnancy test. Verify that the patient has abstained from vasoactive medication and caffeine for 12 hours before the MRI scan. Show the participant the instructional breathing maneuver video and perform a practice session of 60 seconds of paced hyperventilation, followed by a maximal voluntary breath hold with every participant outside of the MRI scanning room and provide feedback on the performance of the hyperventilation.Instruct the participants to resume breathing when they have a strong urge to do so. Increase the repetition and echo time on the MRI console's standard bSSFP sequence and decrease the flip angle. Create two OS series, a baseline labeled OS base, and the continuous acquisition during which the breathing maneuver is performed labeled OS continuous acquisition.In the OS continuous acquisition, increase the TE from one to 1.7. Increase the number of cardiac cycles until the acquisition time is nearly 4.5 minutes. For slice prescription, plan in an end systolic still frame of a long axis view.Prescribe two short axis slices, one at the mid to basal, and the other at the mid to apical ventricular level. Adjust the sequence parameters as required for a given participant. Adjust the average gap in spacing between slices based on the size of the participant's heart and ensure proper slice location.Adjust the field of view to avoid wrap artifacts if necessary. Make every effort to keep the field of view between 360 and 400 millimeters. Adjust the shim volume to be tight around the left ventricle in both the long and short axis views.For sequence acquisition, approve the sequence and run it during the end-expiration breath hold. Ensure that this baseline OS sequence lasts approximately 10 seconds based on the heart rate and MRI scanner. Check both slices of the acquired series and look for any respiratory motion, poor slice location, or the presence of artifacts.For troubleshooting, if the slice location is too basal or too apical, adjust the prescribed slices accordingly. If an artifact is present, check the phase and coating direction. Then make the field of view larger and adjust the shim volume around the left ventricle.For the sequence planning, copy slice position and adjust volume from the OS baseline image as demonstrated earlier. Verify the image and slice positioning, then capture cycle. If applicable, open the livestream window.In the control room, plug a device with the breathing maneuver instructions dot MP3 file into the auxiliary input or place it over the microphone projecting into the MRI scanner. Alternatively, manually guide the participant through the breathing maneuver using a stopwatch for timing and verbally provide instructions through the microphone connected to the MRI speaker system. Simultaneously press play for the OS continuous acquisition sequence on the MRI scanner for sequence acquisition and press play for the dot MP3 breathing instruction file as demonstrated earlier.If manually guiding the participant through the breathing maneuvers, instruct them to breathe in and breathe out, then hold their breath for 10 seconds and to start hyperventilating as soon as they hear the metronome beep. Notify the participant at the 55-second time point of hyperventilation to take a deep breath in, breathe out, and hold their breath. Monitor the participant's performance of the paced hyperventilation through the control room window or MRI scanner camera to ensure adequate performance of the deep breathing.If bellows are used, monitor the amplitude peaks on the respiratory gating viewer. If hyperventilation is not adequately performed after initial guidance, abort the acquisition and repeat the OS continuous acquisition sequence. Monitor for any small breaths taken by participants throughout the breath hold by monitoring the tracing of a respiration belt on the MRI console, or visually through the window or camera.Once the participant starts breathing at the end of the breath hold, stop the acquisition. After the end of the acquisition, ask the participant if they experienced any adverse effects and allowed the participant to breathe normally for three minutes. The global B-MORE data was displayed visually and quantitatively.Visually, pixel-wise color overlay maps were generated to augment quantitative measurements assessing myocardial oxygenation. Myocardial oxygenation reserve assessment depicted a stable global myocardial oxygenation in a healthy volunteer. A decrease in regional myocardial oxygenation was observed in a patient with a left anterior descending stenosis, while a global reduction in myocardial oxygenation was observed in a patient with heart failure.Quantitatively, global B-MORE values were compared between healthy volunteers and patients with obstructive sleep apnea syndrome, coronary artery disease, ischemia with no obstructive coronary artery stenosis, heart failure with preserved ejection fraction, and post-heart transplant. To ensure good OS acquisition overall, there are three things to remember. One, that the images are performed in end-expiration for the OS breath hold.Two, that the images themselves are free of any artifacts. And three, that the technologists are watching in the live scanning window to ensure accurate and adequate performance of the hyperventilation and breath hold. The results of breathing-enhanced oxygenation-sensitive MR can actually easily be combined with other markers from the MR scan such as tissue characteristics and function, and therefore serve as an overall as a very powerful diagnostic test that is cost efficient, safe, and extremely important for clinical decision making.

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Research

JoVE Journal

Medicine

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 ...

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery (SALLI) MRI in Rats

Small animal magnetic resonance imaging is an important tool to study cardiac function and changes in myocardial tissue. The high heart rates of small animals (200 to 600 beats/min) have previously limited the role of CMR imaging. Small animal Look-Locker inversion recovery (SALLI) is a T1 mapping sequence for small animals to overcome this problem 1. T1 maps provide quantitative information about tissue alterations and contrast agent kinetics. It is also possible to detect diffuse myocardial processes such as interstitial fibrosis or edema 1-6. Furthermore, from a single set of image data, it is possible to examine heart function and myocardial scarring by generating cine and inversion recovery-prepared late gadolinium enhancement-type MR images 1.
	The presented video shows step-by-step the procedures to perform small animal CMR imaging. Here it is presented with a healthy Sprague-Dawley rat, however naturally it can be extended to different cardiac small animal models.

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery SALLI MRI in Rats

Contrast enhanced cardiac magnetic resonance (CMR) imaging allows comprehensive in vivo assessment of the heart in small animal models of cardiovascular disease. Here we detail the procedures to perform CMR imaging, reconstruction and analysis using Small Animal Lock-Locker Inversion Recovery (SALLI) in rats.

Small animal magnetic resonance  imaging is an important tool to study cardiac function and changes in myocardial  tissue. The high heart rates of small ...

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Intravital imaging has emerged as an indispensable tool in biological research. In the process, many imaging techniques have been developed to study different biological processes in animals non-invasively. However, a major technical limitation in existing intravital imaging modalities is the inability to combine non-invasive, longitudinal imaging with single-cell resolution capabilities. We show here how transplantation into the anterior chamber of the eye circumvents such significant limitation offering a versatile experimental platform that enables non-invasive, longitudinal imaging with cellular resolution in vivo. We demonstrate the transplantation procedure in the mouse and provide representative results using a model with clinical relevance, namely pancreatic islet transplantation. In addition to enabling direct visualization in a variety of tissues transplanted into the anterior chamber of the eye, this approach provides a platform to screen drugs by performing long-term follow up and monitoring in target tissues. Because of its versatility, tissue/cell transplantation into the anterior chamber of the eye not only benefits transplantation therapies, it extends to other in vivo applications to study physiological and pathophysiological processes such as signal transduction and cancer or autoimmune disease development.

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

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 ...

Intracoronary Acetylcholine Provocation Testing for Assessment of Coronary Vasomotor Disorders

Intracoronary acetylcholine provocation testing (ACH-test) is an established method for assessment of epicardial coronary artery spasm in the catheterization laboratory which was introduced more than 30 years ago. Due to the short half-life of acetylcholine it can only be applied directly into the coronary arteries. Several studies have demonstrated the safety and clinical usefulness of this test. However, acetylcholine testing is only rarely applied in the U.S. or Europe. Nevertheless, it has been shown that 62% of Caucasian patients with stable angina and unobstructed coronary arteries on coronary angiography suffer from coronary vasomotor disorders that can be diagnosed with acetylcholine testing. In recent years it has been appreciated that the ACH-test not only assesses the presence of epicardial spasm but that it can also be useful for the detection of coronary microvascular spam. In such cases no epicardial spasm is seen after injection of acetylcholine but ischemic ECG shifts are present together with a reproduction of the patient's symptoms during the test. This article describes the experience with the ACH-test and its implementation in daily clinical routine.

Intracoronary acetylcholine testing has been established for the assessment of epicardial coronary spasm more than 30 years ago. Recently, the focus has shifted towards the microcirculation and it has been shown that microvascular spasm can be detected using ACH-testing. This article describes the ACH-test and its implementation in daily routine.

Intracoronary acetylcholine provocation testing (ACH-test) is an established method for assessment of epicardial coronary artery spasm in the ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

Interventional Diagnostic Procedure: A Practical Guide for the Assessment of Coronary Vascular Function

Approximately 40% of patients undergoing invasive coronary angiography for investigation of angina are found to have no obstructive coronary artery disease (ANOCA). Abnormal coronary function underlies coronary vasomotion syndromes including coronary endothelial dysfunction, microvascular angina, vasospastic angina, post-PCI angina and myocardial infarction with no obstructive coronary arteries (MINOCA). Each of these endotypes are distinct subgroups, characterized by specific disease mechanisms. Diagnostic criteria and linked therapy for these conditions are now established by expert consensus and clinical guidelines.
Coronary function tests are performed as an adjunctive interventional diagnostic procedure (IDP) in appropriately selected patients during coronary angiography. This aids differentiation of patients according to endotype. The IDP includes two distinct components: a diagnostic guidewire test and a pharmacological coronary reactivity test. The tests last approximately 5 minutes for the former and 10-15 minutes for the latter. Patient safety and staff education are key.
The diagnostic guidewire test measures parameters of coronary flow limitation (fractional flow reserve [FFR], coronary flow reserve [CFR], microvascular resistance [index of microvascular resistance (IMR)], basal resistance index, and vasodilator function [CFR, resistive reserve ratio (RRR)]).
The pharmacological coronary reactivity test measures the vasodilator potential and propensity to vasospasm of both the main coronary arteries and the micro-vessels. It involves intra-coronary infusion of acetylcholine and glyceryl trinitrate (GTN). Acetylcholine is not licensed for parenteral use and is therefore prescribed on a named-patient basis. Vasodilatation is the normal, expected response to infusion of physiological concentrations of acetylcholine. Vascular spasm represents an abnormal response, which supports the diagnosis of vasospastic angina.
The purpose of this practical guide is to provide information on the preparation and administration of the IDP in clinical practice. It discusses some key preparation and safety considerations, as well as tips for procedural success. The IDP supports stratified medicine for a personalized approach to health and wellbeing.

The purpose of this practical guide is to provide information on the preparation and administration of an interventional diagnostic procedure in clinical practice. It discusses some key preparation and safety considerations, as well as tips for procedural success.

Approximately 40% of patients undergoing invasive coronary angiography for investigation of angina are found to have no obstructive coronary artery ...

Measurement of Myocardial Lactate Production for Diagnosis of Coronary Microvascular Spasm

In about a quarter of patients with angina and non-obstructive coronary arteries, no epicardial spasm is noted on coronary arteriography during an angina attack. Since the pressure-rate product is almost identical at rest and the onset of attack in those patients, the decrease in coronary blood flow rather than increased myocardial oxygen consumption is likely to explain myocardial ischemia, indicating a substantial involvement with coronary microvascular spasm (MVS). Myocardial lactate production, which could be defined as a negative myocardial lactate extraction ratio (ratio of the coronary arterial-venous difference in lactate concentration to arterial concentration), is considered indicative of objective evidence to support the emerging myocardial ischemia. Thus, monitoring of the myocardial lactate production and the emergence of chest pain and ischemic electrocardiographic changes during acetylcholine (ACh) provocation testing is of significant value for detecting the entity of MVS. Practically, 1 min after incremental doses of ACh (20, 50, and 100 &#956;g) are administered into the left coronary artery (LCA), paired samples of 1 mL of blood are collected from the LCA ostium and coronary sinus for measurement of lactate concentration by a calibrated automatic lactate analyzer. Then, the development of MVS could be confirmed by negative myocardial lactate extraction ratio despite the absence of angiographically demonstrable epicardial coronary spasm or before its occurrence throughout ACh provocation testing. In conclusion, assessment of myocardial lactate production is essential and valuable for the diagnosis of MVS.

Myocardial lactate production (coronary arterial-venous difference in serum lactate level) during coronary spasm provocation testing is considered as a highly sensitive marker that reflects acetylcholine-induced myocardial ischemia due to microvascular spasm. This article presents the procedures to assess myocardial lactate production for the diagnosis of coronary microvascular spasm.

In about a quarter of patients with angina and non-obstructive coronary arteries, no epicardial spasm is noted on coronary arteriography during an angina ...

In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including Mef2c, Gata4, Tbx5 (MGT), fibroblasts can be reprogrammed into induced cardiomyocytes (iCMs). These iCMs, when generated in situ in an infarcted heart, integrate electrically and mechanically with the surrounding myocardium, leading to a reduction in scar size and an improvement in heart function. Because of the relatively low reprogramming efficiency, purity, and quality of the iCMs, characterization of iCMs remains a challenge. The currently used methods in this field, including flow cytometry, immunocytochemistry, and qPCR, mainly focus on cardiac-specific gene and protein expression but not on the functional maturation of iCMs. Triggered by action potentials, the opening of voltage-gated calcium channels in cardiomyocytes leads to a rapid influx of calcium into the cell. Therefore, quantifying the rate of calcium influx is a promising method to evaluate cardiomyocyte function. Here, the protocol introduces a method to evaluate iCM function by calcium (Ca2+) flux. An &#945;MHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 mouse strain was established by crossing Tg(Myh6-cre)1Jmk/J (referred to as Myh6-Cre below) with Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as Rosa26A-Flox-Stop-Flox-GCaMP3 below) mice. Neonatal cardiac fibroblasts (NCFs) from P0-P2 neonatal mice were isolated and cultured in vitro, and a polycistronic construction of MGT was introduced to NCFs, which led to their reprogramming to iCMs. Because only successfully reprogrammed iCMs will express GCaMP3 reporter, the functional maturation of iCMs can be visually assessed by Ca2+ flux with fluorescence microscopy. Compared with un-reprogrammed NCFs, NCF-iCMs showed significant calcium transient flux and spontaneous contraction, similar to CMs. This protocol describes in detail the mouse strain establishment, isolation and selection of neonatal mice hearts, NCF isolation, production of retrovirus for cardiac reprogramming, iCM induction, the evaluation of iCM Ca2+ flux using our reporter line, and related statistical analysis and data presentation. It is expected that the methods described here will provide a valuable platform to assess the functional maturation of iCMs for cardiac reprogramming studies.

We describe here, the establishment and application of an Tg(Myh6-cre)1Jmk/J /Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as &#945;MHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 below) mouse reporter line for cardiac reprogramming assessment. Neonatal cardiac fibroblasts (NCFs) isolated from the mouse strain are converted into induced cardiomyocytes (iCMs), allowing for convenient and efficient evaluation of reprogramming efficiency and functional maturation of iCMs via calcium (Ca2+) flux.

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including ...