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 population of interest. Use the following general, common exclusion criteria for an OS-CMR with vasoactive breathing maneuvers protocol: general MRI contraindications (e.g., MRI-incompatible devices such as pacemakers or defibrillators, implanted material, or foreign bodies), consumption of caffeine or vasoactive medication in the 12 h prior to the MRI, and age &#60;18 years.</li>
	<li>First, acquire the standard clinical localizer scout and ventricular structure and function images before acquiring the OS acquisitions. Use the bSSFP long-axis cine images to plan the slice positioning of the OS acquisitions. 
	NOTE: A review of standardized clinical CMR protocols is described elsewhere39.</li>
	<li>Baseline breath-hold
	<ol>
		<li>Acquire the first OS-CMR series as a short, baseline breath-hold acquisition to assess image quality and slice location, check for artifacts, as well as serve as a signal intensity baseline.</li>
		<li>Perform a short (~10 s), single cardiac cycle acquisition after the participant has been breathing normally. Ensure that the breath-hold is done on end-expiration.</li>
	</ol>
	</li>
	<li>Continuous acquisition with vasoactive breathing maneuver
	<ol>
		<li>Acquire the second OS-CMR series as a 2 min, continuous acquisition comprised of a short 15 s breath hold and 1 min of paced hyperventilation, followed by a voluntary, maximal breath hold (~45 s). As the continuous acquisition obtains multiple cardiac cycles over 2 min,&#160;modify one additional parameter (the number of cardiac cycles acquired by the acquisition) to make this series a repeated-measures acquisition. 
		NOTE: The minimum required breath-hold length is 30 s, although a breath-hold of 45-60 s is considered the standard.</li>
		<li>Convey the instructions for the vasoactive breathing maneuver to the participants in the MRI scanner by manually directing the participant throughout the breathing maneuver through a microphone connected to the MRI speaker system or through a prerecorded .mp3 file (Supplemental File 1) that can be played for the participant through the MRI speaker system.</li>
		<li>Start the breathing maneuver with a short 15 s, and end the expiration breath hold to acquire one cardiac cycle. Then guide the participant through paced breathing with the use of audible beeps from a metronome at a frequency of 30 breaths/min (one beep indicates breathing in, one beep indicates breathing out). At the 55 s mark of hyperventilation, give a final voice command to &#34;take a deep breath in and then breathe out and hold your breath&#34; to ensure that the breath-hold is performed at an end-expiration level. 
		NOTE: The change in blood CO2 is much more pronounced with the breath-hold at end-expiration (the lung surface is smaller, minimizing the residual diffusion of CO2 into the alveoli).</li>
	</ol>
	</li>
	<li>Image analysis
	<ol>
		<li>To measure the B-MORE, consider the first end-systolic image during breath-hold as time 0 s. Compare the global or regional signal intensity values of the end-systolic image acquired closest to the 30 s time point of the breath-hold to the image signal intensity at the 0 s time point. Report BMORE as a percent change in signal intensity at 30 s compared to time 0 s of the breath-hold.</li>
	</ol>
	</li>
</ol>
2. Pre-scan procedure
<ol>
	<li>Ensure that every participant passes the MRI safety and compatibility questionnaire of the local institution (MRI General Contraindication form), which should include questions on past medical and surgical history and identify the presence of any implant, device, or metallic foreign body inside or at the surgical site of the participant40.</li>
	<li>Obtain a pregnancy test, if applicable.</li>
	<li>Verify that the patient has abstained from vasoactive medication and caffeine in the 12 h prior to the MRI scan.</li>
	<li>Show the participant the instructional breathing maneuver video (Supplemental Video S1).
	<ol>
		<li>Perform a practice session of 60 s 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.</li>
		<li>Instruct the participants that they can simply resume breathing when they have a strong urge to do so. 
		NOTE: See the discussion for points to note and provide feedback on to the participants.</li>
	</ol>
	</li>
</ol>
3. MRI acquisition of oxygenation-sensitive sequences
<ol>
	<li>Modify three parameters from the standard bSSFP sequence on the MRI console: increase repetition time (TR), increase echo time (TE), decrease flip angle (FA). 
	NOTE: The modified values are dependent on THE MRI scanner field strength (Table 2). Increasing TR and TE and decreasing FA results in an increase in T2* or oxygenation sensitivity of the MRI sequence. These modifications will then result in an increase in bandwidth and base resolution of the sequence.</li>
	<li>Create two OS series, a baseline (labeled: OS_base) and the continuous acquisition during which the breathing maneuver is performed (labeled: OS_cont_acq). Leave the baseline OS sequence unchanged. In the OS continuous acquisition, increase the repeated measures from 1 to ~15-40 (depending on the scanner type). Increase the number of cardiac cycles (measures) until the acquisition time is ~2.5 min. 
	NOTE: Two OS-CMR sequences are needed: OS baseline acquisition and OS continuous acquisition with vasoactive breathing maneuvers. The following sections describe these steps.</li>
</ol>
4. OS baseline acquisition
<ol>
	<li>For slice prescription, plan in an end-systolic still frame of a long axis view (two- or four-chamber image). Prescribe two short-axis slices-one at the mid-to-basal and the other at the mid-to-apical ventricular level. See the discussion for points to consider regarding slice location.</li>
	<li>Sequence parameter adjustments
	<ol>
		<li>Adjust the sequence parameters as needed for a given participant. See Table 3 for sequence parameters than can or cannot be changed.</li>
		<li>Adjust the average gap/spacing between slices based on the size of the participant&#39;s heart and ensure proper slice location.</li>
		<li>Adjust the field of view to avoid wrap artifacts if necessary. Make every effort to keep the field of view between 360 mm and 400 mm.</li>
	</ol>
	</li>
	<li>Shim volume
	<ol>
		<li>Adjust the shim volume to be tight around the left ventricle in both the long- and short-axis views.</li>
	</ol>
	</li>
	<li>Sequence acquisition
	<ol>
		<li>Approve the sequence and run it during the end-expiration breath-hold. Ensure that this baseline OS sequence lasts ~10 s, based on the heart rate and MRI scanner.</li>
	</ol>
	</li>
	<li>Image quality check
	<ol>
		<li>Check both slices of the acquired series-look for any respiratory motion, poor slice location, or the presence of artifacts. Repeat the baseline OS sequence until adequate image quality has been obtained.</li>
	</ol>
	</li>
	<li>For troubleshooting, if the slice location is too basal or too apical, adjust the prescribed slice location to be closer to the mid-ventricular level. If there is an artifact present, follow the steps below:
	<ol>
		<li>Check the phase encoding direction.</li>
		<li>Make the field of view larger.</li>
		<li>Adjust the shim volume around the left ventricle.</li>
	</ol>
	</li>
</ol>
5. OS continuous acquisition with vasoactive breathing maneuvers
NOTE: Ensure that every participant has been instructed about the proper performance of the breathing maneuver before they are in the MRI scanner (see section 2).
<ol>
	<li>Sequence planning
	<ol>
		<li>If possible, copy slice position and adjust volume from the OS baseline image or duplicate the baseline OS sequence and, in repeated measurements, increase from 1 to ~15-40 (or close to 2.5 min acquisition time).</li>
	</ol>
	</li>
	<li>Verify the image and slice positioning, and then capture cycle.</li>
	<li>If possible, open the live stream window.</li>
	<li>In the control room, plug a device with the breathing maneuver instructions .mp3 file into the auxiliary input or prepare to hold 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.</li>
	<li>Sequence acquisition
	<ol>
		<li>Simultaneously press play for the OS Continuous Acquisition sequence on the MRI scanner and play for the .mp3 breathing instruction file or start the stopwatch if the participant is being manually instructed.</li>
		<li>If manually guiding the participant through the breathing maneuvers, instruct them to breathe in and breathe out, then hold their breath for 15 seconds (for the short breath-hold), and start hyperventilating as soon as they hear the metronome beep.</li>
		<li>Notify the participant at the 40 s mark of hyperventilation (2:40 on the stopwatch).</li>
		<li>At the 55 s time point of hyperventilation (2:55 on the stopwatch), instruct the participant to &#34;take a deep breath in, breathe out, and hold your breath&#34;. 
		NOTE: The free breathing and hyperventilation images will have motion artifacts. This is expected. However, there should not be any motion artifacts during the breath-hold. It is critically important that the breath-held images are acquired after exhalation (comfortable end-expiratory position). Only a breath-hold after exhalation leads to the rapid increase of blood CO2 during the first 30 seconds of the subsequent breath-hold, with the associated change of coronary blood flow and myocardial oxygenation.</li>
		<li>Monitor the participant&#39;s performance of the paced hyperventilation through the control room window or MRI scanner camera to ensure adequate performance of deep breathing. If bellows are used, then monitor the amplitude peaks on the respiratory gating viewer. If hyperventilation is not being adequately performed after initial guidance, abort the acquisition and repeat the OS continuous acquisition sequence.</li>
		<li>Monitor for any small breaths taken by participants throughout the breath-hold. Do this by monitoring the tracing of a respiration belt on the MRI console or visually through the window/camera.</li>
		<li>Once the participant starts breathing at the end of the breath-hold, stop the acquisition.</li>
		<li>After the end of the acquisition, ask the participant if they experienced any adverse effects and allow the participant to breathe normally for 3 min.</li>
	</ol>
	</li>
	<li>Troubleshooting: repeat acquisition
	<ol>
		<li>If the breathing maneuvers need to be repeated, repeat the baseline OS sequence. 
		NOTE: A period of 2-3 min before repeating the acquisition is required to allow the physiology to return to baseline. Previous data have shown that physiology does not return to baseline after 1 min41.</li>
		<li>If the image quality of the OS baseline sequence is adequate, repeat the OS continuous acquisition and performance of the breathing maneuvers.</li>
	</ol>
	</li>
</ol>
6. Image analysis
<ol>
	<li>Import the OS-CMR image data sets into a DICOM viewer with image contouring capabilities, best with functionality to automatically analyze oxygenation-sensitive signal intensity changes.</li>
	<li>Markers and their measurement
	<ol>
		<li>Acquire a baseline image during a short, baseline breath-hold acquisition before hyperventilation (rest). Compare the first image of the breath-hold (representing the end of the &#34;stress&#34; stimulus) to the baseline image. 
		NOTE: Hyperventilation is a vasoconstrictive stimulus that reduces myocardial oxygenation (Healthy: %&#916;SI &#8776;&#8722;5% to &#8722;10%13).</li>
		<li>Obtain many images (and cardiac cycles) during the breath-hold. Use the first image of the breath-hold as the baseline and compare all following images to this image. 
		NOTE: Breath-hold is a vasodilating stimulus that increases myocardial oxygenation (%&#916;SI &#8776; +5%-15%12,13,14,16).</li>
		<li>Myocardial contours 
		For manual analysis: cardiac phase selection
		<ol>
			<li>As the breath-hold can contain greater than 400 images, analyze only a single phase of each cardiac cycle. As a result, focus the analysis on the end-systolic images of each cardiac cycle.</li>
			<li>Identify the end-systolic images of each cardiac cycle.</li>
			<li>Draw the epicardial and endocardial contours around the myocardium.</li>
			<li>Window the image to look for artifacts, which will appear as either dark (susceptibility) or bright areas due to poor gating in the myocardium. 
			NOTE: Avoid including pixels with partial volume effects from the left and right ventricular blood pools. Most contouring errors occur from the endocardial contour, including pixels with partial volume effects and resultant artificially elevated signal intensity from the left ventricular blood pool. To avoid this, draw the endocardial contour one full pixel inside the myocardium. Similarly, ensure that the epicardial contour is one full pixel inside the myocardium to avoid partial volume effects from the right ventricular blood pool, epicardial fat, or air-lung interface.</li>
			<li>Copy and paste the endocardial and epicardial contours from the first end-systolic image of the cardiac cycle to the end-systolic image of the next cardiac cycle. Adjust the contours as needed. 
			For automated analysis: 
			NOTE: With automated contouring capabilities, if desired, all phases of the cardiac cycle can be contoured and analyzed.</li>
			<li>Check all images to ensure accurate contouring. 
			NOTE: Some commercially available automated contouring capabilities have been trained on data sets that were contoured for volumetric analysis. These contours are prone to partial volume effects as they are meant to border the blood pool and myocardium. OS-CMR contours must be fully inside of the myocardium.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
7. Segmentation for regional analysis
<ol>
	<li>To obtain regional information, identify the anteroseptal and inferoseptal insertion of the right ventricle to divide the myocardium into American Heart Association (AHA) segmentation42.</li>
</ol>
8. Calculating B-MORE
<ol>
	<li>Express B-MORE as a percent change in signal intensity from baseline to vasodilation (see equation 1): 
	<img alt="Equation 1" src="/files/ftp_upload/64149/64149eq01v2.jpg" style="margin: auto;" />&#160; &#160; &#160;(1)</li>
	<li>Calculate the global B-MORE as the global mean myocardial signal intensity of the end-systolic images at 30 s compared to 0 s of the breath-hold8 (see equation 2): 
	<img alt="Equation 2" src="/files/ftp_upload/64149/64149eq02.jpg" style="margin: auto;" />&#160; &#160;&#160;(2)</li>
</ol>

MGF is listed as a holder of United States Patent No. 14/419,877: Inducing and measuring myocardial oxygenation changes as a marker for heart disease; United States Patent No. 15/483,712: Measuring oxygenation changes in tissue as a marker for vascular function; United States Patent No 10,653,394: Measuring oxygenation changes in tissue as a marker for vascular function - continuation; and Canadian Patent CA2020/051776: Method and apparatus for determining biomarkers of vascular function utilizing bold CMR images. EH is listed as a holder of International Patent CA2020/051776: Method and apparatus for determining biomarkers of vascular function utilizing bold CMR images.

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><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 ...

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2.2K Views.  McGill University. Met oxygenatiegevoelige MRI kunnen we de myocardiale oxygenatie in de loop van de tijd volgen, en met behulp van ademhalingsmanoeuvres kunnen we de fysiologie verstoren en daarom deze test gebruiken voor het beoordelen van de microvasculaire functie en de algehele coronaire vasculaire functie. Onze techniek maakt gebruik van een endogeen contrastmiddel bij deoxyhemoglobine en vasoactieve ademhalingsmanoeuvres in plaats van een farmacologisch stressmiddel om veranderingen teweeg te brengen die...

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

Magnetic Resonance Imaging Quantification of Pulmonary Perfusion using Calibrated Arterial Spin Labeling

This demonstrates a MR imaging method to measure the spatial distribution of pulmonary blood flow in healthy subjects 
during normoxia (inspired O2, fraction (FIO2) = 0.21) hypoxia (FIO2 = 0.125), and hyperoxia 
(FIO2 = 1.00). In addition, the physiological responses of the subject are monitored in the MR scan environment. MR images 
were obtained on a 1.5 T GE MRI scanner during a breath hold from a sagittal slice in the right lung at functional residual capacity. An arterial 
spin labeling sequence (ASL-FAIRER) was used to measure the spatial distribution of pulmonary blood flow 1,2 and a multi-echo fast 
gradient echo (mGRE) sequence 3 was used to quantify the regional proton (i.e. H2O) density, allowing the quantification 
of density-normalized perfusion for each voxel (milliliters blood per minute per gram lung tissue). 
With a pneumatic switching valve and facemask equipped with a 2-way non-rebreathing valve, different oxygen concentrations 
were introduced to the subject in the MR scanner through the inspired gas tubing. A metabolic cart collected expiratory gas via expiratory tubing. Mixed expiratory O2 and CO2 concentrations, oxygen consumption, carbon dioxide production, respiratory exchange ratio, 
respiratory frequency and tidal volume were measured. Heart rate and oxygen saturation were monitored using pulse-oximetry. 
Data obtained from a normal subject showed that, as expected, heart rate was higher in hypoxia (60 bpm) than during normoxia (51) or hyperoxia (50) and the arterial oxygen saturation (SpO2) was reduced during hypoxia to 86%. Mean ventilation was 8.31 L/min BTPS during hypoxia, 7.04 L/min during normoxia, and 6.64 L/min during hyperoxia. Tidal volume was 0.76 L during hypoxia, 0.69 L during normoxia, and 0.67 L during hyperoxia. 
Representative quantified ASL data showed that the mean density normalized perfusion was 8.86 ml/min/g during hypoxia, 8.26 ml/min/g during normoxia and 8.46 ml/min/g during hyperoxia, respectively. In this subject, the relative dispersion4, an index of global heterogeneity, was increased in hypoxia (1.07 during hypoxia, 0.85 during normoxia, and 0.87 during hyperoxia) while the fractal dimension (Ds), another index of heterogeneity reflecting vascular branching structure, was unchanged (1.24 during hypoxia, 1.26 during normoxia, and 1.26 during hyperoxia). 
Overview. This protocol will demonstrate the acquisition of data to measure the distribution of pulmonary perfusion noninvasively under conditions of normoxia, hypoxia, and hyperoxia using a magnetic resonance imaging technique known as arterial spin labeling (ASL). 
Rationale: Measurement of pulmonary blood flow and lung proton density using MR technique offers high spatial resolution images which can be quantified and the ability to perform repeated measurements under several different physiological conditions. In human studies, PET, SPECT, and CT are commonly used as the alternative techniques. However, these techniques involve exposure to ionizing radiation, and thus are not suitable for repeated measurements in human subjects.

A MR imaging method to study the distribution of pulmonary blood flow under a variety of physiological conditions, in this case exposure to three different inspired oxygen concentrations: hypoxia, normoxia, and hyperoxia, is described. This technique utilizes human pulmonary physiology research techniques in an MR scanning environment.

This demonstrates a MR imaging method to measure the spatial distribution of pulmonary blood flow in healthy subjects 
during normoxia (inspired O2, ...

MRI Mapping of Cerebrovascular Reactivity via Gas Inhalation Challenges

The brain is a spatially heterogeneous and temporally dynamic organ, with different regions requiring different amount of blood supply at different time. Therefore, the ability of the blood vessels to dilate or constrict, known as Cerebral-Vascular-Reactivity (CVR), represents an important domain of vascular function. An imaging marker representing this dynamic property will provide new information of cerebral vessels under normal and diseased conditions such as stroke, dementia, atherosclerosis, small vessel diseases, brain tumor, traumatic brain injury, and multiple sclerosis. In order to perform this type of measurement in humans, it is necessary to deliver a vasoactive stimulus such as CO2 and/or O2 gas mixture while quantitative brain magnetic resonance images (MRI) are being collected. In this work, we presented a MR compatible gas-delivery system and the associated protocol that allow the delivery of special gas mixtures (e.g., O2, CO2, N2, and their combinations) while the subject is lying inside the MRI scanner. This system is relatively simple, economical, and easy to use, and the experimental protocol allows accurate mapping of CVR in both healthy volunteers and patients with neurological disorders. This approach has the potential to be used in broad clinical applications and in better understanding of brain vascular pathophysiology. In the video, we demonstrate how to set up the system inside an MRI suite and how to perform a complete experiment on a human participant.

Non-invasive imaging of the brain vasculature&#8217;s ability to dilate or constrict may allow a better understanding of cerebrovascular pathophysiology in various neurological diseases. The present report describes a reproducible and patient-comfortable protocol to perform vascular reactivity imaging in humans using magnetic resonance imaging (MRI).

The brain is a spatially heterogeneous and temporally dynamic organ, with different regions requiring different amount of blood supply at different time. ...

Surgical Placement of Catheters for Long-term Cardiovascular Exercise Testing in Swine

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early cardiopulmonary dysfunction is difficult to diagnose, particularly in animal models, as cardiopulmonary function is often measured invasively, requiring anesthesia. As many anesthetic agents are cardiodepressive, subtle changes in cardiovascular function may be masked. In contrast, chronic instrumentation allows for measurement of cardiopulmonary function in the awake state, so that measurements can be obtained under quiet resting conditions, without the effects of anesthesia and acute surgical trauma. Furthermore, when animals are properly trained, measurements can also be obtained during graded treadmill exercise.
Flow probes are placed around the aorta or pulmonary artery for measurement of cardiac output and around the left anterior descending coronary artery for measurement of coronary blood flow. Fluid-filled catheters are implanted in the aorta, pulmonary artery, left atrium, left ventricle and right ventricle for pressure measurement and blood sampling. In addition, a 20 G&#160;catheter is positioned in the anterior interventricular vein to allow coronary venous blood sampling.
After a week of recovery, swine are placed on a motor-driven treadmill, the catheters are connected to pressure and flow meters, and swine are subjected to a five-stage progressive exercise protocol, with each stage lasting 3 min. Hemodynamic signals are continuously recorded and blood samples are taken during the last 30 sec of each exercise stage.
The major advantage of studying chronically instrumented animals is that it allows serial assessment of cardiopulmonary function, not only at rest but also during physical stress such as exercise. Moreover, cardiopulmonary function can be assessed repeatedly during disease development and during chronic treatment, thereby increasing statistical power and hence limiting the number of animals required for a study.

Here we present a protocol to assess cardiopulmonary function in awake swine, at rest and during graded treadmill exercise. Chronic instrumentation allows for repeated hemodynamic measurements uninfluenced by cardiodepressive anesthetic agents.

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early ...

Assessment of Pulmonary Capillary Blood Volume, Membrane Diffusing Capacity, and Intrapulmonary Arteriovenous Anastomoses During Exercise

Exercise is a stress to the pulmonary vasculature. With incremental exercise, the pulmonary diffusing capacity (DLCO) must increase to meet the increased oxygen demand; otherwise, a diffusion limitation may occur. The increase in DLCO with exercise is due to increased capillary blood volume (Vc) and membrane diffusing capacity (Dm). Vc and Dm increase secondary to the recruitment and distension of pulmonary capillaries, increasing the surface area for gas exchange and decreasing pulmonary vascular resistance, thereby attenuating the increase in pulmonary arterial pressure. At the same time, the recruitment of intrapulmonary arteriovenous anastomoses (IPAVA) during exercise may contribute to gas exchange impairment and/or prevent large increases in pulmonary artery pressure.
We describe two techniques to evaluate pulmonary diffusion and circulation at rest and during exercise. The first technique uses multiple-fraction of inspired oxygen (FIO2) DLCO breath holds to determine Vc and Dm at rest and during exercise. Additionally, echocardiography with intravenous agitated saline contrast is used to assess IPAVAs recruitment.
Representative data showed that the DLCO, Vc, and Dm increased with exercise intensity. Echocardiographic data showed no IPAVA recruitment at rest, while contrast bubbles were seen in the left ventricle with exercise, suggesting exercise-induced IPAVA recruitment.
The evaluation of pulmonary capillary blood volume, membrane diffusing capacity, and IPAVA recruitment using echocardiographic methods is useful to characterize the ability of the lung vasculature to adapt to the stress of exercise in health as well as in diseased groups, such as those with pulmonary arterial hypertension and chronic obstructive pulmonary disease.

To assess the pulmonary diffusion and vasculature responses to exercise, we describe the multiple-inspired oxygen diffusion capacity technique to determine capillary blood volume and membrane diffusing capacity, as well as agitated saline contrast echocardiography to assess the recruitment of intrapulmonary arteriovenous anastomoses.

Exercise is a stress to the pulmonary vasculature. With incremental exercise, the pulmonary diffusing capacity (DLCO) must increase to meet the increased ...

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with 'One Stop Shop' Near-infrared Spectroscopy

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic demand. Reactive hyperemia (in response to a brief period of tissue ischemia) is an independent predictor of cardiovascular events and provides important insight into vascular health and vasodilatory capacity. Skeletal muscle oxidative capacity is equally important in health and disease, as it determines the energy supply for myocellular processes. Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess each of these major clinical endpoints (reactive hyperemia, neurovascular coupling, and muscle oxidative capacity) during a single clinic or laboratory visit. Unlike Doppler ultrasound, magnetic resonance images/spectroscopy, or invasive catheter-based flow measurements or muscle biopsies, our approach is less operator-dependent, low-cost, and completely non-invasive. Representative data from our lab taken together with summary data from previously published literature illustrate the utility of each of these end-points. Once this technique is mastered, application to clinical populations will provide important mechanistic insight into exercise intolerance and cardiovascular dysfunction.

Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess reactive hyperemia, neurovascular coupling and skeletal muscle oxidative capacity in a single clinic or laboratory visit.

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic ...

Retinal Vascular Reactivity as Assessed by Optical Coherence Tomography Angiography

The vascular supply to the retina has been shown to dynamically adapt through vasoconstriction and vasodilation to accommodate the metabolic demands of the retina. This process, referred to as retinal vascular reactivity (RVR), is mediated by neurovascular coupling, which is impaired very early in retinal vascular diseases such as diabetic retinopathy. Therefore, a clinically feasible method of assessing vascular function may be of significant interest in both research and clinical settings. Recently, in vivo imaging of the retinal vasculature at the capillary level has been made possible by the FDA approval of optical coherence tomography angiography (OCTA), a noninvasive, minimal risk and dyeless angiography method with capillary level resolution. Concurrently, physiological and pathological changes in RVR have been shown by several investigators. The method shown in this manuscript is designed to investigate RVR using OCTA with no need for alterations to the clinical imaging procedures or device. It demonstrates real time imaging of the retina and retinal vasculature during exposure to hypercapnic or hyperoxic conditions. The exam is easily performed with two personnel in under 30 min with minimal subject discomfort or risk. This method is adaptable to other ophthalmic imaging devices and the applications may vary based on the composition of the gas mixture and patient population. A strength of this method is that it allows for an investigation of retinal vascular function at the capillary level in human subjects in vivo. Limitations of this method are largely those of OCTA and other retinal imaging methods including imaging artifacts and a restricted dynamic range. The results obtained from the method are OCT and OCTA images of the retina. These images are amenable to any analysis that is possible on commercially available OCT or OCTA devices. The general method, however, can be adapted to any form of ophthalmic imaging.

This article describes a method for measuring retinal vasculature reactivity in vivo with human subjects using a gas breathing provocation technique to deliver vasoactive stimuli while acquiring retinal images.

The vascular supply to the retina has been shown to dynamically adapt through vasoconstriction and vasodilation to accommodate the metabolic demands of ...

Neuroscience

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of the fresh gas entering a lung region divided by the region&#8217;s end-expiratory volume &#8213; in the human lung, using only inhaled oxygen as a contrast agent. Regional quantification of specific ventilation has the potential to help identify areas of pathologic lung function. Oxygen in solution in tissue shortens the tissue&#8217;s longitudinal relaxation time (T1), and thus a change in tissue oxygenation can be detected as a change in T1-weighted signal with an inversion recovery acquired image. Following an abrupt change between two concentrations of inspired oxygen, the rate at which lung tissue within a voxel equilibrates to a new steady-state reflects the rate at which resident gas is being replaced by inhaled gas. This rate is determined by specific ventilation. To elicit this sudden change in oxygenation, subjects alternately breathe 20-breath blocks of air (21% oxygen) and 100% oxygen while in the MRI scanner. A stepwise change in inspired oxygen fraction is achieved through use of a custom three-dimensional (3D)-printed flow bypass system with a manual switch during a short end-expiratory breath hold. To detect the corresponding change in T1, a global inversion pulse followed by a single shot fast spin echo sequence was used to acquire two-dimensional T1-weighted images in a 1.5&#160;T MRI scanner, using an eight-element torso coil. Both single slice and multi-slice imaging are possible, with slightly different imaging parameters. Quantification of specific ventilation is achieved by correlating the time-course of signal intensity for each lung voxel with a library of simulated responses to the air/oxygen stimulus. SVI estimations of specific ventilation heterogeneity have been validated against multiple breath washout and proved to accurately determine the heterogeneity of the specific ventilation distribution.

Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...

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 ...

Non-Invasive Monitoring of Microvascular Oxygenation and Reactive Hyperemia using Hybrid, Near-Infrared Diffuse Optical Spectroscopy for Critical Care

The detection of levels of impairment in microvascular oxygen consumption and reactive hyperemia is vital in critical care. However, there are no practical means for a robust and quantitative evaluation. This paper describes a protocol to evaluate these impairments using a hybrid near-infrared diffuse optical device. The device contains modules for near-infrared time-resolved and diffuse correlation spectroscopies and pulse-oximetry. These modules allow the non-invasive, continuous, and real-time measurement of the absolute, microvascular blood/tissue oxygen saturation (StO2) and the blood flow index (BFI) along with the peripheral arterial oxygen saturation (SpO2). This device uses an integrated, computer-controlled tourniquet system to execute a standardized protocol with optical data acquisition from the brachioradialis muscle. The standardized vascular occlusion test (VOT) takes care of the variations in the occlusion duration and pressure reported in the literature, while the automation minimizes inter-operator differences. The protocol we describe focuses on a 3-min occlusion period but the details described in this paper can readily be adapted to other durations and cuff pressures, as well as other muscles. The inclusion of an extended baseline and post-occlusion recovery period measurement allows the quantification of the baseline values for all the parameters and the blood/tissue deoxygenation rate that corresponds to the metabolic rate of oxygen consumption. Once the cuff is released, we characterize the tissue reoxygenation rate, magnitude, and duration of the hyperemic response in BFI and StO2. These latter parameters correspond to the quantification of the reactive hyperemia, which provides information about the endothelial function. Furthermore, the above-mentioned measurements of the absolute concentration of oxygenated and deoxygenated hemoglobin, BFI, the derived metabolic rate of oxygen consumption, StO2, and SpO2 provide a yet-to-be-explored rich data set that can exhibit disease severity, personalized therapeutics, and management interventions.

We describe a protocol to non-invasively and continuously measure absolute microvascular blood flow index and blood oxygen saturation using a multi-modal device based on near-infrared diffuse optics. We then evaluate the metabolic rate of oxygen consumption and reactive hyperemia utilizing a vascular occlusion test.

The detection of levels of impairment in microvascular oxygen consumption and reactive hyperemia is vital in critical care. However, there are no ...

Advanced Lung Imaging for Disease Detection and Monitoring

Phase-Resolved Functional Lung MRI for Pulmonary Ventilation and Perfusion (V/Q) Assessment

Fourier decomposition is a contrast agent-free 1H MRI method for lung perfusion (Q) and ventilation (V) assessment. After image registration, the time series of each voxel is analyzed with regard to the cardiac and breathing frequency components.
Using a standard 2D spoiled gradient-echo sequence with a temporal resolution of ~300 ms, an image-sorting algorithm was developed to produce phase-resolved functional lung imaging (PREFUL) with an increased temporal resolution. Thus, it is feasible to evaluate regional flow volume loops (FVL) during tidal volume breathing and depict the propagation of the pulse wave during the cardiac cycle. This method can be applied at 1.5T or 3T with standard MR hardware without the necessity for sequence programming, as the described protocol can be implemented with the default SPGRE sequence on most systems.
PREFUL ventilation MRI has been validated using 129Xe and 19F gas imaging with good regional agreement. Perfusion-weighted PREFUL MRI has been validated using SPECT as well as dynamic contrast enhanced (DCE) MRI. PREFUL has been tested in a dual center dual vendor setting and is currently applied in several ongoing multicenter trials. Furthermore, it is feasible across a range of field strengths (0.55T-3T) and different age groups, including newborns.
Quantitative V/Q PREFUL MRI has been used in patients with cystic fibrosis, chronic obstructive pulmonary disease, chronic thromboembolic pulmonary hypertension, and corona virus disease-2019 to quantify disease and monitor treatment change after therapy. Furthermore, PREFUL V/Q imaging has been shown to predict transplant loss due to chronic lung allograft dysfunction in patients after lung transplantation. In summary, PREFUL MRI is a validated technique for quantitative ventilation and pulmonary pulse wave/perfusion imaging for regional pulmonary disease detection, quantification, and treatment monitoring with potential added value to the current clinical routine.

Author Spotlight: Enhancing Diagnostic Strategies and Biomarker Development for Comprehensive Lung Function Analysis

Here, we describe the implementation of phase-resolved functional lung MRI as a contrast-agent-free proton MR technique for the assessment of pulmonary ventilation and perfusion dynamics. Validated and applicable across different field strengths and age groups, it could enhance clinical decision-making in the future by aiding in disease quantification and therapy monitoring.

Fourier decomposition is a contrast agent-free 1H MRI method for lung perfusion (Q) and ventilation (V) assessment. After image registration, the time ...

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

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