This work was partly supported by grants from the National Institutes of Health (NIH), under grant numbers R01 MH084021, R01 NS067015, R01 AG042753, NS076588, NS029029-20S1, R21 NS078656; and from National Multiple Sclerosis Society, under grant number of RG4707A2.

NOTE: The protocol was approved by the University of Texas Southwestern Medical Center&#8217;s Institutional Review Board.
1. Diagram of the Gas Delivery System and Preparation Steps before the Experiment
<ol style="margin-left: 40px;">
	<li>Review the diagram of the gas delivery system (Figure 1). Fill a 200 L&#160;Douglas bag (Item #1) with a medical-grade gas mixture containing 5% CO2, 21% O2, and 74% N2.</li>
	<li>Place two diaphragms (Item #4) in the two-way non-rebreathing valve (Item #3) to ensure one way gas flow. Bring this assembled two-way valve and the gas-filled Douglas bag (Item #1) into the magnet room.</li>
	<li>Connect the gas delivery tube (Item #7) to the input end of the two-way valve (Item #3). Attach the gas delivery tube (Item #7) to the side of the head coil for weight support. Connect the other end of the gas delivery tube (Item #7) to the gas-filled Douglas bag (Item #1).</li>
	<li>Connect the mouthpiece (Item #5) to the U-shape tube (Item #12) via an elbow-connector (Item #13 with gas sampling port sealed).</li>
	<li>Connect the gas-sampling tubing (Item #9) to the U-shape tube (Item #12) via another elbow-connector (Item #13).</li>
	<li>Connect a small air-filter (Item #11) to the other end of the gas-sampling tubing (Item #9). Connect the other end of the air-filter (Item #11) to the CO2 (Item #14) monitor.</li>
	<li>In the control room of the MRI suite, turn on CO2 (Item #14) and pulse oximetry (Item #15) monitors. Perform an auto-calibration for the CO2 monitor.</li>
	<li>Connect the monitors to a laptop using USB ports. Open the HyperTerminal software that communicates with the monitors. Synchronize the monitors&#8217; times with a timer by writing down the timer time and corresponding monitors&#8217; times. The differences of the timer time and monitors&#8217; times will be accounted for in data processing (step 4.4).</li>
	<li>Insert one end of a signaling bar into a waveguide so that one end of the bar is inside the magnet room and the other end is in the control room. 
	NOTE: The signaling bar is used for notifying the researcher inside the magnet room during the scan when switching of the three-way valve (Item #2) is needed.</li>
</ol>
2. Procedures during the Experiment
<ol style="margin-left: 40px;">
	<li>Ask the subject to lie on the MRI table but do not put him/her into the magnet bore yet. Instruct the subject to press the nurse-call button if they feel discomfort during the scan. Ask the subject to wipe his/her nose with a piece of cleaning towelette to remove any grease.</li>
	<li>Instruct the subject to breathe by mouth and to establish and maintain a breathing rhythm. Then apply a nose clip (Item #6) on the subject.</li>
	<li>Connect the open end of the U-shape tube (Item #12) to the middle port of the two-way valve (Item #3) via the elbow connector (Item #13).</li>
	<li>Gently place the mouthpiece into the subject&#8217;s mouth so that the subject can breathe through the mouthpiece. Gently attach the finger sensor of the pulse oximetry (Item #15) to the finger tip of the subject.</li>
	<li>Insure that the head of the subject is in the iso-center of the head coil. Operate the MRI table to place him/her inside the magnet bore.</li>
	<li>Ensure that one researcher stays inside the magnet room to monitor the subject and to be prepared to switch the three-way valve on the Douglas bag (Item #1). Ensure that the researcher is wearing earplugs and a headset to block MRI noise.</li>
	<li>Close the magnet room door and, in the control room, check the EtCO2 and arterial oxygen saturation fraction (sO2) parameters displayed on the CO2 (Item #14) and pulse oximetry (Item #15) monitors. Start the recording of the parameters on the laptop.</li>
	<li>Instruct the MRI operator to begin scanning using Blood-Oxygenation-Level-Dependent (BOLD) sequence. For 3T MRI scanner, the BOLD imaging parameters are: TR/TE=1,500/30 msec, flip angle=60&#176;, field of view=220 x 220 mm2, matrix= 64 x 64, 29 slices, thickness=5 mm, no gap between slices, 361 volumes. Review a pre-prepared sheet on which the timing of the valve switching is listed and gently swing the signaling bar when a switch is needed. Pay close attention to the subject&#8217;s physiology, including heart rate, sO2, and EtCO2.</li>
	<li>Now, inside the magnet room, switch on the Douglas bag (Item #1) based on the movement of the signaling bar which controls the type of gas that the subject inspires.</li>
	<li>Continue this procedure for the length of the study. During the 9&#160;min&#160;imaging period, ensure that the valve switching takes place approximately once every minute. Note that the timing of the switch does not have to be exactly precise, as long as the EtCO2 time course is recorded. 
	NOTE: If the subject presses the nurse-call button during the scan, the scan will be aborted and the subject will be moved out of the magnet bore immediately. The researcher will remove the mouth piece and nose clip from the subject.</li>
	<li>Use the intercom to notify the subject that the scan is completed. Pull the MRI table out. Gently remove the nose clip and mouthpiece from the subject while providing cleaning tissue to the subject to wipe any saliva. Gently remove the finger sensor of the pulse oximetry from the subject. The subject can then sit up and get off the MRI table.</li>
</ol>
3. Clean-up Procedures after the Experiment
<ol style="margin-left: 40px;">
	<li>Discard the gas sampling tubing (Item #9), air-filter (Item #11), mouthpiece (Item #5) and nose clip (Item #6).</li>
	<li>Clean the reusable components. Disconnect the two-way valve (Item #3) from the other components and remove the diaphragms (Item #4) from the valve. Soak the two-way valve (Item #3), diaphragm (Item #4) and U-shape tube (Item #12) in a concentrated phosphate-free disinfectant, containing surfactants such as Bacdown detergent disinfectant in a container for 20 min. The dilution ratio of the detergent disinfectant and distilled water is 1:64.</li>
	<li>Rinse the items described in 3.2 thoroughly with distilled water.</li>
	<li>Dry the U-shape tube (Item #12) with compressed air. Place the two-way valve (Item #3) and diaphragms (Item #4) on a clear countertop and allow them dry naturally and completely.</li>
	<li>Empty the Douglas bag. Put away the signaling bar and gray tube.</li>
</ol>
4. Data Analysis to Compute CVR Map
<ol style="margin-left: 40px;">
	<li>Save MRI data in Dicom file format or any other vendor-specific format. Transfer the data to a lab computer and convert the data into volume-by-volume file series, in which each file contains a 3D volume (e.g., BOLD image) corresponding to one time point.</li>
	<li>Pre-process the image data. Perform image pre-processing steps including realignment, normalization, and smoothing using a script that calls library functions provided by the software Statistical Parametric Mapping (SPM). See supplemental code file 1 for an example of the Matlab script.</li>
	<li>Use a script to read the CO2 recording, correct the sampling tubing delay by shifting the time course by a pre-calibrated amount (e.g., 12 sec&#160;in this setup which is determined as the time difference between one breath to the mouth piece and the appearance of that breath on the CO2 recording), and extract EtCO2 which is the envelope (positive peaks) of the raw time series. See supplemental code file 2 for the Matlab script.</li>
	<li>Based on the synchronizing timer, segment the EtCO2 data to keep only the recording from 25 sec&#160;prior to the first image acquisition to 100 sec&#160;after the last image acquisition. The EtCO2 time course is the input function to the vasculature and is used as independent variable in the later-described linear regression analysis.</li>
	<li>Identify the physiological delay between EtCO2 (measured in lung) and MRI signal (measured in the brain) by calculating cross-correlation coefficient (CC) between these two time courses at varying time shifts. The shift value that yields higher CC is considered the optimal time.</li>
	<li>The EtCO2 time course is shifted by the optimal delay and only the time points matching those of MRI signal are preserved, resulting in a time series that are of the same length as the MRI signal.</li>
	<li>Conduct a voxel-by-voxel linear regression using SPM in which the shifted EtCO2 time course is the independent variable and the MRI signal time course is the dependent variable.</li>
	<li>Compute voxel-by-voxel map of CVR 
	<img alt="Equation 1" fo:content-width="2in" src="/files/ftp_upload/52306/52306eq1.jpg" /> 
	where (i,j,k) is the voxel index, &#946;1 is the regression coefficient associated with EtCO2 and &#946;0 is the regression coefficient associated with the constant term. min(EtCO2) is the minimal value of EtCO2 in the time course.</li>
</ol>

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HL and PL are inventors of a provisional patent, owned by UT Southwestern Medical Center, that describes part of the gas delivery system.

This report presented an MR-compatible gas delivery system and a comprehensive experimental protocol that allows the mapping of vascular reactivity in the human brain. A diagram of the gas delivery system is illustrated in Figure 1. All parts inside the MRI scanner room are plastic to ensure their MRI compatibility. The system can be conceptually divided into three sub-systems, including a gas intake sub-system (bag, delivery tube, two-way valve), a breathing interface sub-system (nose clip, mouthpiece, U-shape tube), and a monitoring sub-system (CO2 concentration, oxygen saturation, heart rate, breathing rate). The gas intake sub-system allows the gas to be inhaled to reach the two-way valve. Only inhaled air, but not exhaled air, will flow through this sub-system. The breathing interface sub-system allows the subject to breathe in and out the intended gas. Both inhaled and exhaled gas will flow through this sub-system. The monitoring sub-system should therefore sample the gas at a point along the breathing interface sub-system.
Clinical applications of this technique may include evaluations of brain vascular reserve in neurological diseases such as stroke, atherosclerosis, moyamoya disease, vascular dementia, multiple sclerosis, and brain tumor. The technique can also be used in functional MRI studies to normalize or calibrate fMRI signal for a better quantification of neural activity23,24.
An important feature of the proposed system and experimental protocol is that the gas mixture can be delivered to the subject while causing minimal motion or discomfort. Therefore, it is critical to place the U-shape tube (Item #12) such that it (and the mouthpiece connected to the end of it) naturally falls downwards into the subject&#8217;s mouth. This way, the subject does not need to use his facial muscle to hold or support the mouthpiece. It is also important to be aware that the subject will not be able to talk while the mouthpiece is in their mouth. Therefore, the researcher should avoid talking to the subject with a question tone. Instead, only clear, definitive instructions should be given. Additionally, a researcher should pay close attention to the physiological parameters (e.g., EtCO2, sO2, heart rate, breathing rate) during the entire course of the experiment and respond promptly when one or more of the physiological parameters deviate outside the typical range.
While an exhaustive survey of other gas delivery systems used in the literature is beyond the scope of this article, it is useful to compare the current system to a few commonly used ones17,18. A major difference is that our system uses a mouthpiece to deliver the intended gas while most other systems have used a mask in design. The potential complications of using a mask are two folds. First, a mask occupies a substantial amount of space, and it might not always be feasible to fit the mask into the tight space inside the head coil, considering that, for many subjects, their noses would almost touch the head coil even without a mask. This is especially the case for head coils intended to achieve high sensitivity, which are usually designed to fit tightly to the subject&#8217;s head. A second complication associated with a mask design is that there is large space inside the mask which results in substantial mixing of inhaled and exhaled gas. Consequently, it could affect the accuracy of the measurement of EtCO2, which ideally should be based on exhaled gas only. Accurate EtCO2 is of course important for the reliability of the CVR map. Another major difference of our system in comparison to many other systems is that our system delivers the gas from a bag instead of a gas tank. Therefore, tanks are not needed in the scanner area, saving precious space in the control room of an MRI suite. In our design, we bring the bag before the start of the scan and, following the scan, the bag is emptied, folded, and put away. Finally, compared to several other systems18,21, the current gas delivery system is simpler, requires less training time, and its consumables are less expensive.
It should be pointed out that, although the protocol presented in this report has primarily focused on CO2 inhalation, the presented gas delivery system allows the delivery of other gas mixtures (e.g., any fraction of O2, any fraction of CO2, any fraction of N2, and their combination) to a human for them to breathe while s/he is lying inside the MRI scanner. One can also use the gas delivery system outside the context of MRI, for example in conjunction with electroencephalogram (EEG), magnetoencephalogram (MEG), positron emission tomography (PET), or optimal imaging.
When providing a recommendation of imaging parameters, we have primarily focused on BOLD sequence. Another sequence that can be potentially used in CVR mapping is Arterial Spin Labeling (ASL) MRI, which provides a quantitative measure of cerebral blood flow (CBF) in physiologic units (ml blood per 100 g tissue per min). Therefore, the advantage of ASL-based CVR mapping is that the results are easier to interpret, unlike BOLD signal which reflects a combined effect of blood flow, blood volume as well as possible contributions of brain metabolic alterations during CO2 challenge25-27. However, a limitation of the ASL technique is that its sensitivity is several folds lower than that of BOLD28. As a result, our experience is that, at present, it is highly challenging to obtain an individual-level, voxel-by-voxel CVR map using ASL. Therefore, for application studies of CVR, we mainly use the BOLD sequence and thus also focus on this technique in our recommendations.
One limitation of the present method is that breathing through a mouthpiece with the nose blocked (by a nose clip) is not entirely natural and some subjects (especially patients) may perceive this as a source of discomfort. Breathing with the mouthpiece and nose clip may also exacerbate the feeling of claustrophobia. Additionally, the subject may experience dry mouth due to breathing by mouth only. Therefore, it is recommended that the researcher try his best to complete the experiment swiftly. Finally, it is important to note that, based on the authors&#8217; experience, the potential discomfort mentioned above is transient and will disappear as soon as the experiment is finished.

The brain represents about 2% of the total body weight, but consumes about 20% of the total energy1. Not surprisingly, sufficient and carefully regulated blood supply is critical to meet this high energy demand and for the brain to function properly. Furthermore, brain is a spatially heterogeneous and temporally dynamic organ, with different regions requiring different amount of blood supply at different time. Therefore, dynamic modulation of blood supply represents an important requirement in human brain circulation. Fortunately, it is known that blood vessels are not just rigid pipes and that an important function of blood vessel is to dilate and constrict based on the demand of the brain and physiologic conditions2.
This functional property of the vessel, known as Cerebral-Vascular-Reactivity (CVR), is thought to be a useful indictor of vascular health and may find applications in several neurological conditions such as stroke3, dementia4, atherosclerosis5, small vessel diseases6, brain tumor7, moyamoya disease8, and drug-addiction9. In physiology and anesthesiology literature, it is known that, because CO2 gas is a potent vasodilator, CVR can be assessed by altering the arterial CO2 level (e.g., inhalation of a small amount of CO2) while monitoring vascular responses10-13. In the imaging and radiology field, CVR mapping using MRI is rapidly emerging as a new marker of interest for many basic scientists and clinicians8,14-19. It is usually estimated by examining how much vascular response is induced by a vasoactive challenge. However, there is a need for technical advances in the gas delivery system and standardization of experimental protocol. Delivering special gas mixture to a subject inside the MRI scanner is not trivial and special considerations are required for an MRI-compatible design. Special considerations are required in designing MRI-compatible gas delivery system. These special considerations include: 1) all components must be non-metallic (metal cannot be used inside MRI); 2) the system should work within a small space that the MRI system and its head coil allow; 3) the system should work with a lying-down position (as MRI scanner requires) instead of sitting up, with no discomfort; 4) relevant physiological parameters, such as end-tidal CO2 (EtCO2, an approximation of CO2 content in the arterial blood) and arterial oxygen saturation, should be recorded accurately with seconds of timing accuracy and stored on a computer for analysis use. These issues may limit the scope of applications of CVR mapping.
In this report, we presented an experimental protocol that uses a comprehensive gas delivery system to modulate the content of inspired gas while the subject is lying inside the MRI scanner. Using this approach, the researcher can non-invasively apply a vasoactive stimulus to the participant with minimal discomfort or bulk motion. Physiological parameters and MRI images were recorded during the entire period of approximately 9 min, which consisted of alternating blocks (1 min per block) of room-air and hypercapnic gas breathing. Representative results are presented. Potential applications and limitations are discussed.

<table><tbody><tr><td>Douglas bag&amp;nbsp;</td><td>Harvard Apparatus</td><td>500942</td><td>200 L capacity</td></tr><tr><td>Three-way valve</td><td>Hans Rudolph</td><td>CR1207</td><td>100% plastic</td></tr><tr><td>Two-way non-rebreathing valve</td><td>Hans Rudolph</td><td>CR1480</td><td>22 mm/15 mm ID</td></tr><tr><td>Diaphragm</td><td>Hans Rudolph</td><td>602021-2608</td><td>Size: medium, Type: spiral</td></tr><tr><td>Mouth piece</td><td>Hans Rudolph</td><td>602076</td><td>Silicone, Model #&amp;nbsp;9061</td></tr><tr><td>Nose clip</td><td>Hans Rudolph</td><td>201413</td><td>Plastic foam, Model #9014</td></tr><tr><td>Gas delivery tube</td><td>Vacumed</td><td>1011-108</td><td></td></tr><tr><td>Blue cuff</td><td>Vacumed</td><td>22254</td><td></td></tr><tr><td>Gas sampling tube</td><td>QoSINA</td><td>T4305</td><td>Thin</td></tr><tr><td>Male luer</td><td>QoSINA</td><td>11547</td><td></td></tr><tr><td>Hydrophobic filter</td><td>Philips Medical Systems</td><td>9906-00</td><td>Disposable</td></tr><tr><td>U-shape tube</td><td>Made in-house</td><td></td><td></td></tr><tr><td>Elbow connector</td><td>QoSINA</td><td>51033</td><td></td></tr><tr><td>EtCO&lt;sub&gt;2&lt;/sub&gt; monitor</td><td>Philips Medical Systems</td><td>Model 1265</td><td></td></tr><tr><td>Pulse oximetry&amp;nbsp;</td><td>Invivo</td><td>Expression</td><td>MRI Monitoring Systems</td></tr><tr><td>MRI scanner&amp;nbsp;</td><td>Philips</td><td>Achieva 3.0T TX</td><td></td></tr><tr><td>Disinfectant</td><td>Fisher Scientific</td><td>04-355-13</td><td>Decon BDD Bacdown Detergent Disinfectant</td></tr></tbody></table>

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.

Two types of data are collected with the proposed protocol, physiological recordings and MR images. Figures 2 and 3 show recordings of physiological parameters from a representative subject. Black trace in Figure 2 displays the CO2 time course recorded by the CO2 monitor, which represents the CO2 content in air sampled near the mouthpiece. Note that this trace fluctuates rapidly as a function of time. This is because, during the inhalation phase of the breathing cycle, this recording reflects the CO2 content in the inhaling air and, during the exhalation phase, this recording reflects the CO2 content in the exhaled air. As such, the upper peak of each breathing cycle, referred to as end-tidal CO2 or EtCO2, represents the CO2 content in the lung, which can be approximately used as the CO2 concentration in the arterial blood. Note that CO2 concentration in the arterial blood is the driving force, i.e., input function, of the vasodilatory response. The peaks of the CO2 trace (red curve in Figure 2) were delineated with a detection algorithm which searches for the peak during each breath, in combination with manual inspection and correction. This was followed by a median filtering to remove abrupt peaks due to partial breath and to account for blood mixing during the course of flow from pulmonary vessels to cerebral vessels. The final EtCO2 time course is shown by the green curve in Figure 2 and it is used in the computation of CVR.
Figure 3 shows the time courses of breathing rate, arterial oxygen saturation fraction (sO2), and heart rate. The breathing rate is obtained from the CO2 monitor while sO2 and heart rate are obtained from the pulse oximetry. As can be seen, these parameters do not show a systematic change with the hypercapnia challenge. Note that hypercapnia does cause hyperventilation in the subject, thus the O2 partial pressure in the lung will increase modestly. However, its impact on sO2 is minimal as blood hemoglobin is already largely saturated at room-air breathing and the oxygen dissociation curve is rather flat within that range.
Figure 4 shows representative BOLD MR images at different time of the experiment. The average signal intensity (in arbitrary MR units) is also shown. It can be seen that the BOLD signal in the brain shows an increase with CO2 inhalation. Note that the signal difference between room-air and CO2 periods is on the order of 1-3% in amplitude.
Combining data from physiological recordings and MR images, a voxel-by-voxel CVR map can be computed. Figure 5 shows representative CVR maps (in units of % signal change per mmHg CO2 change) of a healthy subject scanned on five different days, demonstrating an excellent reproducibility of the results. The proposed technique has so far been applied in studies of aging20, Alzheimer&#8217;s Disease4, Multiple Sclerosis21, and exercise training22.
<img alt="Figure 1" src="/files/ftp_upload/52306/52306fig1highres.jpg" /> 
Figure 1.&#160;Diagram of the gas delivery system. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52306/52306fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52306/52306fig2highres.jpg" /> 
Figure 2.&#160;CO2 time course from a representative subject during the experiment. Segments of the breath-by-breath CO2 content trace as recorded by the CO2 monitor are shown for room air breathing period (lower left) and 5% CO2 inhalation period (lower right). Extracted EtCO2 time courses are shown in colored curves. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52306/52306fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52306/52306fig3highres.jpg" /> 
Figure 3.&#160;Recorded physiology parameters from a representative subject during the experiment. (A) Breathing rate (bpm, breath per minute) time course of the subject. (B) sO2 (%) time course of the subject. (C) Heart rate (bpm, beat per minute) time course of the subject. The subject is the same one as that in Figure 1. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52306/52306fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52306/52306fig4highres.jpg" /> 
Figure 4.&#160;Representative BOLD MR images at different time of the experiment. Average signal intensities of the displayed brain slice (axial slice #54 in MNI space) are showed in the bottom row. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52306/52306fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52306/52306fig5highres.jpg" /> 
Figure 5.&#160;Representative CVR map from a representative subject. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52306/52306fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about MRI Mapping of Cerebrovascular Reactivity via Gas Inhalation Challenges at JoVE.com

MRI Mapping of Cerebrovascular Reactivity via Gas Inhalation Challenges

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. ...

mri-mapping-cerebrovascular-reactivity-via-gas-inhalation

Research

JoVE Journal

Medicine

13.9K Views.  University of Texas Southwestern Medical Center. Non-invasive imaging of the brain vasculature’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).

Video: MRI Mapping of Cerebrovascular Reactivity via Gas Inhalation Challenges

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, ...

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

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

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

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

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...

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

Cerebrovascular Reactivity Measurement with Functional Near Infrared Spectroscopy

Cerebrovascular reactivity (CVR) is the capacity of blood vessels in the brain to alter cerebral blood flow (either with dilation or constriction) in response to chemical or physical stimuli. The amount of reactivity in the cerebral microvasculature depends on the integrity of the capacitance vasculature and is the primary function of endothelial cells. CVR is, therefore, an indicator of the microvasculature&#8217;s physiology and overall health. Imaging methods that can measure CVR are available but can be costly, and require magnetic resonance imaging centers and technical expertise. In this study, we used fNIRS technology to monitor changes of oxyhemoglobin (HbO) and deoxyhemoglobin (HbR) in the cerebral microvasculature to assess the CVR of 15 healthy controls (HC) in response to a vasoactive stimulus (inhaled 5% carbon dioxide or CO2). Our results suggest that this is a promising imaging technology that offers a non-invasive, accurate, portable, and cost-effective method of mapping cortical CVR and associated microvasculature function, resulting from a traumatic brain injury or other conditions associated with cerebral microvasculopathy.

Presented here is a protocol for imaging and measurement of cerebrovascular reactivity in humans with functional Near Infrared Spectroscopy (fNIRS). fNIRS is a novel imaging modality that captures the concentration changes of hemoglobin species in the brain&#8217;s outermost cortex under specific stimuli.

Cerebrovascular reactivity (CVR) is the capacity of blood vessels in the brain to alter cerebral blood flow (either with dilation or constriction) in ...

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 ...

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.

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 ...

PET-CT Scanning for Multi-Tracer Study of Cerebral Oxygen and Glucose Metabolism

Multi-Tracer Studies of Brain Oxygen and Glucose Metabolism Using a Time-of-Flight Positron Emission Tomography-Computed Tomography Scanner

The authors have developed a paradigm using positron emission tomography (PET) with multiple radiopharmaceutical tracers that combines measurements of cerebral metabolic rate of glucose (CMRGlc), cerebral metabolic rate of oxygen (CMRO2), cerebral blood flow (CBF), and cerebral blood volume (CBV), culminating in estimates of brain aerobic glycolysis (AG). These in vivo estimates of oxidative and non-oxidative glucose metabolism are pertinent to the study of the human brain in health and disease. The latest positron emission tomography-computed tomography (PET-CT) scanners provide time-of-flight (TOF) imaging and critical improvements in spatial resolution and reduction of artifacts. This has led to significantly improved imaging with lower radiotracer doses.
Optimized methods for the latest PET-CT scanners involve administering a sequence of inhaled 15O-labeled carbon monoxide (CO) and oxygen (O2), intravenous 15O-labeled water (H2O), and 18F-deoxyglucose (FDG)-all within 2-h or 3-h scan sessions that yield high-resolution, quantitative measurements of CMRGlc, CMRO2, CBF, CBV, and AG. This methods paper describes practical aspects of scanning designed for quantifying brain metabolism with tracer kinetic models and arterial blood samples and provides examples of imaging measurements of human brain metabolism.

Quantitative measurements of oxygen and glucose metabolism by PET are established technologies, but details of practical protocols are sparsely described in the literature. This paper presents a practical protocol successfully implemented on a state-of-the-art positron emission tomography-computed tomography scanner.

The authors have developed a paradigm using positron emission tomography (PET) with multiple radiopharmaceutical tracers that combines measurements of ...

Multi-modal Pulmonary Imaging: Using Complementary Information from CT and Hyperpolarized 129Xe MRI to Evaluate Lung Structure-Function

Hyperpolarized 129Xe gas MRI is an emerging technique to evaluate and measure regional lung function including pulmonary gas distribution and gas exchange. Chest computed tomography (CT) still remains the clinical gold standard for imaging of the lungs, though, in part due to the rapid CT protocols that acquire high-resolution images in seconds and the widespread availability of CT scanners. Quantitative approaches have enabled the extraction of structural lung parenchymal, airway and vascular measurements from chest CT that have been evaluated in many clinical research studies. Together, CT and 129Xe MRI provide complementary information that can be used to evaluate regional lung structure and function, resulting in new insights into lung health and disease. 129Xe MR-CT image registration can be performed to measure regional lung structure-function to better understand lung disease pathophysiology, and to perform image-guided pulmonary interventions. Here, a method for 129Xe MRI-CT registration is outlined to support implementation in research or clinical settings. Registration methods and applications that have been employed to date in the literature are also summarized, and suggestions are provided for future directions that may further overcome technical challenges related to 129Xe MR-CT image registration and facilitate broader implementation of regional lung structure-function evaluation.

Multi-modal Pulmonary Imaging: Using Complementary Information from CT and Hyperpolarized 129Xe MRI to Evaluate Lung Structure-Function

CT and 129Xe MRI provide complementary lung structure-function information that can be exploited for regional analysis using image registration. Here, we provide a protocol that builds from the existing literature for 129Xe MR to CT image registration using open-source platforms.

Hyperpolarized 129Xe gas MRI is an emerging technique to evaluate and measure regional lung function including pulmonary gas distribution and gas ...

Dynamic Lung Ventilation Assessment with 3D Phase Resolved Functional Lung MRI

Three-Dimensional Phase Resolved Functional Lung Magnetic Resonance Imaging

Pulmonary magnetic resonance imaging (MRI) offers a variety of radiation-free techniques tailored to assess regional lung ventilation or its surrogates. These techniques encompass direct measurements, exemplified by hyperpolarized gas MRI and fluorinated gas MRI, as well as indirect measurements facilitated by oxygen-enhanced MRI and proton-based Fourier decomposition (FD) MRI. In recent times, there has been substantial progress in the field of FD MRI, which involved improving spatial/temporal resolution, refining sequence design and postprocessing, and developing a comprehensive whole-lung approach.
The two-dimensional (2D) phase-resolved functional lung (PREFUL) MRI stands out as an FD-based approach developed for the comprehensive assessment of regional ventilation and perfusion dynamics, all within a single MR acquisition. Recently, a new advancement has been made with the development of 3D PREFUL to assess dynamic ventilation of the entire lung using 8 min exam with a self-gated sequence.
The 3D PREFUL acquisition involves employing a stack-of-stars spoiled-gradient-echo sequence with a golden angle increment. Following the compressed sensing image reconstruction of approximately 40 breathing phases, all the reconstructed respiratory-resolved images undergo registration onto a fixed breathing phase. Subsequently, the ventilation parameters are extracted from the registered images.
In a study cohort comprising healthy volunteers and patients with chronic obstructive pulmonary disease, the 3D PREFUL ventilation parameters demonstrated strong correlations with measurements obtained from pulmonary function tests. Additionally, the interscan repeatability of the 3D PREFUL technique was deemed to be acceptable, indicating its reliability for repeated assessments of the same individuals.
In summary, 3D PREFUL ventilation MRI provides a whole lung coverage and captures ventilation dynamics with enhanced spatial resolution compared to 2D PREFUL. 3D PREFUL technique offers a cost-effective alternative to hyperpolarized 129Xe MRI, making it an attractive option for patient-friendly evaluation of pulmonary ventilation.

Three-dimensional (3D) phase-resolved functional lung (PREFUL) is a functional magnetic resonance imaging (MRI) technique that allows for quantification of regional ventilation of the whole human lung volume, using tidal breathing and contrast-agent free acquisition for 8 min. Here, we present an MR protocol to collect and analyze 3D PREFUL imaging data.

Pulmonary magnetic resonance imaging (MRI) offers a variety of radiation-free techniques tailored to assess regional lung ventilation or its surrogates. ...