Name: mri mapping of cerebrovascular reactivity via gas inhalation challenges
Uploaded: 2014-12-17T15:00:00
Duration: 9 min 33 s
Description: Watch this Scientific Journal Video about MRI Mapping of Cerebrovascular Reactivity via Gas Inhalation Challenges at JoVE.com

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.

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			<td>www.rudolphkc.com&#160;</td>
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			<td>QoSINA</td>
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			<td>www.qosina.com</td>
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			<td>Philips Medical Systems</td>
			<td>Model 1265</td>
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			<td>Philips</td>
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			<td>Fisher Scientific</td>
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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://www.jove.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://www.jove.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://www.jove.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://www.jove.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://www.jove.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

Microsurgical Venous Pouch Arterial-Bifurcation Aneurysms in the Rabbit Model: Technical Aspects

For ruptured human cerebral aneurysms endovascular embolization has become an equivalent alternative to aneurysm clipping.1 However, large clinical trials have shown disappointing long-term results with unacceptable high rates of aneurysm recanalization and delayed aneurysm rupture.2 To overcome these problems, animal experimental studies are crucial for the development of better endovascular devices.3-5
Several animal models in rats, rabbits, canines and swine are available.6-8 Comparisons of the different animal models showed the superiority of the rabbit model with regard to hemodynamics and comparability of the coagulation system and cost-effectiveness.9-11 
The venous pouch arterial bifurcation model in rabbits is formed by a venous pouch sutured into an artificially created true bifurcation of both common carotid arteries (CCA). The main advantage of this model are true bifurcational hemodynamics.12 The major drawbacks are the sofar high microsurgical technical demands and high morbidity and mortality rates of up to 50%.13 These limitations have resulted in less frequent use of this aneurysm model in the recent years. These shortcomings could be overcome with improved surgical procedures and modified peri- and postoperative analgetic management and anticoagulation.14-16 Our techniques reported in this paper demonstrate this optimized technique for microsurgical creation of arterial bifurcation aneurysms.

An optimized technique for the microsurgical creation of arterial bifurcation aneurysms mimicking bifurcation human cerebral aneurysms is described. A venous pouch is sutured into an artificially created true bifurcation of both common carotid arteries. Facilitated microsurgical techniques and aggressive postoperative anticoagulation and analgesia lead to minimized morbidity rates and high aneurysm patency rates.

For ruptured human cerebral aneurysms endovascular embolization has become an equivalent alternative to aneurysm clipping.1 However, large clinical trials ...

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

Coordinate Mapping of Hyolaryngeal Mechanics in Swallowing

Characterizing hyolaryngeal movement is important to dysphagia research. Prior methods require multiple measurements to obtain one kinematic measurement whereas coordinate mapping of hyolaryngeal mechanics using Modified Barium Swallow (MBS) uses one set of coordinates to calculate multiple variables of interest. For demonstration purposes, ten kinematic measurements were generated from one set of coordinates to determine differences in swallowing two different bolus types. Calculations of hyoid excursion against the vertebrae and mandible are correlated to determine the importance of axes of reference.
To demonstrate coordinate mapping methodology, 40 MBS studies were randomly selected from a dataset of healthy normal subjects with no known swallowing impairment. A 5 ml thin-liquid bolus and a 5 ml pudding swallows were measured from each subject. Nine coordinates, mapping the cranial base, mandible, vertebrae and elements of the hyolaryngeal complex, were recorded at the frames of minimum and maximum hyolaryngeal excursion. Coordinates were mathematically converted into ten variables of hyolaryngeal mechanics.
Inter-rater reliability was evaluated by Intraclass correlation coefficients (ICC). Two-tailed t-tests were used to evaluate differences in kinematics by bolus viscosity. Hyoid excursion measurements against different axes of reference were correlated. Inter-rater reliability among six raters for the 18 coordinates ranged from ICC = 0.90 - 0.97. A slate of ten kinematic measurements was compared by subject between the six raters. One outlier was rejected, and the mean of the remaining reliability scores was ICC = 0.91, 0.84 - 0.96, 95% CI. Two-tailed t-tests with Bonferroni corrections comparing ten kinematic variables (5 ml thin-liquid vs. 5 ml pudding swallows) showed statistically significant differences in hyoid excursion, superior laryngeal movement, and pharyngeal shortening (p &#60; 0.005). Pearson correlations of hyoid excursion measurements from two different axes of reference were: r = 0.62, r2 = 0.38, (thin-liquid); r = 0.52, r2 = 0.27, (pudding).
Obtaining landmark coordinates is a reliable method to generate multiple kinematic variables from video fluoroscopic images useful in dysphagia research.

Coordinate mapping is a method of documenting salient features of hyolaryngeal biomechanics in the pharyngeal phase of swallowing. This methodology uses image analysis software to record coordinates of anatomical landmarks. These coordinates are imported into an excel macro and translated into kinematic variables of interest useful in dysphagia research.

Characterizing hyolaryngeal movement is important to dysphagia research. Prior methods require multiple measurements to obtain one kinematic measurement ...

Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and bronchodilator drugs. Despite the availability of multiple treatments, both diseases are growing public health concerns. The majority of asthma patients are well controlled on current inhaled therapies but a substantial number of patients with severe asthma are not. Asthma affects an estimated 300 million people worldwide and approximately 20 percent have a severe form of the disease. In contrast to asthma, there are few effective therapies for COPD. An estimated 10% of the population has COPD and the trend in death rates is increasing for COPD while decreasing for other major diseases. Although developing drugs for inhaled delivery is challenging, the nose-only inhalation unit enables direct delivery of novel drugs to the lung of rodents for pre-clinical efficacy and safety/toxicology studies. Inhaled drug delivery has multiple advantages for respiratory diseases, where high concentration in the lung improves efficacy and minimizes systemic side effects. Inhaled corticosteroids and bronchodilators benefit from these advantages and inhaled delivery may also hold potential for future biologic therapies. The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs of rodents, key data required before initiating clinical development.

The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs; key data enabling clinical inhaled drug development.

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and ...

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

Novel Methods for Intranasal Administration Under Inhalation Anesthesia to Evaluate Nose-to-Brain Drug Delivery

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain barrier. However, there have been few reports regarding not only the quantitative analysis but also optimal administration conditions and dosing regimens for investigations of nose-to-brain delivery. The limited progress in research on nose-to-brain pathway mechanisms using rodents represents a significant impediment in terms of designing nose-to-brain delivery systems for candidate drugs.
To gain some headway in this regard, we developed and evaluated two novel methods of stable intranasal administration under inhalation anesthesia for experimental animals. We also describe a method for the evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radio-labeled [14C]-inulin (molecular weight: 5,000) as a model substrate of water-soluble macromolecules.
Initially, we developed a pipette-based intranasal administration protocol using temporarily openable masks, which enabled us to perform reliable administration to animals under stable anesthesia. Using this system, [14C]-inulin could be delivered to the brain with little experimental error.
We subsequently developed an intranasal administration protocol entailing reverse cannulation from the airway side through the esophagus, which was developed to minimize the effects of mucociliary clearance (MC). This technique led to significantly higher levels of [14C]-inulin, which was quantitatively detected in the olfactory bulb, cerebrum, and medulla oblongata, than the pipette method. This appears to be because retention of the drug solution in the nasal cavity was substantially increased by active administration using a syringe pump in a direction opposite to the MC into the nasal cavity.
In conclusion, the two methods of intranasal administration developed in this study can be expected to be extremely useful techniques for evaluating pharmacokinetics in rodents. The reverse cannulation method, in particular, could be useful for evaluating the full potential of nose-to-brain delivery of drug candidates.

Here, we describe two novel methods of stable intranasal administration under inhalation anesthesia with minimal physical stress for experimental animals. We also describe a method for quantitative evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radiolabeled [14C]-inulin as a model substrate of water-soluble macromolecules.

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain ...

Use of MRI-ultrasound Fusion to Achieve Targeted Prostate Biopsy

Here, we present a protocol to perform targeted prostate biopsy using a magnetic resonance imaging-ultrasound (MRI/US) fusion system. Prostate cancer has traditionally been diagnosed via transrectal ultrasound (TRUS) biopsy. Though considered the gold standard, TRUS is unable to visualize most prostate cancer lesions and therefore requires sampling of the entire prostate. This biopsy method often undergrades prostate cancer and fails to detect up to 35% of cancers on initial biopsy. Prostate MRI has been shown to have excellent sensitivity in the detection of cancerous lesions, and advancements in MRI technology during the last decade have led to the development of targeted biopsy. In targeted biopsy, a software platform overlays MRI data onto live TRUS images to create a fused MRI/US three-dimensional model of the prostate. Regions suspicious for malignancy on MRI are contoured by a radiologist, uploaded into the fusion system, and then displayed within the live MRI/US fused model. The urologist is then able to directly biopsy these targets. When compared to conventional TRUS biopsy, MRI/US fusion technology has been demonstrated to improve the detection of clinically significant cancer while reducing insignificant cancer detection. This technology, therefore, has the potential to diagnose prostate cancer primarily in men who would benefit from treatment.

Presented herein is a protocol to perform targeted biopsy of the prostate using an MRI-ultrasound fusion system.

Here, we present a protocol to perform targeted prostate biopsy using a magnetic resonance imaging-ultrasound (MRI/US) fusion system. Prostate cancer has ...

A Novel Inhalation Mask System to Deliver High Concentrations of Nitric Oxide Gas in Spontaneously Breathing Subjects

Nitric Oxide (NO) is administered as gas for inhalation to induce selective pulmonary vasodilation. It is a safe therapy, with few potential risks even if administered at high concentration. Inhaled NO gas is routinely used to increase systemic oxygenation in different disease conditions. The administration of high concentrations of NO also exerts a virucidal effect in vitro. Owing to its favorable pharmacodynamic and safety profiles, the familiarity in its use by critical care providers, and the potential for a direct virucidal effect, NO is clinically used in patients with coronavirus disease-2019 (COVID-19). Nevertheless, no device is currently available to easily administer inhaled NO at concentrations higher than 80 parts per million (ppm) at various inspired oxygen fractions, without the need for dedicated, heavy, and costly equipment. The development of a reliable, safe, inexpensive, lightweight, and ventilator-free solution is crucial, particularly for the early treatment of non-intubated patients outside of the intensive care unit (ICU) and in a limited-resource scenario. To overcome such a barrier, a simple system for the non-invasive NO gas administration up to 250 ppm was developed using standard consumables and a scavenging chamber. The method has been proven safe and reliable in delivering a specified NO concentration while limiting nitrogen dioxide levels. This paper aims to provide clinicians and researchers with the necessary information on how to assemble or adapt such a system for research purposes or clinical use in COVID-19 or other diseases in which NO administration might be beneficial.

This simple and highly adaptable system device for the inhalation of high-concentration nitric oxide (NO) gas does not require mechanical ventilators, positive pressure, or high gas flows. Standard medical consumables and a snug-fitting mask are used to safely deliver NO gas to spontaneously breathing subjects.

Nitric Oxide (NO) is administered as gas for inhalation to induce selective pulmonary vasodilation. It is a safe therapy, with few potential risks even if ...

Creation of Two Saccular Elastase-Digested Aneurysms with Different Hemodynamics in One Rabbit

Preclinical animal models with hemodynamic, morphologic, and histologic characteristics close to human intracranial aneurysms play a key role in the understanding of the pathophysiological processes and the development and testing of new therapeutic strategies. This study aims to describe a new rabbit aneurysm model that allows the creation of two elastase-digested saccular aneurysms with different hemodynamic conditions within the same animal.
Five female New Zealand white rabbits with a mean weight of 4.0 (&#177; 0.3) kg and mean age of 25 (&#177;5) weeks underwent microsurgical stump and bifurcation aneurysm creation. One aneurysm (stump) was created by right common carotid artery (CCA) exposure at its origin at the brachiocephalic trunk. A temporary clip was applied at the CCA origin and another, 2 cm above. This segment was treated with a local injection of 100 U of elastase for 20 min. A second aneurysm (bifurcation) was created by suturing an elastase-treated arterial pouch into the end-to-side anastomosis of the right CCA to left CCA. Patency was controlled by fluorescence angiography immediately after creation.
The average duration of surgery was 221 min. The creation of two aneurysms in the same animal was successful in all rabbits without complication. All aneurysms were patent immediately after surgery except for one bifurcation aneurysm, which showed an extreme tissue reaction due to elastase incubation and an immediate intraluminal thrombosis. No mortality was observed during surgery and up to one-month follow-up. Morbidity was limited to a transient vestibular syndrome (one rabbit), which recovered spontaneously within one day.
Demonstrated here for the first time is the feasibility of creating a two-aneurysm rabbit model with stump and bifurcation hemodynamic characteristics and highly degenerated wall conditions. This model allows the study of the natural course and potential treatment strategies on the basis of aneurysm biology under different flow conditions.

This protocol describes the steps for the creation of a rabbit model with two elastase-digested aneurysms with different hemodynamics (stump and bifurcation constellation). This allows the testing of novel endovascular devices in aneurysms with different angioarchitecture and hemodynamic conditions within a single animal.

Preclinical animal models with hemodynamic, morphologic, and histologic characteristics close to human intracranial aneurysms play a key role in the ...