This work was supported by grants from Ministry of Science and Technology, Taiwan, under grant number of MOST-105-2314-B-039-044-MY2.

The Institutional Care and Use Committees (IACUC) of the China Medical University approved all procedures.
1. Animal Preparation and Monitoring
<ol>
	<li>Leave all magnetically susceptible objects such as wallets, keys, credit card, etc. outside the scanner room prior to commencing animal preparation for MRI scanning.</li>
	<li>Initially anesthetize the rat (2-month-old male Sprague-Dawley (SD) rat, 280&#8211;350 g) in an induction box using a mixture of 5% isoflurane (ISO) and oxygen (2 L/min) for 3&#8211;5 min, as necessary.</li>
	<li>When the animal is recumbent and exhibits no response to a tail or toe pinch, discontinue ISO administration and transfer the animal to the scanning room.</li>
	<li>Place the rat in the MRI bed in a head-first prone position, and deliver 2-3% ISO through a nose cone device for maintaining anesthesia.</li>
	<li>Monitor respiration by placing a respiratory pillow sensor under the animal&#39;s torso.</li>
	<li>Connect the sensor to a respiratory system and check for respiration rate between 40-50 beats per minute (bpm).</li>
	<li>For cine-gated PC-MRI acquisition, place one electrode each on the right forepaw and the left hind paw, respectively (Figure 1a).</li>
	<li>Twist the electrocardiography (ECG) cables together.</li>
	<li>Use a head holder with ear bars and a bite bar to secure the animal to restrict head movement.</li>
	<li>Use a warm air heating system or gauze pads to maintain body temperature while in the magnet.</li>
	<li>Ensure that the R-wave is clear on the ECG monitor (Figure 1b), and place the animal in the scanner. There is no need to place the surface coil on the top of the animal&#39;s neck as images are acquired by the volume coil.</li>
</ol>
2. MRI Acquisition
<ol>
	<li>Use 2&#8211;3% ISO to maintain anesthesia during the entire imaging procedure. Continuously monitor physiological responses and keep as constant as possible.</li>
	<li>Start the MRI scans once the animal is placed inside the scanner and continues to be physiologically stable. In this study, use a 7 T small animal MRI system with a gradient strength of 630 mT/m, but other field strengths of small animal MRI systems can be used.</li>
	<li>Select the &#34;Localizer&#34; sequence from the console monitor of the MRI scanner and acquire scout images along all three orientations using any fast image acquisition sequence, e.g., the fast spin echo, to create coronal, axial, and sagittal images. The purpose of these scout images is to determine the imaging planes.</li>
	<li>Ensure that the center of the animal&#39;s head and neck is at the center of the magnet. If necessary, adjust the animal&#39;s position until the correct position is reached. If the animal is repositioned, repeat scanning to obtain scout images.</li>
	<li>Select the &#34;Time-of-flight (TOF) angiogram&#34;&#160;sequence from the console monitor of the MRI scanner and acquire a 2D TOF angiogram first to ascertain the precise anatomical location of the CCA. Use the following scanning parameters: repetition time (TR)/ echo time (TE)=22/4.87 ms, flip angle = 90&#176;, field of view (FOV) = 40 &#215; 40 mm2, matrix size = 256 &#215; 256, slice thickness 0.6 mm, with the number of excitation (NEX) = 1. 
	NOTE: The name of the TOF sequence could be vender-specific. The user can insert these parameters in the console monitor.</li>
	<li>Ensure that the saturation band is &#34;on&#34; and is placed on the top to avoid interference from venous signals. 
	NOTE: For the saturation band, it usually comes with the TOF sequence. If the saturation band does not show on the monitor, please notify the service person.</li>
	<li>After locating the CCA using the TOF angiogram, target the image plane of the PC-MRI to the center of the CCA and orient such that the slice is perpendicular to the direction of blood flow (Figure 2a).</li>
	<li>Ensure that both respiration and ECG gating are connected to the MRI system, showing the clear signal on the monitor computer (Figure 1b), and set the trigger module to be &#34;on&#34; in the &#34;Trigger Mode&#34; from the console monitor of the MRI scanner.</li>
	<li>Confirm that the animal&#39;s physiological responses are stable before initiating the PC-MRI scan from the monitoring computer (Figure 1b). Verify that gating selections are &#34;on&#34; in both the monitor computer and the console monitor of MRI scanner. 
	Note: The physiology monitoring system used in this study is provided by the vender. For most animal scanners, the similar physiology monitoring systems are provided and vender-specific.</li>
	<li>Select the sequence of PC-MRI sequence from the console monitor of the MRI scanner and perform gated PC-MRI scans using the following parameters: TR/TE=15.55/4.51 ms (minimum TR and TE), flip angle=30&#176;, FOV = 40 &#215; 40 mm2, matrix size = 192 &#215; 192, slice thickness = 2 mm, velocity encoding (VENC) = 120 cm/s, with NEX=8. Unidirectional VENC is acquired in the through-plane direction. 
	Note: The scan time is approximately 8.5 min, but the actual scan time may be slightly different among animals due to the variation in cardiac cycles.</li>
	<li>Repeat steps 2.6&#8211;2.9 of image acquisition if the region of interest (ROI) is to be changed to another location in the CCA, such as at the bifurcation&#160;4.</li>
	<li>Remove the animal from the scanner and return it to its recovery&#160;cage when the scan is complete.</li>
	<li>Warm the animal with a heating lamp to maintain body temperature. Keep the lamp at least 15 cm away from the animal to prevent overheating.</li>
	<li>When the animal starts to move and exhibits a response to a tail or toe pinch, turn off the heating lamp.</li>
</ol>
3. Data Processing
<ol>
	<li>Save MRI data in Digital Imaging and Communications in Medicine (DICOM) format or any other vender-specific format. Generate cine series with two types of images: a magnitude image (anatomy image) and a phase image (Figure 2b). 
	NOTE: In some scanners, the third type of image, which could be the magnitude image &#215; phase image or the complex-difference (the complex subtraction between the two acquisitions with different velocity-encoding gradients), is generated. The third image is vendor-dependent.</li>
	<li>Pre-process the image data. Covert the phase image into the velocity map and correct the phase-offset error&#160;5. 
	NOTE: The phase image has an arbitrary MR unit of signal intensity instead of true velocity values, but the MR signal intensity is linearly proportional to velocity. The maximum MRI signal from the phase image is typically assigned as the value of VENC, and the minimum signal is assigned the opposite value of VENC. See supplemental code file 1 for an example of the Matlab script and press the button of &#34;Run&#34;.</li>
	<li>Delineate the ROI carefully by tracing the boundary of the CCA. As the artery may dilate and construct during the various cardiac phases, delineate ROIs for each time frame. Calculate the blood flow by integrating over the artery ROI, i.e., velocity &#215; area. The resulted blood flow of each artery was in the units of mL/s. See supplemental code file 2 for an example of the Matlab script and press the button of &#34;Run&#34;.</li>
</ol>

<ol>
	<li>Zakszewski, E., Schmit, B., Kurpad, S., Budde, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+imaging+in+the+rat+cervical+spinal+cord.">Diffusion imaging in the rat cervical spinal cord.</a> J Vis Exp. (98), (2015).</li><li>Wise, R. G., Al-Shafei, A. I., Carpenter, T. A., Hall, L. D., Huang, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+measurement+of+blood+and+myocardial+velocity+in+the+rat+heart+by+phase+contrast+MRI+using+sparse+q-space+sampling.">Simultaneous measurement of blood and myocardial velocity in the rat heart by phase contrast MRI using sparse q-space sampling.</a> J Magn Reson Imaging. 22 (5), 614-627 (2005).</li><li>Skardal, K., Espe, E. K., Zhang, L., Aronsen, J. M., Sjaastad, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Directional+Evaluation+of+Mitral+Flow+in+the+Rat+Heart+by+Phase-Contrast+Cardiovascular+Magnetic+Resonance.">Three-Directional Evaluation of Mitral Flow in the Rat Heart by Phase-Contrast Cardiovascular Magnetic Resonance.</a> PLoS One. 11 (3), e0150536 (2016).</li><li>Peng, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase-contrast+magnetic+resonance+imaging+for+the+evaluation+of+wall+shear+stress+in+the+common+carotid+artery+of+a+spontaneously+hypertensive+rat+model+at+7T:+Location-specific+change,+regional+distribution+along+the+vascular+circumference,+and+reproducibility+analysis.">Phase-contrast magnetic resonance imaging for the evaluation of wall shear stress in the common carotid artery of a spontaneously hypertensive rat model at 7T: Location-specific change, regional distribution along the vascular circumference, and reproducibility analysis.</a> Magn Reson Imaging. 34 (5), 624-631 (2016).</li><li>Yu, H. Y., Peng, H. H., Wang, J. L., Wen, C. Y., Tseng, W. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+the+pulse+wave+velocity+of+the+descending+aorta+using+axial+velocity+profiles+from+phase-contrast+magnetic+resonance+imaging.">Quantification of the pulse wave velocity of the descending aorta using axial velocity profiles from phase-contrast magnetic resonance imaging.</a> Magn Reson Med. 56 (4), 876-883 (2006).</li><li>Peng, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+phase-contrast+MRI+for+the+quantification+of+whole-brain+cerebral+blood+flow.">Optimization of phase-contrast MRI for the quantification of whole-brain cerebral blood flow.</a> J Magn Reson Imaging. 42 (4), 1126-1133 (2015).</li><li>Peng, S. L., Shih, C. T., Huang, C. W., Chiu, S. C., Shen, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+analysis+of+blood+flow+and+wall+shear+stress+in+the+common+carotid+artery+of+rat+model+by+phase-contrast+MRI.">Optimized analysis of blood flow and wall shear stress in the common carotid artery of rat model by phase-contrast MRI.</a> Sci Rep. 7 (1), 5253 (2017).</li><li>Bozgeyik, Z., Berilgen, S., Ozdemir, H., Tekatas, A., Ogur, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+effects+of+sildenafil+citrate+(viagra)+on+vertebral+artery+blood+flow+in+patients+with+vertebro-basilar+insufficiency.">Evaluation of the effects of sildenafil citrate (viagra) on vertebral artery blood flow in patients with vertebro-basilar insufficiency.</a> Korean J Radiol. 9 (6), 477-480 (2008).</li><li>Swampillai, J., Rakebrandt, F., Morris, K., Jones, C. J., Fraser, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+effects+of+caffeine+and+tobacco+on+arterial+function+and+wave+travel.">Acute effects of caffeine and tobacco on arterial function and wave travel.</a> Eur J Clin Invest. 36 (12), 844-849 (2006).</li><li>Neff, K. W., Horn, P., Schmiedek, P., Duber, C., Dinter, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2D+cine+phase-contrast+MRI+for+volume+flow+evaluation+of+the+brain-supplying+circulation+in+moyamoya+disease.">2D cine phase-contrast MRI for volume flow evaluation of the brain-supplying circulation in moyamoya disease.</a> AJR Am J Roentgenol. 187 (1), W107-W115 (2006).</li><li>Stalder, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+2D+and+3D+phase+contrast+MRI:+optimized+analysis+of+blood+flow+and+vessel+wall+parameters.">Quantitative 2D and 3D phase contrast MRI: optimized analysis of blood flow and vessel wall parameters.</a> Magn Reson Med. 60 (5), 1218-1231 (2008).</li><li>Dall'Armellina, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+method+for+quantification+of+regional+cardiac+function+in+mice+using+phase-contrast+MRI.">Improved method for quantification of regional cardiac function in mice using phase-contrast MRI.</a> Magn Reson Med. 67 (2), 541-551 (2012).</li><li>Peng, S. L., Ravi, H., Sheng, M., Thomas, B. P., Lu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Searching+for+a+truly+&amp;#34;iso-metabolic&amp;#34;+gas+challenge+in+physiological+MRI.">Searching for a truly &amp;#34;iso-metabolic&amp;#34; gas challenge in physiological MRI.</a> J Cereb Blood Flow Metab. 37 (2), 715-725 (2017).</li><li>Liu, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+global+cerebral+metabolic+rate+of+oxygen+(CMRO2)+in+neonates+using+MRI.">Quantitative assessment of global cerebral metabolic rate of oxygen (CMRO2) in neonates using MRI.</a> NMR Biomed. 27 (3), 332-340 (2014).</li><li>Xu, F., Ge, Y., Lu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+quantification+of+whole-brain+cerebral+metabolic+rate+of+oxygen+(CMRO2)+by+MRI.">Noninvasive quantification of whole-brain cerebral metabolic rate of oxygen (CMRO2) by MRI.</a> Magn Reson Med. 62 (1), 141-148 (2009).</li><li>Lotz, J., Meier, C., Leppert, A., Galanski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+flow+measurement+with+phase-contrast+MR+imaging:+basic+facts+and+implementation.">Cardiovascular flow measurement with phase-contrast MR imaging: basic facts and implementation.</a> Radiographics. 22 (3), 651-671 (2002).</li><li>Pelc, N. J., Herfkens, R. J., Shimakawa, A., Enzmann, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+contrast+cine+magnetic+resonance+imaging.">Phase contrast cine magnetic resonance imaging.</a> Magn Reson Q. 7 (4), 229-254 (1991).</li><li>Kim, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+phase-contrast+cine+MRI+using+k-t+SPARSE-SENSE.">Accelerated phase-contrast cine MRI using k-t SPARSE-SENSE.</a> Magn Reson Med. 67 (4), 1054-1064 (2012).</li><li>Valvano, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerating+4D+flow+MRI+by+exploiting+low-rank+matrix+structure+and+hadamard+sparsity.">Accelerating 4D flow MRI by exploiting low-rank matrix structure and hadamard sparsity.</a> Magn Reson Med. 78 (4), 1330-1341 (2017).</li></ol>

There is nothing to disclose.

PC-MRI is a comprehensive approach for non-invasive and longitudinal evaluation of blood flow. We present a protocol for performing PC-MRI of the rat CCA. This procedure is easy to perform in any animal MRI scanner and demonstrates good reproducibility.
The PC-MRI technique has gained increasing popularity in human 10,11 as well as animal studies 4,12. Among these studies, results of Peng et al.4 is of particular interest due to the similarity of their approach, but the major difference in the current work is the use of the spatial resolution of 0.21 mm, compared with that of 0.31 mm in the aforementioned report. The limited spatial resolution significantly reduces the scan time, but the resultant partial volume effect may bias the flow quantification, especially for smaller vessels&#160;6,7. Since measurement accuracy is on the list of priorities, a spatial resolution of 0.21 mm with longer scanning time is suggested in future animal studies.
Non-gated PC-MRI is being used as an alternate method of blood flow measurement in many human studies due to its considerably lesser scanning time 6,13,14,15. However, non-gated PC-MRI is not suggested in animals used for pre-clinical testing as the heart rate of rats can be as high as 400 bpm, leading to the fast alternation between systolic and diastolic phases. Non-gated PC-MRI may miss crucial information during the systolic phase, resulting in relatively lower flow values and higher variations 7; therefore, it can only be used for arriving at rough estimations in animals used for pre-clinical testing.
Many scanning parameters are linked to precise quantifications for PC-MRI data, and VENC is one of them. VENC underestimation causes phase aliasing&#160;16 but a higher VENC value will lead to deterioration in image quality&#160;17. We used a VENC value of 120 cm/s, which is appropriate for normal adult SD rats. When changes in vascular tone are expected, such as different species&#160;4, the VENC value should be optimized such that better images or assessments can be acquired.
Particular attention should be paid to important steps of the protocol to obtain a reliable result. First, to avoid resonant circuits and MRI resonant frequency corruption by the ECG signal, it is suggested to twist the ECG cables together. Second, the majority of small animal MRI scanners incorporate a circulating warm water circuit to maintain the animal&#39;s body temperature while in the magnet. However, the flowing water introduces noise and, therefore, interferes with the ECG signal. Thus, in this gated PC-MRI study, we suggest using a warm air heating system or gauze pads instead of using the warm water circulation system to improve the gating quality.
It should be noted that, in this work, only a 2D PC-MRI sequence was employed for data acquisition. The basic technique of cine-gated 2D PC-MRI has emerged as a promising tool for blood flow quantification due to the advantages of reduced scanning time and easy to be implemented in standard scanners. However, data acquired by the 2D PC-MRI technique is limited due to the lack of volumetric acquisitions and reliable streamline tracking, thereby missing some important information such as turbulent flow. Time-resolved 3D PC-MRI pulse sequences with more state-of-the-art techniques such as accelerated cine PC-MRI with compressed sensing and parallel imaging 18,19 should be implemented in future rat CCA experiments. This improvement will enable the provision of insights into spatial aspects of both velocity distribution and flow structures. Nevertheless, the animal preparation and monitoring protocols presented in this report are still applicable in these 4D PC-MRI techniques.
In conclusion, we demonstrate a simple and reliable procedure that measures blood flow in the rat CCA using non-invasive PC-MRI. Further applications of this imaging method include testing of pharmacological effects, pathophysiological assessment, and evaluation of cerebral hemodynamics.

Magnetic resonance imaging (MRI) is a versatile approach that provides detailed information on internal body structures and physiology, and is increasingly being used for clinical diagnosis and in preclinical animal studies. Animal models are vital for better understanding of considerable clinical implication 1. As animal models considerably differ from humans with respect to anesthesia requirements and physiological parameters, optimization of MRI procedures for such animals assumes importance.
Phase contrast MRI (PC-MRI) is a specialized type of MRI that uses the velocity of flowing spins to quantify flow-related parameters such as blood flow. With PC-MRI, mapping flow patterns in the major arteries using animal models can help shed light on cardiovascular pathologies 2. Moreover, PC-MRI can non-invasively monitor the inherent alternations in blood flow in pathophysiological conditions 3. These observations suggest that PC-MRI is a valuable approach that can be used in animal models of human cardiovascular diseases.
In this report, we describe a method for the quantification of blood flow in the common carotid artery (CCA) of rats. The two CCAs supply the head and neck with oxygenated blood, and the carotid artery disease is a major cause of stroke. Therefore, detecting the early pathology in the CCA is pivotal. This procedure has a duration of approximately 15 min and can be potentially applied to conditions with hemodynamics alterations, such atherosclerosis or stroke.

<table border="0" cellpadding="0" cellspacing="0" width="875">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">7T small animal MRI system</td>
			<td style="width:123px;">Bruker</td>
			<td style="width:344px;"></td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Isoflurane&#160;</td>
			<td>Baxter</td>
			<td>1001936040</td>
			<td>anesthetic</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ECG lead&#160;</td>
			<td>3M</td>
			<td>2269T</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Matlab</td>
			<td>MathWorks</td>
			<td></td>
			<td>sofeware for image processing</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Monitoring and gating system</td>
			<td>SA instruments, Inc</td>
			<td>Model 1030</td>
			<td style="width:193px;"></td>
		</tr>
	</tbody>
</table>

phase contrast magnetic resonance imaging in the rat common carotid artery

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous studies have shown that abnormal blood flow may be associated with systemic vascular risk. Thus, PC-MRI can facilitate the translation of data obtained from animal models of cardiovascular diseases to pertinent clinical investigations. In this report, we describe the procedure for measuring blood flow in the common carotid artery (CCA) of rats using cine-gated PC-MRI and discuss relevant analysis methods. This procedure can be performed in a live, anesthetized animal and does not require euthanasia after the procedure. The proposed scanning parameters yield repeatable measurements for blood flow, indicating excellent reproducibility of the results. The PC-MRI procedure described in this article can be used for pharmacological testing, pathophysiological assessment, and cerebral hemodynamics evaluation.

Correct slice geometry is pivotal for ensuring the success of the PC-MRI experiment. The accurate image plane positioning yields a &#34;round&#34; artery shape (Figure 3a), and as angulation increases, i.e., when it is less perpendicular to the artery, the resulting artery geometry becomes ovoid, leading to larger partial volume effects (Figure 3b). Severe partial volume effects would lead to the overestimation of blood flow&#160;6,7. Therefore, we advocate re-positioning of the image plane if the artery shape is ovoid.
Intra-scan reproducibility of time course changes in blood flow within one cardiac cycle from a representative rat is displayed in Figures 4. As can be seen, blood flow reaches its maximum during the systolic phase and returns to baseline during the diastolic phase for both sections. Figure 5a and 5b show a Bland-Altman plot and a scatter plot, respectively, between two blood measurements in the same session, demonstrating a good correlation between measurements (R2=0.7, P &#60; 0.001). With the proposed scanning parameters, blood flow achieves repeatable measurements, demonstrating excellent reproducibility of the results. This characteristic could prove beneficial in testing pharmacological effects on major arteries&#160;8,9.
As PC-MRI is a noninvasive approach to measure blood flow, it can be advantageous in protocols that require longitudinal monitoring. Figure 6 displays the time course for one cardiac cycle in an animal scanned at ages 2 and 4 months and shows that blood flow in the CCA is significantly age-dependent, suggesting rapid development in rats. These quantitative assessments of blood flow are essential for a better understanding of the circulatory system and may, therefore, become a potentially useful tool in preclinical studies on stroke and atherosclerosis.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57304/57304fig1v2.jpg" /> 
Figure 1: Animal monitoring. (a) ECG electrodes are placed on the right forepaw and the left hind paw, and the respiratory pillow sensor is placed under the animal&#39;s torso. (b) The ECG and respiratory signals are clearly visible on the monitor. <a href="/files/ftp_upload/57304/57304fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57304/57304fig2v2.jpg" /> 
Figure 2: Illustration of the position of PC-MRI scans and representative images. (a) Slice positioning on the reconstructed sagittal and coronal views from the TOF angiogram. The blue line indicates image plane at the level of the midpoint of the CCA. (b) Magnitude and phase images from one time-frame of the cine-series images from a representative animal. Red arrows indicate CCA location. <a href="/files/ftp_upload/57304/57304fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57304/57304fig3v2.jpg" /> 
Figure 3: Representative magnitude images. (a) Imaging plane is perpendicular to the artery and (b) imaging plane is non-perpendicular to the artery. The shape of artery changes from round to ovoid if the imaging plane is not perpendicular to the artery. The area containing the CCA is amplified in the red box. <a href="/files/ftp_upload/57304/57304fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57304/57304fig4v2.jpg" /> 
Figure 4: Intra-scan test of blood flow from a representative rat. <a href="/files/ftp_upload/57304/57304fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57304/57304fig5v2.jpg" /> 
Figure 5: Intra-section reproducibility of CCA blood flow measurements. (a) Bland-Altman plot comparing two blood flow measurements acquired between sections. The solid line represents the mean difference between two measurements while dashed lines depict the 95% confidence interval. (b) Scatter plot of the two blood flow measurements. <a href="/files/ftp_upload/57304/57304fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57304/57304fig6.jpg" /> 
Figure 6: Longitudinal scan in a 2-month and 4-month old animal showing age-dependent changes in blood flow in the CCA.

Watch this Scientific Journal Video about Phase Contrast Magnetic Resonance Imaging in the Rat Common Carotid Artery at JoVE.com

Phase Contrast Magnetic Resonance Imaging in the Rat Common Carotid Artery

The overall goal of this procedure is to measure the blood flow in the rat common carotid artery by using noninvasive phase contrast magnetic resonance imaging.

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous ...

phase-contrast-magnetic-resonance-imaging-rat-common-carotid

Google

Research

JoVE Journal

Medicine

9.4K Views.  Chang Gung Memorial Hospital. The overall goal of this procedure is to measure the blood flow in the rat common carotid artery by using noninvasive phase contrast magnetic resonance imaging.

Video: Phase Contrast Magnetic Resonance Imaging in the Rat Common Carotid Artery

Magnetic Resonance Derived Myocardial Strain Assessment Using Feature Tracking

Purpose: An accurate and practical method to measure parameters like strain in myocardial tissue is of great clinical value, since it has been shown, that strain is a more sensitive and earlier marker for contractile dysfunction than the frequently used parameter EF. Current technologies for CMR are time consuming and difficult to implement in clinical practice. Feature tracking is a technology that can lead to more automization and robustness of quantitative analysis of medical images with less time consumption than comparable methods.
Methods: An automatic or manual input in a single phase serves as an initialization from which the system starts to track the displacement of individual patterns representing anatomical structures over time. The specialty of this method is that the images do not need to be manipulated in any way beforehand like e.g. tagging of CMR images.
Results: The method is very well suited for tracking muscular tissue and with this allowing quantitative elaboration of myocardium and also blood flow.
Conclusions: This new method offers a robust and time saving procedure to quantify myocardial tissue and blood with displacement, velocity and deformation parameters on regular sequences of CMR imaging. It therefore can be implemented in clinical practice.

An accurate and practical method to measure parameters like strain in myocardial tissue is of great clinical value, since it has been shown, that strain is a more sensitive and earlier marker for contractile dysfunction than the frequently used parameter EF.

Purpose: An accurate and practical method to measure parameters like strain in myocardial tissue is of great clinical value, since it has been shown, that ...

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., 9 as well as others.12-14 Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8&plusmn;3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice.

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model creating an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV) has been widely described and proved technically feasible. This construct provokes a consistent cerebral venous hypertension (CVH), and therefore has helped studying the contribution of venous hypertension to formation, clinical symptoms, and prognosis of brain AVMs and dural AVFs. Equivalent mice models have been only scarcely described and have shown trouble with stenosis of the fistula. An established murine model would allow the study of not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases.
We present a model of arteriovenous fistula that produces a durable intracranial venous hypertension in the mouse. Microsurgical anastomosis of the murine CCA and EJV can be difficult due to diminutive anatomy and frequently result in a non-patent fistula. In this step-by-step protocol we address all the important challenges encountered during this procedure. Avoiding excessive retraction of the vein during the exposure, using 11-0 sutures instead of 10-0, and making a carefully planned end-to-side anastomosis are some of the critical steps. Although this method requires advanced microsurgical skills and a longer learning curve that the equivalent in the rat, it can be consistently developed.
This novel model has been designed to integrate transgenic mouse techniques with a previously well-established experimental system that has proved useful to study brain AVMs and dural AVFs. By opening the possibility of using transgenic mice, a broader spectrum of valid models can be achieved and genetic treatments can also be tested. The experimental construct could also be further adapted to the study of other cerebrovascular diseases related with venous hypertension such as migraine, transient global amnesia, transient monocular blindness, etc.

We describe a method for creating a reliable model of cerebral venous hypertension in the adult mouse. This model has been widely described and tested in the rat. This new counterpart in the mice opens the possibility of using genetic modified animals and thereby broadens the applications of the model.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model ...

Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and cellular mechanisms involved in vascular smooth muscle dedifferentiation, neointima formation and vascular remodeling. Male Sprague-Dawley rats are the most frequently employed animals for this model. Female rats are not preferred as female hormones are protective against vascular diseases and thus introduce a variation into this procedure. The left carotid is typically injured with the right carotid serving as a negative control. Left carotid injury is caused by the inflated balloon that denudes the endothelium and distends the vessel wall. Following injury, potential therapeutic strategies such as the use of pharmacological compounds and either gene or shRNA transfer can be evaluated. Typically for gene or shRNA transfer, the injured section of the vessel lumen is locally transduced for 30 min with viral particles encoding either a protein or shRNA for delivery and expression in the injured vessel wall. Neointimal thickening representing proliferative vascular smooth muscle cells usually peaks at 2 weeks after injury. Vessels are mostly harvested at this time point for cellular and molecular analysis of cell signaling pathways as well as gene and protein expression. Vessels can also be harvested at earlier time points to determine the onset of expression and/or activation of a specific protein or pathway, depending on the experimental aims intended. Vessels can be characterized and evaluated using histological staining, immunohistochemistry, protein/mRNA assays, and activity assays. The intact right carotid artery from the same animal is an ideal internal control. Injury-induced changes in molecular and cellular parameters can be evaluated by comparing the injured artery to the internal right control artery. Likewise, therapeutic modalities can be evaluated by comparing the injured and treated artery to the control injured only artery.

This protocol uses a balloon catheter to cause an intraluminal injury on the rat carotid artery and henceforth elicit neointimal hyperplasia. This is a well-established model for studying the mechanisms of vascular remodeling in response to injury. It is also widely used to determine the validity of potential therapeutic approaches.

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

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 ...

Cardiac Magnetic Resonance Imaging at 7 Tesla

CMR at an ultra-high field (magnetic field strength B0&#160;&#8805; 7 Tesla) benefits from the signal-to-noise ratio (SNR) advantage inherent at higher magnetic field strengths and potentially provides improved signal contrast and spatial resolution. While promising results have been achieved, ultra-high field CMR is challenging due to energy deposition constraints and physical phenomena such as transmission field non-uniformities and magnetic field inhomogeneities. In addition, the magneto-hydrodynamic effect renders the synchronization of the data acquisition with the cardiac motion difficult. The challenges are currently addressed by explorations into novel magnetic resonance technology. If all impediments can be overcome, ultra-high field CMR may generate new opportunities for functional CMR, myocardial tissue characterization, microstructure imaging or metabolic imaging. Recognizing this potential, we show that multi-channel radio frequency (RF) coil technology tailored for CMR at 7 Tesla together with higher order B0 shimming and a backup signal for cardiac triggering facilitates high fidelity functional CMR. With the proposed setup, cardiac chamber quantification can be accomplished in examination times similar to those achieved at lower field strengths. To share this experience and to support the dissemination of this expertise, this work describes our setup and protocol tailored for functional CMR at 7 Tesla.

The sensitivity gain inherent to ultrahigh field magnetic resonance holds promise for high spatial resolution imaging of the heart. Here, we describe a protocol customized for functional cardiovascular magnetic resonance (CMR) at 7 Tesla using an advanced multi-channel radio-frequency coil, magnetic field shimming and a triggering concept.

CMR at an ultra-high field (magnetic field strength B0&#160;&#8805; 7 Tesla) benefits from the signal-to-noise ratio (SNR) advantage inherent at higher ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...