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 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.
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.
The Institutional Care and Use Committees (IACUC) of the China Medical University approved all procedures.
1. Animal Preparation and Monitoring
2. MRI Acquisition
3. Data Processing
Correct slice geometry is pivotal for ensuring the success of the PC-MRI experiment. The accurate image plane positioning yields a "round" 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 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 < 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 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.
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's torso. (b) The ECG and respiratory signals are clearly visible on the monitor. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Figure 4: Intra-scan test of blood flow from a representative rat. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Figure 6: Longitudinal scan in a 2-month and 4-month old animal showing age-dependent changes in blood flow in the CCA.
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 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 16 but a higher VENC value will lead to deterioration in image quality 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 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'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.
The authors have nothing to disclose.
This work was supported by grants from Ministry of Science and Technology, Taiwan, under grant number of MOST-105-2314-B-039-044-MY2.
7T small animal MRI system | Bruker | ||
Isoflurane | Baxter | 1001936040 | anesthetic |
ECG lead | 3M | 2269T | |
Matlab | MathWorks | sofeware for image processing | |
Monitoring and gating system | SA instruments, Inc | Model 1030 |