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Assessment of myocardial edema and area at risk in a rat model of myocardial infarction with a faster T2 mapping method Rui Xia, Xi Lu, Bing Zhang, Yuqing Wang, Jichun Liao, Jie Zheng and Fabao Gao Acta Radiol published online 2 September 2014 DOI: 10.1177/0284185114547899 The online version of this article can be found at: http://acr.sagepub.com/content/early/2014/08/31/0284185114547899
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Original Article
Assessment of myocardial edema and area at risk in a rat model of myocardial infarction with a faster T2 mapping method
Acta Radiologica 0(0) 1–6 ! The Foundation Acta Radiologica 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0284185114547899 acr.sagepub.com
Rui Xia1,2, Xi Lu1, Bing Zhang1, Yuqing Wang1, Jichun Liao1, Jie Zheng3 and Fabao Gao1
Abstract Background: The common T2 mapping is not suitable for the use in rat heart with high heart rate, unless data are acquired in multiple cardiac cycles. Purpose: To evaluate a simplified T2 mapping method for faster assessment of myocardial edema and area at risk in a rat model of myocardial infarction. Material and Methods: The simplified T2 mapping method (TR/TE, 1500 ms/10, 20, 30 ms) was implemented at a 7.0T MRI system. The accuracy of T2 mapping was compared with a standard T2 mapping method (TR, 2500 ms, 16 TEs equally spaced from 11 ms to 176 ms) in thigh muscles in rats (n ¼ 6) and a phantom. This method was further evaluated in normal rats (n ¼ 8) and rats with myocardial infarction (n ¼ 8). Late gadolinium enhancement images were also acquired in the rats with myocardial infarction. Results: T2 values of simplified T2 mapping in the muscles and phantom were 27.3 2 ms and 26.5 1.1 ms, which were similar to the T2 values obtained by the standard T2 mapping method (28.1 1.4 ms, P > 0.05; 26.9 1.7 ms). No significant difference in T2 distribution (different segments and slices from base to apex) in the whole heart was found in normal rats (25.6 3.3 ms, P > 0.05). The mean T2 value in the myocardial edema regions of myocardial infarction rats (37 4.9 ms) was significantly higher than that of the normal rats (25.6 3.3 ms, P < 0.001). The T2 value in the myocardial infarction core of myocardial infarction rats (39.9 3.6 ms) was significantly higher than that of area at risk (34.7 2.9 ms, P < 0.001). Conclusion: The simplified myocardial T2 mapping is technically feasible and accurate, and can readily detect myocardial edema and area at risk in rats with high heart rate.
Keywords Cardiac, MR imaging, Experiment Investigations, Edema, Ischemia/Infarction Date received: 6 January 2014; accepted: 10 July 2014
Introduction Cardiovascular magnetic resonance (CMR) is well established and increasingly used in clinical practice for the diagnosis of myocardial ischemia (1). Of all CMR techniques, T2-weighted (T2W) imaging is an important tool for evaluating myocardial edema. When combined with the late gadolinium enhancement (LGE) imaging of irreversible injury, myocardium salvaged area (area at risk [AAR]) can be quantified (2). Rodent models play a key role in developing our understanding of cardiovascular disease and the development of novel therapies (3–5). Accurate non-invasive
1 Molecular Imaging Laboratory, Department of Radiology, West China Hospital, Sichuan University, PR China 2 Department of Radiology, The First Affiliated Hospital of Chongqing Medical University, PR China 3 Mallinckrodt Institute of Radiology, Washington University School of Medicine in St. Louis, MO, USA
Corresponding author: Fabao Gao, Department of Radiology, West China Hospital, Sichuan University No. 37 Guoxuexiang, 610041 Chengdu, China. Email: [email protected]; [email protected]
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assessment of myocardial T2 values in rat models of cardiovascular disease is therefore highly desirable. However, most studies using T2 approaches were achieved on human or big animals (6–10) with relatively low heart rate (300 bpm) (3,11). The standard T2-mapping at a 7.0 T magnetic resonance imaging (MRI) system typically requires acquisition of multiple images with different echo times or TE (typical 16 echo times with 11 ms each and the longest TE is 176 ms) to allow for an accurate fitting of the underlying T2 curve (multi-slice multi-echo [MSME]-T2 mapping). However, this approach is not suitable for the use in a rat heart with high heart rate due to its relatively long data acquisition time within each cardiac cycle (12). In this study, we simplified T2 mapping method at a 7.0 T system with only three TEs and evaluated this method in normal and MI rats. The purpose of this study was to compare: (i) different T2 mapping sequences on thigh muscles of rats and a phantom; and (ii) T2 values of normal myocardium, infarction core area, AAR, and the whole injury myocardium in rats.
Material and Methods Muscle and phantom To determine the accuracy of our simplified T2 mapping sequence, a standard MSME-T2 mapping (repetition time [TR], 2500 ms; 16 TEs equally spaced from 11 ms to 176 ms) sequence on 7.0 T MRI was used as the reference method. The T2 values of thigh muscles of six normal rats were measured. A phantom study was performed by using a vial of water dropped with gadolinium contrast media. The T2 of the vial was designed to close to the T2 of the thigh muscles of rats at a 7.0 T MRI. The simplified T2 mapping sequence with the same parameters used in vivo (see below) was applied, followed by the MSME T2 mapping scan.
Animal model This study was approved by Institutional Animal Care and Use Committee of our local institute. Sixteen male Sprague-Dawley rats (250–350 g) were divided into two groups (normal n ¼ 8, MI n ¼ 8). In order to induce rat myocardial infarction, the rats were first anesthetized with sodium pentobarbital (50 mg/kg) intraperitoneally, and respiration was maintained using a rodent ventilator. Real time electrocardiogram (ECG, SA Instruments Inc., Stony Brook, NY, USA) was monitored throughout the surgery. No surgery was performed in normal rats.
A thoracotomy was then performed. The chest was opened at the fourth intercostal space to expose the heart. The pericardium was opened with forceps, and a 6.0 suture was passed underneath the left anterior descending coronary artery (LAD) at the location 1–2 mm proximal to the ostium of the coronary artery. Coronary occlusion was achieved by tightening the suture. The success of occlusion was confirmed by the pale appearance of the myocardial apex area and the immediate changes in ECG profiles, including a significant increase in the amplitude of the QRS complex and elevation of ST segment.
MRI protocols MRI scans were performed in the normal group and at 24 h after the occlusion in the MI group. All of the MRI protocols were implemented at a 7.0 T MRI (BRUKER BIOSPEC 70/30, Ettlingen, Germany). Each rat was anesthetized with isoflurane (2–3%) in a small container and maintained with the mixture of 100% oxygen and isoflurane (1–2%) during the MRI scan, the body temperature was kept constant using a heating blanket at 37 C monitored with a rectal temperature probe. Each animal was placed prone in a surface coil. The ECG signal was obtained from two subcutaneous copper needles loaded in both left forelimb and hind limb. The respiration signal was acquired from the respiratory pillow (SA Instruments Inc., Stony Brook, NY, USA) under the rat. Scout imaging was first acquired using gradient-echo sequence to localize the short-axis images at the middle level of the left ventricle (LV). The T2 mapping was acquired after keeping respiratory frequency between 30 and 50 cycles per min by changing concentration of isoflurane. Five single-slice simplified T2 mapping images were acquired on the short-axis slices during mid-diastolic phase and end-inspiratory period using both ECG and respiratory gating systems. The imaging parameters included: TR/TE, 1500 ms/10, 20, 30 ms; data acquisition time, 44 ms; matrix size, 192 192; field of view (FOV), 50 50 mm; and slice thickness, 1.5 mm. The last TE of 30 ms was selected based on the approximate T2 value of myocardial tissue at 7.0 T (8). For MI group, LGE imaging was performed by fast imaging with steady precession (FISP)-cine (TR/ TE, 5.2 ms/1.8 ms; flip angle, 25 ; matrix size, 256 256; FOV, 50 50 mm; slice thickness, 1.5 mml 25 frames for each slice) at 10 min after an injection of 0.15 mmol/kg of Gd-DTPA (Magnevist, Bayer Health Care Pharma AG, Berlin, Germany).
Histology of infarction tissue After the MRI scans, three of MI rats were sacrificed with potassium chloride and the hearts were rapidly
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excised. Each heart was cut into five transverse slices from apex to base, each approximately 1.5 mm thick. These slices were then incubated with 1% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma; Saint Louis, MI, USA) for 15 min at 37 C, so that the viable myocardium was stained red and the infarcted area was stained white. The white areas were expressed as a percentage of the whole myocardial tissue of the left ventricle (%LV).
Data were given as mean SD. We compared the results from normal and MI groups with Independent Sample t-test, and differences for T2 values between myocardial infarction cores and AARs were compared by Paired Sample t-test. Different measurements of T2 values between simplified T2 mapping method and MSME-T2-mapping method of thigh muscles were compared by Paired Sample t-test. P values 2 standard deviation (SD) from remote myocardium on a T2 map. The infarction area was defined in areas with signal intensity >5SD from remote myocardium on a LGE image. The area at risk was defined as the difference between the edema area and the corresponding infarction area (2,13,14). To compare with the TTC stain, the LGE positive enhanced areas in the corresponding three rats were added slice-by-slice, respectively, and expressed as a percentage of the whole myocardial tissue of left ventricle (%LV).
Results Muscle and phantom The T2 value in the thigh muscles was 27.3 2 ms by the simplified T2 mapping and 28.1 1.4 ms by the MSME T2 mapping (P > 0.05) (Fig. 1).The T2 value in the phantom was 26.5 1.1 ms by the simplified T2 mapping and 27.1 2.7 ms by the MSME T2 mapping (Fig. 2).
Normal rats Mean normal heart T2 value was 25.6 3.3 ms (Fig. 3). There were no significant differences in T2 values among different segments in normal rats (P > 0.05) (Fig. 4a). In addition, there were no significant differences in T2 values from base to apex in normal rats (P > 0.05) (Fig. 4b).
MI rats Thirty-nine of 40 slices showed delayed enhancement in the LAD regions and were selected to calculated myocardial T2 mappings. The mean T2 value in the LAD regions of MI rats (37 4.9 ms) was significantly higher than that of the normal rats (25.6 3.3 ms, P < 0.001). Twenty-six of 39 slices were found to have AAR and infarction core. The T2 value in the myocardial infarction core of MI rats (39.9 3.6 ms) was significantly higher than that of AAR
Fig. 1. Images of muscle. An example of thigh muscles was scanned with T2W imaging (a), simplified T2 mapping (b), and MSME T2 mapping (c). There was no difference between T2 values of muscles acquired from simplified T2 mapping and the standard MSME T2 mapping (P > 0.05).
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Fig. 2. Images of phantom. A custom-made phantom was scanned with T2W imaging (a), simplified T2 mapping (b), and MSME T2 mapping (c). T2 value acquired from simplified T2 mapping was similar to that from the standard MSME T2 mapping.
Fig. 3. T2W images and T2 mapping image of normal rat heart. T2W images with echo time of 10 ms (a), 20 ms (b), and 30 ms (c) and T2 mapping image of the same slice of a normal rat heart (d).
(34.7 2.9 ms, P < 0.001) (Fig. 5), and both of them was significantly higher than normal rat (25.6 3.3 ms, both P < 0.001). No significant difference was found in the infarcted area defined by LGE and TTC staining for three MI rats (22.5 3% vs. 21.2 2%, n ¼ 3, P > 0.05).
Discussion For the first time, this study examined a simplified myocardial T2 mapping method on 7.0 T MRI for faster evaluation of myocardial edema in rats with high heart
rate. The muscle and phantom studies demonstrated a close agreement between the T2 measured by the simplified method and the standard T2 mapping method on 7.0 T MRI. It was found that myocardial T2 distribution was uniform in the whole heart of normal rats. The MI cores had a highest T2 value, followed by the T2 value of AAR in MI rats, and T2 values in normal rats. The much reduced data acquisition window allowed for 100% success rate for the imaging of myocardial injury area. The mean myocardial T2 value in normal rats was 25.6 3.3 ms that is shorter than T2 values of normal
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Fig. 4. T2 distributions in the whole heart. No difference (P > 0.05) was found among T2 values in different segments (a) and from base to apex (b).
Fig. 5. Images of a rat heart with myocardial infarction. Infarction area (dotted lined, block arrows) was showed on a late gadolinium enhancement (LGE) image (a) and 2,3,5-triphenyltetrazolium chloride (TTC) staining (b). Myocardial edema area (full line, arrows) was observed by an ordinary T2W image (c) and simplified T2 map (d). AAR, the difference between full line area and dotted line area, was showed by overlay a and d (e), and T2 value of the infarction area was bigger than that in AAR.
myocardium of 27 6.3 ms (15) and 31.5 0.7 ms on 4.7 T in previous studies (11). The differences may be due to different magnetic field and different MRI sequences. For the standard T2 mapping method (11), the 20 echoes were collected at different cardiac cycles (the longest TE was 60 ms), While all three echo signals were acquired in one cardiac cycle in simplified T2 mapping (the longest TE was 30 ms), significantly limiting the cardiac motion artifacts. There were no differences in T2 values among different segments and slices (from base to apex) in normal
rats. However, the findings of the current study do not support the previous research by Florian (7), in which T2 increased from base to apex. In another study by Ralf and et al., higher T2 values were observed in the anterior and anteroseptal segments (6). Because these two studies were implemented on humans, the difference in heart structure between human and rat may cause this difference in T2 distribution. In this study, the MI cores showed a higher T2 value than AAR in MI rats. Both these T2 values were higher than that in normal rats. This finding was in agreement
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with findings by Whalen and Jennings who suggested that substantial edema occurred in the acute infarcted region, with minimal edema occurring in the AAR portion with reversible injury (13,16–18). There are several limitations in this study. First, the accuracy of this simplified T2 measurement was not determined by the in vivo study, and myocardial edema was not validated by other reference method, e.g. microscopy. Second, because the longest TE was 30 ms in our simplified T2 mapping method, this approach may not accurately measure T2 values significantly larger than 30 ms. This is one of the reasons that increased noise of the simplified method was observed in the phantom and muscles experiments. Third, defining the area at risk by myocardial edema may overestimate the true area at risk caused by ischemia. Finally, one k-space line was acquired in each cardiac cycle in the simplified T2 mapping method, resulting in a relatively long acquisition time. In conclusion, we simplified a T2 mapping sequence and demonstrated its feasibility for the quantification of T2 distribution for faster assessment of myocardial edema and AAR. This technique may provide a very useful tool for faster evaluation of MI model in rats with high heart rate and monitor the efficacy of the treatment of MI in a small animal model.
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Conflict of interest None declared.
Funding This study was supported by The State Key Program of National Natural Science Foundation of China (81130027), The Twelfth Five Year Science and Technology support program (2012BAI23B08) and The National Key Basic Research Program of China (2011CB935800).
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References 1. Eitel I, Friedrich MG. T2-weighted cardiovascular magnetic resonance in acute cardiac disease. J Cardiovasc Magn Reson 2011;13:13–23. 2. Friedrich MG, Abdel-Aty H, Taylor A, et al. The salvaged area at risk in reperfused acute myocardial infarction as visualized by cardiovascular magnetic resonance. J Am Coll Cardiol 2008;51:1581–1587. 3. Jogiya R, Makowski M, Phinikaridou A, et al. Hyperemic stress myocardial perfusion cardiovascular magnetic resonance in mice at 3 Tesla: initial experience and validation against microspheres. J Cardiovasc Magn Reson 2013;15: 62–71. 4. Geisterfer-Lowrance AA, Christe M, Conner DA, et al. A mouse model of familial hypertrophic cardiomyopathy. Science 1996;272:731–734. 5. Gilson WD, Yang Z, French BA, et al. Measurement of myocardial mechanics in mice before and after infarction
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using multi-slice displacement-encoded MRI with 3D motion encoding. Am J Physiol Heart Circ Physiol 2005;288:H1491–H1497. Wassmuth R, Prothmann M, Utz W, et al. Variability and homogeneity of cardiovascular magnetic resonance myocardial T2-mapping in volunteers compared to patients with edema. J Cardiovasc Magn Reson 2013;15:27–36. von Knobelsdorff-Brenkenhoff F, Prothmann M, Dieringer MA, et al. Myocardial T1 and T2 mapping at 3 T: reference values, influencing factors and implications. J Cardiovasc Magn Reson 2013;15:53–63. Giri S, Chung YC, Merchant A, et al. T2 quantification for improved detection of myocardial edema. J Cardiovasc Magn Reson 2009;11:56–68. Lønborg J, Vejlstrup N, Mathiasen AB, et al. Myocardial area at risk and salvage measured by T2-weighted cardiovascular magnetic resonance: reproducibility and comparison of two T2-weighted protocols. J Cardiovasc Magn Reson 2011;13:50–58. Aletras AH, Tilak GS, Natanzon A, et al. Retrospective determination of the area at risk for reperfused acute myocardial infarction with T2-weighted cardiac magnetic resonance imaging: histopathological and displacement encoding with stimulated echoes (DENSE) functional validations. Circulation 2006;113:1865–1870. Caudron J, Mulder P, Nicol L, et al. MR relaxometry and perfusion of the myocardium in spontaneously hypertensive rat: correlation with histopathology and effect of anti-hypertensive therapy. Eur Radiol 2013;23: 1871–1881. Messroghli DR, Nordmeyer S, Buehrer M, et al. Small animal Look-Locker inversion recovery (SALLI) for simultaneous generation of cardiac T1 mappings and cine and inversion recovery-prepared images at high heart rates: initial experience. Radiology 2011;261:258–265. Friedrich MG, Kim HW, Kim RJ. T2-weighted imaging to assess post-infarct myocardium at risk. JACC Cardiovasc Imaging 2011;4:1014–1021. Abdel-Aty H, Cocker M, Strohm O, et al. Abnormalities in T2-weighted cardiovascular magnetic resonance images of hypertrophic cardiomyopathy: regional distribution and relation to late gadolinium enhancement and severity of hypertrophy. J Magn Reson Imaging 2008;28:242–245. Weis S, Shintani S, Weber A, et al. Src blockade stabilizes a Flk/cadherin complex, reducing edema and tissue injury following myocardial infarction. J Clin Invest 2004;113: 885–894. Whalen DA Jr, Hamilton DG, Ganote CE, et al. Effect of a transient period of ischemia on myocardial cells. I. Effects on cell volume regulation. Am J Pathol 1974;74: 381–397. Jennings RB, Schaper J, Hill ML, et al. Effect of reperfusion late in the phase of reversible ischemic injury. Changes in cell volume, electrolytes, metabolites, and ultrastructure. Circ Res 1985;56:262–278. Croisille P, Kim HW, Kim RJ. Controversies in cardiovascular MR imaging: T2-weighted imaging should not be used to delineate the area at risk in ischemic myocardial injury. Radiology 2012;265:12–22.
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Assessment of myocardial edema and area at risk in a rat model of myocardial infarction with a faster T2 mapping method Rui Xia, Xi Lu, Bing Zhang, Yuqing Wang, Jichun Liao, Jie Zheng and Fabao Gao Acta Radiol published online 2 September 2014 DOI: 10.1177/0284185114547899 The online version of this article can be found at: http://acr.sagepub.com/content/early/2014/08/31/0284185114547899
Published by: http://www.sagepublications.com
On behalf of: Nordic Society of Medical Radiology
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Acta Radiol OnlineFirst, published on September 2, 2014 as doi:10.1177/0284185114547899
Original Article
Assessment of myocardial edema and area at risk in a rat model of myocardial infarction with a faster T2 mapping method
Acta Radiologica 0(0) 1–6 ! The Foundation Acta Radiologica 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0284185114547899 acr.sagepub.com
Rui Xia1,2, Xi Lu1, Bing Zhang1, Yuqing Wang1, Jichun Liao1, Jie Zheng3 and Fabao Gao1
Abstract Background: The common T2 mapping is not suitable for the use in rat heart with high heart rate, unless data are acquired in multiple cardiac cycles. Purpose: To evaluate a simplified T2 mapping method for faster assessment of myocardial edema and area at risk in a rat model of myocardial infarction. Material and Methods: The simplified T2 mapping method (TR/TE, 1500 ms/10, 20, 30 ms) was implemented at a 7.0T MRI system. The accuracy of T2 mapping was compared with a standard T2 mapping method (TR, 2500 ms, 16 TEs equally spaced from 11 ms to 176 ms) in thigh muscles in rats (n ¼ 6) and a phantom. This method was further evaluated in normal rats (n ¼ 8) and rats with myocardial infarction (n ¼ 8). Late gadolinium enhancement images were also acquired in the rats with myocardial infarction. Results: T2 values of simplified T2 mapping in the muscles and phantom were 27.3 2 ms and 26.5 1.1 ms, which were similar to the T2 values obtained by the standard T2 mapping method (28.1 1.4 ms, P > 0.05; 26.9 1.7 ms). No significant difference in T2 distribution (different segments and slices from base to apex) in the whole heart was found in normal rats (25.6 3.3 ms, P > 0.05). The mean T2 value in the myocardial edema regions of myocardial infarction rats (37 4.9 ms) was significantly higher than that of the normal rats (25.6 3.3 ms, P < 0.001). The T2 value in the myocardial infarction core of myocardial infarction rats (39.9 3.6 ms) was significantly higher than that of area at risk (34.7 2.9 ms, P < 0.001). Conclusion: The simplified myocardial T2 mapping is technically feasible and accurate, and can readily detect myocardial edema and area at risk in rats with high heart rate.
Keywords Cardiac, MR imaging, Experiment Investigations, Edema, Ischemia/Infarction Date received: 6 January 2014; accepted: 10 July 2014
Introduction Cardiovascular magnetic resonance (CMR) is well established and increasingly used in clinical practice for the diagnosis of myocardial ischemia (1). Of all CMR techniques, T2-weighted (T2W) imaging is an important tool for evaluating myocardial edema. When combined with the late gadolinium enhancement (LGE) imaging of irreversible injury, myocardium salvaged area (area at risk [AAR]) can be quantified (2). Rodent models play a key role in developing our understanding of cardiovascular disease and the development of novel therapies (3–5). Accurate non-invasive
1 Molecular Imaging Laboratory, Department of Radiology, West China Hospital, Sichuan University, PR China 2 Department of Radiology, The First Affiliated Hospital of Chongqing Medical University, PR China 3 Mallinckrodt Institute of Radiology, Washington University School of Medicine in St. Louis, MO, USA
Corresponding author: Fabao Gao, Department of Radiology, West China Hospital, Sichuan University No. 37 Guoxuexiang, 610041 Chengdu, China. Email: [email protected]; [email protected]
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assessment of myocardial T2 values in rat models of cardiovascular disease is therefore highly desirable. However, most studies using T2 approaches were achieved on human or big animals (6–10) with relatively low heart rate (300 bpm) (3,11). The standard T2-mapping at a 7.0 T magnetic resonance imaging (MRI) system typically requires acquisition of multiple images with different echo times or TE (typical 16 echo times with 11 ms each and the longest TE is 176 ms) to allow for an accurate fitting of the underlying T2 curve (multi-slice multi-echo [MSME]-T2 mapping). However, this approach is not suitable for the use in a rat heart with high heart rate due to its relatively long data acquisition time within each cardiac cycle (12). In this study, we simplified T2 mapping method at a 7.0 T system with only three TEs and evaluated this method in normal and MI rats. The purpose of this study was to compare: (i) different T2 mapping sequences on thigh muscles of rats and a phantom; and (ii) T2 values of normal myocardium, infarction core area, AAR, and the whole injury myocardium in rats.
Material and Methods Muscle and phantom To determine the accuracy of our simplified T2 mapping sequence, a standard MSME-T2 mapping (repetition time [TR], 2500 ms; 16 TEs equally spaced from 11 ms to 176 ms) sequence on 7.0 T MRI was used as the reference method. The T2 values of thigh muscles of six normal rats were measured. A phantom study was performed by using a vial of water dropped with gadolinium contrast media. The T2 of the vial was designed to close to the T2 of the thigh muscles of rats at a 7.0 T MRI. The simplified T2 mapping sequence with the same parameters used in vivo (see below) was applied, followed by the MSME T2 mapping scan.
Animal model This study was approved by Institutional Animal Care and Use Committee of our local institute. Sixteen male Sprague-Dawley rats (250–350 g) were divided into two groups (normal n ¼ 8, MI n ¼ 8). In order to induce rat myocardial infarction, the rats were first anesthetized with sodium pentobarbital (50 mg/kg) intraperitoneally, and respiration was maintained using a rodent ventilator. Real time electrocardiogram (ECG, SA Instruments Inc., Stony Brook, NY, USA) was monitored throughout the surgery. No surgery was performed in normal rats.
A thoracotomy was then performed. The chest was opened at the fourth intercostal space to expose the heart. The pericardium was opened with forceps, and a 6.0 suture was passed underneath the left anterior descending coronary artery (LAD) at the location 1–2 mm proximal to the ostium of the coronary artery. Coronary occlusion was achieved by tightening the suture. The success of occlusion was confirmed by the pale appearance of the myocardial apex area and the immediate changes in ECG profiles, including a significant increase in the amplitude of the QRS complex and elevation of ST segment.
MRI protocols MRI scans were performed in the normal group and at 24 h after the occlusion in the MI group. All of the MRI protocols were implemented at a 7.0 T MRI (BRUKER BIOSPEC 70/30, Ettlingen, Germany). Each rat was anesthetized with isoflurane (2–3%) in a small container and maintained with the mixture of 100% oxygen and isoflurane (1–2%) during the MRI scan, the body temperature was kept constant using a heating blanket at 37 C monitored with a rectal temperature probe. Each animal was placed prone in a surface coil. The ECG signal was obtained from two subcutaneous copper needles loaded in both left forelimb and hind limb. The respiration signal was acquired from the respiratory pillow (SA Instruments Inc., Stony Brook, NY, USA) under the rat. Scout imaging was first acquired using gradient-echo sequence to localize the short-axis images at the middle level of the left ventricle (LV). The T2 mapping was acquired after keeping respiratory frequency between 30 and 50 cycles per min by changing concentration of isoflurane. Five single-slice simplified T2 mapping images were acquired on the short-axis slices during mid-diastolic phase and end-inspiratory period using both ECG and respiratory gating systems. The imaging parameters included: TR/TE, 1500 ms/10, 20, 30 ms; data acquisition time, 44 ms; matrix size, 192 192; field of view (FOV), 50 50 mm; and slice thickness, 1.5 mm. The last TE of 30 ms was selected based on the approximate T2 value of myocardial tissue at 7.0 T (8). For MI group, LGE imaging was performed by fast imaging with steady precession (FISP)-cine (TR/ TE, 5.2 ms/1.8 ms; flip angle, 25 ; matrix size, 256 256; FOV, 50 50 mm; slice thickness, 1.5 mml 25 frames for each slice) at 10 min after an injection of 0.15 mmol/kg of Gd-DTPA (Magnevist, Bayer Health Care Pharma AG, Berlin, Germany).
Histology of infarction tissue After the MRI scans, three of MI rats were sacrificed with potassium chloride and the hearts were rapidly
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excised. Each heart was cut into five transverse slices from apex to base, each approximately 1.5 mm thick. These slices were then incubated with 1% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma; Saint Louis, MI, USA) for 15 min at 37 C, so that the viable myocardium was stained red and the infarcted area was stained white. The white areas were expressed as a percentage of the whole myocardial tissue of the left ventricle (%LV).
Data were given as mean SD. We compared the results from normal and MI groups with Independent Sample t-test, and differences for T2 values between myocardial infarction cores and AARs were compared by Paired Sample t-test. Different measurements of T2 values between simplified T2 mapping method and MSME-T2-mapping method of thigh muscles were compared by Paired Sample t-test. P values 2 standard deviation (SD) from remote myocardium on a T2 map. The infarction area was defined in areas with signal intensity >5SD from remote myocardium on a LGE image. The area at risk was defined as the difference between the edema area and the corresponding infarction area (2,13,14). To compare with the TTC stain, the LGE positive enhanced areas in the corresponding three rats were added slice-by-slice, respectively, and expressed as a percentage of the whole myocardial tissue of left ventricle (%LV).
Results Muscle and phantom The T2 value in the thigh muscles was 27.3 2 ms by the simplified T2 mapping and 28.1 1.4 ms by the MSME T2 mapping (P > 0.05) (Fig. 1).The T2 value in the phantom was 26.5 1.1 ms by the simplified T2 mapping and 27.1 2.7 ms by the MSME T2 mapping (Fig. 2).
Normal rats Mean normal heart T2 value was 25.6 3.3 ms (Fig. 3). There were no significant differences in T2 values among different segments in normal rats (P > 0.05) (Fig. 4a). In addition, there were no significant differences in T2 values from base to apex in normal rats (P > 0.05) (Fig. 4b).
MI rats Thirty-nine of 40 slices showed delayed enhancement in the LAD regions and were selected to calculated myocardial T2 mappings. The mean T2 value in the LAD regions of MI rats (37 4.9 ms) was significantly higher than that of the normal rats (25.6 3.3 ms, P < 0.001). Twenty-six of 39 slices were found to have AAR and infarction core. The T2 value in the myocardial infarction core of MI rats (39.9 3.6 ms) was significantly higher than that of AAR
Fig. 1. Images of muscle. An example of thigh muscles was scanned with T2W imaging (a), simplified T2 mapping (b), and MSME T2 mapping (c). There was no difference between T2 values of muscles acquired from simplified T2 mapping and the standard MSME T2 mapping (P > 0.05).
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Fig. 2. Images of phantom. A custom-made phantom was scanned with T2W imaging (a), simplified T2 mapping (b), and MSME T2 mapping (c). T2 value acquired from simplified T2 mapping was similar to that from the standard MSME T2 mapping.
Fig. 3. T2W images and T2 mapping image of normal rat heart. T2W images with echo time of 10 ms (a), 20 ms (b), and 30 ms (c) and T2 mapping image of the same slice of a normal rat heart (d).
(34.7 2.9 ms, P < 0.001) (Fig. 5), and both of them was significantly higher than normal rat (25.6 3.3 ms, both P < 0.001). No significant difference was found in the infarcted area defined by LGE and TTC staining for three MI rats (22.5 3% vs. 21.2 2%, n ¼ 3, P > 0.05).
Discussion For the first time, this study examined a simplified myocardial T2 mapping method on 7.0 T MRI for faster evaluation of myocardial edema in rats with high heart
rate. The muscle and phantom studies demonstrated a close agreement between the T2 measured by the simplified method and the standard T2 mapping method on 7.0 T MRI. It was found that myocardial T2 distribution was uniform in the whole heart of normal rats. The MI cores had a highest T2 value, followed by the T2 value of AAR in MI rats, and T2 values in normal rats. The much reduced data acquisition window allowed for 100% success rate for the imaging of myocardial injury area. The mean myocardial T2 value in normal rats was 25.6 3.3 ms that is shorter than T2 values of normal
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Fig. 4. T2 distributions in the whole heart. No difference (P > 0.05) was found among T2 values in different segments (a) and from base to apex (b).
Fig. 5. Images of a rat heart with myocardial infarction. Infarction area (dotted lined, block arrows) was showed on a late gadolinium enhancement (LGE) image (a) and 2,3,5-triphenyltetrazolium chloride (TTC) staining (b). Myocardial edema area (full line, arrows) was observed by an ordinary T2W image (c) and simplified T2 map (d). AAR, the difference between full line area and dotted line area, was showed by overlay a and d (e), and T2 value of the infarction area was bigger than that in AAR.
myocardium of 27 6.3 ms (15) and 31.5 0.7 ms on 4.7 T in previous studies (11). The differences may be due to different magnetic field and different MRI sequences. For the standard T2 mapping method (11), the 20 echoes were collected at different cardiac cycles (the longest TE was 60 ms), While all three echo signals were acquired in one cardiac cycle in simplified T2 mapping (the longest TE was 30 ms), significantly limiting the cardiac motion artifacts. There were no differences in T2 values among different segments and slices (from base to apex) in normal
rats. However, the findings of the current study do not support the previous research by Florian (7), in which T2 increased from base to apex. In another study by Ralf and et al., higher T2 values were observed in the anterior and anteroseptal segments (6). Because these two studies were implemented on humans, the difference in heart structure between human and rat may cause this difference in T2 distribution. In this study, the MI cores showed a higher T2 value than AAR in MI rats. Both these T2 values were higher than that in normal rats. This finding was in agreement
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with findings by Whalen and Jennings who suggested that substantial edema occurred in the acute infarcted region, with minimal edema occurring in the AAR portion with reversible injury (13,16–18). There are several limitations in this study. First, the accuracy of this simplified T2 measurement was not determined by the in vivo study, and myocardial edema was not validated by other reference method, e.g. microscopy. Second, because the longest TE was 30 ms in our simplified T2 mapping method, this approach may not accurately measure T2 values significantly larger than 30 ms. This is one of the reasons that increased noise of the simplified method was observed in the phantom and muscles experiments. Third, defining the area at risk by myocardial edema may overestimate the true area at risk caused by ischemia. Finally, one k-space line was acquired in each cardiac cycle in the simplified T2 mapping method, resulting in a relatively long acquisition time. In conclusion, we simplified a T2 mapping sequence and demonstrated its feasibility for the quantification of T2 distribution for faster assessment of myocardial edema and AAR. This technique may provide a very useful tool for faster evaluation of MI model in rats with high heart rate and monitor the efficacy of the treatment of MI in a small animal model.
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Conflict of interest None declared.
Funding This study was supported by The State Key Program of National Natural Science Foundation of China (81130027), The Twelfth Five Year Science and Technology support program (2012BAI23B08) and The National Key Basic Research Program of China (2011CB935800).
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