Capturing acute and chronic myocardial infarction by MRI rotating frame relaxation times in mice in and ex vivo

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Myocardial infarction is one of the most crucial cardiovascular diseases. Acute myocardial infarction is conventionally imaged with T 2 mapping due to its sensitivity related to the correlation times of edema and free-water molecules. Chronic myocardial infarction, which contains fibrosis and scar tissue, is conventionally imaged with MRI with T 1 weighting using contrast agents involved in late gadolinium enhancement and extracellular volume since contrast agent wash out from fibrosis and scar tissue is delayed compared to myocardium. So far, imaging acute myocardial infarcts is related to T 2 mapping, and imaging of scar tissue and fibrosis has been limited to techniques with contrast agent injection. Rotating frame relaxation times T 1ρ and T 2ρ mapping were developed to provide robust measurements with relatively wide B 1 and B 0 range for these quantities. Since rotating frame methods have different correlation times than T 2 and T 1 , these methods can be used to sensitively and specifically characterize both acute and chronic myocardial infarctions. In this study, acute (2 hours) and chronic (7 days after occlusion) myocardial infarcts in and ex vivo mouse models were imaged with rotating frame relaxation time mapping without the use of contrast agents. Methods In vivo imaging protocol contained adiabatic T 1ρ and adiabatic T 2ρ , both with two different HSn pulses, continuous wave T 1ρ and conventional T 2 , together with cine imaging. Mice were imaged 2 hours and 7 days after myocardial infarction. Mice were sacrificed at the 2-hour or at the 7-day time point. Ex vivo measurements contained adiabatic T 1ρ and adiabatic T 2ρ with two different HSn pulses, continuous wave T 1ρ , T 1 and T 2 . After MRI studies, mouse hearts were fixed, and myocardial infarcts were verified using dystrophin and hematoxylin and eosin histology stainings. Results A clear difference between infarcted and normal myocardium was visible at the 2-hour time point in rotating frame relaxation time mapping. Relative relaxation time difference in adiabatic T 2ρ with HS4 pulse might be sensitive to both acute edema reaction and chronic infarction. Also, in vivo and ex vivo results of adiabatic T 1ρ with both HSn pulses and continuous wave T 1ρ measurements showed relative relaxation time, the difference between infarcted and normal myocardium at 2 hours after the occlusion, and the difference increased at the 7-day time point. Conclusion This study showed that rotating frame relaxation time methods have the potential to be a non-invasive MR diagnostic marker for acute and chronic myocardial infarcts. Cardiovascular MRI Rotating frame relaxation times T1ρ T2ρ Myocardial infarction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Cardiovascular diseases (CVD) are the leading cause of death worldwide due to population growth and an aging population [ 1 ]. Myocardial infarction (MI) is one of the most crucial CVDs. MI occurs when the coronary artery is either partially or fully occluded, which is causing disturbance to the perfusion [ 2 ]. Occlusion can lead to a hibernating infarct or complete infarct when the myocardium lacks oxygen, leading to necrotic cell loss and finally to scar formation or, in the worst case, heart failure [ 3 ]. Cardiac magnetic resonance imaging (MRI) provides an accurate assessment of the anatomy and the function of the myocardium [ 4 ]. Functional cardiac MRI is usually done by gradient echo-based cine MRI, where volumetric parameters of the short-axis view of the left ventricle can be calculated [ 4 ]. Late gadolinium enhancement (LGE) is the golden standard to assess MI [ 4 ]. LGE images are acquired so that the area of MI has a brighter tone as compared to remote myocardium in the gray-scale image of myocardium [ 4 , 5 ]. This is due to the weak clearance of gadolinium (Gd) from the MI tissue [ 4 , 5 ]. Increased late Gd accumulation in the MI area has been associated with increased extracellular space [ 6 ]. LGE is a qualitative method to determine MI area; however, LGE has a few drawbacks, including that it is not specific, has challenges in diffuse fibrosis detection, and has limitations in its use for some patients [ 6 ]. The heart and the MI area can also be imaged with conventional endogenous MRI contrasts, such as T 1 - and T 2 -weighted MRI techniques. The T 1 relaxation time constant has been shown to increase in the MI compared to a remote area [ 4 ]. The T 2 relaxation time constant has been used to quantify myocardial edema caused by either inflammation or ischemic insult, and therefore, the T 2 relaxation time constant has been used to determine the edema in acute MI. While conventional T 1 - and T 2 -relaxations occur during free precession after the radio frequency (RF) excitation pulse, rotating frame relaxations occur during RF excitation [ 7 , 8 ]. Relaxation in the rotating frame occurs either along (T 1ρ ) or transversal (T 2ρ ) to the time-dependent effective magnetic field (B eff ) that acts as a spin-lock field. The spin-lock field is a vector sum of the RF pulse magnetic field component (B 1 ) perpendicular to the main magnetic field (B 0 ) and the off-resonance component along B 0 . The amplitudes of B eff and RF pulses are typically between 0.1 and 10 kHz, making rotating frame relaxations sensitive to slow molecular fluctuations close to the corresponding frequency ranges [ 6 , 9 ]. Proton chemical exchange between free water 1 H and 1 H of the exchanging groups of other molecules, usually macromolecules, is typically in the same frequency range [ 6 , 9 ]. T 1ρ rotating frame relaxation time constants can be performed with a continuous wave (CW) RF pulse or adiabatic RF pulse method. In the CW T 1ρ , a composite RF pulse consisting of a 90° hard [ 10 , 11 ] or an adiabatic half passage (AHP) [ 12 ] pulse followed by a CW pulse to lock the spins and then a 90° hard pulse or AHP to return the magnetization (M) back to the B 0 direction. After the composite weighting pulse, the signal can be acquired with a readout sequence. MI area has been determined with CW T 1ρ at multiple time points in mice [ 9 , 13 , 14 ], in swine [ 10 ], and in humans [ 15 ]. A common factor in these studies has been that a significant increase in the T 1ρ relaxation time constant in the MI area compared to remote myocardium has been found with a clear contrast between the MI and remote areas in the T 1ρ relaxation time maps [ 9 , 10 , 13 , 14 ]. Additionally, a good agreement between the MI area defined from CW T 1ρ relaxation time maps and LGE was found in mice [ 9 ], in swine [ 10 ], and in humans [ 15 ]. Another way to perform the T 1ρ rotating frame relaxation time experiments is to use a train of adiabatic RF pulses. The pulses in the train are typically adiabatic full passage (AFP) pulses from the hyperbolic secant (HS) pulse family (Fig. 1 ) [ 16 , 17 ]. Weighting can be tuned by altering the stretching factor n in adiabatic HSn pulses [ 16 , 17 ]. Typically, n is 1 or 4, leading to HS1 and HS4 pulses, respectively (Fig. 1 ) [ 16 , 17 , 18 , 19 ]. The difference between the HS1 and HS4 pulses is in the amplitude and frequency modulation functions [ 16 , 17 ]. HS4 delivers more RF power into tissue than HS1, causing a difference in magnetization decay during the pulse train [ 16 , 17 ]. When the adiabatic condition [ 18 ] is fulfilled, depending on the initial orientation of M according to time-dependent B eff and respect to the B 0 , M decays with T 1ρ , T 2ρ , or a combination of them [ 16 , 19 , 20 ]. However, the choice of parameters of the RF pulses, or choices of the amplitude- and frequency-modulation functions, is affecting the magnetization decay during the pulse train followed by differences in T 1ρ and T 2ρ relaxations [ 17 ]. T 1ρ adiabatic pulses were used to measure T 1ρ relaxation time constants in ex vivo and in vivo in different ischemic, including MI, and non-ischemic CVD patients at 1.5T [ 21 ]. It was found that T 1ρ relaxation time constants were increased in the MI area compared to the rest of the myocardium [ 21 ]. Adiabatic T 1ρ with HS1 pulses have been used to image healthy volunteers at 3T to gain information about the behavior of T 1ρ adiabatic pulses and to get to know the range of T 1ρ relaxation time constants with adiabatic pulses in the normal myocardium [ 22 ]. To the best of our knowledge, neither adiabatic T 1ρ nor T 2ρ methods with different lengths of HSn pulse trains have been used in the imaging of mouse MI. Additionally, very acute MI (2-hour time point after MI) is to our knowledge, rarely used in mouse studies. However, adiabatic T 1ρ and T 2ρ relaxation time contrasts have been used to determine the effect of orientation on the ex vivo heart with respect to the B 0 , and they found that adiabatic T 1ρ and T 2ρ relaxation time constants were not orientation-dependent with respect to the B 0 [ 23 ]. We applied T 1ρ and T 2ρ using adiabatic pulse trains for MI detection in a mouse model. Adiabatic pulses are robust to B 1 and B 0 variations, which makes them potential for T 1ρ and T 2ρ cardiac measurements without contrast agents. In this study, we characterized MI, remote, and intact myocardium by using adiabatic pulse trains at very acute (2 h) and chronic (7 days) time points. The measurements were performed in vivo and ex vivo, and the rotating frame relaxation time maps were visually compared to conventional relaxation time methods, cine-images, dystrophin immunostaining, and hematoxylin and eosin-stained histology sections. Results The MI was observed based on the immobile area in cine images in vivo. The slice for relaxation time measurements was selected based on the largest cross section of the infarct in the cine images. The area of elevated relaxation time constants in adiabatic T 1ρ relaxation time maps correspond to the akinetic area in cine images in vivo (Fig. 2 ). The adiabatic T 1ρ relaxation times measured with both HS1 and HS4 pulses (p < 0.05) and CW (p < 0.05) were significantly elevated at the MI area already at the 2-hour time point compared to controls (Table 1). At day 7 after MI, adiabatic T 1ρ relaxation times with both HS1 and HS4 pulses (p < 0.05) and CW (p < 0.05) were clearly elevated at the MI area compared to controls (Table 1). The RRTD values of the adiabatic T 1ρ relaxation times with both the HS1 and HS4 pulse was elevated after the MI compared to controls (p < 0.05) (Fig. 3 ). The RRTD values of the adiabatic T 2ρ relaxation times with the HS4 pulse was elevated but not significantly after the MI compared to controls (p = 0.06) (Fig. 3 ). However, RRTD values of the adiabatic T 2ρ relaxation times with the HS1 pulse was decreased after the MI from 2-hour to 7-day time point (p = 0.1) (Fig. 3 ). A non-significant difference in RRTD values was found with T 2 between time points (p = 0.17) (Fig. 3 ); however, a significant increase of T 2 relaxation time was found between controls and 7-day time point MI area (p < 0.05) (Table 1). A clear difference in relaxation times between MI and remote myocardium was found visually, with most of the relaxation times ex vivo. The relaxation time difference between MI and remote area after 2 hours of MI is visible with adiabatic T 1ρ with both HS1 and HS4 pulses and CW T 1ρ (Fig. 4 ). Additionally, the contrast is enhanced with adiabatic T 1ρ with both HS1 and HS4 pulses and CW T 1ρ from 2-hour to 7-day time point after MI (Fig. 5 ). The visual interpretation is supported by the RRTD values, which were increased from 2-hour time point to 7-day time point with adiabatic T 1ρ , with HS1 (p < 0.05) and HS4 (p < 0.05) pulses, and CW T 1ρ (p < 0.05) (Fig. 6 ). Adiabatic T 1ρ with HS1 (p < 0.05) and HS4 (p < 0.05), CW T 1ρ (p < 0.05), and T 1 (p < 0.05) relaxation time constants were significantly increased when comparing the intact hearts to MI area of 7-day time point (Table 2). Also, CW T 1ρ relaxation time constant was significantly increased already after 2-hour time point in the MI area as compared to intact hearts (Table 2). A significant RRTD increase was observed in adiabatic T 1ρ with HS1 pulse 7 days after MI compared to acute time point (p < 0.05) (Fig. 6 ). Table 3 shows the decrease of EF as a function of time, which indicates the stiffness of myocardium due to MI. The variation from the nominal value was found to be ± 11% for B 1 field homogeneity. Histological Verification At the 2-hour time point, HE staining was not able to visualize the MI area. Unlike the HE staining, the dystrophin immunohistostaining sensitively evaluates the immunohistochemical expression of dystrophin and shows the early stage of MI by the loss of sarcolemmal dystrophin staining and the focal loss of the fishnet pattern in the MI area [ 37 ]. In contrast to HE staining, the dystrophin immunohistostaining showed a clear MI area at both time points (Fig. 7 ). Additionally, the damage caused by hypoxia in the myocardium was indicated by the partial loss of sarcolemmal dystrophin staining and focal loss of the fishnet patterns in the left ventricle at both time points (Fig. 7 ). At 7-day time point, the HE staining was able to reveal interstitial edema with increased eosinophilia of cardiac myocytes in the MI area (Fig. 7 ). Differing from this, the dystrophin immunohistostaining showed a complete loss of sarcolemmal dystrophin staining, which indicates the fibrosis in the MI area (Fig. 7 ). By a visual comparison between the different relaxation time maps, cine-images, and both stainings, our findings were visually indicating that the increased relaxation time constants in the relaxation time maps and the abnormal left ventricle movement in the cine-images were seen in the same areas as the damaged tissue in histologically stained sections. Discussion Rotating frame relaxation time constants were characterized in the mouse heart in vivo and ex vivo after 2 hours and 7 days of myocardial infarct. Adiabatic T 1ρ with both HS1 and HS4 pulses and adiabatic T 2ρ with HS4 showed high contrast differences between the infarct and remote areas after 2 hours and 7 days of MI in vivo. Ex vivo measurements of adiabatic T 1ρ with both HS1 and HS4 pulses, CW T 1ρ and T 1 showed the highest RRTDs compared to T 2 , and adiabatic T 2ρ , measurements. The contrast difference between MI and remote areas was already increased 2 hours after the MI with adiabatic T 1ρ with both HS1 and HS4 pulses and CW T 1ρ and continued to increase 7 days after MI in vivo. The increase was expected since T 1ρ relaxation time is known to be sensitive to the formation of granulation tissue, which has been previously found in mouse MI studies [ 9 , 13 , 14 , 28 ]. Seven days after MI was considered a chronic phase of MI, although the time point is still fairly early for scar tissue formation. It has been shown that MI consists of 90% necrotic tissue after 2 days of MI, transforming into granulation tissue and finally into scar tissue after 14 days of permanent occlusion in a mouse model [ 25 ]. At this point, when the presence of scar tissue is more sufficient, the T 1ρ relaxation time constant has been found to be elevated [ 9 , 13 , 14 , 28 ]. Additionally, T 1ρ relaxation is selectively sensitive to low-frequency macromolecular interactions and slow molecular motions (long correlation times), while conventional T 1 relaxation is selectively sensitive to Larmor frequency and T 2 is non-selective for low-frequency motions [ 26 ]. These differences may explain the higher RRTD values in both adiabatic and CW T 1ρ relaxation time methods compared to T 1 and T 2 . The significant increase in T 1ρ after 7 days compared to the non-significant increase after 3 days of LAD occlusion [ 13 ] indicated that the increased T 1ρ relaxation time constant might first react to the edema and inflammation, but it reacts more to later MI development phases after the acute phase of MI since T 1ρ is speculated to be affected by collagen, the interstitial water content, and the exchange of protons between hydroxyl and amide groups [ 9 , 13 , 14 , 27 , 28 ]. An interesting finding is that the RRTD of adiabatic T 2ρ with HS4 pulse is increasing as a function of time after MI and has the highest RRTD value 7 days after the MI. This might be explained by the sensitivity to the increased amount of free water caused by edema and the formation of granulation tissue. Additionally, there is an enormous difference between HS1 and HS4 pulses in T 2ρ RRTD values, as seen in Fig. 3 , which indicates that T 2ρ with HS4 pulse is able to create more contrast between MI and remote areas in both time points compared to T 2ρ with HS1 pulse. Furthermore, MI was barely visible in T 2ρ relaxation time mapping with either pulses ex vivo. These might be explained by the differences in blood contribution and the increased amount of free water in the living myocardium, with other physiological differences in the myocardium after the MI. Additionally, tissue fixation decreases the relaxation time values ex vivo compared to in vivo. The difference between HS1 and HS4 can be explained by the difference in stretching factor affecting on amplitude and frequency modulation functions and furthermore magnetization trajectories and the signal decay during the pulse train. HS1 and HS4 T 1ρ relaxation time constants increase simultaneously but less intensively than adiabatic T 2ρ with HS4 pulse. Ex vivo measurements of CW T 1ρ and adiabatic T 1ρ with both HS1 and HS4 pulse wave forms showed the highest RRTDs with a significant RRTD increase from the 2-hour time point to 7 days after the MI. Significant increase in relaxation time constants support the findings of the in vivo measurements. The findings of in vivo and ex vivo adiabatic T 1ρ relaxation time constants are similar to previous CW T 1ρ findings [ 9 , 13 , 14 , 21 ], indicating that T 1ρ is more sensitive to scar formation than to the acute edema reaction, whereas our finding of elevated adiabatic T 2ρ with HS4 pulse in acute phase indicates T 2ρ sensitivity to acute biological tissue changes after MI. The previous studies showed a shorter T 1 relaxation time ex vivo than in vivo at 9.4 T [ 29 , 30 ]. T 1 relaxation is related to protein-water interactions, and tissue fixation leads to protein crosslinking that reduces tissue T 1 relaxation time [ 31 ]. The T 1 relaxation time is increasing in the area of MI, suggesting that the area of MI consists of protein-water interactions. However, several factors can affect the T 1 relaxation time, including the concentration of fixative PFA, the storage solution of 15% sucrose, and magnetic field strength [ 32 , 33 , 34 ]. These factors may also affect the rotating frame relaxation times ex vivo. However, these variables were kept as constant as possible for all the ex vivo measurements of this study. MI areas were verified with two histological methods: HE staining and dystrophin immunostaining. The HE staining method was initially performed to verify MI at both time points (2 hours and 7 days after MI); however, it showed differences between MI and remote tissues only after 7 days of MI. HE staining was found to be an inefficient method for differentiating MI and remote myocardium 2 hours after LAD occlusion. In acute myocardial ischemia, where acute edema reaction is dominant, the reaction is characterized by the swelling of cells and damage to the sarcolemma and membrane-associated proteins; however, to our knowledge, HE is not a specific histological method to determine acute edema reaction. Dystrophin immunohistochemical staining showed clear differences between the remote and MI tissue after 2 hours and after 7 days of infarction. This finding is relevant, since dystrophin is an efficient staining method for differentiating the MI area from remote tissue at the early stages of infarction due to the loss of sarcolemmal dystrophin staining in infarcted areas [ 35 , 36 , 37 , 38 ]. Cardiomyocytes contain a group of proteins called dystrophin-associated proteins, which, with dystrophin, form a complex that is involved in stabilizing the plasma membrane [ 37 ]. Additionally, dystrophin links the laminin receptor and dystrophin complex to the intracellular cytoskeletal talin and actin, and in the case of MI, sub-sarcolemma bleb formation is followed by the loss of dystrophin from the membrane [ 37 ]. Importantly, here visually, both stainings - dystrophin staining more sensitively compared to HE staining - detected the same areas in the myocardium as the increased relaxation time constants in the maps and the abnormal left ventricle movement in the cine-images. However, the exact matching and comparison of a 4 µm histological section to a 1 mm thick MRI slice is difficult, and to our knowledge, there is no perfect method to do the comparison between cardiac MRI and histological sections. Thus, this limitation needs to be considered when comparing the cardiac MRI and histological sections. This preclinical mouse study had some limitations. First, the number of mice could be larger to gain more statistical power for both in vivo and ex vivo measurements. Additionally, some motion artefacts due to failure or partial failure of the ECG and respiratory triggering, might have caused some deviation from the results by affecting the quality of the weighted images. Secondly, due to timing difficulties and the tail vein injections, it was not possible to include LGE imaging in the imaging protocol. Additionally, our in vivo T1 and HS1 pulse measurements were not measured due to technical difficulties and too long anesthesia time for mice. Thirdly, the exact matching between the histological section and relaxation time constant maps is challenging; however, the visual correlation between MRI and histology gives qualitative verification. Fourthly, the 7-day time point after MI has been used as a chronic phase of mouse MI, however, that time period might not be enough for the formation of scar tissue, and therefore, a later time point after the MI is needed for detecting the actual scar formation. The study would benefit of sham-operated mice to find the effect of surgical operation on the relaxation times. Despite these limitations of the study, all the results are promising. Rotating frame relaxation time methods can determine MI area in the very acute phase (T 2ρ ) and chronic phase of MI (T 1ρ ) in an in vivo mouse model, which indicates the potential of these endogenous contrasts for infarct visualization without contrast agents. However, further studies are needed to confirm the results of this study. As a conclusion, this in vivo MI study of mouse heart shows that HS4 adiabatic T 2ρ relaxation time mapping determines both very acute, 2-hour, edema reaction and MI after 7 days of LAD occlusion. Additionally, adiabatic T 1ρ with HS4 pulse can be mapped in the mouse model, and the contrast differences were increased after 7 days of LAD occlusion compared to the 2-hour time point. The characterization of CW T 1ρ and adiabatic T 1ρ with both HS1 and HS4 pulses relaxation time mapping provides possibilities for follow-up studies of acute MI and might provide potential diagnostic markers for tissue damage in MI. Additionally, dystrophin-based immunohistochemical staining showed clear differences between very acute infarct and normal tissue. Methods Fourteen male mice (C57BL/6J, obtained from the Animal Center of the University of Eastern Finland) weighing 20–30 g were divided into two groups: the first group with 10 left anterior descending (LAD) artery occluded mice and the second group with 4 control mice with intact hearts. In vivo measurements were successfully performed in 11 mice (7 mice for the 2-hour time point, 5 mice with also the 7-day time point, and 4 controls). Three mice could not tolerate the imaging anesthesia at 2 hours after the LAD operation. Two out of these three mice had MI at a 2-hour time point and were included in the ex vivo measurements. Additionally, 2 mice were sacrificed at the 2-hour time point after in vivo MRI for ex vivo measurements. Therefore, the ex vivo measurements were performed in all 14 hearts. All animal experiments were performed according to the national and international guidelines for laboratory animal use and under license ESAVI-270-04.10.07-2017 approved by the National Animal Experiment Board of Finland. All procedures performed in this study were in accordance with the ethical standards of the National Animal Experiment Board in Finland. All the experiments conformed to the Animal Research Reporting In Vivo Experiments (ARRIVE) guidelines. LAD Occlusion A novel surgical procedure was performed to occlude the LAD artery [ 9 ]. Briefly, inhalation anesthesia was induced with 4% isoflurane and maintained with 1.5-2% isoflurane in mixture of oxygen and nitrogen, 30%/70% respectively. The chest was opened at the 4th intercostal space of the sternum in the surgical procedure. The heart was partly pulled out and the pericardial sac was removed to perform the LAD occlusion. The ligation was performed, the heart was returned to its original position and the chest was closed between the 3rd and 4th ribs. Analgesia was given on the operation day and the following two days: buprenorphine (0.3 mg/ml) (Temgesic 0.05–0.1 mg/kg) and carprofen (5 mg/ml) (Rimadyl 5 mg/kg) for analgesia subcutaneously. Animal handling in MRI Mice were anesthetized and kept under inhalation anesthesia during the MRI similarly as described for maintenance during the LAD occlusion operation. A pad with circulating warm water maintained the body temperature of the animal close to 37°C. Mice were placed in a prone position with the heart positioned at the magnet's isocenter. Electrocardiography (ECG) was monitored using needle electrodes from forepaws and respiration with a pneumatic pillow using a small animal gating device (Small Animal Instruments Inc., NY, USA). Cardiac and respiratory motion artifacts were minimized by double triggering with ECG and respiration. MRI MRI data were acquired with a horizontal 9.4 T Varian/Agilent DirectDrive (Agilent Inc., Palo Alto, CA, USA) system with 31 cm bore size, equipped with a Varian/Agilent DirectDrive console (Agilent Inc., Palo Alto, CA, USA) and with gradient set bore diameter of 12 cm with maximum gradient amplitude of 600 m/Tm. A volume quadrature RF transceiver coil of 35 mm diameter (Rapid Biomed GmbH, Rimpar, Germany) was used for the measurements. MR cine images were acquired by ECG triggered gradient echo sequence (repetition time (TR) = 10 ms and echo time (TE) = 1.3 ms, 10–15 frames per heart cycle, Field-of-view = 30 x 30 mm 2 , matrix size = 256 x 256). Cine images were used to select the short axis slice close to apex for in vivo relaxation time measurements. CW T 1ρ [ 23 ], adiabatic T 1ρ [ 16 , 24 ], T 2 , and adiabatic T 2ρ [ 19 ] were used in this study. For in vivo relaxation time measurements, a TurboFLASH gradient echo sequence (TR between the excitations = 3.1 ms, TE = 1.6 ms, flip angle = 25°, data matrix of 128 × 128 with 1 mm slice thickness and averages = 1) was used as a readout sequence. A delay of at least 2 seconds was applied after each acquisition, followed by respiration triggering. The scanning times for T 1ρ , T 2 , and T 2ρ measurements were 4 minutes each. B 1 field homogeneity was measured to check the data quality. B 1 measurements were also performed using the matrix size = 128 × 64, using a hard pulse with a nominal power of 625 Hz with durations of 0, 0.125, 0.25, 0.375, 0.5, 0.625, 0.75, 0.875, and 1.0 ms. The scan time for B 1 measurements was 1 minute. An illustrative figure of the differences between CW T 1ρ , adiabatic T 1ρ , and adiabatic T 2ρ is shown in Fig. 1 . For CW T 1ρ measurements, an AHP (duration = 4000 µs and RF power = 1250 Hz) was used to flip M to the xy-plane, followed by a CW spin-lock pulse with durations of 0, 18, 36, and 54 ms and RF pulse power = 625 Hz to create a spin-lock field, and an AHP to return the magnetization to the z-axis before signal readout. The phases of the AHPs and CW pulse formed a continuous function in the pulse train (Fig. 1 ). In the adiabatic T 1ρ and adiabatic T 2ρ measurements, the number of HS pulses (durations of 4525 µs, RF power 625 Hz) was 0, 8, 16, 24, and 32 in the pulse train (Fig. 1 ). In T 2ρ , an AHP pulse (duration = 4000 µs, RF power = 2500 Hz) to tip the magnetization to the xy-plane was placed before, and a similar but time reversed AHP pulse after the AFP pulse train (Fig. 1 ). In both the HS1 and HS4 methods, the spin-lock field was 625 Hz. All the ex vivo measurements were done at a vertical 9.4 T Varian/Agilent DirectDrive (Agilent Inc., Palo Alto, CA, USA) using a volume RF transceiver coil of diameter 10 mm (Rapid Biomedical GmbH, Rimpar, Germany) and a VnmrJ3.1 Varian/Agilent DirectDrive console. The hearts were placed in an 8-mm NMR glass tube and filled with perfluoropolyether (Galden HS 240, Solvay Solexis, Italy), which is a fluorinated heat transfer fluid that gives no 1 H signal in MRI. Ex vivo imaging protocols included T 1 , adiabatic T 1ρ (with both HS1 and HS4 pulses), adiabatic T 2ρ (with both HS1 and HS4 pulses), CW T 1ρ , and T 2 methods. Adiabatic T 1ρ , adiabatic T 2ρ , CW T 1ρ , and T 2 ex vivo measurements were done using the same parameters as in vivo measurements. T 1 measurements were conducted using inversion recovery with inversion times of 0.01, 0.25, 0.5, 1, and 2 seconds. For all the ex vivo measurements, a fast spin echo readout sequence (TR = 2000 ms, effective TE = 9.42 ms, averages = 4 with data matrix = 192 × 128, FOV = 10 mm x 10 mm, resolution 52 µm x 78 µm, echo train length = 4, and slice thickness = 0.70 mm) was used. Histology After the last imaging time point, the mice were sacrificed with CO2, and the hearts were collected for histopathology. Eleven animals (10 occluded and 1 control) were sacrificed after MR measurements for histology. The excised hearts were rinsed with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde (PFA) in 7.5% sucrose for 4 hours. PFA was replaced with 15% sucrose (overnight – 2 weeks). Hearts were dehydrated with increasing concentrations of ethanol (50% − 100%) and xylene, then embedded in paraffin. Sections of 4 µm were prepared from the paraffin blocks. The tissue sections were deparaffinized with xylene and rehydrated with alcohol (100% − 50%) followed by standard hematoxylin and eosin (HE) staining. In addition, for dystrophin immunostaining, sections were rinsed in Triton X-100 for 10 minutes after rehydration. The sections were then placed in a 0.01 M citrate buffer solution, and pretreatment was performed in a microwave oven for 10 minutes. The sections were treated with a peroxidase block for 10 minutes, followed by a protein block for 60 minutes. The sections were incubated for 1 hour with dystrophin antibody (Rabbit Polyclonal Antibody, Thermo Scientific, USA) at room temperature and washed with PBS for 5 minutes. Incubation of sections occurred with a biotin-labeled secondary antibody (Anti-Rabbit IgG Biotinylated Antibody, Vectastain Vector Laboratories) for 30 minutes, followed by washing in PBS for 5 minutes. Then, the sections were incubated with streptavidin peroxidase complex (ABC Kit, Vectastain, Vector Laboratories) for 30 minutes, followed by washing in PBS for 5 minutes. DAB chromogen (Liquid DAB Substrate Kit, Invitrogen) was added to all the sections for 2–3 minutes, followed by washing in water. Counter-staining was performed with Harris hematoxylin for 14 seconds. The sections were dehydrated in alcohol (50% − 100%) and in xylene. The mounting was performed with a permount mounting medium. Images were taken using a light microscope (Olympus AX70). Data Analysis Pixel-by-pixel analysis was performed to reconstruct the maps from the signal intensities using Aedes ( http://aedes.uef.fi/ ) in MATLAB (Mathworks Inc., Natick, CA, USA). Regions of interest (ROIs) were manually drawn in infarcted and remote myocardium to calculate the relative relaxation time difference (RRTD). RRTD was calculated to compare the relaxation times in two different ROIs in that relaxation time map. The infarct area was selected visually based on histology and cine images. The area opposite the infarct site, i.e., ventricular septum, was selected as a remote myocardium. RRTDs in LAD occluded mice were calculated as (T(Infarct) - T(Remote)) / T(Remote), where T is adiabatic T 1ρ , adiabatic T 2ρ , CW T 1ρ , T 2 or T 1 relaxation time averaged over infarct or remote area for both in vivo and ex vivo measurements. RRTDs in control mice in vivo were calculated as (T(Whole myocardium) - T(Chest muscles)) / T(Chest muscles). Results are given as the mean ± standard deviation. For the calculation of ejection fraction (EF), we defined end systolic and end diastolic volumes in the left ventricle. These volumes were drawn based on the endocardial border of the left ventricle in cine images. The statistical analysis was performed by one-way ANOVA, t-test, and Bonferroni's multiple comparison. A corrected significance level of p < 0.05 was considered significant. Abbreviations B eff Effective radio frequency field CMR Cardiovascular magnetic resonance CW Continuous wave HS Hyperbolic secant LGE Late gadolinium enhancement LAD Left anterior descending (artery) MI Myocardial infarction M Magnetization PBS Phosphate buffered saline PFA Paraformaldehyde RRTD Relative relaxation time difference ROI Region of interest T 1ρ Longitudinal rotating frame relaxation time T RAFF Relaxation along the fictitious field Declarations Research involving Human Participants and/or Animals: All animal experiments were performed according to the national and international guidelines for laboratory animal use and under license ESAVI-270-04.10.07-2017 approved by the National Animal Experiment Board of Finland. All procedures performed in this study were in accordance with the ethical standards of the National Animal Experiment Board in Finland (Eläinkoelautakunta, ELLA). Data availability: The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request. Disclosure of potential conflicts of interest: The authors declare that they have no conflicts of interests. Funding: The authors thank the following funding agency for providing funding to be used as a personal grant: Sigrid Juselius Foundation, Finnish Foundation for Cardiovascular Research, Finnish Cultural Foundation, Emil Aaltonen Foundation, State Research Funding and GeneCellNano Flagship Program. Authors' contributions: EYH, HL and MAK did programming, magnetic resonance imaging, analyzed the data and wrote the manuscript. SL and TH contributed to surgery, histology and tissue processing. TL and SYH contributed to the design, data analysis, writing and editing of the manuscript. All the authors have read and approved the manuscript. Acknowledgements The authors thank laboratory and MR maintenance staff members Maarit Pulkkinen and Jari Nissinen for help and support during the measurements. This work/Part of the work was carried out with the support of Kuopio Biomedical Imaging Unit, University of Eastern Finland, Kuopio, Finland (part of Biocenter Kuopio, Finnish Biomedical Imaging Node, and EuroBioImaging). References Virani SS et al. Circulation. 2021;143(8):e254–743. Thygesen K, Alpert JS, Jaffe AS, Simoons ML, Chaitman BR, White HD, Writing Group on the Joint ESC/ACCF/AHA/WHF Task Force for the Universal Definition of Myocardial Infarction & ESC Committee for Practice Guidelines (CPG) et al. Third Universal definition of myocardial infarction. Eur Heart J. 2012 Oct;33(20):2551-2567 Falk E, Shah PK, Fuster V (1995) Coronary plaque disruption. Circulation 92(3):657-71. https://doi.org/10.1161/01.CIR.92.3.657. Raijah PS et al. Cardaic MRI: State of the Art. 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Magn Reson Med 77(2):774-778. https://doi.org/10.1002/mrm.26140. Korb JP, Bryant RG (2002) Magnetic field dependence of proton spin‐lattice relaxation times. Magn Reson Med 48(1):21-6. https://doi.org/10.1002/mrm.10185. Rooney WD, Johnson G, Li X, Cohen ER, Kim SG, Ugurbil K, Springer CS Jr (2007) Magnetic field and tissue dependencies of human brain longitudinal 1H2O relaxation in vivo. Magn Reson Med 57(2):308-18. https://doi.org/10.1002/mrm.21122. Rinck PA, Fischer HW, Vander Elst L, Van Haverbeke Y, Muller RN (1988) Field‐cycling relaxometry: medical applications. Radiology 168(3):843-9. https://doi.org/10.1148/radiology.168.3.3406414. Thomsen H, Held H (1995) Immunohistochemical Detection of C5b-9 (m) in Myocardium: An Aid in Distinguishing Infarction-induced Ischemic Heart Muscle Necrosis From Other Forms of Lethal Myocardial Injury. Forensic Sci Int 71(2):87-95. https://doi.org/10.1016/0379-0738(94)01640-Q. Ouyang J, Guzman M, Desoto-Lapaix F, Pincus MR, Wieczorek R (2009) Utility of Desmin and a Masson's Trichrome Method to Detect Early Acute Myocardial Infarction in Autopsy Tissues. Int J Clin Exp Pathol 3(1):98-105. Hashmi S, Al-Salam S (2013) Loss of Dystrophin Staining in Cardiomyocytes: A Novel Method for Detection Early Myocardial Infarction. Int J Clin Exp Pathol 6(2):249-57. Tables Tables are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Tab1eyhnew1.tif Table 1: Relaxation times from infarcted and remote areas of MI group (n=7 in the 2-hour time point and n=5 in the 7-day time point) and control group (n=4) in vivo. The statistical analysis was performed by t-test and Bonferroni's multiple comparison correction between control and other areas (* = P<0.05). Tab2eyhnew1.tif Table 2: MR relaxation times from infarcted area, remote areas (n=5 in the 2-hour time point and n=5 in the 7-day time point) and control hearts (n=4) ex vivo. The statistical analysis was performed by t-test and Bonferroni's multiple comparison correction between control and other areas (* = P<0.05). Tab3eyhnew.tif Table 3: Functional parameters of the hearts of intact control group, 2 hours and 7 days after myocardial infarct. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4774734","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":342168937,"identity":"8d79ceab-fcc4-4ebf-9283-9ba3b34aea82","order_by":0,"name":"Elias Ylä-Herttuala","email":"data:image/png;base64,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","orcid":"","institution":"University of Eastern Finland","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Elias","middleName":"","lastName":"Ylä-Herttuala","suffix":""},{"id":342168938,"identity":"9848a336-430e-4046-a50f-2fa4e92c9f76","order_by":1,"name":"Muhammad Arsalan Khan","email":"","orcid":"","institution":"University of Eastern Finland","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"Arsalan","lastName":"Khan","suffix":""},{"id":342168939,"identity":"a5822e07-d04f-454c-87a8-24052f392dc0","order_by":2,"name":"Svetlana Laidinen","email":"","orcid":"","institution":"University of Eastern Finland","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Svetlana","middleName":"","lastName":"Laidinen","suffix":""},{"id":342168940,"identity":"607abd72-e2c0-4262-a1e0-09ce5132d595","order_by":3,"name":"Tommi Heikura","email":"","orcid":"","institution":"University of Eastern Finland","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tommi","middleName":"","lastName":"Heikura","suffix":""},{"id":342168941,"identity":"839055c5-e975-47b4-91a1-3c6a81ceca95","order_by":4,"name":"Seppo Ylä-Herttuala","email":"","orcid":"","institution":"University of Eastern Finland","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Seppo","middleName":"","lastName":"Ylä-Herttuala","suffix":""},{"id":342168942,"identity":"3c2a64d2-70c7-4279-861b-dfed362254d6","order_by":5,"name":"Timo Liimatainen","email":"","orcid":"","institution":"University of Oulu","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Timo","middleName":"","lastName":"Liimatainen","suffix":""},{"id":342168943,"identity":"16bfafcc-9079-4047-8651-1fa3b9d27f09","order_by":6,"name":"Hanne Laakso","email":"","orcid":"","institution":"University of Eastern Finland","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hanne","middleName":"","lastName":"Laakso","suffix":""}],"badges":[],"createdAt":"2024-07-20 23:08:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4774734/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4774734/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":63804521,"identity":"c05537ea-6770-4eec-a6dd-d923a95e76d2","added_by":"auto","created_at":"2024-09-02 13:30:20","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":561169,"visible":true,"origin":"","legend":"\u003cp\u003eA schematic view of rotating frame magnetization preparations applied between the ECG trigger and readout sequence. A) continuous wave (CW) \u003cem\u003eT\u003c/em\u003e\u003csub\u003e1ρ\u003c/sub\u003e, B) adiabatic \u003cem\u003eT\u003c/em\u003e\u003csub\u003e1ρ\u003c/sub\u003e, and C) adiabatic \u003cem\u003eT\u003c/em\u003e\u003csub\u003e2ρ\u003c/sub\u003e. In adiabatic \u003cem\u003eT\u003c/em\u003e\u003csub\u003e1ρ\u003c/sub\u003e and \u003cem\u003eT\u003c/em\u003e\u003csub\u003e2ρ\u003c/sub\u003e pulse sequences, a train of adiabatic full-passage RF pulses is used. The amplitude and frequency modulations of HS1 and HS4 RF pulses are shown in an inset [19]. AHP: adiabatic half passage, AFP: adiabatic full passage, TSL: time-spin lock.\u003c/p\u003e","description":"","filename":"Figuusi1pulses.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4774734/v1/4ac20ccbb318494d7c54d5f2.jpg"},{"id":63804524,"identity":"02dd396b-8b83-4b7e-9bcb-77c1491a5511","added_by":"auto","created_at":"2024-09-02 13:30:21","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3998232,"visible":true,"origin":"","legend":"\u003cp\u003eIn vivo rotating frame relaxation time CW T\u003csub\u003e1ρ\u003c/sub\u003e, adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e with HS4 pulse, adiabatic T\u003csub\u003e2ρ\u003c/sub\u003e with HS4 pulse) maps. (A-D) At the 2-hour time point (E-H) at the 7-day time point including (D) cine MRI in diastole after 2 hours and (H) after 7 days after the MI. Longer relaxation times were observed after 7 days after the MI than 2-hour time point at the infarct area. White arrows indicate the site of myocardial infarction.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig2eyhCineBignew.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4774734/v1/f15d926ce3361b7fb3f3e056.jpg"},{"id":63807987,"identity":"bd42ff2f-d941-43f3-b424-b77a0779a39b","added_by":"auto","created_at":"2024-09-02 13:46:21","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":407472,"visible":true,"origin":"","legend":"\u003cp\u003eIn vivo RRTD values (Mean ± SD). The values are calculated from adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e, CW T\u003csub\u003e1ρ\u003c/sub\u003e, T\u003csub\u003e2\u003c/sub\u003e and adiabatic T\u003csub\u003e2ρ\u003c/sub\u003e relaxation time maps in vivo (controls, n=4, LAD occluded mice for the 2-hour time point n=7 and for the 7-day time point n=5). The data is shown as mean ± standard deviation. (*P\u0026lt;0.05, one-way ANOVA, Bonferroni's multiple comparison tests, t-tests).\u003c/p\u003e","description":"","filename":"Fig3eyhnew.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4774734/v1/7d874fb32b40ff1ba7dfc4ac.jpg"},{"id":63804527,"identity":"29e585cc-0331-45f7-9fe2-7195080eb1bb","added_by":"auto","created_at":"2024-09-02 13:30:21","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3794707,"visible":true,"origin":"","legend":"\u003cp\u003eEx vivo relaxation time maps after 2 hours of LAD occlusion of (A) adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e with HS1 pulse, (B) adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e with HS4 pulse, (C) continuous wave T\u003csub\u003e1ρ\u003c/sub\u003e, (D) adiabatic T\u003csub\u003e2ρ\u003c/sub\u003e with HS1 pulse, (E) adiabatic T\u003csub\u003e2ρ\u003c/sub\u003e with HS4 pulse, (F) T2 and (F) T1. Outline of selected ROI of infarct is presented with black line in (C) and it was used in all relaxation time maps. Longer relaxation times were observed also in the outlined infarct area than in normal myocardium.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig4eyh.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4774734/v1/e136f178e5d944d870a9b6cd.jpg"},{"id":63804528,"identity":"92ed1e7f-640b-4391-bf5b-7f12f649b394","added_by":"auto","created_at":"2024-09-02 13:30:21","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3054761,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative ex vivo relaxation maps after 7 days of LAD of (A) adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e with HS1 pulse, (B) adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e with HS4 pulse, (C) continuous wave T\u003csub\u003e1ρ\u003c/sub\u003e, (D) adiabatic T\u003csub\u003e2ρ\u003c/sub\u003e with HS1 pulse, (E) adiabatic T\u003csub\u003e2ρ\u003c/sub\u003e with HS4 pulse, (F) T2 and (F) T1. Outline of selected ROI of infarct is presented with black line in (C) and it was used in all relaxation time maps. Relaxation time maps showed longer relaxation times in an outlined infarct site than in normal myocardium.\u003c/p\u003e","description":"","filename":"Fig5eyh.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4774734/v1/9f584305136bd122bd91cfcd.jpg"},{"id":63804526,"identity":"299a5fd0-f44c-4ea8-b7bd-57af0985d72c","added_by":"auto","created_at":"2024-09-02 13:30:21","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":56006,"visible":true,"origin":"","legend":"\u003cp\u003eEx vivo RRTD values (Mean ± SD). The values are calculated from T\u003csub\u003e1\u003c/sub\u003e, T\u003csub\u003e2\u003c/sub\u003e, T\u003csub\u003e1ρ\u003c/sub\u003e, T\u003csub\u003e1ρ HS1\u003c/sub\u003e, T\u003csub\u003e1ρ HS4\u003c/sub\u003e, T\u003csub\u003e2ρ HS1\u003c/sub\u003e, and T\u003csub\u003e2ρ HS4\u003c/sub\u003e relaxation time maps ex vivo (n=10). The data is shown as mean ± standard deviation. (*P\u0026lt;0.05, one-way ANOVA, Bonferroni's multiple comparison tests, t-tests).\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig6eyh.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4774734/v1/cd58b2eed9c05219151f025b.jpg"},{"id":63804531,"identity":"b83613df-e246-47e5-a831-a6f13d982c64","added_by":"auto","created_at":"2024-09-02 13:30:21","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":24629047,"visible":true,"origin":"","legend":"\u003cp\u003eHistology images of myocardium at both time points taken by light microscope (A) hematoxylin – eosin stain after 2 hours (4x magnification), (B) dystrophin immunostaining after 2 hours (4x magnification), (C) dystrophin immunostaining after 2 hours (10x magnification), (D) hematoxylin – eosin stain after 7 days (4x magnification), (E) dystrophin immunostaining after 7 days (4x magnification) and (F) dystrophin immunostaining after 7 days (10x magnification), where the black arrow is pointing the infarction area. Histology images (G-I) are from intact heart with dystrophin immunostaining so that (G) is 1.25x magnification, (H) is 4x magnification and (I) is 10x magnification.\u003c/p\u003e","description":"","filename":"Fig7eyhnew1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4774734/v1/63f1e5ffd3ec991a3e685879.jpg"},{"id":74220415,"identity":"e2228ba4-66d7-4a57-8c64-94a6c1f10230","added_by":"auto","created_at":"2025-01-20 06:32:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":37202307,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4774734/v1/31ef0f97-b8fb-42ce-b8af-bb86b312356a.pdf"},{"id":63804519,"identity":"b29ba4fe-3300-4bdf-8823-76911fc57d6e","added_by":"auto","created_at":"2024-09-02 13:30:20","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":43340,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1: \u003c/strong\u003eRelaxation times from infarcted and remote areas of MI group (n=7 in the 2-hour time point and n=5 in the 7-day time point) and control group (n=4) in vivo.\u003cstrong\u003e \u003c/strong\u003eThe statistical analysis was performed by t-test and Bonferroni's multiple comparison correction between control and other areas (* = P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Tab1eyhnew1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4774734/v1/ded7ae99ddd99a68c81a1dc0.tif"},{"id":63806203,"identity":"abaf9a3d-52eb-454e-900c-a2ddc8e41ef5","added_by":"auto","created_at":"2024-09-02 13:38:21","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":94118,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 2: \u003c/strong\u003eMR relaxation times from infarcted area, remote areas (n=5 in the 2-hour time point and n=5 in the 7-day time point) and control hearts (n=4) ex vivo. The statistical analysis was performed by t-test and Bonferroni's multiple comparison correction between control and other areas (* = P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Tab2eyhnew1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4774734/v1/82ad4cc92279c0d7c0cbfa8f.tif"},{"id":63806205,"identity":"ff04a7b0-67a6-4bf2-a8fe-a6eb83607c34","added_by":"auto","created_at":"2024-09-02 13:38:21","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":13970,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 3: \u003c/strong\u003eFunctional parameters of the hearts of intact control group, 2 hours and 7 days after myocardial infarct.\u003c/p\u003e","description":"","filename":"Tab3eyhnew.tif","url":"https://assets-eu.researchsquare.com/files/rs-4774734/v1/960cec7ca967b29a09f99713.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Capturing acute and chronic myocardial infarction by MRI rotating frame relaxation times in mice in and ex vivo","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCardiovascular diseases (CVD) are the leading cause of death worldwide due to population growth and an aging population [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Myocardial infarction (MI) is one of the most crucial CVDs. MI occurs when the coronary artery is either partially or fully occluded, which is causing disturbance to the perfusion [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Occlusion can lead to a hibernating infarct or complete infarct when the myocardium lacks oxygen, leading to necrotic cell loss and finally to scar formation or, in the worst case, heart failure [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCardiac magnetic resonance imaging (MRI) provides an accurate assessment of the anatomy and the function of the myocardium [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Functional cardiac MRI is usually done by gradient echo-based cine MRI, where volumetric parameters of the short-axis view of the left ventricle can be calculated [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Late gadolinium enhancement (LGE) is the golden standard to assess MI [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. LGE images are acquired so that the area of MI has a brighter tone as compared to remote myocardium in the gray-scale image of myocardium [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This is due to the weak clearance of gadolinium (Gd) from the MI tissue [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Increased late Gd accumulation in the MI area has been associated with increased extracellular space [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. LGE is a qualitative method to determine MI area; however, LGE has a few drawbacks, including that it is not specific, has challenges in diffuse fibrosis detection, and has limitations in its use for some patients [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe heart and the MI area can also be imaged with conventional endogenous MRI contrasts, such as T\u003csub\u003e1\u003c/sub\u003e- and T\u003csub\u003e2\u003c/sub\u003e-weighted MRI techniques. The T\u003csub\u003e1\u003c/sub\u003e relaxation time constant has been shown to increase in the MI compared to a remote area [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The T\u003csub\u003e2\u003c/sub\u003e relaxation time constant has been used to quantify myocardial edema caused by either inflammation or ischemic insult, and therefore, the T\u003csub\u003e2\u003c/sub\u003e relaxation time constant has been used to determine the edema in acute MI. While conventional T\u003csub\u003e1\u003c/sub\u003e- and T\u003csub\u003e2\u003c/sub\u003e-relaxations occur during free precession after the radio frequency (RF) excitation pulse, rotating frame relaxations occur during RF excitation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Relaxation in the rotating frame occurs either along (T\u003csub\u003e1ρ\u003c/sub\u003e) or transversal (T\u003csub\u003e2ρ\u003c/sub\u003e) to the time-dependent effective magnetic field (B\u003csub\u003eeff\u003c/sub\u003e) that acts as a spin-lock field. The spin-lock field is a vector sum of the RF pulse magnetic field component (B\u003csub\u003e1\u003c/sub\u003e) perpendicular to the main magnetic field (B\u003csub\u003e0\u003c/sub\u003e) and the off-resonance component along B\u003csub\u003e0\u003c/sub\u003e. The amplitudes of B\u003csub\u003eeff\u003c/sub\u003e and RF pulses are typically between 0.1 and 10 kHz, making rotating frame relaxations sensitive to slow molecular fluctuations close to the corresponding frequency ranges [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Proton chemical exchange between free water \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e1\u003c/sup\u003eH of the exchanging groups of other molecules, usually macromolecules, is typically in the same frequency range [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eT\u003csub\u003e1ρ\u003c/sub\u003e rotating frame relaxation time constants can be performed with a continuous wave (CW) RF pulse or adiabatic RF pulse method. In the CW T\u003csub\u003e1ρ\u003c/sub\u003e, a composite RF pulse consisting of a 90\u0026deg; hard [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] or an adiabatic half passage (AHP) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] pulse followed by a CW pulse to lock the spins and then a 90\u0026deg; hard pulse or AHP to return the magnetization (M) back to the B\u003csub\u003e0\u003c/sub\u003e direction. After the composite weighting pulse, the signal can be acquired with a readout sequence. MI area has been determined with CW T\u003csub\u003e1ρ\u003c/sub\u003e at multiple time points in mice [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], in swine [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and in humans [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. A common factor in these studies has been that a significant increase in the T\u003csub\u003e1ρ\u003c/sub\u003e relaxation time constant in the MI area compared to remote myocardium has been found with a clear contrast between the MI and remote areas in the T\u003csub\u003e1ρ\u003c/sub\u003e relaxation time maps [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Additionally, a good agreement between the MI area defined from CW T\u003csub\u003e1ρ\u003c/sub\u003e relaxation time maps and LGE was found in mice [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], in swine [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and in humans [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnother way to perform the T\u003csub\u003e1ρ\u003c/sub\u003e rotating frame relaxation time experiments is to use a train of adiabatic RF pulses. The pulses in the train are typically adiabatic full passage (AFP) pulses from the hyperbolic secant (HS) pulse family (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Weighting can be tuned by altering the stretching factor n in adiabatic HSn pulses [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Typically, n is 1 or 4, leading to HS1 and HS4 pulses, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The difference between the HS1 and HS4 pulses is in the amplitude and frequency modulation functions [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. HS4 delivers more RF power into tissue than HS1, causing a difference in magnetization decay during the pulse train [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. When the adiabatic condition [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] is fulfilled, depending on the initial orientation of M according to time-dependent B\u003csub\u003eeff\u003c/sub\u003e and respect to the B\u003csub\u003e0\u003c/sub\u003e, M decays with T\u003csub\u003e1ρ\u003c/sub\u003e, T\u003csub\u003e2ρ\u003c/sub\u003e, or a combination of them [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, the choice of parameters of the RF pulses, or choices of the amplitude- and frequency-modulation functions, is affecting the magnetization decay during the pulse train followed by differences in T\u003csub\u003e1ρ\u003c/sub\u003e and T\u003csub\u003e2ρ\u003c/sub\u003e relaxations [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eT\u003csub\u003e1ρ\u003c/sub\u003e adiabatic pulses were used to measure T\u003csub\u003e1ρ\u003c/sub\u003e relaxation time constants in ex vivo and in vivo in different ischemic, including MI, and non-ischemic CVD patients at 1.5T [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. It was found that T\u003csub\u003e1ρ\u003c/sub\u003e relaxation time constants were increased in the MI area compared to the rest of the myocardium [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e with HS1 pulses have been used to image healthy volunteers at 3T to gain information about the behavior of T\u003csub\u003e1ρ\u003c/sub\u003e adiabatic pulses and to get to know the range of T\u003csub\u003e1ρ\u003c/sub\u003e relaxation time constants with adiabatic pulses in the normal myocardium [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. To the best of our knowledge, neither adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e nor T\u003csub\u003e2ρ\u003c/sub\u003e methods with different lengths of HSn pulse trains have been used in the imaging of mouse MI. Additionally, very acute MI (2-hour time point after MI) is to our knowledge, rarely used in mouse studies. However, adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e and T\u003csub\u003e2ρ\u003c/sub\u003e relaxation time contrasts have been used to determine the effect of orientation on the ex vivo heart with respect to the B\u003csub\u003e0\u003c/sub\u003e, and they found that adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e and T\u003csub\u003e2ρ\u003c/sub\u003e relaxation time constants were not orientation-dependent with respect to the B\u003csub\u003e0\u003c/sub\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe applied T\u003csub\u003e1ρ\u003c/sub\u003e and T\u003csub\u003e2ρ\u003c/sub\u003e using adiabatic pulse trains for MI detection in a mouse model. Adiabatic pulses are robust to B\u003csub\u003e1\u003c/sub\u003e and B\u003csub\u003e0\u003c/sub\u003e variations, which makes them potential for T\u003csub\u003e1ρ\u003c/sub\u003e and T\u003csub\u003e2ρ\u003c/sub\u003e cardiac measurements without contrast agents. In this study, we characterized MI, remote, and intact myocardium by using adiabatic pulse trains at very acute (2 h) and chronic (7 days) time points. The measurements were performed in vivo and ex vivo, and the rotating frame relaxation time maps were visually compared to conventional relaxation time methods, cine-images, dystrophin immunostaining, and hematoxylin and eosin-stained \u003cspan refid=\"Sec9\" class=\"InternalRef\"\u003ehistology\u003c/span\u003e sections.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe MI was observed based on the immobile area in cine images in vivo. The slice for relaxation time measurements was selected based on the largest cross section of the infarct in the cine images. The area of elevated relaxation time constants in adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e relaxation time maps correspond to the akinetic area in cine images in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e relaxation times measured with both HS1 and HS4 pulses (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and CW (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were significantly elevated at the MI area already at the 2-hour time point compared to controls (Table\u0026nbsp;1). At day 7 after MI, adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e relaxation times with both HS1 and HS4 pulses (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and CW (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were clearly elevated at the MI area compared to controls (Table\u0026nbsp;1). The RRTD values of the adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e relaxation times with both the HS1 and HS4 pulse was elevated after the MI compared to controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The RRTD values of the adiabatic T\u003csub\u003e2ρ\u003c/sub\u003e relaxation times with the HS4 pulse was elevated but not significantly after the MI compared to controls (p\u0026thinsp;=\u0026thinsp;0.06) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). However, RRTD values of the adiabatic T\u003csub\u003e2ρ\u003c/sub\u003e relaxation times with the HS1 pulse was decreased after the MI from 2-hour to 7-day time point (p\u0026thinsp;=\u0026thinsp;0.1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). A non-significant difference in RRTD values was found with T\u003csub\u003e2\u003c/sub\u003e between time points (p\u0026thinsp;=\u0026thinsp;0.17) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e); however, a significant increase of T\u003csub\u003e2\u003c/sub\u003e relaxation time was found between controls and 7-day time point MI area (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA clear difference in relaxation times between MI and remote myocardium was found visually, with most of the relaxation times ex vivo. The relaxation time difference between MI and remote area after 2 hours of MI is visible with adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e with both HS1 and HS4 pulses and CW T\u003csub\u003e1ρ\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Additionally, the contrast is enhanced with adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e with both HS1 and HS4 pulses and CW T\u003csub\u003e1ρ\u003c/sub\u003e from 2-hour to 7-day time point after MI (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The visual interpretation is supported by the RRTD values, which were increased from 2-hour time point to 7-day time point with adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e, with HS1 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and HS4 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) pulses, and CW T\u003csub\u003e1ρ\u003c/sub\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e with HS1 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and HS4 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), CW T\u003csub\u003e1ρ\u003c/sub\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and T\u003csub\u003e1\u003c/sub\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) relaxation time constants were significantly increased when comparing the intact hearts to MI area of 7-day time point (Table\u0026nbsp;2). Also, CW T\u003csub\u003e1ρ\u003c/sub\u003e relaxation time constant was significantly increased already after 2-hour time point in the MI area as compared to intact hearts (Table\u0026nbsp;2). A significant RRTD increase was observed in adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e with HS1 pulse 7 days after MI compared to acute time point (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Table\u0026nbsp;3 shows the decrease of EF as a function of time, which indicates the stiffness of myocardium due to MI. The variation from the nominal value was found to be \u0026plusmn;\u0026thinsp;11% for B\u003csub\u003e1\u003c/sub\u003e field homogeneity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eHistological Verification\u003c/h3\u003e\n\u003cp\u003eAt the 2-hour time point, HE staining was not able to visualize the MI area. Unlike the HE staining, the dystrophin immunohistostaining sensitively evaluates the immunohistochemical expression of dystrophin and shows the early stage of MI by the loss of sarcolemmal dystrophin staining and the focal loss of the fishnet pattern in the MI area [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In contrast to HE staining, the dystrophin immunohistostaining showed a clear MI area at both time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Additionally, the damage caused by hypoxia in the myocardium was indicated by the partial loss of sarcolemmal dystrophin staining and focal loss of the fishnet patterns in the left ventricle at both time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). At 7-day time point, the HE staining was able to reveal interstitial edema with increased eosinophilia of cardiac myocytes in the MI area (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Differing from this, the dystrophin immunohistostaining showed a complete loss of sarcolemmal dystrophin staining, which indicates the fibrosis in the MI area (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). By a visual comparison between the different relaxation time maps, cine-images, and both stainings, our findings were visually indicating that the increased relaxation time constants in the relaxation time maps and the abnormal left ventricle movement in the cine-images were seen in the same areas as the damaged tissue in histologically stained sections.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eRotating frame relaxation time constants were characterized in the mouse heart in vivo and ex vivo after 2 hours and 7 days of myocardial infarct. Adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e with both HS1 and HS4 pulses and adiabatic T\u003csub\u003e2ρ\u003c/sub\u003e with HS4 showed high contrast differences between the infarct and remote areas after 2 hours and 7 days of MI in vivo. Ex vivo measurements of adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e with both HS1 and HS4 pulses, CW T\u003csub\u003e1ρ\u003c/sub\u003e and T\u003csub\u003e1\u003c/sub\u003e showed the highest RRTDs compared to T\u003csub\u003e2\u003c/sub\u003e, and adiabatic T\u003csub\u003e2ρ\u003c/sub\u003e, measurements.\u003c/p\u003e \u003cp\u003eThe contrast difference between MI and remote areas was already increased 2 hours after the MI with adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e with both HS1 and HS4 pulses and CW T\u003csub\u003e1ρ\u003c/sub\u003e and continued to increase 7 days after MI in vivo. The increase was expected since T\u003csub\u003e1ρ\u003c/sub\u003e relaxation time is known to be sensitive to the formation of granulation tissue, which has been previously found in mouse MI studies [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Seven days after MI was considered a chronic phase of MI, although the time point is still fairly early for scar tissue formation. It has been shown that MI consists of 90% necrotic tissue after 2 days of MI, transforming into granulation tissue and finally into scar tissue after 14 days of permanent occlusion in a mouse model [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. At this point, when the presence of scar tissue is more sufficient, the T\u003csub\u003e1ρ\u003c/sub\u003e relaxation time constant has been found to be elevated [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Additionally, T\u003csub\u003e1ρ\u003c/sub\u003e relaxation is selectively sensitive to low-frequency macromolecular interactions and slow molecular motions (long correlation times), while conventional T\u003csub\u003e1\u003c/sub\u003e relaxation is selectively sensitive to Larmor frequency and T\u003csub\u003e2\u003c/sub\u003e is non-selective for low-frequency motions [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. These differences may explain the higher RRTD values in both adiabatic and CW T\u003csub\u003e1ρ\u003c/sub\u003e relaxation time methods compared to T\u003csub\u003e1\u003c/sub\u003e and T\u003csub\u003e2\u003c/sub\u003e. The significant increase in T\u003csub\u003e1ρ\u003c/sub\u003e after 7 days compared to the non-significant increase after 3 days of LAD occlusion [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] indicated that the increased T\u003csub\u003e1ρ\u003c/sub\u003e relaxation time constant might first react to the edema and inflammation, but it reacts more to later MI development phases after the acute phase of MI since T\u003csub\u003e1ρ\u003c/sub\u003e is speculated to be affected by collagen, the interstitial water content, and the exchange of protons between hydroxyl and amide groups [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAn interesting finding is that the RRTD of adiabatic T\u003csub\u003e2ρ\u003c/sub\u003e with HS4 pulse is increasing as a function of time after MI and has the highest RRTD value 7 days after the MI. This might be explained by the sensitivity to the increased amount of free water caused by edema and the formation of granulation tissue. Additionally, there is an enormous difference between HS1 and HS4 pulses in T\u003csub\u003e2ρ\u003c/sub\u003e RRTD values, as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, which indicates that T\u003csub\u003e2ρ\u003c/sub\u003e with HS4 pulse is able to create more contrast between MI and remote areas in both time points compared to T\u003csub\u003e2ρ\u003c/sub\u003e with HS1 pulse. Furthermore, MI was barely visible in T\u003csub\u003e2ρ\u003c/sub\u003e relaxation time mapping with either pulses ex vivo. These might be explained by the differences in blood contribution and the increased amount of free water in the living myocardium, with other physiological differences in the myocardium after the MI. Additionally, tissue fixation decreases the relaxation time values ex vivo compared to in vivo. The difference between HS1 and HS4 can be explained by the difference in stretching factor affecting on amplitude and frequency modulation functions and furthermore magnetization trajectories and the signal decay during the pulse train. HS1 and HS4 T\u003csub\u003e1ρ\u003c/sub\u003e relaxation time constants increase simultaneously but less intensively than adiabatic T\u003csub\u003e2ρ\u003c/sub\u003e with HS4 pulse.\u003c/p\u003e \u003cp\u003eEx vivo measurements of CW T\u003csub\u003e1ρ\u003c/sub\u003e and adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e with both HS1 and HS4 pulse wave forms showed the highest RRTDs with a significant RRTD increase from the 2-hour time point to 7 days after the MI. Significant increase in relaxation time constants support the findings of the in vivo measurements. The findings of in vivo and ex vivo adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e relaxation time constants are similar to previous CW T\u003csub\u003e1ρ\u003c/sub\u003e findings [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], indicating that T\u003csub\u003e1ρ\u003c/sub\u003e is more sensitive to scar formation than to the acute edema reaction, whereas our finding of elevated adiabatic T\u003csub\u003e2ρ\u003c/sub\u003e with HS4 pulse in acute phase indicates T\u003csub\u003e2ρ\u003c/sub\u003e sensitivity to acute biological tissue changes after MI.\u003c/p\u003e \u003cp\u003eThe previous studies showed a shorter T\u003csub\u003e1\u003c/sub\u003e relaxation time ex vivo than in vivo at 9.4 T [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. T\u003csub\u003e1\u003c/sub\u003e relaxation is related to protein-water interactions, and tissue fixation leads to protein crosslinking that reduces tissue T\u003csub\u003e1\u003c/sub\u003e relaxation time [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The T\u003csub\u003e1\u003c/sub\u003e relaxation time is increasing in the area of MI, suggesting that the area of MI consists of protein-water interactions. However, several factors can affect the T\u003csub\u003e1\u003c/sub\u003e relaxation time, including the concentration of fixative PFA, the storage solution of 15% sucrose, and magnetic field strength [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. These factors may also affect the rotating frame relaxation times ex vivo. However, these variables were kept as constant as possible for all the ex vivo measurements of this study.\u003c/p\u003e \u003cp\u003eMI areas were verified with two histological methods: HE staining and dystrophin immunostaining. The HE staining method was initially performed to verify MI at both time points (2 hours and 7 days after MI); however, it showed differences between MI and remote tissues only after 7 days of MI. HE staining was found to be an inefficient method for differentiating MI and remote myocardium 2 hours after LAD occlusion. In acute myocardial ischemia, where acute edema reaction is dominant, the reaction is characterized by the swelling of cells and damage to the sarcolemma and membrane-associated proteins; however, to our knowledge, HE is not a specific histological method to determine acute edema reaction.\u003c/p\u003e \u003cp\u003eDystrophin immunohistochemical staining showed clear differences between the remote and MI tissue after 2 hours and after 7 days of infarction. This finding is relevant, since dystrophin is an efficient staining method for differentiating the MI area from remote tissue at the early stages of infarction due to the loss of sarcolemmal dystrophin staining in infarcted areas [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Cardiomyocytes contain a group of proteins called dystrophin-associated proteins, which, with dystrophin, form a complex that is involved in stabilizing the plasma membrane [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Additionally, dystrophin links the laminin receptor and dystrophin complex to the intracellular cytoskeletal talin and actin, and in the case of MI, sub-sarcolemma bleb formation is followed by the loss of dystrophin from the membrane [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Importantly, here visually, both stainings - dystrophin staining more sensitively compared to HE staining - detected the same areas in the myocardium as the increased relaxation time constants in the maps and the abnormal left ventricle movement in the cine-images. However, the exact matching and comparison of a 4 \u0026micro;m histological section to a 1 mm thick MRI slice is difficult, and to our knowledge, there is no perfect method to do the comparison between cardiac MRI and histological sections. Thus, this limitation needs to be considered when comparing the cardiac MRI and histological sections.\u003c/p\u003e \u003cp\u003eThis preclinical mouse study had some limitations. First, the number of mice could be larger to gain more statistical power for both in vivo and ex vivo measurements. Additionally, some motion artefacts due to failure or partial failure of the ECG and respiratory triggering, might have caused some deviation from the results by affecting the quality of the weighted images. Secondly, due to timing difficulties and the tail vein injections, it was not possible to include LGE imaging in the imaging protocol. Additionally, our in vivo T1 and HS1 pulse measurements were not measured due to technical difficulties and too long anesthesia time for mice. Thirdly, the exact matching between the histological section and relaxation time constant maps is challenging; however, the visual correlation between MRI and histology gives qualitative verification. Fourthly, the 7-day time point after MI has been used as a chronic phase of mouse MI, however, that time period might not be enough for the formation of scar tissue, and therefore, a later time point after the MI is needed for detecting the actual scar formation. The study would benefit of sham-operated mice to find the effect of surgical operation on the relaxation times. Despite these limitations of the study, all the results are promising. Rotating frame relaxation time methods can determine MI area in the very acute phase (T\u003csub\u003e2ρ\u003c/sub\u003e) and chronic phase of MI (T\u003csub\u003e1ρ\u003c/sub\u003e) in an in vivo mouse model, which indicates the potential of these endogenous contrasts for infarct visualization without contrast agents. However, further studies are needed to confirm the results of this study.\u003c/p\u003e \u003cp\u003eAs a conclusion, this in vivo MI study of mouse heart shows that HS4 adiabatic T\u003csub\u003e2ρ\u003c/sub\u003e relaxation time mapping determines both very acute, 2-hour, edema reaction and MI after 7 days of LAD occlusion. Additionally, adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e with HS4 pulse can be mapped in the mouse model, and the contrast differences were increased after 7 days of LAD occlusion compared to the 2-hour time point. The characterization of CW T\u003csub\u003e1ρ\u003c/sub\u003e and adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e with both HS1 and HS4 pulses relaxation time mapping provides possibilities for follow-up studies of acute MI and might provide potential diagnostic markers for tissue damage in MI. Additionally, dystrophin-based immunohistochemical staining showed clear differences between very acute infarct and normal tissue.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eFourteen male mice (C57BL/6J, obtained from the Animal Center of the University of Eastern Finland) weighing 20\u0026ndash;30 g were divided into two groups: the first group with 10 left anterior descending (LAD) artery occluded mice and the second group with 4 control mice with intact hearts. In vivo measurements were successfully performed in 11 mice (7 mice for the 2-hour time point, 5 mice with also the 7-day time point, and 4 controls). Three mice could not tolerate the imaging anesthesia at 2 hours after the LAD operation. Two out of these three mice had MI at a 2-hour time point and were included in the ex vivo measurements. Additionally, 2 mice were sacrificed at the 2-hour time point after in vivo MRI for ex vivo measurements. Therefore, the ex vivo measurements were performed in all 14 hearts. All animal experiments were performed according to the national and international guidelines for laboratory animal use and under license ESAVI-270-04.10.07-2017 approved by the National Animal Experiment Board of Finland. All procedures performed in this study were in accordance with the ethical standards of the National Animal Experiment Board in Finland. All the experiments conformed to the Animal Research Reporting In Vivo Experiments (ARRIVE) guidelines.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eLAD Occlusion\u003c/h2\u003e \u003cp\u003eA novel surgical procedure was performed to occlude the LAD artery [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Briefly, inhalation anesthesia was induced with 4% isoflurane and maintained with 1.5-2% isoflurane in mixture of oxygen and nitrogen, 30%/70% respectively. The chest was opened at the 4th intercostal space of the sternum in the surgical procedure. The heart was partly pulled out and the pericardial sac was removed to perform the LAD occlusion. The ligation was performed, the heart was returned to its original position and the chest was closed between the 3rd and 4th ribs. Analgesia was given on the operation day and the following two days: buprenorphine (0.3 mg/ml) (Temgesic 0.05\u0026ndash;0.1 mg/kg) and carprofen (5 mg/ml) (Rimadyl 5 mg/kg) for analgesia subcutaneously.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAnimal handling in MRI\u003c/h2\u003e \u003cp\u003eMice were anesthetized and kept under inhalation anesthesia during the MRI similarly as described for maintenance during the LAD occlusion operation. A pad with circulating warm water maintained the body temperature of the animal close to 37\u0026deg;C. Mice were placed in a prone position with the heart positioned at the magnet's isocenter. Electrocardiography (ECG) was monitored using needle electrodes from forepaws and respiration with a pneumatic pillow using a small animal gating device (Small Animal Instruments Inc., NY, USA). Cardiac and respiratory motion artifacts were minimized by double triggering with ECG and respiration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMRI\u003c/h2\u003e \u003cp\u003eMRI data were acquired with a horizontal 9.4 T Varian/Agilent DirectDrive (Agilent Inc., Palo Alto, CA, USA) system with 31 cm bore size, equipped with a Varian/Agilent DirectDrive console (Agilent Inc., Palo Alto, CA, USA) and with gradient set bore diameter of 12 cm with maximum gradient amplitude of 600 m/Tm. A volume quadrature RF transceiver coil of 35 mm diameter (Rapid Biomed GmbH, Rimpar, Germany) was used for the measurements. MR cine images were acquired by ECG triggered gradient echo sequence (repetition time (TR)\u0026thinsp;=\u0026thinsp;10 ms and echo time (TE)\u0026thinsp;=\u0026thinsp;1.3 ms, 10\u0026ndash;15 frames per heart cycle, Field-of-view\u0026thinsp;=\u0026thinsp;30 x 30 mm\u003csup\u003e2\u003c/sup\u003e, matrix size\u0026thinsp;=\u0026thinsp;256 x 256). Cine images were used to select the short axis slice close to apex for in vivo relaxation time measurements.\u003c/p\u003e \u003cp\u003eCW T\u003csub\u003e1ρ\u003c/sub\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], T\u003csub\u003e2\u003c/sub\u003e, and adiabatic T\u003csub\u003e2ρ\u003c/sub\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] were used in this study. For in vivo relaxation time measurements, a TurboFLASH gradient echo sequence (TR between the excitations\u0026thinsp;=\u0026thinsp;3.1 ms, TE\u0026thinsp;=\u0026thinsp;1.6 ms, flip angle\u0026thinsp;=\u0026thinsp;25\u0026deg;, data matrix of 128 \u0026times; 128 with 1 mm slice thickness and averages\u0026thinsp;=\u0026thinsp;1) was used as a readout sequence. A delay of at least 2 seconds was applied after each acquisition, followed by respiration triggering. The scanning times for T\u003csub\u003e1ρ\u003c/sub\u003e, T\u003csub\u003e2\u003c/sub\u003e, and T\u003csub\u003e2ρ\u003c/sub\u003e measurements were 4 minutes each. B\u003csub\u003e1\u003c/sub\u003e field homogeneity was measured to check the data quality. B\u003csub\u003e1\u003c/sub\u003e measurements were also performed using the matrix size\u0026thinsp;=\u0026thinsp;128 \u0026times; 64, using a hard pulse with a nominal power of 625 Hz with durations of 0, 0.125, 0.25, 0.375, 0.5, 0.625, 0.75, 0.875, and 1.0 ms. The scan time for B\u003csub\u003e1\u003c/sub\u003e measurements was 1 minute.\u003c/p\u003e \u003cp\u003eAn illustrative figure of the differences between CW T\u003csub\u003e1ρ\u003c/sub\u003e, adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e, and adiabatic T\u003csub\u003e2ρ\u003c/sub\u003e is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. For CW T\u003csub\u003e1ρ\u003c/sub\u003e measurements, an AHP (duration\u0026thinsp;=\u0026thinsp;4000 \u0026micro;s and RF power\u0026thinsp;=\u0026thinsp;1250 Hz) was used to flip M to the xy-plane, followed by a CW spin-lock pulse with durations of 0, 18, 36, and 54 ms and RF pulse power\u0026thinsp;=\u0026thinsp;625 Hz to create a spin-lock field, and an AHP to return the magnetization to the z-axis before signal readout. The phases of the AHPs and CW pulse formed a continuous function in the pulse train (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e and adiabatic T\u003csub\u003e2ρ\u003c/sub\u003e measurements, the number of HS pulses (durations of 4525 \u0026micro;s, RF power 625 Hz) was 0, 8, 16, 24, and 32 in the pulse train (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In T\u003csub\u003e2ρ\u003c/sub\u003e, an AHP pulse (duration\u0026thinsp;=\u0026thinsp;4000 \u0026micro;s, RF power\u0026thinsp;=\u0026thinsp;2500 Hz) to tip the magnetization to the xy-plane was placed before, and a similar but time reversed AHP pulse after the AFP pulse train (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In both the HS1 and HS4 methods, the spin-lock field was 625 Hz.\u003c/p\u003e \u003cp\u003eAll the ex vivo measurements were done at a vertical 9.4 T Varian/Agilent DirectDrive (Agilent Inc., Palo Alto, CA, USA) using a volume RF transceiver coil of diameter 10 mm (Rapid Biomedical GmbH, Rimpar, Germany) and a VnmrJ3.1 Varian/Agilent DirectDrive console. The hearts were placed in an 8-mm NMR glass tube and filled with perfluoropolyether (Galden HS 240, Solvay Solexis, Italy), which is a fluorinated heat transfer fluid that gives no \u003csup\u003e1\u003c/sup\u003eH signal in MRI. Ex vivo imaging protocols included T\u003csub\u003e1\u003c/sub\u003e, adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e (with both HS1 and HS4 pulses), adiabatic T\u003csub\u003e2ρ\u003c/sub\u003e (with both HS1 and HS4 pulses), CW T\u003csub\u003e1ρ\u003c/sub\u003e, and T\u003csub\u003e2\u003c/sub\u003e methods. Adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e, adiabatic T\u003csub\u003e2ρ\u003c/sub\u003e, CW T\u003csub\u003e1ρ\u003c/sub\u003e, and T\u003csub\u003e2\u003c/sub\u003e ex vivo measurements were done using the same parameters as in vivo measurements. T\u003csub\u003e1\u003c/sub\u003e measurements were conducted using inversion recovery with inversion times of 0.01, 0.25, 0.5, 1, and 2 seconds.\u003c/p\u003e \u003cp\u003eFor all the ex vivo measurements, a fast spin echo readout sequence (TR\u0026thinsp;=\u0026thinsp;2000 ms, effective TE\u0026thinsp;=\u0026thinsp;9.42 ms, averages\u0026thinsp;=\u0026thinsp;4 with data matrix\u0026thinsp;=\u0026thinsp;192 \u0026times; 128, FOV\u0026thinsp;=\u0026thinsp;10 mm x 10 mm, resolution 52 \u0026micro;m x 78 \u0026micro;m, echo train length\u0026thinsp;=\u0026thinsp;4, and slice thickness\u0026thinsp;=\u0026thinsp;0.70 mm) was used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eHistology\u003c/h2\u003e \u003cp\u003eAfter the last imaging time point, the mice were sacrificed with CO2, and the hearts were collected for histopathology. Eleven animals (10 occluded and 1 control) were sacrificed after MR measurements for histology. The excised hearts were rinsed with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde (PFA) in 7.5% sucrose for 4 hours. PFA was replaced with 15% sucrose (overnight \u0026ndash; 2 weeks). Hearts were dehydrated with increasing concentrations of ethanol (50% \u0026minus;\u0026thinsp;100%) and xylene, then embedded in paraffin. Sections of 4 \u0026micro;m were prepared from the paraffin blocks.\u003c/p\u003e \u003cp\u003eThe tissue sections were deparaffinized with xylene and rehydrated with alcohol (100% \u0026minus;\u0026thinsp;50%) followed by standard hematoxylin and eosin (HE) staining. In addition, for dystrophin immunostaining, sections were rinsed in Triton X-100 for 10 minutes after rehydration. The sections were then placed in a 0.01 M citrate buffer solution, and pretreatment was performed in a microwave oven for 10 minutes. The sections were treated with a peroxidase block for 10 minutes, followed by a protein block for 60 minutes. The sections were incubated for 1 hour with dystrophin antibody (Rabbit Polyclonal Antibody, Thermo Scientific, USA) at room temperature and washed with PBS for 5 minutes. Incubation of sections occurred with a biotin-labeled secondary antibody (Anti-Rabbit IgG Biotinylated Antibody, Vectastain Vector Laboratories) for 30 minutes, followed by washing in PBS for 5 minutes. Then, the sections were incubated with streptavidin peroxidase complex (ABC Kit, Vectastain, Vector Laboratories) for 30 minutes, followed by washing in PBS for 5 minutes. DAB chromogen (Liquid DAB Substrate Kit, Invitrogen) was added to all the sections for 2\u0026ndash;3 minutes, followed by washing in water. Counter-staining was performed with Harris hematoxylin for 14 seconds. The sections were dehydrated in alcohol (50% \u0026minus;\u0026thinsp;100%) and in xylene. The mounting was performed with a permount mounting medium. Images were taken using a light microscope (Olympus AX70).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eData Analysis\u003c/h2\u003e \u003cp\u003ePixel-by-pixel analysis was performed to reconstruct the maps from the signal intensities using Aedes (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://aedes.uef.fi/\u003c/span\u003e\u003cspan address=\"http://aedes.uef.fi/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) in MATLAB (Mathworks Inc., Natick, CA, USA). Regions of interest (ROIs) were manually drawn in infarcted and remote myocardium to calculate the relative relaxation time difference (RRTD). RRTD was calculated to compare the relaxation times in two different ROIs in that relaxation time map. The infarct area was selected visually based on histology and cine images. The area opposite the infarct site, i.e., ventricular septum, was selected as a remote myocardium. RRTDs in LAD occluded mice were calculated as (T(Infarct) - T(Remote)) / T(Remote), where T is adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e, adiabatic T\u003csub\u003e2ρ\u003c/sub\u003e, CW T\u003csub\u003e1ρ\u003c/sub\u003e, T\u003csub\u003e2\u003c/sub\u003e or T\u003csub\u003e1\u003c/sub\u003e relaxation time averaged over infarct or remote area for both in vivo and ex vivo measurements. RRTDs in control mice in vivo were calculated as (T(Whole myocardium) - T(Chest muscles)) / T(Chest muscles). Results are given as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. For the calculation of ejection fraction (EF), we defined end systolic and end diastolic volumes in the left ventricle. These volumes were drawn based on the endocardial border of the left ventricle in cine images. The statistical analysis was performed by one-way ANOVA, t-test, and Bonferroni's multiple comparison. A corrected significance level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.502590673575128%\" valign=\"top\"\u003e\n \u003cp\u003eB\u003csub\u003eeff\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"78.49740932642487%\" valign=\"top\"\u003e\n \u003cp\u003eEffective radio frequency field\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.502590673575128%\" valign=\"top\"\u003e\n \u003cp\u003eCMR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"78.49740932642487%\" valign=\"top\"\u003e\n \u003cp\u003eCardiovascular magnetic resonance\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.502590673575128%\" valign=\"top\"\u003e\n \u003cp\u003eCW\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"78.49740932642487%\" valign=\"top\"\u003e\n \u003cp\u003eContinuous wave\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.502590673575128%\" valign=\"top\"\u003e\n \u003cp\u003eHS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"78.49740932642487%\" valign=\"top\"\u003e\n \u003cp\u003eHyperbolic secant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.502590673575128%\" valign=\"top\"\u003e\n \u003cp\u003eLGE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"78.49740932642487%\" valign=\"top\"\u003e\n \u003cp\u003eLate gadolinium enhancement\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.502590673575128%\" valign=\"top\"\u003e\n \u003cp\u003eLAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"78.49740932642487%\" valign=\"top\"\u003e\n \u003cp\u003eLeft anterior descending (artery)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.502590673575128%\" valign=\"top\"\u003e\n \u003cp\u003eMI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"78.49740932642487%\" valign=\"top\"\u003e\n \u003cp\u003eMyocardial infarction\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.502590673575128%\" valign=\"top\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"78.49740932642487%\" valign=\"top\"\u003e\n \u003cp\u003eMagnetization\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.502590673575128%\" valign=\"top\"\u003e\n \u003cp\u003ePBS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"78.49740932642487%\" valign=\"top\"\u003e\n \u003cp\u003ePhosphate buffered saline\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.502590673575128%\" valign=\"top\"\u003e\n \u003cp\u003ePFA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"78.49740932642487%\" valign=\"top\"\u003e\n \u003cp\u003eParaformaldehyde\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.502590673575128%\" valign=\"top\"\u003e\n \u003cp\u003eRRTD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"78.49740932642487%\" valign=\"top\"\u003e\n \u003cp\u003eRelative relaxation time difference\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.502590673575128%\" valign=\"top\"\u003e\n \u003cp\u003eROI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"78.49740932642487%\" valign=\"top\"\u003e\n \u003cp\u003eRegion of interest\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.502590673575128%\" valign=\"top\"\u003e\n \u003cp\u003eT\u003csub\u003e1\u0026rho;\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"78.49740932642487%\" valign=\"top\"\u003e\n \u003cp\u003eLongitudinal rotating frame relaxation time\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.502590673575128%\" valign=\"top\"\u003e\n \u003cp\u003eT\u003csub\u003eRAFF\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"78.49740932642487%\" valign=\"top\"\u003e\n \u003cp\u003eRelaxation along the fictitious field\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cu\u003eResearch involving Human Participants and/or Animals:\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were performed according to the national and international guidelines for laboratory animal use and under license\u0026nbsp;ESAVI-270-04.10.07-2017\u0026nbsp;approved by the National Animal Experiment Board of Finland. All procedures performed in this study were in accordance with the ethical standards of the\u0026nbsp;National Animal Experiment Board in Finland (El\u0026auml;inkoelautakunta, ELLA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eData availability:\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eDisclosure of potential conflicts of interest:\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eFunding:\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the following funding agency for providing funding to be used as a personal grant: Sigrid Juselius Foundation, Finnish Foundation for Cardiovascular Research, Finnish Cultural Foundation, Emil Aaltonen Foundation, State Research Funding and\u0026nbsp;GeneCellNano Flagship Program.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eAuthors\u0026apos; contributions:\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEYH, HL and MAK did programming, magnetic resonance imaging, analyzed the data and wrote the manuscript.\u0026nbsp;SL and TH\u0026nbsp;contributed to surgery, histology and tissue processing. TL and SYH\u0026nbsp;contributed to the design, data analysis, writing and editing of the manuscript. All the authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank laboratory and MR maintenance staff members Maarit Pulkkinen and Jari Nissinen for help and support during the measurements.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThis work/Part of the work was carried out with the support of Kuopio Biomedical Imaging Unit, University of Eastern Finland, Kuopio, Finland (part of Biocenter Kuopio, Finnish Biomedical Imaging Node, and EuroBioImaging).\u003c/em\u003e\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eVirani SS et al. 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Magn Reson Med 57(2):308-18. https://doi.org/10.1002/mrm.21122.\u003c/li\u003e\n\u003cli\u003eRinck PA, Fischer HW, Vander Elst L, Van Haverbeke Y, Muller RN (1988) Field‐cycling relaxometry: medical applications. Radiology 168(3):843-9. https://doi.org/10.1148/radiology.168.3.3406414.\u003c/li\u003e\n\u003cli\u003eThomsen H, Held H (1995) Immunohistochemical Detection of C5b-9 (m) in Myocardium: An Aid in Distinguishing Infarction-induced Ischemic Heart Muscle Necrosis From Other Forms of Lethal Myocardial Injury. Forensic Sci Int 71(2):87-95. https://doi.org/10.1016/0379-0738(94)01640-Q.\u003c/li\u003e\n\u003cli\u003eOuyang J, Guzman M, Desoto-Lapaix F, Pincus MR, Wieczorek R (2009) Utility of Desmin and a Masson\u0026apos;s Trichrome Method to Detect Early Acute Myocardial Infarction in Autopsy Tissues. Int J Clin Exp Pathol 3(1):98-105.\u003c/li\u003e\n\u003cli\u003eHashmi S, Al-Salam S (2013) Loss of Dystrophin Staining in Cardiomyocytes: A Novel Method for Detection Early Myocardial Infarction. Int J Clin Exp Pathol 6(2):249-57.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cardiovascular MRI, Rotating frame relaxation times, T1ρ, T2ρ, Myocardial infarction","lastPublishedDoi":"10.21203/rs.3.rs-4774734/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4774734/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCardiovascular diseases are the leading cause of death worldwide due to population growth and aging. Myocardial infarction is one of the most crucial cardiovascular diseases. Acute myocardial infarction is conventionally imaged with T\u003csub\u003e2\u003c/sub\u003e mapping due to its sensitivity related to the correlation times of edema and free-water molecules. Chronic myocardial infarction, which contains fibrosis and scar tissue, is conventionally imaged with MRI with T\u003csub\u003e1\u003c/sub\u003e weighting using contrast agents involved in late gadolinium enhancement and extracellular volume since contrast agent wash out from fibrosis and scar tissue is delayed compared to myocardium. So far, imaging acute myocardial infarcts is related to T\u003csub\u003e2\u003c/sub\u003e mapping, and imaging of scar tissue and fibrosis has been limited to techniques with contrast agent injection. Rotating frame relaxation times T\u003csub\u003e1ρ\u003c/sub\u003e and T\u003csub\u003e2ρ\u003c/sub\u003e mapping were developed to provide robust measurements with relatively wide B\u003csub\u003e1\u003c/sub\u003e and B\u003csub\u003e0\u003c/sub\u003e range for these quantities. Since rotating frame methods have different correlation times than T\u003csub\u003e2\u003c/sub\u003e and T\u003csub\u003e1\u003c/sub\u003e, these methods can be used to sensitively and specifically characterize both acute and chronic myocardial infarctions. In this study, acute (2 hours) and chronic (7 days after occlusion) myocardial infarcts in and ex vivo mouse models were imaged with rotating frame relaxation time mapping without the use of contrast agents.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn vivo imaging protocol contained adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e and adiabatic T\u003csub\u003e2ρ\u003c/sub\u003e, both with two different HSn pulses, continuous wave T\u003csub\u003e1ρ\u003c/sub\u003e and conventional T\u003csub\u003e2\u003c/sub\u003e, together with cine imaging. Mice were imaged 2 hours and 7 days after myocardial infarction. Mice were sacrificed at the 2-hour or at the 7-day time point. Ex vivo measurements contained adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e and adiabatic T\u003csub\u003e2ρ\u003c/sub\u003e with two different HSn pulses, continuous wave T\u003csub\u003e1ρ\u003c/sub\u003e, T\u003csub\u003e1\u003c/sub\u003e and T\u003csub\u003e2\u003c/sub\u003e. After MRI studies, mouse hearts were fixed, and myocardial infarcts were verified using dystrophin and hematoxylin and eosin histology stainings.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eA clear difference between infarcted and normal myocardium was visible at the 2-hour time point in rotating frame relaxation time mapping. Relative relaxation time difference in adiabatic T\u003csub\u003e2ρ\u003c/sub\u003e with HS4 pulse might be sensitive to both acute edema reaction and chronic infarction. Also, in vivo and ex vivo results of adiabatic T\u003csub\u003e1ρ\u003c/sub\u003e with both HSn pulses and continuous wave T\u003csub\u003e1ρ\u003c/sub\u003e measurements showed relative relaxation time, the difference between infarcted and normal myocardium at 2 hours after the occlusion, and the difference increased at the 7-day time point.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study showed that rotating frame relaxation time methods have the potential to be a non-invasive MR diagnostic marker for acute and chronic myocardial infarcts.\u003c/p\u003e","manuscriptTitle":"Capturing acute and chronic myocardial infarction by MRI rotating frame relaxation times in mice in and ex vivo","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-02 13:30:16","doi":"10.21203/rs.3.rs-4774734/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ef9b2eb0-9fdb-4038-9e24-5ef0a3df23bc","owner":[],"postedDate":"September 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-20T06:23:52+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-02 13:30:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4774734","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4774734","identity":"rs-4774734","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}