Author + information
- Received October 19, 2014
- Revision received November 5, 2014
- Accepted November 6, 2014
- Published online February 3, 2015.
- Rodrigo Fernández-Jiménez, MD∗,†,
- Javier Sánchez-González, PhD∗,‡,
- Jaume Agüero, MD∗,
- Jaime García-Prieto, BSc∗,
- Gonzalo J. López-Martín, Tech∗,
- José M. García-Ruiz, MD∗,
- Antonio Molina-Iracheta, DVM∗,
- Xavier Rosselló, MD∗,
- Leticia Fernández-Friera, MD, PhD∗,§,
- Gonzalo Pizarro, MD∗,‖,
- Ana García-Álvarez, MD, PhD∗,
- Erica Dall'Armellina, MD, DPhil¶,
- Carlos Macaya, MD, PhD†,
- Robin P. Choudhury, DM¶,
- Valentin Fuster, MD, PhD∗,# and
- Borja Ibáñez, MD, PhD∗,†∗ ()
- ∗Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain
- †Hospital Universitario Clínico San Carlos, Madrid, Spain
- ‡Philips Healthcare, Madrid, Spain
- §Hospital Universitario Montepríncipe, Madrid, Spain
- ‖Hospital Universitario Quirón Universidad Europea de Madrid, Madrid, Spain
- ¶Oxford Acute Vascular Imaging Centre, Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom
- #The Zena and Michael A. Wiener Cardiovascular Institute, Icahn School of Medicine at Mount Sinai, New York, New York
- ↵∗Reprint requests and correspondence:
Dr. Borja Ibáñez, Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Melchor Fernández Almagro 3, 28029 Madrid, Spain.
Background It is widely accepted that edema occurs early in the ischemic zone and persists in stable form for at least 1 week after myocardial ischemia/reperfusion. However, there are no longitudinal studies covering from very early (minutes) to late (1 week) reperfusion stages confirming this phenomenon.
Objectives This study sought to perform a comprehensive longitudinal imaging and histological characterization of the edematous reaction after experimental myocardial ischemia/reperfusion.
Methods The study population consisted of 25 instrumented Large-White pigs (30 kg to 40 kg). Closed-chest 40-min ischemia/reperfusion was performed in 20 pigs, which were sacrificed at 120 min (n = 5), 24 h (n = 5), 4 days (n = 5), and 7 days (n = 5) after reperfusion and processed for histological quantification of myocardial water content. Cardiac magnetic resonance (CMR) scans with T2-weighted short-tau inversion recovery and T2-mapping sequences were performed at every follow-up stage until sacrifice. Five additional pigs sacrificed after baseline CMR served as controls.
Results In all pigs, reperfusion was associated with a significant increase in T2 relaxation times in the ischemic region. On 24-h CMR, ischemic myocardium T2 times returned to normal values (similar to those seen pre-infarction). Thereafter, ischemic myocardium-T2 times in CMR performed on days 4 and 7 after reperfusion progressively and systematically increased. On day 7 CMR, T2 relaxation times were as high as those observed at reperfusion. Myocardial water content analysis in the ischemic region showed a parallel bimodal pattern: 2 high water content peaks at reperfusion and at day 7, and a significant decrease at 24 h.
Conclusions Contrary to the accepted view, myocardial edema during the first week after ischemia/reperfusion follows a bimodal pattern. The initial wave appears abruptly upon reperfusion and dissipates at 24 h. Conversely, the deferred wave of edema appears progressively days after ischemia/reperfusion and is maximal around day 7 after reperfusion.
Tissue characterization after myocardial ischemia/reperfusion (I/R) is of great scientific and clinical value. After myocardial I/R, there is an intense edematous reaction (due to abnormal fluid accumulation in the interstitial and/or cardiomyocyte compartments) in the post-ischemic myocardium (1–5). Cardiac magnetic resonance (CMR) is a noninvasive technique that allows accurate tissue characterization of the myocardium (6). In particular, T2-weighted (T2W) and T2-mapping CMR sequences have the potential to identify tissues with high water content (7). Few experimental studies have correlated post-I/R T2-CMR data with myocardial water content (2,8), and these validations were undertaken at different times after reperfusion. Many recent experimental and clinical studies have used these CMR sequences to retrospectively evaluate post-myocardial infarction edema on the basis of the assumptions that myocardial edema appears early after I/R, persists in a stable form for at least 1 week (9,10), and is accurately visualized by CMR. However, the time chosen for the CMR examination varies significantly among studies, from 1 day (9,10) up to several weeks (9–16) after reperfusion. In addition, post-I/R T2W signal intensity and T2 relaxation time are affected by other factors besides water content: T2-CMR results can be modulated independently by hemorrhage (17,18), microvascular obstruction (19), and even cardioprotective therapies (20–22). There is, therefore, intense debate about the accuracy of CMR-based methods for detecting, quantifying, and tracking the post-infarction edematous reaction (7,23). Given the growing use of CMR technology to quantify post-I/R edema in clinical trials (24,25), a comprehensive characterization of the time course of post-I/R myocardial edema, including evaluation of both CMR and histological reference standards, is needed (22–24,26–28).
The present study aimed to comprehensively characterize myocardial edema and reperfusion-related tissue changes after I/R, covering from early to late reperfusion stages. For this, we performed a full CMR and histopathological study in a large animal (pig) model of I/R.
Experiments were performed in castrated male Large-White pigs weighing 30 kg to 40 kg. A total of 25 pigs completed the full protocol and comprised the study population. The study was approved by the Institutional Animal Research Committee and conducted in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals. The study design is summarized in Figure 1. Five pigs (Group 1) served as controls and were sacrificed with no intervention other than baseline CMR. In 20 pigs, reperfused acute myocardial infarction (I/R) was induced experimentally by closed-chest 40-min left anterior descending coronary artery occlusion. These pigs were sacrificed at 120 min (n = 5, Group 2), 24 h (n = 5, Group 3), 4 days (n = 5, Group 4), and 7 days (n = 5, Group 5) after reperfusion. CMR scans, including T2W short-tau inversion recovery (STIR), T2-mapping, and delayed enhancement sequences, were performed at every follow-up stage until sacrifice (i.e., animals sacrificed on day 7 underwent CMR examinations at baseline, 120 min, 24 h, day 4, and day 7). After the last follow-up CMR scan, animals were immediately euthanized, and myocardial tissue samples from ischemic and remote areas were rapidly collected for evaluation of water content by histology.
Myocardial infarction procedure
The I/R protocol has been detailed elsewhere (29). Anesthesia was induced by intramuscular injection of ketamine (20 mg/kg), xylazine (2 mg/kg), and midazolam (0.5 mg/kg), and maintained by continuous intravenous infusion of ketamine (2 mg/kg/h), xylazine (0.2 mg/kg/h), and midazolam (0.2 mg/kg/h). Animals were intubated and mechanically ventilated with oxygen (fraction of inspired O2: 28%). Central venous and arterial lines were inserted, and a single bolus of unfractionated heparin (300 mg/kg) was administered at the onset of instrumentation. The left anterior descending coronary artery, immediately distal to the origin of the first diagonal branch, was occluded for 40 min with an angioplasty balloon introduced via the percutaneous femoral route using the Seldinger technique. Balloon location and maintenance of inflation were monitored angiographically. After balloon deflation, a coronary angiogram was recorded to confirm patency of the coronary artery. A continuous infusion of amiodarone (300 mg/h) was maintained during the procedure in all pigs to prevent malignant ventricular arrhythmias. In cases of ventricular fibrillation, a biphasic defibrillator was used to deliver nonsynchronized shocks.
A baseline CMR scan was performed immediately before myocardial infarction and subsequent CMR scans were performed at post-infarction follow-up time points until sacrifice. All studies were performed in a Philips 3-T Achieva Tx whole-body scanner (Philips Healthcare, Best, the Netherlands) equipped with a 32-element phased-array cardiac coil. The imaging protocol included a standard segmented cine steady-state free-precession (SSFP) sequence to provide high-quality anatomical references, a T2-weighted triple inversion-recovery (T2W-STIR) sequence, a T2-turbo spin echo (TSE) mapping sequence, and a late gadolinium enhancement sequence. The imaging parameters for the SSFP sequence were field of view (FOV) of 280 × 280 mm, slice thickness 6 mm with no gaps, repetition time (TR) 2.8 ms, echo time (TE) 1.4 ms, flip angle 45°, cardiac phases 30, voxel size 1.8 × 1.8 mm, and 3 number of excitations (NEX). Imaging parameters for the T2W-STIR sequence were FOV 280 × 280 mm, slice thickness 6 mm, TR 2 heartbeats, TE 80 ms, voxel size 1.4 × 1.95 mm, delay 210 ms, end-diastolic acquisition, echo-train length 16, and 2 NEX. The imaging parameters for the T2-TSE mapping were FOV 300 × 300 mm, slice thickness 8 mm, TR 2 heartbeats, and 10 echo times ranging from 4.9 to 49.0 ms. Delayed enhancement imaging was performed 10 to 15 min after intravenous administration of 0.20 mmol of gadopentetate dimeglumine contrast agent per kg of body weight (30) using an inversion-recovery spoiled turbo field echo (IR-T1TFE) sequence with the following parameters: FOV of 280 × 280 mm, voxel size 1.6 × 1.6 mm, end-diastolic acquisition, thickness 6 mm with no gap, TR 5.6 ms, TE 2.8 ms, inversion delay time optimized to null normal myocardium, and 2 NEX. SSFP, T2W-STIR, and IR-T1TFE sequences were performed to acquire 13 to 15 contiguous short-axis slices covering the heart from the base to the apex. To track T2 relaxation time changes across time, T2 maps in all studies were acquired in midapical ventricular short-axis slices corresponding to the same anatomical level.
CMR data analysis
CMR images were analyzed using dedicated software (MR Extended Work Space 2.6, Philips Healthcare, and QMass MR 7.5, Medis, Leiden, the Netherlands) by 2 observers experienced in CMR analysis. T2 maps were automatically generated on the acquisition scanner by fitting the signal intensity of all echo times to a monoexponential decay curve at each pixel with a maximum likelihood expectation maximization algorithm. T2 relaxation maps were quantitatively analyzed by placing a wide transmural region of interest (ROI) at the ischemic and remote areas of the corresponding slice in all studies. Hypointense areas suggestive of microvascular obstruction or hemorrhage were included in the ROI for T2 quantification purposes. Delayed gadolinium-enhanced regions were defined as >50% of maximum myocardial signal intensity (full width at half maximum) with manual adjustment when needed. If present, a central core of hypointense signal within the area of increased signal was included as late gadolinium-enhanced myocardium. Regional transmurality of contrast enhancement was evaluated in the same segments where ROIs for T2 quantification were placed with a scheme on the basis of the spatial extent of delayed enhancement tissue within each segment (31). Segments with more than 75% hyperenhancement were considered segments with transmural enhancement.
Quantification of myocardial water content by histology
Paired myocardial samples were collected within minutes of euthanasia from the infarcted and remote myocardia of all pigs. Tissue samples were immediately blotted to remove surface moisture and introduced into laboratory crystal containers previously weighed on a high-precision scale. The containers were weighed before and after drying for 48 h at 100°C in a desiccating oven. Tissue water content was calculated as follows: water content (%) = ([wet weight − dry weight]/wet weight) ×100. An empty container was weighed before and after desiccation as an additional calibration control.
Normal distribution of each data subset was checked using graphical methods and a Shapiro-Wilk test. For quantitative variables showing a normal distribution, data are expressed as mean ± SD. Leven’s test was performed to check the homogeneity of variances. A 1-way analysis of variance was conducted for comparison of myocardial water content among groups (from animals sacrificed at different time points). To take repeated measures into account, a generalized mixed model was conducted for comparison of T2 relaxation times among different time points. As this study was exploratory, all pairwise comparisons were explored, adjusting p values for multiple comparisons using the Holm-Bonferroni method. In all cases, data from the ischemic and remote myocardium (i.e., myocardial water content and T2 relaxation time values) were separately analyzed. All statistical analyses were performed using commercially available software (Stata version 12.0, StataCorp, College Station, Texas).
Natural evolution of myocardial edema during the first week after I/R
Baseline (i.e., before ischemia) mean T2 relaxation times were 47.2 ± 2.6 ms and 46.3 ± 1.7 ms for the midapical anteroseptal and posterolateral left ventricular walls, respectively. In all pigs, early reperfusion (120-min CMR) was associated with a sharp and significant increase in T2 relaxation time above baseline, in the former ischemic area (midapical anteroseptal ventricular wall). T2 relaxation times returned to baseline values at 24 h post-I/R in all animals, but subsequently increased progressively, reaching post-I/R values on day 7 similar to those observed during early reperfusion. Albeit slight, a linear trend for a progressive increase in T2 relaxation times across different time points was observed in the remote myocardium. Figure 2A shows mean changes in T2 relaxation time in the ischemic myocardium as well as a representative example of 1 animal serially scanned at all time points. Measurements of T2 relaxation time in the ischemic and remote myocardium at different time points after I/R are summarized in Table 1. Changes observed in T2W-STIR and T2-TSE mapping were consistent in all animals, as seen in Figure 3, which shows images from 8 pigs scanned at the different time points. The transmural extent of infarction was >80% in all evaluated segments containing the ROIs for T2 relaxation time quantification.
Myocardial water content
Myocardial water content in noninfarcted myocardium (from animals in group 1) was 79.4 ± 0.6% and 79.4 ± 0.7% for the midapical anteroseptal and posterolateral left ventricular walls, respectively. In the ischemic myocardium, an abrupt increase in water content was detected at early reperfusion. Consistent with the CMR data, there was a systematic and significant decrease in tissue water content in the formerly ischemic region at 24 h, followed by a subsequent increase over the following days to reach values on day 7 similar to those observed at early reperfusion. A linear trend for a slight, but progressive increase in water content across different time points was observed in the remote myocardium. Time courses for absolute differences in water content between ischemic and remote myocardium are shown in Figure 2B. Table 2 summarizes measurements of water content in the ischemic and remote myocardium at different time points after I/R.
The present experimental study challenges the accepted view of the development of post-ischemia/reperfusion myocardial edema. Through state-of-the-art CMR analysis and histological validation in a pig model of I/R, we show that the edematous reaction during the first week after reperfusion is not stable, instead following a bimodal pattern (Central Illustration). The first wave appears abruptly upon reperfusion and dissipates at 24 h. Conversely, the second wave of edema appears progressively days after I/R and increases to a maximum on post-reperfusion day 7. To the best of our knowledge, this is the first study to comprehensively characterize the time course of myocardial edema during the first week after I/R, covering from very early to late reperfusion stages. Because edema has been perceived as both stable and persistent during at least 1 week after myocardial I/R, it has been used increasingly both clinically and in the setting of clinical trials as a marker of “ischemic memory.” Therefore, our findings that neither of these assumptions is accurate will have important translational implications.
As with most organs, water is a major component of healthy cardiac tissue. In steady-state conditions, myocardial water content is stable and mostly intracellular, with only a very small interstitial component contained within the extracellular matrix. Cardiac edema occurs in numerous pathological conditions in which this homeostasis is disrupted, and affects both fluid accumulation outside cells (interstitial edema) and within cardiomyocytes (cellular edema). In the context of myocardial infarction, edema appears initially in the form of cardiomyocyte swelling during the early stages of ischemia (5). Myocardial edema is then significantly exacerbated upon restoration of blood flow to the ischemic region. This increase is due to increased cell swelling (3) and, more importantly, to interstitial edema secondary to reactive hyperemia and leakage from damaged capillaries when the hydrostatic pressure is restored upon reperfusion (1,4).
CMR has emerged as a noninvasive technology that allows characterization of cardiac tissue after I/R (6), with T2-weighted (T2W) CMR sequences especially suited to detecting high water content in post-ischemic edematous cardiac muscle (7). Under the accepted dogma that myocardial edema appears early after I/R and is present for at least 1 week (9,10,24), numerous experimental and clinical studies have used T2W-CMR to retrospectively evaluate the edematous reaction associated with myocardial infarction. Although visually attractive, T2W imaging is subject to several technical limitations and does not offer quantitative T2 measurements that would allow for comparisons between different studies (32,33). Recently developed quantitative T2 relaxation maps (T2 mapping) have been proposed to overcome at least some of the limitations for the detection and quantification of myocardial edema (34,35). However, T2-mapping sequences have inherent limitations, are time-consuming, and are thus mostly used as a research tool and require further validation. Large animal models of I/R offer an ideal platform for such a validation (36), and, due to its anatomical and physiological similarities to the human heart, the pig is one of the most reliable models for studying I/R-related processes.
The disparate time points examined in different experimental and clinical studies are an important source of confusion in CMR evaluation of post-I/R edema. As demonstrated in the present study, because T2 values in the ischemic myocardium fluctuate significantly during the first week after reperfusion, the timing of post-infarction imaging is critically important for noninvasive assessment of myocardial edema. In a previous study, Foltz et al. (37,38) suggested a similar myocardial T2 relaxation time course in a pig model of I/R, with CMR scans at days 0, 2, and 7 after reperfusion. However, this study lacked histological validation of myocardial water content, and the observed T2 value fluctuations were interpreted as reflecting the oxidative denaturation of hemoglobin to methemoglobin (17,39) rather than fluctuations in myocardial water content. The histological validation in the present study demonstrates the consistent appearance of 2 consecutive waves of edema during the first week after I/R, a groundbreaking concept in the field.
The first wave of edema appears soon after reperfusion and dissipates at 24 h. Interestingly, water content within the ischemic myocardium did not return to normal values, whereas T2 relaxation time in the ischemic ventricular wall dropped to baseline values. It is plausible that the decrease in T2 relaxation time observed at 24 h is due to at least 2 components: the classically described paramagnetic effect of hemoglobin denaturation products and the sharp decrease in myocardial water content at 24 h post-reperfusion reported here.
The second wave of edema appeared progressively in the days after I/R and was maximal at day 7. Interestingly, T2 abnormalities and increased water content in the ischemic region were ultimately as impressive as those observed at early reperfusion. Further studies are needed to elucidate the physiopathology underlying this bimodal edematous reaction after I/R. It is intuitive to argue that the first and second waves of post-I/R edema are related to different pathological phenomena, although this has not been demonstrated in the present work. Whereas the first wave seems to be directly related to reperfusion, the pathophysiology underlying the second wave is more challenging to decipher. We speculate that tissue changes during the first week of infarction (removal of cardiomyocyte debris from the extracellular compartment and its replacement by water, collagen homeostasis, and healing of tissue/inflammation, among others) could play a role in this second edematous reaction.
The data presented here might have implications for understanding the role of CMR in retrospective quantification of the post-infarction area at risk. Given that this study was not designed to correlate the actual anatomical area at risk (perfusion defect during ischemia) with the extension of CMR-visualized edema, any conclusions in this regard are speculative and distract from this study’s main objective. Future studies should specifically evaluate the impact of the dual edema phenomenon on the role of CMR to accurately quantify area at risk.
The identification of the time course of post-I/R myocardial edema has important biological, diagnostic, prognostic, and therapeutic implications, and opens a route to further exploration of factors influencing this phenomenon.
Extrapolation of the results of this experimental study to the clinic should be done with caution. The intensity and time course of bimodal post-I/R edema may be modified by several factors, such as the duration of ischemia, pre-existence of collateral flow, and even the application of peri-reperfusion therapies to attenuate I/R damage. Nonetheless, the use of a large animal model is of great translational value, especially considering the difficulty of performing such a comprehensive serial CMR study (including 1 examination immediately upon reperfusion) in patients. The data presented in this study are robust and consistent, and the pig is one of the most clinically translatable large animal models for the study of I/R issues, because (unlike other mammals) its coronary artery anatomy and distribution are similar to those of humans (40) and it has minimal pre-existing coronary collateral flow (41). In addition, as shown here with the direct quantification of myocardial water content, experimental studies offer the possibility of histological validation.
In this study, the ROIs for quantification of T2 relaxation time were placed in the entire wall thickness, then were carefully and individually adjusted by hand to avoid the right and left ventricular cavities. Therefore, ROIs might include different myocardial states (i.e., hemorrhage, microvascular obstruction). We took this approach to mimic the histological water content evaluation, which was performed in the entire wall thickness. Given the parallel courses of T2 relaxation times and water content, we believe that the possible inclusion of different myocardial states had little effect on the results, although it might have had some influence on the differences in absolute T2 relaxation times between our study and others that used a different methodological approach to select ROIs.
Contrary to the accepted view, the present work consistently shows that edematous reaction during the first week after ischemia/reperfusion is not stable, but follows a bimodal pattern. The first wave of edema appears abruptly upon reperfusion and dissipates at 24 h. Conversely, the second wave appears progressively days after ischemia/reperfusion and is maximal around day 7 after reperfusion.
COMPETENCY IN MEDICAL KNOWLEDGE: Myocardial edema that develops after acute infarction is a bimodal phenomenon with diagnostic, prognostic, and therapeutic implications.
TRANSLATIONAL OUTLOOK: Temporal variation in post-ischemic reperfusion edema should be considered in studies of cardiac magnetic resonance imaging for quantification of jeopardized myocardium.
The authors are indebted to the contributions of Maria Del Trigo, Carlos Galán-Arriola, and David Sanz-Rosa. The authors also thank Tamara Córdoba, Oscar Sanz, Eugenio Fernández, and other members of the CNIC animal facility and farm for outstanding animal care and support, and Simon Bartlett (from the CNIC) for providing English editing.
This work was supported by a competitive grant from the Ministry of Economy and Competitiveness (MINECO), Fondo Europeo de Desarrolo Regional (FEDER) Carlos III Institute of Health–Fondo de Investigación Sanitaria (PI13/01979), and in part by FP7-PEOPLE-2013-ITN Next Generation Training in Cardiovascular Research and Innovation–Cardionext. This study forms part of a Master Research Agreement between CNIC and Philips Healthcare. QMass software use was partially supported by a scientific collaboration with Medis Medical Imaging Systems BV. The Spanish Ministry of Economy and Competitiveness and the Pro-CNIC Foundation support the CNIC. Dr. Fernández-Jiménez is a recipient of a Rio Hortega fellowship from the Ministry of Economy and Competitiveness through the Instituto de Salud Carlos III; and has received an FICNIC fellowship from the Fundació Jesús Serra, the Fundación Interhospitalaria de Investigación Cardiovascular, and the CNIC. Dr. Sánchez-González is an employee of Philips Healthcare. Dr. Aguero is an FP7-PEOPLE-2013-ITN-Cardionext fellow. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. Derek Yellon, PhD, DSc, served as Guest Editor for this paper.
- Abbreviations and Acronyms
- cardiac magnetic resonance
- field of view
- number of excitations
- region of interest
- short-tau inversion recovery
- echo time
- repetition time
- T2 weighted
- Received October 19, 2014.
- Revision received November 5, 2014.
- Accepted November 6, 2014.
- 2015 American College of Cardiology Foundation
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