animal models of tissue characterization of area at risk, edema and fibrosis

CARDIAC MAGNETIC RESONANCE (E NAGEL AND V PUNTMANN, SECTION EDITORS)

Animal Models of Tissue Characterization of Area at Risk,Edema and Fibrosis

Rodrigo Fernández-Jiménez & Leticia Fernández-Friera &

Javier Sánchez-González & Borja Ibáñez

Published online: 13 February 2014# Springer Science+Business Media New York 2014

Abstract Myocardial in vivo tissue characterization is ofgreat importance because it can provide meaningful informa-tion to understand pathophysiological processes underlyingdifferent cardiac diseases. Ex vivo histologic analyses oftissue samples have been classically considered the gold stan-dard in the study of tissue properties and its composition.However, over the past decade, there has been a growinginterest in the in vivo myocardial characterization with differ-ent imaging techniques, which can potentially be translatedinto the clinics in order to make an early diagnosis andevaluate serial changes, opening the possibility of dynamicevaluation. Animal models have become an essential tool toachieve this goal. This article aims at concisely reviewingrecent and significant developments in the field of imagingtechniques—mostly cardiac magnetic resonance—in relevantanimal models of tissue characterization of area at risk, edema,and fibrosis.

Keywords Magnetic resonance . Animal model . Area atrisk . Edema . Fibrosis

Introduction

In vitro tissue characterization techniques have been classical-ly considered as the gold standard for assessment of cardiacdiseases and treatments. Although these methods are veryuseful for a profound analysis of tissue composition, theyhave limitations such as changes in tissue composition be-cause of manipulation, poorly suited for serial measurementsand inability to translate it into the required follow-up clinicalfield. Conversely, several imaging modalities have been de-veloped for the in vivo characterization of cardiac tissueproperties such as positron emission tomography (PET) [1]or computed tomography (CT) [2]. However, cardiac magnet-ic resonance (CMR) is the technique that has captured most ofthe attention in preclinical and clinical research, since it offersa combination of high spatial resolution and high soft tissuecontrast with no ionizing radiation. These features entitleCMR as an ideal tool for imaging the heart in terms ofmyocardial structure and function, becoming a reference stan-dardmodality for the tissue characterization in general, and forevaluation of the heart in particular, in preclinical and clinicalresearch, including small/large animals and humans [3].

Myocardial tissue characterization in the context of a myo-cardial infarction or a suspected cardiomyopathy is one of themost frequent clinical indications to perform a CMR. SpecificMR sequences addressing a comprehensive evaluation ofheart structure and function are currently implemented in mostclinical protocols [4], including late gadolinium enhancementsequences, which allow visualization and quantification ofmacroscopic areas of scar tissue [5]. However, an intenseclinical and research interest is found today in the develop-ment of CMR sequences, which are dependent on native

This article is part of the Topical Collection on Cardiac MagneticResonance

R. Fernández-Jiménez : L. Fernández-Friera :J. Sánchez-González : B. IbáñezCentro Nacional de Investigaciones Cardiovasculares Carlos III(CNIC), Madrid, Spain

R. Fernández-Jiménez : B. IbáñezHospital Universitario Clínico San Carlos, Madrid, Spain

L. Fernández-FrieraHospital Universitario Montepríncipe, Madrid, Spain

J. Sánchez-GonzálezPhilips Healthcare, Madrid, Spain

B. Ibáñez (*)Department of Epidemiology, Atherothrombosis and Imaging,Centro Nacional de Investigaciones Cardiovasculares Carlos III(CNIC), Melchor Fernández Almagro, 3, 28029 Madrid, Spaine-mail: [email protected]

Curr Cardiovasc Imaging Rep (2014) 7:9259DOI 10.1007/s12410-014-9259-z

myocardial signal characteristics—because of change in itscomposition—rather than in exogenous contrast-based im-ages, to demonstrate pathophysiology and tissue characteriza-tion in cardiac disease [6].

Special attention has been focused in distinguishing andquantifying several main components of myocardial tissue(water, collagen, fat) that can be altered in different clinicalscenarios, mostly in myocardial infarction and cardiomyopa-thies. In fact, changes in water content have been demonstrat-ed after an ischemic event and other myocardial forms ofinflammation such as myocarditis or sepsis, making possiblethe identification and delineation of the area of a myocardialinsult [7]. Conversely, increased amounts of fibrosis, main-ly—but not restricted to—collagen, has been also extensivelyshown after myocardial infarction and in other cardiomyopa-thies in the form of macroscopic areas of fibrosis (scar), aswell as more recently in several cardiac diseases in the form ofdiffuse microscopic fibrosis, the former being associated withpoor prognosis and mortality [8-12] and to be better correlatedwith actual infarct size than other biomarkers [13]. Finally,changes in the amount of myocardial, epicardial, or pericardialfat have been also shown in a wide spectrum of heart andsystemic diseases, being a marker as well of adverse outcomes[14-16].

Some ex vivo models have been proposed as valid tools forthe development of novel MR sequences in this regard [17].However, beyond its value in the development of new imag-ing algorithms, relevant animal models can further provide,with a human, closer profound understanding of disease-related mechanisms and pathophysiology, important informa-tion before experimental-to-clinical leap, and histologic vali-dation of new imaging methods and sequences. The purposeof the present review is to bring an updated overview of themost recent works involving small and large animal modelsusing imaging technology—mainly CMR—for the in vivotissue characterization of myocardial area at risk, edema, andfibrosis.

Animal Models of Tissue Characterization of Area at Risk

The myocardial territory, which becomes ischemic after oc-clusion of its supplying coronary artery, defines the regionpotentially at risk for necrosis, named as area at risk (thereaf-ter, AAR). Modern percutaneous revascularization techniquesand pharmacologic interventions aim to salvage areas of re-versibly acute injured myocardium, thus, limiting infarct sizeand improving prognosis [18-21]. Testing the efficacy ofcardioprotective therapies benefits from measuring both in-farct size and AAR to calculate the normalized amount ofmyocardial salvage, which is probably a better measure oftherapeutic efficacy than the absolute infarct size (Fig. 1) [22,23]. Thus, there is considerable interest in finding a

reproducible in vivo imaging method, which accurately re-flects the AAR. CMR has become a paradigm of this researchbeing one of the imaging techniques that have generated morepublished articles and controversies over recent past years onthis topic.

Hyperintense areas on T2-weightedMR imaging have beenclassically proposed as having good correlation with AAR inanimal models [24••, 26], being among the most popularsequences used in humans to retrospectively delineate theischemic myocardium (ie, to delineate area of former ischemiaafter the acute episode). Signal intensity on T2-weighted MRsequences appears to be linearly related to myocardial watercontent, which seems to be increased over the ischemic terri-tory in the form of myocardial edema [27-29]. However, allthese assumptions come from wide experimental studies andfewer human studies [30], most of them with small samplesizes and mixed patient population. As a result of this greatheterogeneity, if these hyperintense areas accurately track theAAR or just the infarcted region, or merely something relatedto it, is still a matter of controversy [31••, 32••]. Moreover, T2-weighted imaging is often limited by a low signal-to-noiseratio, motion artifacts, incomplete blood suppression, and coilsensitivity related-issues of surface coils [33]. On top, noconsensus exists in regards to how this data has to be optimallyacquired and quantitatively analyzed.

Newer quantitative MR methods as T1 and T2 mapping,which allow direct measurement of intrinsic tissue propertiesin the form of T1 and T2 relaxation times, respectively, areless dependent on confounders affecting signal intensity andcould overcome some of T2-weighted imaging limitations[34, 35] (Fig. 2). In this way, in a recent animal research in adog model of ischemia/reperfusion injury [36], the accuracyfor quantifying AAR with clinically available T1- and T2-mapping sequences, compared with microsphere blood flowanalysis as a reference standard was demonstrated. However,pre-reperfusion delineation of AAR is likely the most accurateapproach to delineate the anatomic myocardium at risk in the

Fig. 1 Diagram representing both the AAR (blue delimited region),defined as the hypoperfused myocardial distal to a coronary occlusion;and the salvaged myocardium (green area), defined as the differencebetween the myocardial AAR and the necrotic area (pink area)

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experimental setting since reperfusion induces tissue changesafter reperfusion that can modify the extension of formerischemic insult. Pathophysiological responses after acutemyocardial infarction include edema, hemorrhage, and micro-vascular obstruction along with cellular damage, which prob-ably have counter-acting effects on T2 and T1 relaxationtimes, as recently demonstrated in a porcine model of myo-cardial infarction [37].

Thus, in a recent experimental work it compared the accu-racy of early postreperfusion CMR and prereperfusion multi-detector computed tomography (MDCT) imaging to measurethe size of the AAR, using pathology as a reference techniquein a porcine acute myocardial infarction model [38]. It wasshown that pathology best correlated with measurementsmade by MDCT. However, AAR measurements obtained byseveral T2-weigthed sequences (T2-STIR and T2-ACUTE)resulted in a modest correlat ion with pathologyoverestimating the anatomic extension of AAR. In our labo-ratory, we have been able to reproduce prereperfusion MDCTto measure AAR (Fig. 3, unpublished data) with similarresults. However, this approach is hardly expected to betranslated into humans, and available information suggeststhat postreperfusion T2-weighted imaging (or any otherpostreperfusion sequence or technique) aimed to measureAAR may have some limitations depending on the timing ofimage acquisition after ischemic insult.

On top, novel CMR methods have been recently added tothe AAR identification and characterization repertoire. MRspectroscopy (MRS) and MR-based molecular imagingmethods have shown promise for evaluating cardiac metabo-lism in the setting of ischemia/reperfusion. For example,phosphorus-31 MRS can assess high-energy phosphate con-tent and energy reserve in the heart in animal and humansubjects [39]. In a rat model of myocardial infarction, wholeheart PCr content was inversely correlated with infarct size,whereas ATP distribution provided a profile of viable myo-cardium around the infarction reflecting remodeling of theheart [40]. In rats undergoing ligation of the left anteriordescending coronary artery, 1H-MRS proved lower creatinemyocardium content compared with controls [41]. In addition,dynamic nuclear polarization has been recently shown toallow a signal increase of more than 10,000-fold, openingthe door to new understanding of the metabolic processes inthe heart [42]. Experiments performed in the globally ische-mic, isolated rat heart have demonstrated that hyperpolarized13C-pyruvate can show the glycolytic switch characteristic ofmyocardial ischemia [43, 44]. In addition, hyperpolarizedexperiments in pig model of ischemia/reperfusion showedthe potential of 13C-bicarbonate and (1-13C) alanine mapsto distinguish between stunned myocardium and not viabletissue [45]. Modified blood oxygen level-dependent (BOLD)sequence, which signal intensity changes primarily from

Fig. 2 Direct measurement of intrinsic tissue properties in the form of T1 mapping (Panel A) and T2 mapping (Panel B) in a pig model of ischemia/reperfusion injury. Note abnormal T1 and T2 relaxation times over anterior ventricular wall

Fig. 3 In vivo delineation of areaat risk at the mid-ventricular levelusing prereperfusion multi-detector computed tomography(Panel A, white arrows) andpostreperfusion T2-weightedSTIR magnetic resonancesequence (Panel B, white arrows)in the same pig submitted totransient percutaneous occlusionof the left anterior descendingcoronary artery

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alterations in blood oxygen saturation (and so, from changesin myocardial perfusion) has been just proposed as able todetect ischemic myocardium in a dog model of severe coro-nary stenosis, even earlier in time than the appearance ofedema [46]. Tracking of upregulated vascular cell and inter-cellular adhesionmolecules (VCAM and ICAM, respectively)following ischemia using targeted microparticles of iron ox-ide, which shorten T2 and T2* relaxation times, have alsoshown promise for the detection and localization of recentischemia. Thus, they have been reported as suitable for theearly identification of ischemic/damage organs [47, 48] aswell as myocardial AAR [49] in small animal models, al-though its translation into the clinic does not seem to bearound the corner. Finally, re-invented balanced steady-statefree precession (bSSFP) sequence with T2 preparation, hasbeen also recently described as potentially able to detectmyocardial edema in both porcine model and patients withreperfused acute myocardial infarction, due to its sensitivity toT1, T2, and magnetization transfer effects [50-52].

Despite all these experiments and some clinical studies[30], important questions remain unclear. As previously men-tioned, comprehensive study of the time course of myocardialedema and its visualization by CMR from the just earlyreperfusion is of critical importance and not fully addressedat the moment. Knowing the optimal time to accurately mea-sure AAR would allow standardization of experimental andclinical protocols, avoiding conflicting data published over theliterature. Large animal models of ischemia/reperfusion in-volving CMR studies become an ideal platform to go in depthinto this phenomenon. Among them, pig is one of the mosttranslational and reliable model to study ischemia/reperfusion-related issues since shows, unlike other mammals but similarto humans, analogous coronary artery anatomy and distribu-tion [53, 54], and minimal pre-existing coronary collateralflow [55••, 56].

Animal Models of Tissue Characterizationof Infarction-Unrelated Myocardial Edema

Experimental myocardial edema is mostly generated througha surgical (small and large animal models) or percutaneous(only large animal models) ischemia/reperfusion procedureinvolving transient occlusion of a coronary artery. This pro-cedure has been described elsewhere [23], and its edema-related consequences have been briefly reviewed along previ-ous section.

Different procedures inducing infarct-independent increaseof myocardial water content in animal models have been alsodescribed, trying to reproduce other forms of myocardialedema. Sepsis triggered by sublethal injection of lipopolysac-charide [57, 58] or mechanical-induced peritonitis [59] hasbeen shown to augment water content in the myocardium due

to increased vascular permeability in mice. Global acute myo-cardial edema has been induced in a canine model by transientelevation of coronary sinus pressure [60]; whereas local myo-cardial edema has been described in the setting of experimen-tal autoimmune myocarditis in CD69-defficient mice, whichis characterized by an infiltration of inflammatory cells intothe myocardium, fibrosis, edema, and necrosis, leading toventricular wall dysfunction and heart failure [61].

Same CMR sequences described in previous section forAAR identification have been investigated for tissue charac-terization of noninfarct models of myocardial edema sinceshare identical target; that is myocardial edema and so, chang-es in water tissue content. However, some imaging techniquesare briefly pointed in this section since are specifically focusedin tracking changes in the amount and conformation proper-ties of the tissue water content itself, beyond the ischemicview.

Diffusion imaging provides information of water mole-cules displacement using motion sensitive gradients [62]adding complementary information to T2 weighted for edemadefinition (Fig. 4). Although limited experiments have beenreported in animal models, different publications in humansubjects have proposed this technique as a valuable tool foredema identification mostly using this diffusion informationas a black-blood technique obtaining better blood suppressionthan conventional T2-weighted images [63].

Sodium chemical shift imaging (23Na-CSI) has been re-ported as able to assess the sodium gradient and cell mem-brane integrity in order to visualize areas of myocardial edemain isolated rat hearts [64]. Because of expansion of the extra-cellular space in interstitial edema, imaging intra- and extra-cellular sodium separately may provide an alternative ap-proach to assess myocardial edema. In this way, an elevatedtotal sodium signal has been observed after myocardial infarc-tion both in small and large animals [65, 66], and humans [67].In a very recent research, using a noninfarcted isolated heartmodel of extracellular edema and 23Na-CSI, it has been shownthat viable edematous myocardium with cell membranes stillintact is characterized by increased extracellular sodium butnormal intracellular sodium since viable cells are able tomaintain a normal sodium gradient [68]. Although promising,translation into clinically feasible seems to be again furtheraway.

Animal Models of Tissue Characterization of MyocardialFibrosis

Myocardial fibrosis is a common phenomenon that is ob-served in different stages in a wide variety of heart diseases.Collagen accumulation and increase in extracellular volume isbasically shown in the myocardium in 2 ways: macroscopicfibrosis in the form of a visible based scar and microscopic


fibrosis in the form of diffuse global fibrosis. Myocardialfibrosis has become one of the hot topics at the moment,generating a huge amount of publications in animal modelsand humans over the last recent years.

Macroscopic fibrosis in the form of a scar (Fig. 5) has beenclassically and extensively described after myocardial infarc-tion, and in other cardiomyopathies [69, 70], based on delayedenhancement sequences both in CMR and CT [71], being its

size and distribution intimately associated with heart remod-eling, prognosis, and mortality of patients [69, 71-75]. Smalland large animal models of myocardial infarction are nowbeing frequently used for precl inical test ing ofcardioprotective therapies [23], development, and progressin imaging techniques [76], and study of scarring-relatedarrhythmias [77, 78]. Moreover, animal models closely repro-ducing human atrial arrhythmias are becoming a nice platform

Fig. 4 In vivo diffusion-weighted images (Panels A, B, C and D) from apig submitted to myocardial infarction. All images show a very brightregion corresponding to edema within the area at risk, with different

diffusion weighted b values (0, 50, 150, 500 s/mm2). Image of theventricle at same level using a T2W-STIR sequence for comparisonbetween both techniques (Panel E)

Fig. 5 Macroscopic fibrosis inthe form of scar localized at themid-ventricular anterior wall onmagnetic resonance images usinglate gadolinium enhancement(Panels A and B), which is alsodemonstrated on pathologicalspecimen (Panels C and D)


to develop and improve new and classic ablation techniques,which can potentially be translated to clinical trials, along withgoing in depth with arrhythmia-related imaging, mechanisms,and pathophysiology [79-81].

Although conventional late gadolinium enhancementCMR sequences are able to identify macroscopic fibrosis inconditions such as myocardial infarction and other cardiomy-opathies as previously described, they are less suitable forin vivo detection of microscopic diffuse myocardial fibrosissince the myocardium is globally affected by smaller collagendeposits and there may be little normal myocardium to com-pare with. However, relying on quantification of the T1 relax-ation times after contrast enhancement equilibrium, the myo-cardial extracellular volume fraction has been proposed ashaving a good correlation with the amount of diffuse fibrosis

[82, 83]. Fibrosis shortens the postcontrast longitudinal relax-ation time (and so, T1) properties of the myocardium and thisfeature may be tracked by specific pulse sequences. Thus, ashorter myocardial T1 after administration of gadolinium-based contrast agent indicates extracellular matrix expansionand is associated with accumulation of connective tissue in themyocardium. Promising noncontrast CMR methods for thediscrimination of normal and diffusely diseased myocardiumare emerging as the recently described T1 native [84].

Animal models have become critical for validation ofnewer CMR methods, and research in fibrosis imaging is notan exception. Diffuse myocardial fibrosis over the left ventri-cle has been generated in small and large animals mostlybased on different approaches such as placement of aorticconstrictive banding [85] or drug administration [86, 87],

Fig. 6 Cardiac contrastcomputed tomography - sagittalview showing surgical placementof a banding at the ascendingaorta (black asterisk) of a pig(Panel A). Histologic mid-ventricular short axis slice of thesame animal with Picrosirius redstaining demonstrating leftventricular hypertrophy anddiffuse interstitial fibrosis (PanelB, red fibers; courtesy of DamianSanchez-Quintana)

Fig. 7 Contrast computedtomography images showingnormal pulmonary vein anatomy(Panel A) and a significantstenosis of the inferior pulmonaryvein (Panel B) in a pig model ofpulmonary hypertension.Histologic images of the rightventricle of the same pig usingPicrosirius red (Panel C) andMasson trichrome (Panel D)stainings showing myofiberdisarray and diffuse myocardialfibrosis over the right ventricle(courtesy of Damian Sanchez-Quintana)


overall inducing pressure-overload left ventricular hypertro-phy (Fig. 6); although other animal models reproducing spe-cific cardiomyopathies have been also reported [88]. Particu-lar modified sequences for generating T1 maps, and so de-tecting diffuse fibrosis, have been developed and are neededin small animals due to their high heart rates [89]. Althoughnot so well studied, generation of animal models with diffusemyocardial fibrosis in the right ventricle (Fig. 7) is an inter-esting and promising approach for investigating its forgottendiseases and for achieving new treatments for pulmonaryhypertension [90, 91].

Novel imaging techniques have been recently describedadding new information about myocardial fibrosis. Diffusiontensor magnetic resonance imaging (DT-MRI) is a diffusion-weighted technique that uses the directionality information ofwater diffusion to track myofibers orientation and organiza-tion [92] (Fig. 8). This myofiber organization can bemeasuredwith a DTI parameter called fractional anisotropy that repre-sents the capability of water molecules to move more easily in1 direction compared with the others. Thus, DT-MRI hasdemonstrated a reduction of fractional anisotropy values inthe infarcted and border zones compared with the remotemyocardium showing good agreement with histology findingsin a pig model [93, 94]. New approaches using molecularimaging have emerged to in vivo visualize and quantify in-flammatory molecules involved in the formation of scar tissuein the postinfarction myocardium [95]. For example, targetingmyofibroblasts or metalloproteinases using specific ligandshave been proposed for imaging left ventricular remodelingin a murine model of myocardial infarction [96]. Thus, thepossibility to identify early stages of postinfarction healing

may allow the development of new therapies focused inpreventing irreversible changes.

Conclusions

Characterization of myocardial tissue changes with CMRrepresents a valuable tool to define the presence and stage ofa particular heart disease, and to serially and noninvasivelymonitor both animal models and patients for progression ofdisease or therapeutic efficacy. However, despite great devel-opment in the last years, this topic is far from been closed,remaining important questions to be answered. Early detectionas well as timing and mechanistics of these tissue changes areof great importance and interest, although little and controver-sial information only is published at present. Full animalmodels become the ideal tool for the in vivo development ofnewer imaging methods, which would potentially allow an-swering these questions since ability for follow-up acquisi-tions and feasibility of validation and translation to the clinic.

Compliance with Ethics Guidelines

Conflict of Interest Rodrigo Fernández-Jiménez declares that he has noconflict of interest. Leticia Fernández-Friera declares that he has noconflict of interest. Javier Sánchez-González declares that he has noconflict of interest. Borja Ibáñez declares that he has no conflict of interest.

Human and Animal Rights and Informed Consent This article doesnot contain any studies with human or animal subjects performed by anyof the authors.

Fig. 8 Diffusion tensor 3Dreconstruction imaging of anex vivo pig heart showing themyofiber tracks form a long axisand short axis view (Panel A).Two-dimensional cross sectionsof the same ventricle at 3 differentlevels. Note circumferentialorganization of the myofibers(courtesy of Prof Jesús Ruíz-Cabello)


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animal models of tissue characterization of area at risk, edema and fibrosis

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