Volumetric assessment of myocardial viability in rats using 3D double contrast enhanced T1 and T2-weighted MRI

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MAGMA (2005)

DOI 10.1007/s10334-005-0016-9 RESEARCH ARTICLE

C. Chapon F. Franconi L. Lemaire L. Marescaux P. Legras B. Denizot J-J Le Jeune

Volumetric assessment of myocardial viability in rats using 3D double contrast enhanced T1 and T2-weighted MRI

Received: 30 November 2004 Accepted: 20 October 2005

C. Chapon·L. Lemaire (

B

⁾

B. Denizot·J-J Le Jeune INSERM U 646, Ing´enierie de la Vectorisation Particulaire, 10, rue Andr´e Boquel, 49100 Angers, France

E-mail: [email protected] Tel.: +33-0241-735855

Fax.: +33-0241-735853 F. Franconi

Service Commun d’Analyses

Spectroscopiques, Universit´e d’Angers, Angers, France

L. Marescaux

Ecole Nationale Vétérinaire, Unité de Pathologie Chirurgicale, Nantes, France P. Legras

Service Commun Animalerie Hospitalo-Universitaire, Universit´e d’Angers, Angers, France

C. Chapon·L. Lemaire B. Denizot·J-J Le Jeune

Universit´e d’Angers, Angers, France

Abstract Objective: Volumetric evaluation of the myocardial

viability post-infarction in rats using 3D in vivo MR imaging at 7 T using injection of an extracellular

paramagnetic contrast agent and intravascular superparamagnetic iron oxide nanoparticles in the same imaging session.Materials and methods: Five hours after induction of permanent myocardial infarction in rats (n=6), 3D in vivo T1- and T2-weighted MR Imaging was performed prior to and after Gd-DOTA injection (0.2 mmol/kg) and prior to and after nanoparticle injection (5 mg Fe/kg) to assess infarct size and myocardial viability.

Results: 3D MR Imaging using a successive contrast agent injection showed a difference of infarct size after Gd-DOTA injection on

T1-weighted images compared to the one measured on T2-weighted images after Gd-DOTA and nanoparticle injection.Conclusion: The use of 3D T1- and T2-weighted MR Imaging using a double contrast

agents protocol made possible the accurate characterization of myocardial infarction volume and allowed the detection of myocardial viability post-infarction in rats.

Keywords 3D MRI·Myocardial infarction·Viability·Contrast agents·Rat

Introduction

The development of high resolution cardiac MR imaging associated to contrast agents allowed investigators to evolve new preclinical methods for the detection and quantitation of myocardial infarction and to evaluate myocardial viability. Indeed, these viable territo- ries which present transient ischemic dysfunction are of major interest because they are known as predictor

parameters for outcome and survival if reperfusion occurs [1–3]. The MRI mapping of those potentially salvageable peri-infarction zones has been assessed by the use of a protocol using two different contrast agents in terms of distribution with, on one hand, the combination of necrosis-specific porphyrin-based and extracellular nonspecific contrast media [4, 5] and on the other hand the combination of a non-specific diffusive positive T1 contrast agent (Gd-DOTA) and of a vascular negative T2 contrast media

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(superparamagnetic iron oxide nanoparticles) [6]. These studies and most of MR cardiac studies were performed using monoslice or multislice 2D techniques but 3D MR techniques are required for covering the complete heart and for accurately characterizing the myocardial infarction, allowing quantitative measurements of necrosis and viable areas.

However, the approach using two contrast agents different in terms of distribution and in terms of contrast requires both T1- and T2-weighted 3D MR acquisition of myocardium. T1-weighted MR imaging is currently performed both in humans [7–10] and in animals [11, 12] but T2-weighted 3D MR imaging of myocardium or vessels is less common.

The purpose of this study was to characterize accurately the myocardial infarction and evaluate the myocardial viability post-infarction using in vivo 3D T1- and T2- weighted MR imaging in rats associated to a double contrast protocol using Gd-DOTA injection followed by iron oxide nanoparticle injection in the same imaging session.

Methods

MR contrast media

SPIO nanoparticles were prepared and puriﬁed as previously de- scribed [13]. The average size of the magnetite crystal was 12 nm and the hydrodynamic diameter of the particles was 50 nm.

Magnetic relaxivities at 7 T were:r1=1.2 mmol⁻¹l⁻¹s⁻¹and r2=247 mmol⁻¹l⁻¹s⁻¹[14]. Relaxivities of commercial Gado- linium-DOTA (Dotarem, Guerbet, France) also used in this study werer1=2.6 mmol⁻¹l⁻¹s⁻¹andr2=4.9 mmol⁻¹l⁻¹s⁻¹ at 7 T [14].

Experimental protocol

Animal care and use was in strict accordance with the French Ministry of Agriculture regulations. Female Wistar rats weigh- ing 250±30 g were anaesthetized by spontaneous inhalation of a mixture of isoﬂurane (5–1.5%) and oxygen (3 l/min.). Perma- nent ligature with surgical suture of the anterior branch of the left coronary artery was performed according to Selye [15].

Five hours after coronary ligature, six rats received an IV injection of Gd-DOTA (0.2 mmol/kg) followed, 20 min later, by an IV-injection of SPIO nanoparticles (5 mg Fe kg⁻¹), both administered via the tail vein using two 27 G catheters.

MR imaging

In vivo MR imaging experiments were performed on a Bru- ker Avance DRX 300 equipped with a vertical superwide-bore magnet operating at 7 T (maximum shielded gradient strength 144 mT/m, birdcage resonator of 64 mm diameter).

Rats were anaesthetized with the same procedure used for surgery. ECG was recorded with a set of two AgCl electrodes

(KLEAR-TRACE, CAS Medical Systems) connected to an electrocardiographic monitor (Rapid Biomedical, W ¨urzburg, Germany) to provide a trigger pulse at the R-wave signal. Scout Gradient echo images were acquired in order to obtain the short- axis plane of the heart.

3D T1- and T2-weighted sequences were ECG triggered and the trigger delay was chosen to obtain diastolic images in order to minimize heart’s motion artifacts. A ﬁeld-of-view (FOV) of 30×30×15 mm and a matrix size of 64×64×16 were employed leading to an in-plane resolution of 468 and 937µm in the third dimension.

3D T1-weighted gradient echo sequence was performed using a minimum TR = 10.3 ms, a TE = 2.2 ms, a ﬂip angle of 30–50^◦adjusted to provide the best contrast between myocardial wall and cavity, and four averages. 3D T2-weighted RARE sequence was performed using a minimum TR of 1,000 ms, an effective TE of 32.5 ms with a train of 16 echoes and 4 averages.

The acquisition time was close to 8 min for the T1-weighted sequence and 4 min for the T2-weighted sequence, depending on the heart rate of the animal.

Reference T1- and T2-weighted images were collected prior to any contrast agent injection. Then T1-weighted images were acquired 15 min after Gd-DOTA injection to correspond to the Gd late-enhancement phase with the highest contrast-to-noise ratio between infarcted and non-infarcted tissue. Afterwards, T2-weighted images were performed prior to and just after nanoparticle injection.

MR image analysis and statistics

Images were analyzed using the Paravision 2.1 software (Bruker, Wissembourg, France). Left ventricle volumes were determined manually on all images and infarct volumes only on post-contrast short axis images using a window leveling. Infarct sizes were calculated as the percentage of infarct volume to the left ventricular wall volume and data were expressed as mean ± SEM (standard error of the mean). Inter- and intra-observer analyses were performed to assess the reproducibility of volume measurements.

Volume variations were compared by repeated-measures analysis of variance. Values ofP <0.05 were considered statis- tically signiﬁcant. Maximum intensity projections (MIP) were reconstructed from post-contrast images zero-ﬁlled to 128× 128×64.

Postmortem measurements

Hearts were sliced into 2-mm-thick sections and were incubated in 2% triphenyltetrazolium chloride (TTC) in phosphate buffer (pH 7.4) for 10 min at 37^◦C. The size of the infarcted region was measured as the percentage of left ventricle on TTC stained images and was compared to MRI measurements.

Results

Figure 1 shows 3D MR images of rat myocardium 5 h post permanent ligature of the left coronary. According to the

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Fig. 1 Short axis, sagittal and coronal view images of myocardium prior to contrast agent injection on

(a) T1-weighted and (b) T2-weighted images and (c) following Gd-DOTA injection on T1-weighted images and (d) following Gd-DOTA and nanoparticles administration on T2-weighted images. Thedotted lines represent the epicardic and endocardic outlines. Thearrows point at the infarct area on post-Gd-DOTA images

volumetric acquisition, data can be reformatted along any axis to improve visualization and volumetric analysis of myocardial infarction. Left ventricle at end diastole was visualized in the short and long axis views prior to any contrast agent injection on 3D-T1 (Fig. 1a) and 3D-T2- weighted images (Fig. 1b). Injection of Gd-DOTA and nanoparticles improved the visualization of myocardial infarction on 3D T1 (Fig. 1c) and T2-weighted images (Fig. 1d).

Intra- and inter-observer variabilities for T1- and T2- weighted MRI and TTC infarct and left ventricle wall measurements are given in Table 1.

Measurements of left ventricle wall volumes were consistent on T1 and T2 images prior contrast agent injection

with a variation of 5.8±1.9% (P=0.25) which reached 14.4±2.0% (P=0.29) after contrast agent injections.

Difference between the infarct volume measurements de- ﬁned on T1 and T2 images after contrast agent injection reached 24.5±3.6% (P=0.005) (Table 2).

Figure 2 shows the T1 and T2 MR infarct volumes versus TTC infarct volumes. Although no statistical evidence has been demonstrated, dependence can be observed between the infarct volumes measured using T2-weighted images and TTC (Fig. 2), TTC sizes always being under- estimated compared to MR sizes. No clear dependence was point out between T1 measurement and TTC.

An overestimation of infarct volume was observed on post-contrast T1-weighted images compared to the

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Table 1 Reproducibility of measurements of the left ventricle wall and infarct volumes: intra- and inter-observers agreements between measurements performed using pre-injection MR T1-weighted (MR pre-T1), pre-injection MR T2-weighted (MR pre-T2), post-Gd-DOTA injection T1-weighted (MR post-T1), post-Gd-DOTA and nanoparticle injection T2-weighted images (MR post-T2) and TTC staining post-mortem images (TTC)

Intra-observers Inter-observers

Left ventricle Infarct Left ventricle Infarct

MR pre-T1 2.7±0.8% X 12.7±3.4% X

MR pre-T2 4.6±1.2% X 15.7±2.4%* X

MR post-T1 12.3±4.3%* 7.3±1.4% 12.7±2.4%* 8.7±2.8%*

MR post-T2 16.0±5.8%* 7.5±4.3% 18.0±4.1%* 18.1±4.6%

TTC 1.1±0.3% 2.7±0.7% 21.3±7.6% 22.1±3.8%

*P <0.05

Table 2 Coherence of measurements of the (a) left ventricle wall volumes performed using MR T1- and T2-weighted pre- and post-contrast images and (b) infarct volumes performed using post Gd-DOTA T1-weighted MR images compared to post-SPIO T2-weighted MR images

*P <0.05

measurements on post-contrast T2-weighted images for the three rats presenting the smaller infarct size using TTC measurements (Fig. 2). However, according to the difﬁculty to measure T2-post contrast left ventricle volume (Table 1), the infarct volumes expressed as voxel and not as percentage of left ventricle must be considered. In- deed, the volume of infarct enhanced following Gd-DOTA injection was always larger than the one demarcated after nanoparticle injection (from 10.49 to 61.34%) (Table 1).

Post-mortem analysis of histological slices showed two different animal groups: group I rats did not present transmural infarct whereas group II rats did (Fig. 3). The infarct volume defined on post-Gd-DOTA T1-weighted 3D data set were always larger than the values defined on the post-SPIO T2-weighted 3D data set (P=0.047) and this overestimation ranged from 20 to 51% (average 32.7%) in group I rats (non-transmural infarct defined by histology) and from 2 to 8% (average 4.8%) in group II rats (transmural infarct defined by histology) (Fig. 3). In the non-transmural group I (as illustrated on frame a, rat 2),

the post-Gd-DOTA enhancement pattern was inhomoge- neous due to regional differences in the contrast washout time.

Figure 4 illustrates the infarct delineation overestimation by Gd-DOTA compared to SPIO nanoparticles using maximum intensity projection (MIP) reconstruction. Indeed, the infarct appeared transmural and large on T1-weighted image post Gd-DOTA injection but non- transmural and reduced in size on T2-weighted image post SPIO nanoparticles injection.

Discussion

The present study shows the 3D assessment of the myocardial infarction using in vivo cardiac T1- and T2-weighted MR imaging at high magnetic ﬁeld in rats. The use of an extracellular contrast agent (Gd-DOTA) in combination with vascular superparamagnetic nanoparticles made

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Fig. 2 Myocardial infarction volume (expressed as % left ventricle wall volume) measured on T1-weighted images post-Gd-DOTA (aopened symbols) and T2 weighted images post-Gd-DOTA and nanoparticles (bclosed symbols) compared to the one measured by TTC staining (c)

post-mortem in six rats.Dotted linerepresents the equation of the ideal correlation between MRI and TTC measurements (y=x).Grey symbolsdifference (expressed as voxels) between infarct volume measurements on T1-w. and T2-w. images.

Short axis view images on (a) T1-weighted (b) T2-weighted and (c) TTC images in rat 1 (non-transmural infarct) and in rat 2 (transmural infarct)

possible to portray the infarct volume and to highlight the difference of tagging of those two contrast agents in the myocardium after ischemia.

3D cardiac MR imaging has several advantages compared to 2D MR assessment of myocardial infarction as the possibility to reformat the data along any axis for improved visualization (Fig. 1) and volumetric analysis (Figs. 2, 3) and to include a higher signal-to-noise ratio

per acquisition time than multislice technique and a higher possible resolution in the third dimension.

To date, only few 3D T1-weighted studies in rodents, mainly in mice, have been performed to explore anatomy such as coronary vessels [11] or atherosclerotic plaque [12].

These studies presented images with a higher spatial resolution than the one used in the present study but required longer acquisition times (respectively, 60 and 30 min). As

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Fig. 3 Difference between infarct volumes deﬁned from 3D-T1 weighted images and 3D-T2 weighted images as a function of his- tologically deﬁned volumes. (group I: non transmural infarcts, group II: transmural infarcts; infarcts are delineated bydotted lines)

Fig. 4 Reconstruction from 3D data using maximum intensity projection.aReconstruction from 3D T1-weighted sequence post- injection of Gd-DOTA showing a massive and transmural infarct.

bReconstruction from 3D T2-weighted sequence post-injection of Gd-DOTA and SPIO nanoparticles where theblack areawithin the highlighted area on frame (a) revealed a vascularized area (1trans- mural infarcted area; 2non-transmural infarcted area Gd-DOTA enhanced;3vascularised area targeted by SPIO nanoparticles)

the aim of our study was to assess the myocardial viability using contrast enhanced imaging, the acquisition time had to be kept short enough to perform the double contrast protocol including four MR sequences and two contrast agent injection.

Double contrast protocols using either a combination of necrosis-specific porphyrin-based contrast agents and nonspecific Gd-DTPA [4] or the combination of vascular SPIO and diffusive Gd-DOTA [6] have shown the ability to define peri-infarction zone after myocardial infarction. In- deed, the injured but still vascularized peri-infarcted zone is not only targeted by the diffusive Gd-DOTA, but also

by SPIO particles which allow detailed visualization of the non-perfused myocardium [16, 17].

The reproducibility of our method was validated by an inter and intra-observers analysis of left ventricle wall and infarct volumes measurements (Table 1). The coherence between left ventricle wall volume measurements using T1- and T2-weighted pre- and post-contrast images was in reasonable agreement, except for T2-weighted post- injection images versus the other contrast images, epicardic and endocardic contours being very difﬁcult to deﬁne on T2-post contrast images due to the absence of signal in the still vascularized myocardium (Table 2).

The poor correlation between TTC staining and T2 infarct volume measurements was explained by the difﬁ- culty to determine accurate volumes especially for TTC staining due the slice thickness. Besides, the number of animals would have to be increased to establish statistical results. However, measurements determined from T1 and T2 data (Table 2, Fig. 2) showed a difference of infarct volumes measured using T1-weighted images following Gd-DTPA injection compared to the ones measured using T2-weighted images post SPIO nanoparticle injection.

Due to the vascular biodistribution of the SPIO nanoparticles and to the extracellular diffusion of the Gd-DTPA, that difference may correspond to the presence of suffer- ing but still vascularized myocardium in peri-infarction zone and appeared to be dependant on the infarct extent (Fig. 3). Indeed, the difference reached ca 32% in rats with non-transmural infarcts (group I) whereas it was un- changed (ca 5%) in rats with transmural infarcts (group II). Such a result is consistent with previous studies [18, 20]

which showed that most of the ischemic myocardium in large infarcted hearts is irreversibly injured. However, it is not excluded that the blooming effect associated to SPIO nanoparticles on T2-weighted images may be responsible, in part, for the larger myocardial areas tagged by SPIO nanoparticles.

In conclusion, the use of 3D T1- and T2-weighted MRI associated to a double contrast protocol using diffusive gadolinium chelate and vascular SPIO nanoparticles facil- itated volumetric characterization of myocardial infarction and allowed the detection of a signiﬁcant difference between both techniques. The MIP reconstruction from 3D data clearly showed the larger area enhancing in T1- weighted imaging after Gd-DOTA injection compared to the area that did not show a signal drop in T2-weighted imaging after injection of the intravascular nanoparticles, especially when non-transmural infarcts were addressed.

AcknowledgementsThe authors thank M. Moreau for synthesis of iron oxide nanoparticles, J. Roux and D. Gilbert for the housing and care of the animals used in this study. Sources of support: This work is part of the “Imagerie du Petit Animal” project supported by grant 8BG02H from CNRS-INSERM and C. Chapon is supported in part by a grant from “Angers Agglom´eration D´eveloppement”.

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