EXPERIMENTAL

MR relaxometry and perfusion of the myocardium in spontaneouslyhypertensive rat: correlation with histopathology and effectof anti-hypertensive therapy

Jérôme Caudron & Paul Mulder & Lionel Nicol &Vincent Richard & Christian Thuillez &

Jean-Nicolas Dacher

Received: 18 November 2012 /Accepted: 16 January 2013 /Published online: 17 April 2013# European Society of Radiology 2013

AbstractObjectives To investigate myocardial relaxation times andperfusion values in spontaneously hypertensive rats (SHRs)at various stages of the disease, with or without anti-fibrotictherapy, and to correlate magnetic resonance imaging (MRI)findings with histopathological myocardial fibrosis and cap-illary density.Methods Five groups of rats underwent MRI at 4.7 T. Theywere either untreated or treated with an aldosterone-synthaseinhibitor. T1, T2 and T2* relaxation times were determinedand myocardial perfusion was quantified from an arterial spinlabelling sequence. MR relaxation times and perfusion valueswere compared with the fibrotic content and capillary densityof the myocardium obtained at histology after euthanasia.Results T1 values significantly increased during the courseof hypertensive disease, and correlated with myocardialfibrosis (R=0.71, P<0.001); T2 values also increased butwere weakly correlated with myocardial fibrosis (R=0.27,P=0.047). Myocardial perfusion and capillary density sig-nificantly decreased with hypertensive disease but they didnot correlate. Following prolonged treatment, we observed atrend associating T1 decrease and improved perfusion com-pared with untreated SHRs.

Conclusions Myocardial T1 and T2 values increase withhypertensive disease, whereas myocardial perfusion de-creases. The correlation between T1 values and collagendensity suggests that the former could be considered as anon-invasive marker of myocardial fibrosis.Key Points• MR is increasingly used to assess alteration in myocardialtissue content.

• MR relaxometry and perfusion can be assessed in ratswithout exogenous contrast agents.

•Myocardial T1 and T2 values significantly increase duringthe course of hypertensive heart disease.

• T1 values correlate significantly with myocardial collagencontent.

•Myocardial perfusion values decrease with hypertensivedisease.

Keywords Cardiac MRI . Myocardial fibrosis . MRrelaxometry . Arterial spin labelling . Spontaneouslyhypertensive rat

AbbreviationsASL Arterial spin labellingCMR Cardiac magnetic resonanceHF Heart failureLGE Late gadolinium enhancementLV Left ventricleSHR Spontaneously hypertensive rat

Introduction

Heart failure (HF), resulting from ischaemic or non-ischaemiccardiomyopathy, is one of the most important causes of deathworldwide [1]. Myocardial fibrosis [2–4] and angiogenesis [5]

J. Caudron : P. Mulder : L. Nicol :V. Richard :C. Thuillez :J.-N. DacherInserm U1096, Rouen, France

J. Caudron (*) : P. Mulder : L. Nicol :V. Richard : C. Thuillez :J.-N. DacherInstitute for Research and Innovation in Biomedicine,University of Rouen, 22 Blvd Gambetta,76183 Rouen, Francee-mail: jerome.caudron@chu-rouen.fr

J. Caudron : J.-N. DacherDepartment of Radiology, Rouen UniversityHospital, Rouen, France

Eur Radiol (2013) 23:1871–1881DOI 10.1007/s00330-013-2801-6

are key determinants of the onset and progression of HF. In thissetting, many therapies target the collagen cycle and are knownto inhibit myocardial fibrosis, mainly through their action onthe renin-angiotensin-aldosterone system [6, 7]. Therapeuticagents active on angiogenesis and/or arteriogenesis are alsopromising in this condition [8].

In clinical practice, assessing myocardial fibrosis andperfusion could be useful to better understand, diagnoseand manage HF patients, and various methods are availablefor that purpose. Histopathology is the reference method forthe evaluation of fibrosis. However, endomyocardial biop-sies carry the risk of severe complications, hence they arenot routinely performed in humans [9]. Many methods—such as biology (collagen-derived serum peptides) [10],nuclear medicine (targeted radiopharmaceuticals) [11],echocardiography (ultrasound backscatter) [12] or opticalimaging [13]—have been successively evaluated with vari-ous results. However, late gadolinium enhancement (LGE)is currently considered as the reference method for theevaluation of myocardial fibrotic content [4, 14]. Thus,cardiac magnetic resonance (CMR) imaging is more andmore commonly performed in the management of HF pa-tients, mainly for evaluating prognosis [15]. LGE sequencesare limited in cases of diffuse myocardial fibrosis [4], suchas that found in hypertension or aortic stenosis. In thesecases, T1 mapping sequences allow quantitative measure-ments of the T1 values and can be performed with orwithout injection or gadolinium (Gd) chelate. Previous invivo studies based on Gd-enhanced T1 mapping sequencesshowed that T1 relaxation times of the myocardium weredecreased in the case of diffuse fibrosis, both in humans [16,17] and animals [18]. On the other hand, ex vivo animalstudies showed that T1 relaxation time increased in thefibrotic myocardium [19–21], but this method has receivedfew investigations in humans [22] and, to our knowledge,no study has evaluated unenhanced T1 mapping in vivo andits possible correlation with histopathology. T2 relaxationtime is considered as a marker of tissue water content[23, 24], and quantitative measurements are currently eval-uated in the context of acute coronary syndromes [24, 25],whereas T2* is routinely used to detect myocardial ironoverload [26]. As far as we know, few studies haveevaluated T2 and T2* relaxation times in the context ofmyocardial fibrosis [27].

Finally, the evaluation of perfusion is of interest in mostcardiomyopathies [8] and could bring prognostic and thera-peutic implications. CMR allows a unique evaluation ofperfusion based on the arterial spin labelling (ASL) method[28], but the natural history of myocardial blood flow incase of diffuse fibrosis, as well as the effect of anti-hypertensive therapy, remain unknown.

In this in vivo animal study, we investigated myocardialrelaxation times and perfusion values in spontaneously

hypertensive rats (SHRs) at various stages of disease, withor without treatment by an anti-fibrotic therapy. Then, wecorrelated CMR findings with myocardial fibrosis and cap-illary density as evaluated by histopathology.

Materials and methods

This experimental investigation conforms to the EuropeanGuidelines for care and use of laboratory animals and wasapproved by the local animal experimentation EthicsCommittee.

Animal model

Spontaneously hypertensive rats are characterised by a pro-gressive diffuse myocardial fibrosis [29]. Five groups wereinvestigated, including 15 rats each:

& Group 1: Normotensive Wistar Kyoto (WKY) controls,12 weeks old, no treatment.

& Group 2: SHRs, 12 weeks old, no treatment.& Group 3: SHRs, 24 weeks old, no treatment.& Group 4: SHRs, 24 weeks old, 12 weeks of treatment.& Group 5: SHRs, 24 weeks old, 1 week of treatment, in

order to differentiate acute from chronic effects of theanti-hypertensive therapy on CMR parameters. Indeed,short treatment does not influence the collagen contentof the myocardium but could alter perfusion, while long-term treatment influences both collagen content andperfusion.

Anti-hypertensive therapy

In this study we used an aldosterone synthase inhibitor(FAD286, 10 mg/kg/day; Novartis, USA). Our previousresults have suggested that this treatment could be an effec-tive alternative to mineralocorticoid-receptor antagonists incongestive HF [30].

CMR protocol

All CMR examinations were performed on a small animal4.7-Tesla MRI system (Bruker, Ettlingen, Germany).

Animal preparation

General anaesthesia was performed using an intra-peritonealinjection of methohexital (50 mg/kg). Rats were positioned inthe supine position. A dedicated surface 1H transmitter andreceiver quadrature coil of 86 mm diameter was used.Cardiac synchronisation was obtained by insertion of twosubcutaneous electrodes in the animal fore-paws. Respiration

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was monitored using a pressure sensor placed under the abdo-men. The physiological body temperature was maintainedusing a pad with circulating hot water.

CMR sequences

& Myocardial perfusion and T1 mapping

Perfusion was evaluated on a short axis slice located at mid-left ventricular cavity, with an ASL sequence [28]. The se-quence is an electrocardiography (ECG)- and respiratory-gated spin labelling gradient echo that provides a high-resolution perfusion map.

Global and slice-selective inversion recovery T1 mapswere acquired. The T1 relaxation time observed after theslice-selective inversion recovery was modified when com-pared with the second measurement performed after globalmagnetisation inversion. The latter provides myocardial T1relaxation time cleared of the perfusion effect of theinflowing spins. For each of the two relaxation curvesobtained, a series of 32 gradient echoes was acquired aftereach inversion pulse (one echo per RR interval, saturationcorrection performed in the dedicated software). Acquisitionparameters were: TR=14 ms; TE=2 ms; flip angle approx-imately 15°; field of view=50×50 mm; matrix size 128×64;slice-thickness=3 mm; voxel size=0.4×0.8×3 mm3.Results are presented on three parametric images: globaland slice-selective T1 maps, and perfusion map (Fig. 1).Using a previously described quantitative model [28], myo-cardial blood flow was inferred.

& T2 mapping

We used prospectively ECG-gated spin-echo sequences.An initial black blood preparation with adiabatic pulse wasused to null the perfusion effect. A fat saturation was alsoapplied. Echo-times ranged 3–60 ms every 3 ms (20 echoes)with TR=1,500 ms, slice thickness=1.5 mm, field of view=50×50 mm, matrix size 128×256, voxel size=0.4×0.2×1.5 mm3. The slice position was the same as that of T1mapping. An automatic fitting was applied on the exponentialdecreasing signal curve and results were displayed on a para-metric image (Fig. 2).

& T2* mapping

T2* sequences were performed using prospectively ECG-gated gradient echo sequences. Black blood and fat saturationpreparations were applied. Echo times ranged 2.7–42.3 ms,every 3.6 ms (12 echoes). Other acquisition parameters were:TR=1,500 ms; flip angle=30°; field of view=50×50 mm; ma-trix size=256×256; voxel size=0.2×0.2×1.5 mm3. Slice posi-tioning was the same as for T1 mapping. An automatic fittingwas applied on the exponential decreasing signal curve obtainedand results were displayed on a parametric image (Fig. 2).

& Cine-MRI

To evaluate myocardial function (i.e. ventricular volumesand ejection fraction), we used the recently describedIntragate method [31]. This method does not require con-ventional ECG and respiratory gating. This is a modifiedFLASH sequence with an in-slice navigator echo for retro-spective gating, derived from the half-echo following theslice selective pulse, as described by Heijman et al. [31].Cardiac cycle images are retrospectively reordered and al-low cine images without any cardiac or respiratory motionartefacts. The operator determines the number of cardiacphases prior to the acquisition. In this study, we used 25phases per RR interval and seven short-axis slices. Otherparameters were as follows: TR=68.5 ms; slice thickness=1.6 mm; field of view=55×55 mm; matrix size=256×256;voxel size=0.2×0.2×1.6 mm3.

Data analysis

A single operator blinded to animal age and treatment groupsprocessed all images. Perfusion and relaxation time maps wereprocessed with dedicated software (Paravision 5; Bruker,Ettlingen, Germany). Global region of interest (ROI) was man-ually drawn on parametric images. Functional measurements(left ventricle [LV] volumes, mass and ejection fraction [EF])were obtained with the CAAS software (CAAS MRV FARM2.0.1; Pie Medical Imaging, Maastricht, The Netherlands) andwere calculated after semi-automatic delineation of the endo-cardial and epicardial borders of the LV.

Left ventricular haemodynamics

At the end of the determined periods, the surviving rats ofeach group were anaesthetised with sodium methohexital(50 mg/kg i.p.). The right carotid artery was cannulated witha micromanometer-tipped catheter (SPR 407; MillarInstruments, Houston, USA) for the recording of arterialblood pressure, and the catheter was then advanced intothe LV for the recording of LV pressures, maximal andminimal dP/dt, and the relaxation constant Τ [32].

Histopathology

All animals were euthanized after cardiac catheterisationand the heart was immediately explanted. Right and leftatria and ventricles were weighed separately and a sectionof the LV was immersed in fixative solution. After fixation,the sections were dehydrated and embedded in paraffin.From these sections, 5-μm-thick slices were obtained atmid-LV level and stained with Sirius Red for quantitative eval-uation of collagen [32]. LV collagen densitywas determined andexpressed as the surface occupied by collagen divided by the

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surface of the image. Capillary density was determined andexpressed as the ratio of capillary/cardiomyocytes in the imagesurface as previously described [33]. A blinded operatorprocessed all images (Image Pro-Plus, AxioVision, or AdobePhotoshop image analysis software).

Statistical analysis

Data are presented as mean ± SD. The Student’s t-test (two-tailed) was used to compare two groups of independent

samples. For multiple comparisons, one-way analysisof variance was used followed, in case of significance,by a two-sided Student-Newman-Keuls test for pairwisecomparison of subgroups. Pearson coefficient of corre-lation was used to evaluate the correlation betweenCMR data and histopathology, i.e. relaxation timesvalues versus collagen density and perfusion versuscapillary density. Values of P<0.05 were consideredsignificant. All statistical analyses were performed usingMedCalc for Windows, version 11.3.2.0 (MedCalcSoftware, Mariakerke, Belgium).

Fig. 1 Representative short axis slice of T1 and perfusion maps with(a) a fitted curve representing longitudinal magnetisation signal inten-sity recovery versus inversion time; b T1 map after selective inversionrecovery; c T1 map after global inversion recovery from which T1

value of the myocardium was deduced; d perfusion map inferred fromT1 maps, the arterial spin labelling method allowing quantification ofmyocardial blood flow

Fig. 2 Representative short-axis slice of T2 and T2* mapswith (a) a fitted curverepresenting the decrease of thetransverse magnetisation signalfor T2 spin echo sequencesversus echo times; b T2 map; ca fitted curve representing thedecrease of the myocardialsignal with T2* gradient echosequences versus echo times; dT2* map

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Results

Inclusion rate

Twelve rats were investigated in group 1 (one animal diedimmediately after anaesthesia and anaesthesia failed in twocases); 14 rats in group 2 (one death due to anaesthesia); 14in group 3 (one failure of anaesthesia); 14 in group 4 (onefailure of anaesthesia); and 15 in group 5. No rat died afterCMR examination.

CMR data

Left ventricular function

CMR data are shown in Table 1. A progressive increase ofLVEF and LV mass was demonstrated in SHR during thecourse of hypertensive disease. Left ventricular volumes de-creased in 12-week-old SHRs but increased in 24-week-olduntreated SHRs. This late increase was prevented by long-term (12 weeks) but not by short-term (1 week) treatment.

Myocardial relaxation times

All data regarding relaxometry are shown in Table 2 andFig. 3.

T1 relaxation All T1 maps were interpretable. Paravertebralmuscles and blood pool T1 values did not differ among the

different groups. Compared with normotensive controls,myocardial T1 times were significantly increased in allSHR groups. Twenty-four-week-old SHR myocardial T1values significantly exceeded those of their 12-week-oldcounterparts. Neither short- nor long-term treatment didsignificantly affect myocardial T1 values. However, animalswith prolonged treatment tended to have lower T1 values(1,245±20 ms) than untreated 24-week-old SHRs (T1=1,258±18 ms, P=0.07).

T2 relaxation Paravertebral muscles T2 values did not differamong the different groups. Compared with normotensivecontrols, T2 values were not different in 12-week-old SHRs,but they were significantly increased in all 24-week SHRgroups (P=0.006). Neither short-term nor long-term treat-ment did significantly affected myocardial T2 values.

T2* relaxation Paravertebral muscles or myocardial T2*values did not differ among the different groups.

LV perfusion

All data regarding LV myocardial perfusion are summarisedin Table 2 and Fig. 3. A progressive significant decrease wasnoted in SHRs between 12 and 24 weeks of age. In com-parison to the controls (myocardial blood flow=8.10±0.55 ml/100 g/min), untreated 24-week-old SHRs had de-creased perfusion values (7.03±0.98 ml/100 g/min) (P=

Table 1 Left ventricular functional parameters by CMR

Controls SHRs 12 weeks SHRs 24 weeks SHRs 24 weeks SHRs 24 weeks12 weeks Untreated Untreated Long treatment Short treatment

LV volumes (μl)

LVEDV 413±46 354±51* 387±24 334±49*‡ 383±42

LVESV 156±21 120±24* 93±29*, ** 71±27*, **, *** 96±29*, **

Stroke volume 257±33 241±35 295±31*, ** 263±31*** 286±28**

LV volumes index (μl/g)

LVEDV 1.06±0.10 1.07±0.14 0.96±0.07*, ** 0.87±0.12*, ** 0.96±0.11 **

LVESV 0.40±0.05 0.36±0.07 0.23±0.07*, ** 0.18±0.07*, ** 0.24±0.07*, **

Stroke volume 0.66±0.07 0.73±0.10 0.73±0.09 0.68±0.07 0.71±0.07

LV output (ml/min) 96.8±11.1 81.3±10.6* 98.1±15.5 ** 87.4±8.8* 98.0±9.5**

LV output index (ml/g/min) 0.25±0.03 0.25±0.03 0.24±0.04 0.23±0.02 0.25±0.02

LVEF (%) 62±3 66±4 76±7*, ** 79±6*, ** 75±6*, **

LV mass (mg) 539±48 629±26* 829±73*, ** 725±60*, **, *** 829±70*, **

LV mass index (mg/g) 1.38±0.10 1.91±0.08* 2.07±0.24*, ** 1.88±0.18*, *** 2.07±0.14*, **

Data are expressed as mean ± SD

LV left ventricle, LVEDV left ventricular end diastolic volume, LVESV left ventricular end systolic volume, LVEF left ventricular ejection fraction

*P<0.05 vs controls, **P<0.05 vs untreated 12-week-old SHRs, ***P<0.05 vs untreated 24-week-old SHRs, for pairwise comparison whenANOVA test is positive

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0.013). This decreased perfusion was partly, but not signif-icantly prevented by treatment, whatever short or long.When compared with the untreated 24-week-old SHRs, thelong-treatment group showed a trend to higher perfusionvalues (7.44±0.67 ml/100 g/min, P=0.27). Finally, myocar-dial perfusion values did not correlate with the relaxationtimes of the myocardium.

Left ventricular haemodynamics

Haemodynamic data are summarised in Table 3. All SHRgroups had significantly elevated blood pressure when com-pared with the controls (P<0.001), with a progressive in-crease in diastolic and systolic blood pressure from 12 to24 weeks of age. Long-term treatment reduced systolic anddiastolic blood pressure in comparison to short-term treat-ment and no treatment groups, but not significantly.Relaxation and compliance abnormalities appeared in 12-week-old animals and worsened in 24-week-old rats. Longtreatment significantly improved these parameters in com-parison to untreated animals as well as those with shorttreatment.

Histopathology

Histopathology results are shown in Table 3 and Figs. 3, 4and 5.

LV hypertrophy

LV mass increased in all 24-week-old SHR groups whencompared with the controls (P<0.001). Compared with

untreated SHRs, long-term treatment significantly loweredLV mass.

Collagen density

Collagen quantification showed significant differences be-tween animals groups. In comparison to controls, hyperten-sion in SHRs was associated with a significant, time-dependent increase in collagen density, which was reducedby long-term treatment. A significant correlation was foundbetween T1 values and collagen density as evaluated byhistology (R=0.71, P<0.001) (Figs. 4 and 5). On the con-trary, T2 values correlated weakly with myocardial collagen(R=0.27, P=0.047) and T2* values did not (Fig. 4).

Capillary density

Capillary density decreased slightly but significantly duringthe course of hypertensive disease in SHRs without treat-ment and with short-term treatment (P=0.008). Long-termtreatment was associated with a reduction in capillary scar-city (Fig. 3). No significant correlation was found betweenthe capillary density and myocardial blood flow.

Discussion

Left ventricular function

Our MR study demonstrated an increased myocardial mass,concordant with the increased weight of the LVas evaluatedafter animal euthanasia. This increased LV mass was asso-ciated with decreased LV volumes and increased LVEF, all

Table 2 Relaxation and perfusion by CMR

Controls SHRs 12 weeks SHRs 24 weeks SHRs 24 weeks SHRs 24 weeks12 weeks Untreated Untreated Long treatment Short treatment

T1 (ms)

Myocardium 1,214±13 1,237±13* 1,258±18*, ** 1,245±20* 1,259±25*, **

Blood 1,711±31 1,693±19 1,695±29 1,702±28 1,712±36

Paravertebral muscle 1,377±29 1,379±20 1,380±17 1,379±20 1,385±23

T2 (ms)

Myocardium 31.5±0.7 31.9±1.6 32.9±1.1* 33.2±1.8* 33.0±1.1*

Paravertebral muscle 28.7±1.0 29.2±1.0 29.1±1.1 28.9±1.9 29.0±0.6

T2* (ms)

Myocardium 21.8±1.4 20.9±1.7 21.9±2.1 21.4±1.4 21.5±1.6

Paravertebral muscle 20.4±1.3 20.8±1.7 21.9±2.3 21.3±1.4 20.5±1.4

LV perfusion (ml/100 g/min) 8.10±0.55 7.84±0.82 7.03±0.98* 7.44±0.67 7.35±0.96

Data are expressed as mean ± SD

LV left ventricle

*P<0.05 vs controls, **P<0.05 vs untreated 12-week-old SHRs, for pairwise comparison when ANOVA test is positive

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features of concentric hypertrophy resulting from theafterload elevation of hypertensive heart disease. However,there was a trend showing elevated absolute LV volumes

and decreased LVEF in 24-week-old SHRs whether animalshad received no treatment or short treatment. This certainlyreflects an early alteration of LV function, which could

Fig. 3 Charts with bars representing for each group the mean valueswith 95 % confidence interval of: a T1 values; b T2 values; c T2*values; d perfusion values; e collagen density; f capillary density.Group 1=controls; Group 2=untreated 12-week-old SHRs; Group 3=untreated 24-week-old SHRs; Group 4=24-week-old SHRs with long

treatment; Group 5=24-week-old SHRs with short treatment. *P<0.05vs controls, †P<0.05 vs untreated 12 week-old SHRs, ‡P<0.05 vsuntreated 24-week-old SHRs, for pairwise comparison when ANOVAtest is positive

Table 3 Haemodynamics, pathology and histopathology results

Controls SHRs 12 weeks SHRs 24 weeks SHRs 24 weeks SHRs 24 weeks12 weeks Untreated Untreated Long treatment Short treatment

Body weight (g) 390±19 330±8* 400±26** 385±22** 401±10 **, ***

Haemodynamics

Heart rate (bpm) 386±19 416±21 399±39 398±36 402±36

SBP (mmHg) 114±9 199±15* 235±34*, ** 207±33*, *** 236±28*, **

DBP (mmHg) 85±20 146±10* 167±25*, ** 154±17* 171±16*, **

LVESP (mmHg) 119±17 183±15* 234±37*, ** 196±35*, *** 231±35*, **

LVEDP (mmHg) 0.3±0.2 1.6±0.6 5.3±2.5*, ** 2.7±1.0** 4.5±1.4*, **

Tau 3.2±0.4 6.7±0.6* 8.4±1.4*, ** 7.2±0.7*, *** 7.7±0.9*, **

LVEDPVR 0.4±0.2 1.5±0.5* 2.9±0.7*, ** 1.8±0.4*, *** 2.5±0.6*, **

LV weight (μg) 858±92 881±30 1,151±59*, ** 1,018±77*, **, *** 1,130±56*, **

Collagen density (%) 1.18±0.51 1.67±0.40 2.75±0.91*, ** 2.13±0.77* 2.65±0.88*, **

Capillary density 1.22±0.09 1.22±0.11 1.12±0.09*, ** 1.23±0.11 *** 1.13±0.08*, **

Data are expressed as mean ± SD

bpm beats per minute, SBP systolic blood pressure, DBP diastolic blood pressure, LVEDP left ventricular end diastolic pressure, LVESP leftventricular end systolic pressure, LVEDPVR left ventricular end diastolic pressure volume ratio

*P<0.05 vs controls, **P<0.05 vs untreated 12-week-old SHRs, ***P<0.05 vs untreated 24-week-old SHRs, for pairwise comparison whenANOVA test is positive

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ultimately lead to HF. CMR was able to demonstrate asignificant reduction of LV hypertrophy with long-termanti-aldosterone therapy. However, an underestimation ofLV mass by CMR was found in comparison to post-

mortem LV weight. This is probably related firstly to thefixed number of short axis slices used in this study (sevenslices) that was sometimes insufficient to cover the wholeLVand secondly to the fact that, when assessing LV systolicfunction, the LV papillary muscles were included in LVcavity (i.e. excluded from the LV mass).

Myocardial relaxation times

CMR LGE sequences are increasingly performed for eval-uating focal areas of fibrosis in the myocardium in patientswith either ischaemic or non-ischaemic cardiomyopathy[14, 15]. However, these sequences are limited in patientswith diffuse fibrosis. Indeed, LGE sequences are based oncontrast differences between normal and abnormal myocar-dium. Consequently, in case of diffuse disease, differentiat-ing normal from abnormal myocardium is uneasy with thespatial and contrast resolution of current systems. To avoidthis limitation, quantitative measurements of the relaxation

Fig. 4 Scatter diagrams illustrating the relationship between the col-lagen rate as evaluated by histopathology and a T1 values, b T2 values,c T2* values. Collagen rate correlated moderately with T1 values (R=0.71, P<0.001), weakly with T2 values (R=0.27, P=0.047), and didnot correlate with T2* values. Empty circles indicate controls; filledcircle untreated 12-week-old SHRs; empty triangles untreated 24-week-old SHRs; filled triangles 24-week-old SHRs with long treat-ment; squares 24-week-old SHRs with short treatment

Fig. 5 Representative examples of T1 map and associated histologicalimage (Picro-Sirius Red staining) in three rats: untreated 24-week-oldSHRs (top, T1=1,282 ms, collagen rate=3.2 %); untreated 12-week-oldSHRs (middle, T1=1,232 ms, collagen rate=1.6 %); control (bottom,T1=1,213 ms, collagen rate=1.2 %)

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times represent a new option [16–22]. In the present studywe have investigated the myocardial T1, T2 and T2* valuesin an animal model of diffuse fibrosis.

T1 relaxation

A significant increase of T1 values was found during thenatural history of hypertensive heart disease. These resultsaccord with previous ex vivo studies [19–21]. Interestingly,the range of T1 values found in ex vivo studies with a 4.7-Tsystem were close to our results [21]. Differences of T1relaxation times were maximal between Wistar and 24-week SHRs. A difference related to species could be arguedbut seems unlikely since the SHR lineage derives fromhypertensive Wistar rats that have been crossed together[29]. Moreover, T1 relaxation times increased within theSHR from 12 to 24 weeks of age.

T1 values correlated with the collagen content of themyocardium, in accordance with ex vivo studies. The rea-sons for such elevation could be related to both an increaseof the water content and collagen macromolecules, assuggested by ex vivo studies [19–21]. The increase of thewater content in old SHRs was previously reported [21] andis the consequence of various factors: firstly the progressiveleft ventricular hypertrophy associating increased extra-cellular volume and modification of the cellular components(cardiomyocytes growing scarce while myofibroblasts mul-tiply); secondly the chronic inflammation as a consequenceof microcirculation/angiogenesis changes in agreement withthe lowest capillary density found in the oldest SHR in thisstudy; thirdly the hydrophilic nature of the macromoleculesof collagen, increasing the water content of the myocardium.It should be noted that the significant correlation betweenT1 values and collagen content is certainly multifactorialand not specific for fibrosis. However, this drawback shouldbe weighted by the simplicity of this measurement in com-parison to the recently described methods involving Gdchelate injection [16–18]. Indeed, although the enhancedT1 mapping method theoretically allows the calculation ofthe extracellular volume fraction, this method has somelimitations: elevation of extracellular volume fraction doesnot only reflect an increase of the collagen content;relaxivity of the chosen contrast medium strongly influencesT1 values [34]; the effect of glomerular filtration rate re-mains unclear; finally routine application of the recentlydescribed state equilibrium method seems difficult [17].Consequently, even when taking into account the limitationsof the non-enhanced method, we believe that its simplicitycould be of potential interest in humans, as corroborated bya recently published study [35] demonstrating a significantincrease of myocardial T1 values in hypertrophic and dilatedcardiomyopathies, whether LGE was present or not.

T2 relaxation

As for T1 relaxation, a significant increase of T2 values wasfound during the natural history of hypertensive heart dis-ease, corroborating the results of ex vivo studies [19–21].Again, the range of T2 values was close to the values foundat 4.7 T ex vivo. Contrary to T1 relaxation, few data regard-ing T2 relaxation have been reported in the setting of myo-cardial fibrosis. Recently, Bun et al. [27] reported the valueof in vivo T2 measurement for myocardial fibrosis assess-ment in diabetic mice at 11.75 T. They found that myocar-dial T2 was significantly lower in diabetic mice than incontrols after 8 weeks of streptozotocin-induced diabetesand that there was a good correlation between T2 valuesand collagen content. The discrepancies between the resultsfound in our study/ex vivo studies and the study by Bun etal. could be related to multiple experimental differences:animal models (hypertensive rats vs diabetic mice), medica-tions (anti-hypertensive therapy vs streptozotocin induceddiabetes), heart rate (300–400 in rat vs 500–600 in mice),magnetic field (4.7 T in our study vs 11.75 T, with T2*effect clearly more pronounced at 11.75 T). Finally, theeffect of water content, discussed for T1 relaxation, alsoimpacts T2 relaxation and in none of the studies was eval-uated the water content of the myocardium. This will neces-sitate further research to precisely highlight the linksbetween in vivo MR relaxation times and collagen/watercontent of the myocardium.

Myocardial perfusion

Myocardial perfusion is a major determinant of HF progres-sion and therapeutics targeting angiogenesis and/orarteriogenesis could be advantageous in this situation inhumans [5, 8]. Thus an absolute quantification of myocar-dial blood flow could be important for evaluating the naturalhistory of the disease and the effects of therapeutics. TheASL method used in this study allowed quantitative assess-ment of myocardial blood flow without Gd injection [27,28] and to our knowledge this is the first study monitoringthe effect of anti-hypertensive therapy on LV perfusion.Progressive decrease of the LV myocardial perfusion wasfound during the natural history of hypertensive heart dis-ease. As early as 12 weeks of age, perfusion was decreased,but not significantly if compared with controls. In 24-week-old SHRs, the alteration of perfusion was significant butremained moderate. The capillary density was also signifi-cantly decreased during the course of hypertensive heartdisease. However, capillary density did not correlate withCMR perfusion values. This could be the consequence ofminor capillary scarcity in 24-week SHRs, and the effect ofvasoreactivity. An evaluation of the coronary reserve could

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be useful in this setting but was beyond the scope of thepresent study.

Effect of aldosterone-synthase inhibitor

In this experience we demonstrated the efficiency of a longtreatment by aldosterone-synthase inhibitor in preventingLV hypertrophy, fibrosis and capillary rarefaction. This re-sult was correlated by MRI, which showed less increased T1relaxation times and less decreased perfusion. On the con-trary, animals with a short treatment did not significantlydiffer from age-matched untreated SHRs.

Study limitations

Firstly, although significant differences were demonstratedfor T1 and, to a lesser extent, T2 values between the differ-ent groups, these differences were relatively minor and highstandard deviation values were recorded. Consequently,using relaxation times in individuals could not be discrim-inating enough. However, those limited differences could beexplained by an assessment performed too early in the 24-week-old SHRs. Forty-eight-week-old SHRs in a pre-HFstage could have shown more pronounced T1 and T2 dif-ferences with both controls and young SHRs.

Secondly, the effect of myocardial perfusion on T1 valuescan be discussed. However, calculation of myocardial T1values was based on a high-resolution T1 mapping sequencewith 32 different inversion times and a global adiabaticinversion pulse allowing to restrain the effect of perfusionon myocardial T1 values. Moreover, no correlation wasfound between myocardial T1 and perfusion in this study.Thirdly, we could not investigate Gd-enhanced T1 mappingsequences for maximal time of anaesthesia limited the num-ber of MR sequences. The use of T1 maps after Gd injectionallows evaluating the extracellular volume fraction of themyocardium using pre- and post-contrast T1 maps [17, 18].Comparison between unenhanced and enhanced methods inhypertensive heart disease requires further research.

Fourthly, as previously mentioned, we did not assess thewater content of the myocardium, which is clearly a majordeterminant of relaxation changes.

Fifthly, potential flaws in the T2 mapping protocolshould be mentioned: (1) motion artefacts could occur dueto the time of the echo train (60 ms) compared with themean heart rate period of rats (175 ms in this study); how-ever, the time of echo train was calculated to be at leastequal to twice the value of the T2, for better accuracy of themeasurement; (2) the TR (=1,500 ms in this study) could beconsidered too short to avoid signal variation arising fromlongitudinal recovery.

Finally, modifications of relaxation times are indirectmarkers of myocardial fibrosis. Specific contrast media couldbring added value but they cannot be used routinely [36, 37].

In conclusion, this study performed in spontaneouslyhypertensive rats demonstrated an increase of T1 and T2relaxation time values and a decrease of myocardial perfu-sion during the course of the hypertensive heart disease. T1values correlated with myocardial collagen content as eval-uated by histopathology. Moreover, the use of an anti-hypertensive therapy showed a trend to reduce myocardialT1 values and improve myocardial perfusion. Therefore,unenhanced CMR relaxometry and perfusion has potentialfor monitoring in vivo myocardial tissue changes in hyper-tensive heart disease.

Acknowledgments This work received a research award from theSociété Française de Radiologie, 2011 Annual Meeting

This work was presented as a scientific presentation during the 2011RSNA Annual Meeting (SSK03-03).

The authors are grateful to Novartis Institutes for BioMedical Re-search (East Hanover, New Jersey, USA) that provided the aldosteronesynthase inhibitor FAD286 used in the present study.

Supported by a research grant, “Médaille d’Or des Hôpitaux deRouen”.

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