Echocardiography, a non-invasive method for the assessment of cardiac function and morphology in preclinical drug toxicology and safety pharmacolo

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Echocardiography, a non-invasive method for the assessment of cardiac
function and morphology in preclinical drug toxicology and safety pharmacology
Article in Expert Opinion on Drug Metabolism & Toxicology · July 2008
DOI: 10.1517/17425255.4.6.681 · Source: PubMed
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Review
1.
Introduction
2.
The different modes
of echocardiography
Echocardiography, a non-invasive
method for the assessment of
cardiac function and morphology
in preclinical drug toxicology
and safety pharmacology
3.
Use of echocardiography
4.
Application of echocardiography
in preclinical safety
pharmacology and toxicology
5.
Value of echocardiography
in experimental toxicology
or safety pharmacology
as a method of refinement
6.
Conclusion
7.
Expert opinion
Gilles Hanton†, Véronique Eder, Gael Rochefort, Pierre Bonnet &
Jean-Marc Hyvelin
†Pfizer
Global Research and Development, Department of Toxicology and Comparative Medicine,
Z.I. Pocé sur Cisse, BP 159, F-37401 Amboise Cedex, France
Background: Echocardiography (EC) is a method used for the investigation of
cardiac morphology and function. Two-dimensional EC gives a visualisation
of the morphology of the heart. M-mode EC allows heart function to be
monitored. Pulsed Doppler EC is the method of choice to measure blood
flows. Objective: To describe the information EC can provide for cardiovascular investigation in laboratory animals, with a special focus on
the potential helpfulness of EC in preclinical toxicology and safety pharmacology. Methods: This review includes publications describing the methodology
of EC and its application to several animal species used in biological
experimentation. Results/conclusion: EC has been established in dogs,
monkeys, rodents, rabbits and pigs. As demonstrated by experiments in
different species, EC can be particularly helpful in toxicology and safety
pharmacology, based on the amount of information it can give on the
causes and consequences of drug adverse effects on the cardiovascular
system. Furthermore, EC does not require any surgery and is therefore a key
refinement compared to invasive methods generally used for investigating
the cardiovascular function in laboratory animals. Despite some limitations
of the method (the need for trained people, time required for an accurate
EC recording, lack of current validation), EC should be further developed
in preclinical toxicology and safety pharmacology.
Keywords: cardiac, Doppler, echocardiography, laboratory animal, toxicity, vascular
Expert Opin. Drug Metab. Toxicol. (2008) 4(6):681-696
1.
Introduction
Echocardiography (EC) is a method used for the investigation of cardiac
morphology and function [1]. A transducer is placed on the chest of the subject
and emits ultrasounds that are reflected by the cardiac structures and surrounding
tissues. The reflected ultrasounds are received by the transducer and then processed
by the echographic device in order to form an image on a screen. The fraction
of the ultrasounds that are reflected characterises the echogenicity and depends
on the physical properties of the tissues. Bones and air have a strong echogenicity
and appear in white on the screen. Liquids such as blood have a weak echogenicity
and appear as black areas corresponding to the cardiac cavities and lumen of
large vessels. Fibrous tissues and muscles have an intermediate echogenicity and
appear as grey structures corresponding to cardiac valves and the wall of heart
and main vessels.
10.1517/17425250802106271 © 2008 Informa UK Ltd ISSN 1742-5255
681
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Echocardiography, a non-invasive method for the assessment of cardiac function and morphology in preclinical drug
toxicology and safety pharmacology
5 MHz transducer
Figure 1. Schematic representation of bidimensional (2-D)
echocardiography in parasternal incidence, showing a
longitudinal section of the heart by the ultrasound beam.
Septum
Aortic valves
Aorta
Left ventricle
Mitral valves
Left atrium
Figure 2. Example of a 2-D longitudinal section of the heart
in a dog by 2-D echocardiography.
echographic device [2,3]. The transducer emits a planar beam
of ultrasounds in which the cardiac structures are visualised.
Depending on the position of the transducer on the chest of
the animal, different heart sections are obtained. By changing
the orientation of the transducer progressively, the operator
can scan the heart in successive sectors from which the walls,
the cavities, cardiac valves and main vessels are visualised.
The changes in their structures are followed over a number
of cardiac beats. An ECG that is recorded simultaneously
allows one to follow the cardiac cycle. 2-D images can then
be frozen either in ventricular systole, which correspond to
the beginning of the QRS complex (corresponding to
ventricle depolarisation) or in diastole, at the end of the
T wave (corresponding to ventricular repolarisation).
In 2-D sections, anatomical abnormalities can be detected
and a number of measurements can be taken. 2-D EC views
allow placing guidance lines for M-mode EC or Doppler EC
across cardiac structures.
The 2-D EC examination is usually performed in two
different incidences. In parasternal incidence, the transducer is
placed on the right side (dog and marmoset) or left side (rat and
mice) of the sternum of the animal, generally standing or
placed in a sling [2,4,5] and the cardiac structures are visualised
in two different sections. A long axis (longitudinal) section is
carried out across the left and right ventricles, left atrium and
aorta (Figure 1). This section is used for the examination of the
septum, free wall and cavity of left ventricle, mitral valves,
cavity and wall of the left atrium and the aortic root (Figure 2).
A short axis section allows visualisation of the heart in
successive transverse sections, from the apex to the upper part
of the heart (Figure 3). The section at the upper level allows
visualisation of the aortic and pulmonary artery trunk and
measurement of the pulmonary artery diameter (Figure 4).
For apical incidence, the transducer is placed on the
left side of the animal at the level of the cardiac apex [6].
The best images are obtained when the animal is lying
on its left side. The four cardiac cavities are visualised
simultaneously (Figure 5) and their areas can be measured
in systole and diastole. The volumes of the left and
right ventricles in systole (LVVs and RVVs) and diastole
(LVVd and RVVd) are calculated using the Simpson
method, which integrates the surfaces of the ventricles
areas from apex to valve plane. The stroke volume (SV)
is calculated by Equation 1:
(1)
There are three modes of EC, which are usually performed
successively for a complete examination of the cardiac
structures, their movements over time and blood flows.
2.
The different modes of echocardiography
2.1
Two-dimensional echocardiography
and the ejection fraction (EF) of the left ventricle is
derived from these volumes by Equation 2:
(2)
EF = SV / LVVd
Two-dimensional echocardiography (2-D EC) gives a view
of the morphology of the heart on the screen of the
682
SV = LVVd − LVVs
EF indicates the fraction of the ventricular diastolic
volume that is ejected at each beat and is therefore considered
Expert Opin. Drug Metab. Toxicol. (2008) 4(6)
Hanton, Eder, Rochefort, Bonnet & Hyvelin
D.
C.
PM
RV
LVO
LV
RVO
PMV
AMV
CH
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E
D
E.
C
B.
B
A
RV
TV
PPM
RA
LV
LA
RV
NC RC
LC
PV
APM
A.
LAu
RV
LV
Figure 3. Schematic representation of bidimensional (2-D) echocardiography in parasternal incidence, showing the different
levels of the transverse sections of the heart. Extracted from Thomas, 1984 [6].
RV
AO
PA
Figure 4. Transverse 2-D section of the upper part of the heart
in a marmoset, showing the right ventricle (RV), the pulmonary
artery (PA) and the aorta (AO). In this view, the Doppler window
can be positioned for recording the pulmonary flow.
as a key indicator of the ventricular contractile function. By
measuring myocardial and cavity area of the left ventricle, it
is also possible to evaluate its mass [7].
These calculation of ventricle volume from a single 2-D
section are based on assumption of their geometry, and the
accuracy of volume evaluation can be improved using
3-dimensional EC, in which the heart is scanned in successive
2-D sections and the volume computerised by ventricular
surface reconstruction [8,9]. The method is of particular
interest for the evaluation of volume and contractile function
of the right ventricle because its eccentric and complicated
morphology does not allow accurate calculation from an
unique 2-D section [10,11].
2-D EC imaging can be improved by the use of contrast
EC. Microbubbles which exhibit a high degree of echogenicity
are injected intravenously and enhance the ultrasound
backscatter of perfused tissues [12]. Contrast-enhanced EC
can be used to image blood perfusion in the heart. It
helps in detecting intracardiac shunts and valvular
regurgitation, in improving endocardial border detection
and in measuring cardiac output [13]. The method is
Expert Opin. Drug Metab. Toxicol. (2008) 4(6)
683
Echocardiography, a non-invasive method for the assessment of cardiac function and morphology in preclinical drug
toxicology and safety pharmacology
(5)
PWTI =(PWTd – PWTs)/PWTd
It is also possible to evaluate the volume (V) of the left
ventricle in diastole (LVVd) and systole (LVVs) using the
Teicholz formula (Equation 6).
LV
(6)
RV
V = 7D /(2.4 + D)
3
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RA
LA
Figure 5. Visualisation of the four cardiac cavities in 2-D
echocardiography, apical incidence in a marmoset showing,
the right ventricle (RV), the right atrium (RA), the left
ventricle (LV) and the left atrium (LA). In this view, the Doppler
window can be positioned for recording the mitral flow.
particularly useful for quantitative evaluation of myocardial
perfusion (Figure 6) [14,15].
2.2
Time-motion EC
In time-motion (M-mode), the position and movements of
the cardiac structures crossed by the guidance line are
displayed for a few cycles. In this way, M-mode EC allows
the observer to visualise changes in heart morphology over
the cardiac cycle and to monitor cardiac function [16].
M-mode EC is recorded from a 2-D view of the heart in
either short axis or long-axis section [5,17,18].
In a short axis section, the upper part of the ventricle is
visualised at the level of the papillary muscles close to the
chordae tendinae. The guideline is positioned between
the papillary muscles and the movements of the cardiac
walls and septum are recorded (Figure 7). Left ventricular
end-diastolic (LVDd) and end-systolic (LVDs) diameters are
measured at the time of maximum diastolic and minimum
systolic dimensions (Figure 8). Left ventricular fractional
shortening (LVFS) is calculated by Equation 3:
(3)
LVFS = (LVDd − LVDs)/LVDd
and gives an index of left ventricle systolic function.
Thicknesses of the interventricular septum (IVSd and IVSs)
and of the left ventricular posterior wall (PWTd and PWTs)
are measured in diastole and systole. The thickening index is
calculated for the septum (STI) and wall (PWTI) by
Equations 4 and 5:
(4)
STI =(IVSd – IVSs)/IVSd
684
where D = LVDd or LVDs, respectively, for LVVd
and LVVs. SV and EF can then be derived from LVVd and
LVVs [19]. Based on the dimensions of the left ventricle
cavity and thickness of its free wall and septum, the mass of
the ventricle can be estimated [7].
The mean slope of the systolic wave of the free wall of
ventricle is calculated between the onset and the peak of the
wave, whereas the maximal slope is measured as a tangent of
the wave at its onset (Figure 8). These slopes are further
indices of left ventricle contractile function.
In long axis section, an area of the heart giving a clear
longitudinal view of the cavities and walls, in particular
these of the left heart is selected, and the guidance line is
usually placed at two levels. For the evaluation of the
ventricular function and morphology, the guidance line is
positioned at the tips of the mitral valves and the movements
of the cardiac walls and septum are recorded. The same
parameters as those measured or calculated from M-mode
recordings in short axis section can be obtained from the
long axis section. In addition, the guidance line can be
placed in long axis section in the upper part of the heart,
across the aortic root and left atrium, for recording of their
movements (Figure 9). Aortic diameter is measured in systole
and diastole (ADs and ADd).
Depending on the quality of the short- or long-axis view,
the movements of the right ventricular can also be assessed.
The thickness of the right ventricle wall in diastole and
systole (RVTd and RVTs) and diameter of its cavity (RVDd
and RVDs) can be measured.
2.3
Doppler echocardiography
In pulsed Doppler EC, blood velocity is measured at the
level of a window selected in a 2-D section [20]. The spectrum
of distribution of the velocities of the red blood cells and
their variations over the cardiac cycle are recorded as
successive waves produced by the pulsatile flows. The
brightness of the spectrum indicates the number of red
blood cells at each velocity. The waves appear positive on
the screen when the blood is flowing to the transducer and
the waves are negative when the blood is flowing in the
opposite direction. By measuring the speed of blood motion
in vessels and cardiac cavities, pulsed Doppler EC allows the
assessment of flows patterns and, consequently, of the systolic
and diastolic cardiac function, pressure gradients across
valves and orifices, pressure changes in the cardiac chambers
Expert Opin. Drug Metab. Toxicol. (2008) 4(6)
Hanton, Eder, Rochefort, Bonnet & Hyvelin
A.
B.
S
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LV
D.
C.
LV
Figure 6. Contrast echocardiography (B and D) coupled with M-mode echocardiography (A and C) in rats. A. M-mode in a
normal rat, showing the movements of the septum (S) and left ventricle posterior wall (LV). B. Bidimensional contrast echocardiography
in the same rat as in (A), showing a longitudinal section of the heart with homogeneous myocardial perfusion (the whole heart appear
as grey). C. M-mode in a rat with experimental infarct (coronary ligation). Movements of septum and left ventricle wall are decreased.
A notching appears on the movements of the ventricle wall. D. Contrast echocardiography in the same rat as in (C). A black crescent
(arrow) can be seen on the apex of the heart, indicating an ischemic area.
Right ventricle
Septum
Mitral valve
Left ventricle
Left ventricle posterior wall
Figure 7. Transventricular M-mode echocardiography in a
dog. The guidance line has been placed at the level of the tip
of the mitral valves in a longitudinal section (Figure 2) or between
the chordae tendinae in a transverse section (Section C on
Figure 3). The M-mode recording shows the changes over time
in the right ventricle cavity, the interventricular septum, the
left ventricle cavity and posterior wall of the left ventricle.
and large vessels and vascular resistance. Physiological and
pathological changes in pulmonary, aortic and atrioventricular flows can be investigated [1,21,22]. Doppler EC is
also a reliable method for measurement of stroke volume
and minute cardiac output [23-25].
The atrio-ventricular flows are assessed with pulsed
Doppler from a four-cavity section obtained in apical
incidence. The Doppler windows are placed downstream of
the flows, below the mitral or the tricuspid valves (Figure 5).
The flows are recorded and two positive waves occur at each
cardiac beat (Figure 10). The rapid inflow E wave corresponds
to the passive filling of the ventricle occurring during its
diastole and is recorded during the isoelectric section of the
ECG, between T and P waves. The A wave corresponds to
the ventricular filling associated with atrial contraction and
occurs at the time of the P wave. Peak velocities (Vmax) of
E and A waves, their ratio (E/A), E wave deceleration and
the integral of velocity over time (VTI) of the two waves
together are recorded and serve as indices of ventricle
diastolic and/or atrial systolic functions. In particular, E/A
Expert Opin. Drug Metab. Toxicol. (2008) 4(6)
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Echocardiography, a non-invasive method for the assessment of cardiac function and morphology in preclinical drug
toxicology and safety pharmacology
STd
LVIDd
STs
PWVM
LVIDs
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PWVm
LVPWd
LVPWs
Figure 8. Schematic representation of a transventricular M-mode recording showing the different parameters measured.
(7)
SVPul ⫽ VTIPul ⫻ p ⫻ (DPul / 4 )
2
(8)
SVAo = VTIao × π × (D Ao / 4 )
2
The cardiac output (CO) is calculated by Equation 9:
(9)
AO
CO = SV × heart rate
LA
Figure 9. M-mode echocardiography of the upper part of
the heart in a marmoset. The guidance line is positioned across
the aorta and left atrium (Section E on Figure 3) The movements of
aorta (AO) and left atrium (LA) marmoset, are recorded over time.
gives an indication of the relative contribution of ventricular
diastole and atrial systole to the ventricular filling.
Pulsed Doppler EC is also used to assess arterial flows.
The aortic flow is recorded from a modified section in apical
incidence showing the four cavities and the aortic root. The
pulmonary flow is recorded from a short axis section of the
upper part of the heart, in right parasternal incidence (Figure 4).
The Doppler window for aortic or pulmonary flows is placed
just above (downstream of ) the sigmoid valves. For either
flow, a negative wave is recorded at each beat, from which
Vmax, VTI, pre-ejection time, acceleration time (AT) and
ejection time (ET) can be measured (Figure 11).
The SV is calculated from the pulmonary (SVPul) or
aortic flows (SVAo) by Equations 7 and 8.
686
In colour Doppler EC, the flows in the cavities and
great vessels are visualised in real time from a 2-D section,
based on a colour code. The flow appears in blue when
the blood is flowing toward the transducer or in red when
blood is flowing in the opposite direction (Figure 12).
The brightness of blue or red colour indicates the velocity
of the blood. Colour Doppler EC shows a number of qualitative blood flow changes, for example, laminar versus
turbulent flows or abnormal timing and location of blood
flows. Colour Doppler is therefore a useful tool for the
assessment of disturbed flow patterns associated with valve
insufficiency or stenosis [21]. On a four-cavity apical view of
the heart, the atrioventricular flows during ventricular
diastole and atrial systole and the arterial flows during
ventricular systole are observed by colour Doppler at each
cardiac beat. Images can be frozen and used to determine
abnormalities in the flow patterns.
Doppler tissue imaging evaluates the velocity of
the movements of the cardiac walls and valves and in
this way gives a direct evaluation of the cardiac contractile
function [26]. On an apical view of the heart in 2-D EC, the
Doppler window is placed on the free wall of the ventricle
at the level of the valve. On the tissue Doppler velocity
spectrum, three waves are identified [27]. The positive
wave (Sm wave) corresponds to ventricular systole and
Expert Opin. Drug Metab. Toxicol. (2008) 4(6)
Hanton, Eder, Rochefort, Bonnet & Hyvelin
ventricle. The lag between Sm wave and Ea wave evaluates
the time of isovolumic relaxation of the ventricle (TIVR)
and the lag between Aa wave and the following Sm
wave evaluates the time of isovolumic contraction (TIVC).
The Tei index is then calculated by Equation 10.
(10)
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E
(TIVC − TIVR )/Sm duration
A
Figure 10. Pulsed Doppler recording of the mitral flow in
a marmoset. Presence of two waves on the velocity spectrum:
E wave (E) during the isoelectric inter-beat interval of the ECG and
A wave (A) at the time of the P wave of the ECG.
and gives information on the global ventricular
performance [28,29]. The Q-Sm time measured from the
onset of Q wave of ECG to the onset of the Sm wave of the
tricuspid annulus is used to evaluate the electromechanical
coupling of the right ventricle [27].
In addition, the ratio of the amplitudes of the E wave of
the mitral flow measured in pulsed Doppler and of the Ea
wave measured in tissue Doppler (E/Ea) is a marker of the
left ventricle filling pressure and is frequently used in human
EC examination [30]. The E/Ea ratio can be applied in
animals, although its usefulness may be limited by the fusion
of the E and A waves of the mitral flow occurring as a
consequence of tachycardia, mainly in small species such as
rats and marmosets.
3.
b
d
a
c
Figure 11. Pulsed Doppler recording of the aortic flow in a
marmoset. Measurement of pre-ejection time from the Q wave
of the ECG (a) to the onset of the Doppler velocity spectrum
(b), acceleration time from the onset to the peak of the velocity
spectrum (c) and ejection time from the onset to the end of the
velocity spectrum (d).
two negative waves correspond, respectively, to the early,
passive filling of the ventricle due to its relaxation (Ea wave)
and to the late filling of the ventricle associated with atrial
contraction (Aa wave) (Figure 13). The ratio Ea/Aa is
calculated as an index of the diastolic function of the
Use of echocardiography
EC has been widely used in humans to investigate cardiac
physiology and pathology [31] and to evaluate the
pharmacological effects of drugs [32,33].
EC has a number of applications in laboratory animals, in
particular in dogs, but also rodents, rabbit and nonhuman
primates. The frequency of the ultrasound should be
adapted to the size of the animals. A 3.5 – 5 MHz transducer
is used in Beagle dogs and Cynomolgus monkeys [5,9,34],
7.5 – 15 MHz transducer in rats, guinea-pigs, rabbits
and small monkeys [35-38] and 15 MHz in mice [39].
In dogs, the method of EC recording is well established
and has been described in a number of papers, together with
values in normal subjects [4,6,18,20,40]. Changes in cardiac
dimensions have been followed in growing dogs with
M-mode EC [41], whilst Doppler parameters in normal dogs
and influencing factors have been established [42,43].
In canine or feline veterinary practice, EC is routinely
used and can assist in the diagnosis of cardiac morphological
alterations or dysfunction [2,17]. M-mode and Doppler EC
enabled testing the efficacy of ACE inhibition, digitalis
therapy, inodilation and venodilation in the treatment of
dogs affected by mitral regurgitation [44]. EC is also used as
an investigational tool in dog experimentation. In particular,
EC has several applications in evaluating impairment of the
cardiac function in canine models of heart disease and in
testing the beneficial effects of drugs. EC was applied in
canine models of partial left ventriculectomy or congestive
heart failure. In these animals, 2-D and M-mode EC allowed
the following of drug-induced increases in size and volume
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Echocardiography, a non-invasive method for the assessment of cardiac function and morphology in preclinical drug
toxicology and safety pharmacology
Figure 12. Colour Doppler image in an apical view of a
marmoset heart, showing in red the blood flow from left
atrium to left ventricle.
Sm
Ea
Aa
Figure 13. Doppler tissue imaging of mitral valve of a rat.
Three waves are identified and correspond to the movement of
the valve at the time left ventricle systole (Sm), ventricle diastole
(Ea) and left atrium systole (Aa).
of the cavities associated with a decrease in EF and in
fractional shortening [45-48]. Effects of enalapril in dogs with
doxorubicin-induced cardiomyopathy have also been assessed
using EC [49,50]. In dogs with myocardial failure, treatment
with milrinone at doses of 0.3, 0.5, 0.75 or 1 mg/kg
produced a marked improvement of left ventricle function
as indicated by a dose-related increase in fractional
shortening, which at the high dose reached ∼ 1.6-fold the
control value [51]. Using Doppler tissue imaging, it is possible
to evaluate the impairment of left ventricular diastolic
function in dogs with cardiac ischaemia, as indicated by a
688
decrease in Ea wave of the mitral annulus [52]. Contrast EC
was used to assess the changes in regional myocardial
perfusion in a dog model for coronary artery bypass
surgery [13]. Performance of contrast Doppler EC also
improved the measurement of haemodynamic changes
associated with aortic stenosis in boxer dogs [12]. Moreover,
EC is considered as a key method of investigation in
preclinical toxicology in dogs [5,34,53].
In monkeys, EC can be used to evaluate the cardiac
consequences of several diseases. Doppler EC showed
systolic and diastolic abnormalities of both ventricles in
baboons (Papio hamadryas) affected by Chagas disease [54].
Rhesus monkeys (Macaca mulata) infected with simian
immunodeficiency virus had changes in indices of ventricular
contractility derived from EC measurements, which
correlated with postmortem evidence of myocardial
pathology [55]. Development of cardiomyopathy in a colony
of owl monkeys (Aotus nancymae) was monitored through
echocardiographic markers of changes in size and contractile
function of the left ventricle [56]. Similarly, heart failure
associated with ageing in squirrel monkeys (Saimiri sp.)
was assessed through the changes in left heart dimensions
measured in 2-D EC [57]. The different modes of EC
were also shown to allow assessment of the effects of cardiovascular compounds or irradiation [9,58]. In particular,
3-dimensional EC allowed the demonstration of a doserelated decrease in EF in Cynomolgus monkeys treated
with the β-adrenergic blocker metroprolol (-19 and -24%
at 10 min after treatment with 0.1 and 0.3 m/kg, respectively,
when compared to pretreatment values). Less marked effects
were found in monkeys receiving 0.1 and 0.3 mg/kg of
the calcium channel blocker verapamil (-10 and -19%
change in ejection fraction, respectively) [9]. Based on EC
usefulness in primate experimentation, reference values
have been established in normal Rhesus [59] and
Cynomolgus monkeys (Macaca fascicularis) [60]. The
authors of this article have recently set up EC in
marmosets (Callithrix jacchus) [35] are considered promising
species for biological research, in particular in toxicology
and pharmacology [61].
The different EC modes have been established in rats
and extensively used for assessing changes in heart
structures and function in experimental models of
cardiac
disease
and
effects
of
pharmacological
interventions [62]. In particular, progressive impairment
of cardiac contractility and haemodynamic function
has been monitored in spontaneously hypertensive (SHR)
rats and in spontaneously hypertensive heart failure
prone (SHHF) rats [37,62].
Progressive impairment of pulmonary artery flow patterns
and right ventricle dilation occurring in rats with pulmonary
hypertension (PAH), induced by chronic hypoxia or
after monocrotaline treatment, have been determined
using pulsed Doppler and M-mode EC [27]. These methods
Expert Opin. Drug Metab. Toxicol. (2008) 4(6)
Hanton, Eder, Rochefort, Bonnet & Hyvelin
Table 1. Effects of candesartan and cilazapril on left ventricle function in a model of myocardial infarction in rats,
after 4 weeks of oral treatment: comparison of echocardiographic and invasive method data [68].
Ratio of mean values in treated animals versus controls
Candesartan
1 mg/kg/day
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Echocardiography
Cardiac catherisation
10 mg/kg/day
Cilazapril
1 mg/kg/day
10 mg/kg/day
FS
1.77
1.85
1.92
1.92
EF
1.29
1.39
1.32
1.32
E/A
0.31
0.30
0.33
0.29
LVEDP
0.32
0.36
0.27
0.27
E/A: Ratio of amplitudes of E and A wave of the mitral flow; EF: Ejection fraction of the left ventricle; FS: Fractional shortening of the left ventricle;
LVEDP: Left ventricle end diastolic pressure.
provided evidence for pulmonary outflow impairment, with
a midsystolic notch of the Doppler pulmonary spectrum,
a thickening of the right ventricle anterior wall, a decreased
acceleration time and a reduction of right ventricle fractional
shortening. Doppler tissue imaging was found to be an
accurate method for evaluating the right ventricle diastolic
dysfunction and the impairment of electro-mechanical
coupling of the right ventricle occurring in these PAH
models [27]. EC also allowed the investigation of the changes
in the left ventricle systolic and diastolic function associated
with myocardial degeneration in streptozotocin-induced
diabetic rats, together with the beneficial effect of sulindac,
an aldose reductase inhibitor [63,64].
A number of experimentations focussed on the use of EC
in assessing the morphological and functional cardiac changes
in rats with experimental myocardial infarction [65].
Measurement of infarct size with 2-D EC was in good
agreement with histopathological examination [66]. Pulsed
Doppler measurement of E/A and Doppler tissue imaging
measurement of Ea/Aa at the level of the mitral valve were
shown to accurately detect rats with an increased left
ventricle end diastolic pressure as a marker of the cardiac
failure resulting from myocardial infarction [67]. EC was
used to demonstrate the protective effects of the calcium
channel blocker diltiazem, the ACE inhibitor cilazapril and
the angiotensin II receptor antagonist candesartan against
cardiac dysfunction and morphological changes associated
with experimental myocardial infarction in rats [68,69].
In this model of myocardial infarction, the treatment
with cilazapril or candesartan for 4 weeks at doses of 1 or
10 mg/kg increased the left ventricle fractional shortening
and EF and decreased the E/A ratio of the mitral flow,
when compared to untreated rats [68]. This indicated that
both the systolic and diastolic functions were improved by
the treatment. The left ventricle end diastolic pressure
measured by cardiac catherisation (via carotid artery) was
markedly decreased in treated rats, indicating an improvement
of the pumping ability of the left ventricle. Therefore, there
was a good consistency between data obtain by EC or
by invasive methods (Table 1).
Doppler and M-mode EC monitoring also demonstrated
the beneficial effects of the ACE inhibitor perindopril in
preventing ventricular dilation and the decrease in cardiac
contractility and cardiac output occurring in an experimental
rat model of aortic regurgitation [70].
In mice, EC was also used in a number of experiments
for assessing heart function and structure [71] and was
demonstrated to correlate with invasive measures [39]. Left
ventricle mass of the mice has been estimated from
measurement of ventricle areas in 2-D EC or from
measurements of cavity and wall dimensions in M-mode,
the former method giving the most accurate data when
compared to postmortem measurements [7]. A combination
of M-mode, pulsed and colour Doppler EC allowed
investigators to determine the impairment in myocardial
function and intra-cardiac flows in cardiomyopathic
mice [72]. Effects of ketamine, xylazine, isoprenaline,
atropine and atenolol on cardiac contractility were determined
with M-mode EC for assessing the importance of changes
in autonomic balance during anaesthesia in mice [73].
As in other species, contrast EC is a method of choice
for evaluating myocardial perfusion in mice and to assess
the effects of vasodilators [74]. Using pulsed and colour
Doppler, it is possible to image the major branches of the
coronary arteries in mice and to measure corresponding
blood flows [75].
EC was less frequently applied in other laboratory animal
species than in dogs, monkeys, rats or mice. However, in
rabbits, pulsed and Doppler tissue imaging have been
established and can assess the depressive effects of xylazine
and ketamine on cardiac function [38]. Tissue Doppler also
gave an accurate evaluation of the myocardial function in a
model of adriamycin-induced cardiomyopathy in rabbits [76].
Pulsed Doppler and M-mode EC were used to assess the
changes in SV and ventricle contractility in rabbits with
experimental thrombo-embolism [77].
Expert Opin. Drug Metab. Toxicol. (2008) 4(6)
689
Echocardiography, a non-invasive method for the assessment of cardiac function and morphology in preclinical drug
toxicology and safety pharmacology
In guinea-pigs, impairment of cardiac function after
coronary ligation was successfully assessed by 2-D and
M-mode EC [36], which were applied for evaluating the
effects of the calcium-sensitizer levosimendan on systolic
and diastolic function [78].
The technique of EC has also been established in
mini pigs [79].
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4. Application of echocardiography in
preclinical safety pharmacology and
toxicology
Adverse effects on heart morphology and function may
occur with a number of drugs and have dramatic consequences or even lead to death. Therefore, it is critical
to have a thorough preclinical evaluation of the cardiac risk,
in which EC will find a number of applications [80].
EC is frequently used to assess toxicity of drugs in humans
and in particular, Doppler EC is the gold method to assess
the functional consequences of anthracycline’s cardiotoxic
effects [81,82]. In contrast, this technique has not yet been
routinely used for the preclinical toxicological evaluation of
drugs despite its potential interest, mainly in dogs [83].
Using 2-D EC, it is possible to visualise and evaluate
morphological changes induced by drug treatment, such as
myocardium hypertrophy or cardiac chamber dilation [84].
Furthermore, the functional consequences of treatmentinduced arrhythmia or cardiac lesions in laboratory animals
can be assessed by M-mode or Doppler EC. Changes
in haemodynamic parameters (SV and flows patterns) and
indicators of cardiac contraction (ejection fraction, fractional
shortening and velocities of cardiac structures movements)
would allow evaluation of the degree of cardiac function
impairment. Doppler EC and M-mode EC were used to
demonstrate the depression in cardiac contractile function
and haemodynamics produced by the anaesthetics, medetomidine and xylazine, in dogs [85]. These EC techniques were
also shown to be helpful in evaluating cardiac impairment
in dogs treated with doxorubicin [48,86,87]. In particular,
the treatment of dogs with 6 doses of doxorubicin at
3-week intervals produced a progressive decrease in mean
fractional shortening, which was reduced to ∼ 25%
of its initial value, at the end of the treatment and in the
mean E/A ratio of the mitral flow [85]. These changes
are consistent with a progressive impairment of the systolic
and diastolic function of the left ventricle. In dogs treated
with five doses, the changes in fractional shortening
were less marked than after six doses and no change were
seen in E/A ratio, showing a relationship between the
total cumulative dose and the impairment of the left ventricle
function. Moreover, when data from both dosing regimens
were analysed at the individual level, the magnitude
of the decrease in fractional shortening correlated well
with the degree of myocardial damage evaluated by
histopathological examination.
690
EC was also recently used to assess the effects of
dex-fenfluramine, an agonist of serotoninergic 2B receptors,
on PAH development in rats exposed to chronic hypoxia [88].
At the end of a 4-week treatment period, the compound
had no effect on PAH associated with the hypoxic conditions.
However, 2 weeks after the end of the dexfenfluramine
treatment, changes in EC parameters of pulmonary flow
and right ventricle function indicated an aggravation of
the PAH compared to control animals exposed to hypoxia
and treated with vehicle alone.
Colour Doppler EC was used to visualise tricuspid,
mitral, pulmonary and aortic regurgitant jets in rats with
valvulopathy produced by treatment with serotonin
or pergolide, a dopamine agonist. A good correlation was
found between regurgitation evaluated by colour Doppler
and valvular thickening measured at histopathology [89].
EC is also of prime interest in assessing the adverse
effects of cardiovascular drugs. Toxic effects of such drugs
on the heart or blood vessels are often due to exaggerated
pharmacological effects [90-92], resulting in marked changes
in the cardiovascular function. M-mode and Doppler EC
can quantify drug-induced changes in the patterns of cardiac
contraction, flows, SV and cardiac output and in this way
help to clarify the pathogenesis of cardiac or arterial lesions.
For example, M-mode and Doppler EC were used to assess the
cardiac effects of the potassium channel opener minoxidil and
the PDE3 inhibitors UK-61,260 and milrinone in dogs [93-96].
The changes in heart function and haemodynamics produced
by these vasodilatory and/or inotropic drugs help in understanding the mechanism of development of the left
ventricular and right atrial lesions that are well know adverse
effects of these compounds [97]. Major effects detected by
EC were a decrease in LVVd due to the decrease in
diastolic time associated with tachycardia and an increase
in fractional shortening and in EF characterising the reflex
stimulation of the cardiac contractility in response to
hypotension [93]. In addition, milrinone had a direct effect
on cardiac contractility [96], which explained an increase in
fractional shortening similar to that seen after minodidil,
despite a smaller increase in heart rate after milrinone,
indicating a lower compensatory cardiac stimulation. Overall
the changes were dose related and were consistent with the
pharmacological effects of the drugs (Table 2).
EC was also used to evaluate the effects of minoxidil
on the cardiac function of marmosets and in this way
to compare the pathogenesis of the cardiac lesion induced
in this species by a potent vasodilator with similar data
generated in dogs [98]. As in dogs, a major effect of treatment
on EC parameters consisted of an increase in fractional
shortening (1.44-fold increase 1 h after dose compared
to predose value) indicative of a compensatory cardiac
stimulation, after treatment with 150 mg/kg. The magnitude
of the change was similar to that produced by 2 mg/kg
of minoxidil in dogs (Table 2) showing that the marmoset
is less sensitive than the dog to the cardiovascular effects
Expert Opin. Drug Metab. Toxicol. (2008) 4(6)
Hanton, Eder, Rochefort, Bonnet & Hyvelin
Table 2. Effects on minoxidil [93] and milrinone [96]
on echocardiographic parameters in the dog.
Ratio of mean values recorded after a single dose
versus values recorded before treatment
Minoxidil
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Milrinone
Dose (mg/kg)
Heart rate
LVVd
FS
Control
1.08
0.95
1.08
1
2.13
0.73
1.30
2
2.08
0.66
1.55
Control
0.98
0.98
0.97
0.5
1.39
0.67
1.42
1
1.46
0.77
1.49
of this compound, although the pathogenesis of the cardiac
lesion is similar in both species.
5. Value of echocardiography in
experimental toxicology or safety
pharmacology as a method of refinement
In contrast to most methods presently used in animal
experimentation to investigate cardiac function or blood
flows, EC is non-invasive and does not necessitate surgery. It
requires only a gentle restraint and, in some species, sedation
or light anaesthesia. EC does not induce any pain and no or
minimal stress. It does not alter the cardiovascular function
that it seeks to measure [99]. Similarly, EC has no or little
interference with the measurement of other parameters
recorded in toxicity or pharmacology studies and has no
effect on the health status of the animal. EC measurements
are easily repeatable and therefore allow subsequent follow-up
in the same animal. However, rats and marmosets should be
sedated or slightly anaesthetised for EC recording, and the drugs
used for this purpose may have some effect on cardiac function
and/or blood pressure. Therefore, sedation or anaesthesia
may possibly interfere with the effects of the compound
tested in the toxicology or pharmacology studies.
6.
Conclusion
EC is well established in different species of laboratory
animals, in particular those used routinely in preclinical
toxicology. A number of experiments have demonstrated
that EC is a highly valuable tool for assessing the cause or
the consequences of drug adverse effects on the cardiovascular
system. In a number of examples, EC could replace invasive
methods and complement other cardiovascular investigations,
like ECG or histopathology.
7.
Expert opinion
EC is a key method for cardiologic examination in veterinary
and human medicine, including human toxicology. It was
extensively used in animal models of diseases, in particular
models of cardiomyopathy induced by toxic doses of
antracyclines. EC also provides a reliable tool for assessing
pharmacological and therapeutic effects of drugs in animals.
In contrast, EC has rarely been used in preclinical toxicology,
possibly because of some technical constraints. However, a
number of studies indicate that EC can accurately measure
drug effects on cardiac function, their dose-relationship
and time course. EC data are consistent with the known
pharmacological effects of the drug and correlate well
with invasive measurements. Moreover, a few experiments
demonstrated that EC has successfully assessed cardiovascular
adverse events in laboratory animals and have found a good
consistency between morphological changes evaluated by
EC and lesions seen at histopathological examination.
The currently available literature on EC applications in
humans and laboratory animals indicate therefore that this
technology can generate key data in preclinical toxicology.
Each EC examination provides a panel of quantitative and
qualitative information on drug adverse effects on cardiac
morphology and function. In particular, simultaneous recording
of changes in cardiac structures, contractility and haemodynamics
allow the establishment of correlations between the different
drug adverse effects and to follow the evolution of cardiac
toxic hits over time. Both the development and reversibility
of cardiac adverse effects can be determined. Moreover, EC has
a key ethical value, since it is a major refinement compared
to invasive methods generally used to assess drug effects on
the heart. We therefore strongly recommend developing EC
as an investigative tool in preclinical toxicology laboratories.
As EC recording investigates the same parameters of
cardiac function and morphology in laboratory animals and
humans, changes seen in preclinical toxicology can also be
investigated in humans. When changes in EC end points are
seen in animals, the clinicians can therefore select the
cardiovascular examinations to be done for assessing the cardiac
effects in clinical trials and ensure in this way the safety of
volunteers or patients. In addition, EC will help in understanding
the mechanism of development of cardiac lesions and in
distinguishing cardiac toxicity due to direct action of the
compound on the cardiac structures or function from adverse
events due to exaggerated pharmacological effects. In this later
case, the clinicians can monitor the volunteers or patients to
ensure that the changes in cardiac function causing the
lesions in laboratory animals do not occur in humans.
The different EC modes give complementary data and are
recommended to be used simultaneously for a full evaluation
of drug effects. 2-D EC gives information on heart structures
and allows an accurate calculation of ejection fraction.
M-mode gives further information on cardiac dimensions
and ventricular systolic function though calculation of
fractional shortening. Thus, EF and fractional shortening,
although derived from different EC modes, are both an
index of cardiac contractility and changes in these parameters
should be compared when assessing drug effects. Contrast
Expert Opin. Drug Metab. Toxicol. (2008) 4(6)
691
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Echocardiography, a non-invasive method for the assessment of cardiac function and morphology in preclinical drug
toxicology and safety pharmacology
EC indicating the changes in myocardial perfusion can be
associated to M-mode for evaluating the consequences of
ischaemia on cardiac contractility (Figure 6). Doppler tissue
imaging is an additional method for evaluating systolic
function and has the advantage of being relatively
independent of cardiac load [26]. Using pulsed Doppler it is
possible to calculate a number of parameters characterising
the flows through the atrio-ventricular and arterial orifices
and then to evaluate the haemodynamic consequences of
drug effects on myocardial function. Combination of
M-mode and Doppler EC can therefore be used either to
evaluate the consequences of drug-induced cardiac lesions or
to investigate the pathogenesis of cardiac lesions produced
by exaggerated cardiovascular pharmacological effects. Colour
Doppler gives a picture of the flow patterns and has a
particular interest for detecting disturbances resulting from
valvular abnormalities, which are serious adverse effects of a
number of drugs, in particular antiobesity compounds [100].
Contrast EC is the method of choice for evaluating changes
in myocardial perfusion and therefore can be used as an
application for assessing cardiac ischaemia produced by
overdoses of different drugs, in particular vasodilators [97].
However, for providing reliable and accurate results, EC
needs to be carried out by highly experienced and trained
people who frequently perform EC examination. Therefore,
some laboratories may consider it not worthwhile to ensure
continuous training of staff for EC recording. In this case, it
is possible to contract out EC examination in dogs to a local
veterinary cardiologist who routinely uses EC in his/her
practice. He/she can perform the EC examinations in the
toxicology facilities of the pharmaceutical laboratory and
provide data to the local scientists for interpretation.
However, even when performed by experienced people,
EC is overall less accurate than invasive methods and
some variability in the results can be found between different
experimenters or between different measurements of
the same experimenter. It is therefore critical to have all
EC examinations of a toxicity study carried out by the
same experimenter [101]. It is also important to repeat
the measurements at each experimental time point and
to calculate the mean. Each measurement should be taken
at least in triplicate and ideally in quintuplicate.
As a consequence of the number of parameters that can
be recorded and the need for repeated measurement at each
time point, EC is time consuming and a complete examination takes at least 20 min. It is therefore critical to adapt
the study design to the performance of the EC recording.
A staggered start of the drug treatment can generally allow
692
the investigator to perform all the scheduled EC examinations
in a study involving a large number of animals.
At present, EC has only been used in a limited number
of exploratory toxicity studies and if used in regulatory
studies, it should be considered as a non-validated add-on.
However, we expect that if the method is further developed
as an investigative tool in toxicology, it will enter a validation
process and will be recognised by authorities.
For accurate EC recording, the animal involved should be
quiet and motionless, standing or lying, depending on the
incidence of the ultrasound beam. EC can thus be easily
performed in dogs without any sedative medication, since
these animals accept handling and restrain during the
recording. Monkeys, rabbits and rodents should generally be
anesthetised or at least sedated, which may interfere with
the cardiac function and its sensitivity to drug action. It is
therefore critical to establish a sedation process that has the
minimum possible interaction with the cardiovascular
function. The optimal method of sedation remains to be
established and probably will vary in each species depending
on the strain, pre-existing cardiac disease or compound to
be tested [102]. EC examination without any medication in
primates and rodents will require training of the animals
and consequently be time consuming. Moreover, even when
EC recording is technically feasible on non-sedated animals,
the stress induced by restraint will probably affect the
measured cardiac parameters. EC recording in non-sedated
primates or rodents does therefore not appear to be
recommended in toxicity studies.
Despite some limitations, EC can therefore be considered
a key method for preclinical investigation of drug adverse
effects and has a major ethical value. Pharmaceutical
companies and contract research operations should develop
and validate EC in the laboratory animal species they use in
toxicology. Due to its technical constraints, EC can probably
not be recorded routinely in all toxicity studies. As a first
step, the method can be applied to exploratory studies
assessing the mechanism of development of cardiac lesions
or their consequences on the cardiac function. As a later
step, when the echocardiographic technology is adequately
validated, it could be used in regulatory studies, when there
is a possible risk of cardiovascular adverse effects.
Acknowledgements
We thank J Leaney for her critical review of the manuscript
and F Besse for his help in formatting the figures in
this article.
Expert Opin. Drug Metab. Toxicol. (2008) 4(6)
Hanton, Eder, Rochefort, Bonnet & Hyvelin
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Expert Opin. Drug Metab. Toxicol. (2008) 4(6)
Gilles Hanton†1 DVM DABT,
Véronique Eder2 MD PhD,
Gael Rochefort2 PhD, Pierre Bonnet2 MD PhD &
Jean-Marc Hyvelin2 PhD
†Author for correspondence
1Associate Research Fellow
Pfizer Global Research and Development,
Department of Toxicology and
Comparative Medicine,
Z.I. Pocé sur Cisse, BP 159,
F-37401 Amboise Cedex, France
Tel: +33 2 47 23 77 27; Fax: +33 2 47 23 79 39;
E-mail: [email protected]
2University François Rabelais,
LABPART, EA3852,
Faculty of Medicine,
IFR 135, Boulevard Tonnellé,
Tours, France
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