ORIGINAL PAPER

Non-invasive in vivo measurement of cardiac output in C57BL/6mice using high frequency transthoracic ultrasound: evaluationof gender and body weight effects

Elisabet Domınguez • Jesus Ruberte •

Jose Rıos • Rosa Novellas • Maria Montserrat Rivera del

Alamo • Marc Navarro • Yvonne Espada

Received: 21 January 2014 / Accepted: 16 May 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Even though mice are being increasingly used

as models for human cardiovascular diseases, non-invasive

monitoring of cardiovascular parameters such as cardiac

output (CO) in this species is challenging. In most cases,

the effects of gender and body weight (BW) on these

parameters have not been studied. The objective of this

study was to provide normal reference values for CO in

C57BL/6 mice, and to describe possible gender and/or BW

associated differences between them. We used 30-MHz

transthoracic Doppler ultrasound to measure hemodynamic

parameters in the ascending aorta [heart rate (HR), stroke

volume (SV), stroke index (SI), CO, and cardiac index

(CI)] in ten anesthetized mice of either sex. No differences

were found for HR, SV, and CO. Both SI and CI were

statistically lower in males. However, after normalization

for BW, these differences disappeared. These results sug-

gest that if comparisons of cardiovascular parameters are to

be made between male and female mice, values should be

standardized for BW.

Keywords Cardiac output � Mice � High frequency

ultrasound � Gender effect � Body weight effect

Introduction

The mouse has been increasingly used as model of human

cardiovascular diseases in the last years because of the

extensive knowledge of its genome and the availability of

mouse embryonic stem cell technology [1]. These advances

have led to the need for in vivo approaches to reliably

assess the murine cardiac function and anatomy. However,

the small size of mice limits the use of traditional imaging

devices in hemodynamic studies [2, 3]. Conventional

clinical ultrasound systems with frequencies up to 15 MHz

have been employed in cardiac studies in mice to calculate

cardiac dimensions, ventricular systolic and diastolic

functional parameters, and cardiac output (CO) [4–6].

However, their reduced B-Mode spatial resolution restricts

the visualization of small structures such as the ascending

aorta (AAo) [7, 8]. High-frequency ultrasound [referred as

ultrasound biomicroscope (UBM)] has been proved to be

useful in the evaluation of cardiac structures in mice due to

its high spatial resolution and adequate penetration [7–9].

Traditionally, invasive techniques (i.e. flow probes

positioned around the AAo) have been used in mice to

measure the CO [10]. However, these procedures disturb

the normal cardiac physiology, are technically challenging

and are not suitable for serial assessment [6, 11]. Other

E. Domınguez (&) � R. Novellas � M. M. R. del Alamo �Y. Espada

Departament de Medicina i Cirurgia Animals, Facultat de

Veterinaria, Universitat Autonoma de Barcelona, Edifici V,

Campus, 08193 Barcelona, Spain

e-mail: elisabet.dominguezm@gmail.com

J. Ruberte � M. Navarro

Departament de Sanitat i Anatomia Animals, Facultat de

Veterinaria, Universitat Autonoma de Barcelona, Barcelona,

Spain

J. Ruberte � M. Navarro

Mouse Imaging Plattform, Centre de Biotecnologia i Terapia

Genica (CBATEG), Universitat Autonoma de Barcelona,

Barcelona, Spain

J. Rıos

Unitat de Bioestadıstica, Facultat de Medicina, Universitat

Autonoma de Barcelona, Barcelona, Spain

J. Rıos

IDIBAPS (Institut d’investigacions Biomediques August Pi i

Sunyer), Hospital Clınic Barcelona, Barcelona, Spain

123

Int J Cardiovasc Imaging

DOI 10.1007/s10554-014-0454-4

advanced imaging techniques, such as cardiac magnetic

resonance imaging (MRI) allow the quantification of ven-

tricular volumes, and consequently of CO [12]. However,

MRI studies are time consuming, and performing serial

imaging studies of a large number of animals could be

difficult, and excessively expensive [13].

In human medicine, transthoracic echocardiography is

routinely used to calculate the CO [14]. Recently, a high

frequency echocardiographic approach to CO has also been

validated in mice [15]. This study demonstrated that CO

could be accurately estimated in mice using echocardiog-

raphy. Other authors have described the use of UBM in the

evaluation of CO and other cardiovascular parameters in

pregnant mice and mice models of atherosclerosis [16, 17].

The effects of age, strain, and genotype on CO values have

not been completely investigated in mice [17, 18]. On the

other hand, even if general scientific guidelines recommend

performing studies in animals of both sexes, most of the

research projects are conducted just in males [19–21].

Thus, the gender effect and the relationship between gender

and body weight (BW) are not always taken in consider-

ation when assessing the phenotype in genetically modified

mice [18]. This lack of information may lead to erroneous

consideration that differences observed are strain or gen-

ome related [21].

The objectives of this study were: (1) To evaluate the

use of high frequency Doppler ultrasound in the measure-

ment of hemodynamic parameters in the AAo of intact

anesthetized mice of either sex; (2) To provide normal

reference values for stroke volume, stroke index, cardiac

output, and cardiac index in mice; and (3) To describe

possible gender related differences in these parameters and

the effect of body weight in these differences.

Materials and methods

The experimental protocol was approved by the Ethical

Commission of Animal and Human Experimentation of the

Universitat Autonoma de Barcelona (authorization number

CEEAH 724/08; DMAH 4666). The study was conducted

in accordance with the guidelines of the Spanish Govern-

ment (RD 1201/05) and the European Union (Directive

86/609/EEC) on the protection of animals used for scien-

tific purposes.

Mice

Thirty two-month-old inbred C57BL/6 mice were included

in the study (13 males and 17 females; Servicio de Es-

tabulacion de Ratones, SER-CBATEG, Universitat Auto-

noma de Barcelona, Spain). Their body weights ranged

from 17 to 25 g. Mice were housed in a room under 12-h

dark/12-h light cycles, at constant temperature (20–21 �C)

and humidity (50–60 %). Animals were fed ad libitum with

a commercial standard diet (Teklad 2018S Harlan Teklad,

Blackthorn, UK) and had free access to water. Three male

mice were studied with micro-CT to assess the anatomic

relations of the ascending aorta in the thorax. Twenty-

seven animals (10 male and 17 female mice) were included

in the ultrasonographic study.

Micro-CT

With the aim to assess the best anatomic approach to the

ascending aorta, a pilot micro-CT study of the thorax was

conducted in three mice. These animals were injected with

0.05 ml intraperitoneal heparin (Heparina Sodica Rovi,

5,000 UI/5 ml. Laboratorios Farmaceuticos Rovi S.A,

Madrid, Spain). After 10 min, mice were placed in a closed

induction chamber and were euthanized with an overdose

of inhaled anesthesia (isoflurane, Isoflo� Abbot Laborato-

ries. Veterinaria Esteve, Barcelona, Spain) in an attempt to

avoid anatomical disturbances associated with other

methods of euthanasia such as cervical dislocation. After

that, an abdominal laparotomy was performed. The

abdominal aorta was cannulated and manually injected

with 2 ml of a solidifying silicone contrast agent (Microfil�

MV-122; Flow Tech, Carver, MA, USA). After polymeri-

zation at 4 �C for several hours, the mice were scanned

with a micro-CT eXplore Locus (GE Healthcare, London,

Ontario, Canada). The images were acquired at 45 lm

isotropic resolution, using settings of 80 kV, 450 lA and

2,000 ms. Volumetric images of the thorax and 3D volume

rendering were viewed and reconstructed using MicroView

2.2 software package (GE Healthcare, London, Ontario,

Canada).

Transthoracic echocardiography

A high frequency ultrasound system (Vevo� 770, Visu-

alSonics, Toronto, Canada) equipped with a 30 MHz

mechanical transducer (RMV-707B) was used. The

transducer had a central frequency of 30 MHz, focal

length of 12.7 mm, and frame rate of 50 Hz. The max-

imum field of view of B-Mode images was 20 9 20 mm

with spatial resolution of 115 lm (lateral) by 55 lm

(axial). The maximum Doppler pulse repetition fre-

quency was 96 kHz, corresponding to a maximum un-

aliased velocity of 120 cm/s. In this study, the axial

dimension of Doppler sample volume was less than

1 mm and Doppler angles were always kept under 50

degrees.

Mice were scanned between 9 and 11 AM without

previous fasting. Anesthesia was initiated in an induction

chamber with 3 % isoflurane in oxygen and was

Int J Cardiovasc Imaging

123

maintained with a face mask (1.5 % isoflurane). Animals

were placed in dorsal recumbency on a heating pad (SA-

11093 Mouse Plattform, Visualsonics, Toronto, Canada)

with the limbs taped to ECG electrodes for heart rate

(HR) and respiratory rate monitoring. Body temperature

was controlled via a rectal thermometer (Indus Instru-

ments, Houston, USA). The hair from the chest was

removed using a chemical hair remover (Nair hair

removal cream, Church& Dwight Canada Corp. Missis-

sauga, Ontario, Canada) and artificial tears (Viscotears�,

Novartis Farmaceutica, S.A. Barcelona, Spain) were

applied over the cornea to avoid dryness. A pre-warmed

ultrasound gel (Transonic-Gel�, Telic S.A. Barcelona,

Spain) was spread over the chest to provide a coupling

medium. The ultrasonographic study was initiated after

stabilization of the mice, when the heart rate was stable

over 400 bpm.

A pilot study was conducted in a group of seven female

mice to determine the best acoustic window to interrogate

the AAo. Two different views were tested: right and left

parasternal longitudinal approaches.

After that, the definitive ultrasonographic study was

performed in ten male and ten female mice. On B-Mode,

the lumen of the AAo was visualized by means of a long

axis view. The scan head was then rotated 90� to obtain a

short axis view of the vessel. A cine loop was recorded at

this level and a systolic frame was used to calculate the

diameter of the lumen (D, mm) 0.5–1.5 mm downstream of

the aortic valve. The area of the vessel (A, mm2) was

calculated applying the following formula [22]:

A = (p 9 D2)/4. Then, the long axis view of the AAo was

visualized again. The scan head and the heating pad were

regulated and orientated to obtain a Doppler angle below

50�. The uniform insonation technique (entire lumen of the

vessel incorporated in the sample volume) was employed

in the examination and characterization of the spectral

Doppler waveforms in the AAo [23]. The vascular protocol

available in the software of the UBM (Visual Sonics Car-

diac Measurements, Toronto, Canada) was used to record

numerical data from the Doppler waveforms. From

Doppler spectra, HR and VTI (mm) were extracted and

averaged over three cardiac cycles based on manual or

automatic mean velocity tracing. Stroke volume (SV, mm3.

SV = VTI 9 A), stroke index (SI, mm3/kg BW), CO (ml/

min. CO = SV 9 HR), and cardiac index (CI, ml/min kg

BW) were also calculated and averaged in three cardiac

cycles.

Statistical analysis

A descriptive analysis of the numerical ultrasonographic

data in both male (N = 10) and female (N = 10) mice was

performed (results expressed as mean ± SD). Inferential

analyses were made by a covariance analysis (ANCOVA).

These models were used to evaluate the differences

attributable to the gender, adjusted by the body weight and

the results are expressed as 95 % confidence interval of

mean, 95 % CI. Two different models were applied in this

analysis. Model 1 (dependent variable = l ? gender)

described the independent effect of the gender on each of

the dependent variables evaluated (D, A, HR, VTI, SV, SI,

CO, and CI). Model 2 (dependent variable = l ? gen-

der ? BW) described the dependent variables using gender

as independent factor adjusted by BW.

Intra-observer coefficient of variation for manual VTI

measurements was calculated from repeated measurements

performed by one observer. The average VTI over three

cardiac cycles was calculated twice in a static frame

obtained from a mouse on two different days. The same

protocol was applied on a total of ten mice. Variability was

expressed as the percentage of the absolute difference

between two measurements divided by the mean value of

the two measurements [24]. VTI Lin’s concordance coef-

ficient (LCC) and its corresponding 95 % confidence

interval (CI) was used as a measure of agreement between

two operator’s VTI measurements made offline with the

ultrasound machine software. Two operators calculated

VTI values independently in at least three cardiac cycles

for every mouse. The coefficient of concordance for VTI

was classified then as poor (\0.21), fair (0.21–0.40),

moderate (0.41–0.60), substantial (0.61–0.80), or almost

perfect (0.81–1.00) concordance.

The statistical package SPSS version 18 for Windows

was used considering a 5 % bilateral Type I Error. A

P value B0.05 was considered statistically significant.

Results

Figure 1 show the results of the pilot micro-CT study. The

AAo was identified on the right hemithorax running par-

allel to the sternum and thoracic spine. Based on the ana-

tomic information obtained with micro-CT, the right

parasternal longitudinal approach was considered the most

appropriate for the ultrasonographic scans. Long axis and

short axis B-Mode images were visually compared with

those obtained on CT to confirm the accurate anatomic

approach to the AAO (Fig. 2a–d).

The anesthetic protocol allowed to perform a proper

ultrasound examination in all mice, maintaining both HR

and body temperature under physiological values [25]. The

study was performed in a total of 26 animals (10 male and

16 female mice). A female mouse of the pilot study was

eliminated because of the impossibility to obtain a high

quality Doppler waveform in the AAo. Male BW was

26.12 ± 2.33 g and female BW was 18.29 ± 1.07 g.

Int J Cardiovasc Imaging

123

The right parasternal longitudinal ultrasonographic

approach was confirmed to be the most useful access

to image the AAo in both B-Mode and pulsed Doppler

ultrasound. No vascular or perivascular abnormalities

were observed in any of the scanned animals. The

luminal diameter of the AAo was 1.54 ± 0.13 and

1.56 ± 0.12 mm in male and female mice respectively.

The corresponding luminal areas were 1.88 ± 0.29 and

1.93 ± 0.29 mm2 in male and female mice

respectively.

Doppler waveforms were obtained with Doppler angles

below 458 in all the scanned animals. The AAo showed a

plug flow velocity profile with a high resistance flow pat-

tern (Fig. 2e).

Male HR was 448 ± 57 bpm and female HR was

446 ± 56 bpm. Table 1 shows the descriptive result values

(Mean ± SD) for D, A, HR, VTI, SV, SI, CO, and CI in

both genders.

Using the two statistical models previously described,

no statistically significant effects related to gender or BW

were observed in D, A, HR, VTI, SV, or CO (Table 2).

Using the first model, both SI and CI were statistically

lower in male than in female mice. However, after nor-

malization for BW, these differences disappeared

(Table 2).

Intra-observer coefficient of variation for manual VTI

measurement was 4.95 %. Lin’s concordance coefficient

for VTI values was classified as almost perfect (0.94; 95 %

CI 0.91, 0.96) between the two observers.

Discussion

Transthoracic echocardiography is one of the imaging

techniques most commonly used in phenotyping the car-

diovascular system in mice [26]. Different studies have

proven its usefulness and reliability, especially when

devices adapted to the small size and the normal physiol-

ogy of these animals are employed [7, 27].

Different non-invasive approaches have been proposed

to calculate the CO in mice [5, 15]. In this study, we pre-

ferred to perform measurements in the AAo rather than in

the pulmonary artery because this technique has been val-

idated in previous studies in mice and it makes possible to

obtain high quality images with Doppler angles below 45�in all cases [5, 8, 28]. Based on the results of the pilot study

performed in seven female mice, we decided to use the

right parasternal longitudinal view because it was consid-

ered the easiest and most reproducible in the assessment of

the left ventricle outflow tract [8]. On the other hand,

M-Mode ultrasonographic calculations of CO, based in the

left ventricular volume (LVED) at end diastole and end

systole (LVES) were avoided because overestimation of

CO has been noted when applying this formula [15].

The diameter of the AAo, used in the calculation of the

luminal area and therefore in the measurement of the stroke

volume, was similar to those reported in previous studies

using male mice of the same strain and similar age [29], and

did not differ from other studies on 1.5 month-old CD-1

mice [18]. Values of VTI in the AAo were similar to those of

Fig. 1 Micro-CT images after injection of Microfil. Ventral views.

a 3D reconstruction showing the approximated location where the

ultrasound transducer was placed to obtain the long axis or sagittal

(yellow arrow), and the short axis or transverse (green arrow)

sections. b Sagittal (yellow) and transverse (green) sections of the

mouse. 1 Ascending aorta, 2 descending aorta, 3 brachiocephalic

trunk, 4 left subclavian artery, 5 right ventricle, 6 left ventricle, 7 right

cranial vena cava, 8 left cranial vena cava, 9 jugular veins, 10

subclavian veins, 11 Third sternebra

Int J Cardiovasc Imaging

123

Fig. 2 a Long axis B-Mode view of the ascending aorta in a mouse.

Cranial is to the right. b Corresponding micro-CT image after

injection of Microfil. Sagittal section, cranial is to the right. c The

transducer was rotated 90� to obtain a short axis view of the lumen

0.5–1.5 mm downstream from the aortic valve. Measurements of the

luminal diameter (D) were made at this location. d Corresponding

micro-CT image after injection of Microfil. Transverse section.

e Doppler waveform of the ascending aorta in a mouse showing a

plug flow velocity profile with high resistance flow pattern. VTI

(area under the yellow lines) was calculated averaging three cardiac

cycles. 1 Ascending aorta, 2 descending aorta, 3 brachiocephalic

trunk, 4 left atrium, 5 left ventricle, 6 right atrium, 7 right ventricle,

8 aortic valve, 9 pulmonary trunk, 10 left cranial vena cava, 11 right

cranial vena cava, 12 Sternum (third sternebra), 13 thymus, 14 left

main bronchus, 15 right main bronchus, 16 third rib. VTI velocity

time integral

Int J Cardiovasc Imaging

123

Stoyanova [29], who reported a normal value of

55 ± 10 mm (mean ± SD) in C57BL/6 control mice. In

contrast, SV was slightly higher than the values presented

until now in mice of the same strain and related age [29, 30].

These differences can be due to the anesthetic protocol used

in the former case, where 2,2,2-tribromoethanol 2.5 % was

employed. In our case, isoflurane was preferred because it

causes less hemodynamic detrimental effects than other

anesthetics [31]. On the other hand, tribromoethanol pro-

duces several side effects in mice and has been recom-

mended only for acute terminal studies in laboratory

animals [32]. No substantial differences exist between the

present experimental protocol and those of Baumann [30],

therefore, differences in the results of both studies may be

explained by individual variance [31].

Similarly, in the present study, the results for CO were

slightly higher than previously reported data [11, 29, 30].

This difference is not surprising considering that CO was

calculated multiplying the SV by the HR. As HR is similar

in all these studies and was maintained under normal

physiological values for anesthetized mice [8, 26, 33], the

differences may be explained by the higher SV [34].

In human medicine, gender-dependent differences in

structural and functional cardiovascular characteristics

have been observed [35–38]. Previous studies in mice

showed differences between male and female in cardio-

vascular parameters, such as left ventricular dimensions at

end diastole or end systole, and left ventricle wall thickness

[30]. In that case, even after normalization for BW, ven-

tricular dimensions appeared still larger in females. In

contrast, other study performed in CD1 mice reported no

gender-related differences in several cardiovascular char-

acteristics, including CO and CI [18]. In the present study,

only SI and CI seemed to be greater in female than in male

mice. However, when BW was added as a covariate, the

trend for sex differences did not achieve statistical signif-

icance. This effect has been observed in measurements of

diastolic and systolic left ventricle mass in mice, and may

reflect the variation in BW between both genders [39].

When working with genetically modified mice such as

knockout mice, animals of the same littermate are com-

monly selected allowing the formation of groups with the

same age and genetic background. Comparisons between

wild type and mutant mice of the same littermate are

desirable. However, differences in BW related to gender or

genetic background may be observed within these groups

and may interfere in the interpretation of the results. The

results of the present study suggest that, if male and female

are to be compared, values should be standardized for BW

to avoid erroneous interpretation of the results. This may

also be considered when comparing mutant and wild type

mice belonging to the same littermate if BW is signifi-

cantly different between them. An additional approach,

evaluating the influence of body surface area, might be

contemplated in future studies.

One of the limitations of this study was the need for

general anesthesia. Although non-invasive echocardiogra-

phy may be performed in trained conscious mice, the results

in awaked animals may be affected by increased sympa-

thetic tone and contractile function secondary to stress [33].

Inhalatory anesthesia with isoflurane was therefore selected,

because it is one of the agents that produce less detrimental

effects in the cardiovascular function in mice [5, 33].

All the measurements were performed by the same

experienced observers, reducing the variability in the

results. Unfortunately, intra-observer variability evaluated

in the same animal on different days could not be deter-

mined because the internal legislation of our institution

precludes this type of experiment. In the future, additional

studies evaluating day-to-day and inter-observer variability

need to be performed.

Another limitation was that the study was performed in a

relatively low number of young adult mice of a single

strain. We selected C57BL/6 inbred mice because this

strain is one of the most commonly used in international

mouse phenotyping consortia and because it represents the

background for a broad range of transgenic mice [40–42].

This limitation makes extrapolations to other strains or age

groups difficult. Therefore, cardiovascular values used as

reference ranges in research protocols should be carefully

selected before making inaccurate comparisons.

Conclusions

Non-invasive measurement of the CO in the AAo can be

easily performed in anesthetized mice by means of high

Table 1 Descriptive numerical values obtained in the AAo of adult

C57BL/6 mice by means of high frequency transthoracic cardiac

ultrasound

Male (Mean ± SD) Female (Mean ± SD)

D (mm) 1.54 ± 0.13 1.56 ± 0.12

A (mm2) 1.88 ± 0.29 1.93 ± 0.29

HR (bpm) 448 ± 57 446 ± 56

VTI (mm) 38.97 ± 10.41 41.30 ± 9.30

SV (mm3) 73.36 ± 24.17 79.79 ± 21.24

SI (mm3/kg BW) 2,847.87 ± 1,127.12 4,370.90 ± 1,284.03

CO (ml/min) 32.10 ± 8.43 35.27 ± 9.33

CI (ml/min kg BW) 1,240.83 ± 384.73 1,925.61 ± 527.64

A area, AAo ascending aorta, BW body weight, CI cardiac index, CO

cardiac output, D diameter, HR heart rate, SI stroke index, SV stroke

volume, VTI velocity time integral

Int J Cardiovasc Imaging

123

frequency transthoracic ultrasound. Results obtained in

this study may serve as reference values for male and

female young adult C57BL/6 mice. No gender related

differences were observed in the diameter, area, stroke

volume, stroke index, cardiac output, and cardiac index,

measured in the AAo, when values were standardized by

body weight.

Acknowledgments This study was supported by a grant (Beca de

Formacion de Profesorado Universitario-FPU) of the Ministerio de

Educacion, Cultura y Deporte del Gobierno de Espana.

Conflict of interest None.

References

1. Paigen K (1995) A miracle enough: the power of mice. Nat Med

1:215–220

2. Doevendans PA, Daemen MJ, de Muinck ED, Smits JF (1998)

Cardiovascular phenotyping in mice. Cardiovasc Res 39:34–49

3. Fitzgerald SM, Gan L, Wickman A, Bergstrom G (2003) Car-

diovascular and renal phenotyping of genetically modified mice:

a challenge for traditional physiology. Clin Exp Pharmacol

Physiol 30:207–216

4. Tanaka N, Dalton N, Mao L, Rockman HA, Peterson KL, Gott-

shall KR et al (1996) Transthoracic echocardiography in models

of cardiac disease in the mouse. Circulation 94:1109–1117

5. Yang XP, Liu YH, Rhaleb NE, Kurihara N, Kim HE, Carretero

OA (1999) Echocardiographic assessment of cardiac function in

conscious and anesthetized mice. Am J Physiol 277:H1967–

H1974

6. Collins KA, Korcarz CE, Lang RM (2003) Use of echocardiog-

raphy for the phenotypic assessment of genetically altered mice.

Physiol Genomics 13:227–239

7. Zhou YQ, Foster FS, Qu DW, Zhang M, Harasiewicz KA, Ad-

amson SL (2002) Applications for multifrequency ultrasound

biomicroscopy in mice from implantation to adulthood. Physiol

Genomics 10:113–126

8. Zhou YQ, Foster FS, Nieman BJ, Davidson L, Chen XJ, Henk-

elman RM (2004) Comprehensive transthoracic cardiac imaging

in mice using ultrasound biomicroscopy with anatomical confir-

mation by magnetic resonance imaging. Physiol Genomics

18:232–244

9. McVeigh ER (2006) Emerging imaging techniques. Circ Res

98:879–886

10. Hartley CJ, Taffet GE, Reddy AK, Entman ML, Michael LH

(2002) Noninvasive cardiovascular phenotyping in mice. ILAR J

43:147–158

11. Janssen B, Debets J, Leenders P, Smits J (2002) Chronic mea-

surement of cardiac output in conscious mice. Am J Physiol

282:R928–R935

12. Carlsson M, Andersson R, Bloch KM, Steding-Ehrenborg K,

Mosen H, Stahlberg F et al (2012) Cardiac output and cardiac

index measured with cardiovascular magnetic resonance in

healthy subjects, elite athletes and patients with congestive heart

failure. J Cardiovasc Magn Reson 28(14):51

13. Schneider JE, Wiesmann F, Lygate CA, Neubauer S (2006) How

to perform an accurate assessment of cardiac function in mice

using high-resolution magnetic resonance imaging. J Cardiovasc

Magn Reson 8:693–701

14. Huntsman LL, Stewart DK, Barnes SR, Franklin SB, Colocousis

JS, Hessel EA (1983) Noninvasive Doppler determination of

cardiac output in man. Clinical validation. Circulation

67:593–602

15. Tournoux F, Petersen B, Thibault H, Zou L, Raher MJ, Kurtz B

et al (2011) Validation of noninvasive measurements of cardiac

Table 2 Mean values and 95 % IC for diameter (D), area (A), heart rate (HR), velocity time integral (VTI), stroke volume (SV), cardiac output

(CO), stroke index (SI), and cardiac index (CI) in male (M) and female (F) mice

Variable Gender Model 1 mean (95 % IC) p value Model 1 Model 2 mean (95 % IC) p value Model 2

D (mm) M 1.54 (1.47–1.62) p(sex) = 0.690 1.58 (1.44–1.71) p(sex) = 0.511

p(BW) = 0.561F 1.56 (1.51–1.62) 1.54 (1.46–1.63)

A (mm2) M 1.88 (1.71–2.05) p(sex) = 0.752 1.95 (1.70–2.19) p(sex) = 0.436

p(BW) = 0.466F 1.93 (1.79–2.07) 1.89 (1.72–2.05)

HR (bpm) M 448 (415–481) p(sex) = 0.650 439 (375–503) p(sex) = 0.883

p(BW) = 0.737F 446 (419–473) 452 (406–498)

VTI (mm) M 38.97 (32.85–45.09) p(sex) = 0.545 44.57 (27.5–61.64) p(sex) = 0.582

p(BW) = 0.405F 41.30 (36.89–45.71) 37.80 (29.91–45.68)

SV (mm3) M 73.36 (59.15–87.57) p(sex) = 0.469 88.78 (48.56–128.99) p(sex) = 0.527

p(BW) = 0.319F 79.79 (69.72–89.87) 70.16 (50.82–89.50)

CO (ml/min) M 32.10 (27.14–37.06) p(sex) = 0.350 33.74 (19.43–48.05) p(sex) = 0.963

p(BW) = 0.768F 35.27 (30.84–39.69) 34.24 (26.22–42.26)

SI (mm3/kg BW) M 2,847.87 (2,185.13–3,510.61) p(sex) = 0.001 4,357.54 (2,540.83–6,174.26) p(sex) = 0.481

p(BW) = 0.350F 4,370.90 (3,761.72–4,980.08) 3,427.35 (2,540.53–4,314.17)

CI (ml/min kg BW) M 1,240.83 (1,014.62–1,467.05) p(sex) \ 0.001 1,645.04 (996.47–2,293.61) p(sex) = 0.955

p(BW) = 0.123F 1,925.61 (1,675.28–2,175.94) 1,672.98 (1,287.84–2,058.12)

No statistical significant effect was observed in D, A, HR, VTI, SV and CO secondary to differences in sex or body weight (BW) with either

model. When Model 1 was applied (dependent variable = l ? gender), statistical significant differences were found related to the sex in SI and

CI. However, when Model 2 was applied (dependent variable = l ? gender ? BW) these differences disappeared

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output in mice using echocardiography. J Am Soc Echocardiogr

24:465–470

16. Khankin EV, Hacker MR, Zelop CM, Karumanchi SA, Rana S

(2012) Intravital high-frequency ultrasonography to evaluate

cardiovascular and uteroplacental blood flow in mouse preg-

nancy. Pregnancy Hypertens 2:84–92

17. Tomita H, Hagaman J, Friedman MH, Maeda N (2012) Rela-

tionship between hemodynamics and atherosclerosis in aortic

arches of apolipoprotein E-null mice on 129S6/SvEvTac and

C57BL/6 J genetic backgrounds. Atherosclerosis 220:78–85

18. Stypmann J, Engelen MA, Epping C, van Rijen HV, Milberg P,

Bruch C et al (2006) Age and gender related reference values for

transthoracic Doppler-echocardiography in the anesthetized CD1

mouse. Int J Cardiovasc Imaging 22:353–362

19. National Research Council (US) of the National Academies

(2011) Guide for care and use of laboratory animals. The

National Academies Press, Washington, pp 155–195

20. Cahill L (2006) Why sex matters for neuroscience. Nat Rev

Neurosci 7:477–484

21. Holdcroft A (2007) Integrating the dimensions of sex and gender

into basic life sciences research: methodologic and ethical issues.

Gend Med 4(Suppl B):S64–S74

22. Riesen S, Schmid V, Gaschen L, Busato A, Lang J (2002)

Doppler measurement of splanchnic blood flow during digestion

in unsedated normal dogs. Vet Radiol Ultrasound 43:554–560

23. Szatmari V, Sotonyi P, Voros K (2001) Normal duplex Doppler

waveforms of major abdominal blood vessels in dogs: a review.

Vet Radiol Ultrasound 42:93–107

24. Ni M, Zhang M, Ding SF, Chen WQ, Zhang Y (2008) Micro-

ultrasound imaging assessment of carotid plaque characteristics

in apolipoprotein-E knockout mice. Atherosclerosis 197:64–71

25. Fox JG, Stephen WB, Davisson MT, Newcomer CE, Quimby

FW, Smith AL (2007) The mouse in biomedical research. Else-

vier, US

26. Wu J, Bu L, Gong H, Jiang G, Li L, Ma H et al (2010) Effects of

heart rate and anesthetic timing on high-resolution echocardio-

graphic assessment under isoflurane anesthesia in mice. J Ultra-

sound Med 29:1771–1778

27. Foster FS, Zhang MY, Zhou YQ, Liu G, Mehi J, Cherin E et al

(2002) A new ultrasound instrument for in vivo microimaging of

mice. Ultrasound Med Biol 28:1165–1172

28. Feintuch A, Ruengsakulrach P, Lin A, Zhang J, Zhou YQ, Bishop

J et al (2007) Hemodynamics in the mouse aortic arch as assessed

by MRI, ultrasound, and numerical modeling. Am J Physiol Circ

Physiol 292:H884–H892

29. Stoyanova E, Trudel M, Felfly H, Garcia D, Cloutier G (2007)

Characterization of circulatory disorders in beta-thalassemic mice

by noninvasive ultrasound biomicroscopy. Physiol Genomics

29:84–90

30. Baumann PQ, Sobel BE, Tarikuz Zaman AK, Schneider DJ

(2008) Gender-dependent differences in echocardiographic

characteristics of murine hearts. Echocardiography 25:739–748

31. Janssen BJ, De Celle T, Debets JJ, Brouns AE, Callahan MF,

Smith TL (2004) Effects of anesthetics on systemic hemody-

namics in mice. Am J Physiol Circ Physiol 287:H1618–H1624

32. Meyer RE, Fish RE (2005) A review of tribromoethanol anes-

thesia for production of genetically engineered mice and rats. Lab

Anim 34:47–52

33. Roth DM, Swaney JS, Dalton ND, Gilpin EA, Ross J Jr (2002)

Impact of anesthesia on cardiac function during echocardiogra-

phy in mice. Am J Physiol Circ Physiol 282:H2134–H2140

34. Klabunde RE (2005) Cardiovascular physiology concepts. Lip-

pincott Williams & Wilkins, Philadelphia

35. Pearce WH, Slaughter MS, LeMaire S, Salyapongse AN, Fein-

glass J, McCarthy WJ et al (1993) Aortic diameter as a function

of age, gender, and body surface area. Surgery 114:691–697

36. Salton CJ, Chuang ML, O’Donnell CJ, Kupka MJ, Larson MG,

Kissinger KV et al (2002) Gender differences and normal left

ventricular anatomy in an adult population free of hypertension.

A cardiovascular magnetic resonance study of the Framingham

Heart Study Offspring cohort. J Am Coll Cardiol 39:1055–1060

37. Mao SS, Ahmadi N, Shah B, Beckmann D, Chen A, Ngo L et al

(2008) Normal thoracic aorta diameter on cardiac computed

tomography in healthy asymptomatic adults: impact of age and

gender. Acad Radiol 15:827–834

38. Wolak A, Gransar H, Thomson LE, Friedman JD, Hachamovitch

R, Gutstein A et al (2008) Aortic size assessment by noncontrast

cardiac computed tomography: normal limits by age, gender, and

body surface area. JACC Cardiovasc Imaging 1:200–209

39. Rottman JN, Ni G, Khoo M, Wang Z, Zhang W, Anderson ME

et al (2003) Temporal changes in ventricular function assessed

echocardiographically in conscious and anesthetized mice. J Am

Soc Echocardiogr 16:1150–1157

40. Breslow JL (1996) Mouse models of atherosclerosis. Science

272:685–688

41. Daugherty A, Cassis LA (2004) Mouse models of abdominal

aortic aneurysms. Arterioscler Thromb Vasc Biol 24:429–434

42. Westrick RJ, Winn ME, Eitzman DT (2007) Murine models of

vascular thrombosis. Arterioscler Thromb Vasc Biol 27:2079–

2093

Int J Cardiovasc Imaging

123