In vivo assessment of fibrosis in murine liver using transient microelastography: a feasibility study

Cécile Bastard1,2, Matteo Bosisio1,3, Meriem Yorgov4, Hélène Gilgenkrantz4, Laurent Sandrin1

1Echosens, Research and Development Department, Paris, FRANCE. 2UMRS INSERM U930 / CNRS ERL3106 / Université de Tours, Tours, FRANCE.

3Laboratoire d’Imagerie Paramétrique, CNRS UMR7623 / Université de Paris VI, Paris, FRANCE. 4Institut Cochin, UMRS INSERM U567 / CNRS UMR8104 / Université de Paris V, Paris, FRANCE.

cecile.bastard@echosens.com

Abstract— Recently, transient elastography has been successfully applied to assess fibrosis in the liver of human patients with chronic viral hepatitis C. In small animal experimentation, investigations on antifibrogenic substances often involve large cohorts of animals and require massive euthanasia for lack of non-invasive technique for monitoring the progression of the pathology. In this study, the potential of transient microelastography (TME) as a non-invasive technique to assess fibrosis stage in murine liver in vivo was investigated on a murine model of experimental fibrosis. The results of elastographic measurements show that the Young’s modulus is higher for experimental fibrosis mice (E = 18.2 ± 3.7 kPa) than for untreated mice (E = 6.8 ± 2.2 kPa) and control mice (E = 3.6 ± 1.2 kPa). A Spearman correlation performed between the picrosirius red staining extent and the Young’s modulus gives a Spearman’s coefficient equal to 0.88 (P-value < 0.01). TME could be a valuable non-invasive tool to assess the evolution and the treatment response of fibrosis in murine models in vivo without proceeding to euthanasia.

Transient elastography; mice; liver; fibrosis

I. INTRODUCTION

A. Background Over the past decades, the mouse has emerged as one of

the best model organisms for experimental studies of human diseases and drug testing [1]. Concerning liver pathologies, mice have been used for instance in numerous investigations involving antifibrogenic substances [2]. More generally, amongst the large range of diseases for which murine models are employed, some of them are prone to generate side effects on the liver. Recently, elastography, a tissue characterization technique used to measure the elasticity of biological tissues, has been introduced as a novel diagnostic tool in oncology and hepatology[3]. Several studies have shown that in human patients liver stiffness measured using transient elastography (Fibroscan®, Echosens, France) is correlated to liver fibrosis in various liver pathologies such as hepatites B and C [4]. Elastography is now implemented on commercial ultrasound scanners and is used for instance for breast tumors classification [5] or prostate cancer imaging

[6]. In the area of small animal experimentation, the use of elastographic techniques has been reported by several groups with magnetic resonance elastography (MRE) [7, 8] and static ultrasound elastography [9, 10]. However the techniques proposed remain expensive concerning MRE, and are sometimes invasive [9] and unsuitable for in vivo applications in the case of static elastography. The use of elastographic techniques on small animals is therefore a challenging area of investigation.

B. Purpose Here, transient microelastography (TME), a new non-

invasive method dedicated to small animals and based on ultrasound transient elastography [11] is proposed to quantify murine liver stiffness in vivo. The technique is first tested on elasticity phantoms and then applied on a murine model of experimental fibrosis.

II. METHODS

A. Experimental setup Transient elastography (TE) is a quantitative ultrasound

elastography technique which consists in following with ultrasound the propagation of a low frequency shear wave generated in a tissue by an external vibrator. This technique allows the measurement of the shear wave velocity and thus the determination of tissue stiffness. The novel transient microelastography device was composed of a microprobe, a dedicated high frequency electronic system and a laptop computer to control the ultrasound system and analyze the data. The microprobe contained an ultrasound transducer used both as receiver and emitter. This transducer was mounted on a mechanical vibrator and was also used as a piston to generate a low frequency shear wave. The electronic system was fully programmable and enabled to sample the radiofrequency data at a frequency of 200 MHz with a 12-bit precision. The induced displacement waves were computed from the radiofrequency data using an autocorrelation method [12] and derived versus depth in

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10.1109/ULTSYM.2009.0349

Figure 1. 3 mm diameter transducer used in TME

Figure 2. Liver stiffness measurement in a mouse liver using TME.

order to provide a strain image. The shear wave velocity was then computed using a time of flight method in a region of interested located between 2 and 6 mm from the piston. Assuming that the liver is a homogeneous, isotropic and purely elastic medium, the Young’s modulus can be obtained from the shear wave velocity using the formula E = 3ρVs² where ρ and Vs are the mass density and the shear wave velocity, respectively.

To adapt transient elastography to the measurement of elasticity at a short distance from the source, we increased the ultrasound frequency to 10 MHz and the shear wave frequency to 300 Hz. The diameter of the ultrasound transducer was also reduced to 3 mm (Figure 1). The choice of a small piston diameter permits to overcome the problem of the geometrical diffraction of the source. Moreover, the increase of the shear wave frequency permits to reduce the influence of the near field term which could lead to an overestimation of the shear wave velocity [13].

During the propagation of the low frequency shear wave, the radiofrequency data were acquired at a pulse repetition frequency of 15000 Hz. The system enables to measure stiffness values between 0.5 kPa and 200 kPa.

B. Phantoms The measurements were performed on two tissue-

mimicking phantoms made of a mixture of styrene-ethylene/butylenestyrene (SEBS) copolymer and mineral oil [14]. A 35–70 µm silica powder was used for acoustic scattering. The elasticity of a phantom is dependant on the percentage of SEBS copolymer used for its preparation. The first phantom (phant.1) contained 4 % of SEBS and the second phantom (phant.2) contained 6 % of SEBS.

C. Animals Nine mice of the same genetic background (cluster of

differentiation 1) aged of 9 ± 6 months were divided into three groups i.e. a control group composed of three untreated mice, a second group composed of three mice who received biweekly

injections of sole excipient (paraffin oil) and finally a group of three experimental fibrosis mice obtained by biweekly intraperitoneal injections of tetrachloride (CCl4) diluted in paraffin oil. After seven weeks (fourteen injections), the mice were anesthetized with isoflurane (0.75-1% in oxygen) and TME measurements were performed (Figure 2). Two weeks and four injections later, the animals were sacrificed and blood samples were collected for alanine transaminase (ALT) testing. The livers were harvested, fixed in 4% neutral-buffered formalin and embedded in paraffin for further histological analyses. After picrosirius red staining, the samples were stained with haematoxylin and eosin for light microscopy quantitative evaluation of the fibrosis content.

III. RESULTS

A. Phantom study To validate the technique of transient microelastography, a

series of 10 measurements were performed on each phantom using the novel transient microelastography device and a conventional transient elastography device i.e. the Fibroscan® (Echosens, Paris, France). The Fibroscan® computes the Young’s modulus in a region of interest located between 25 mm and 65 mm from the piston. For each series of measurements, the median is kept as representative and the interquartile range (IQR) is computed. The results presented in table I show that the two techniques are in good accordance.

TABLE I. YOUNG’S MODULUS MEASUREMENTS USING FIBROSCAN AND TRANSIENT MICROELASTOGRAPHY

E (IQR) (kPa)

Fibroscan® Transient microelastography

Phant.1 8.2 (0.0) 9.9 (0.3)

Phant.2 16.5 (0.0) 22.3 (1.1)

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B. Animal study The results of elastographic measurements show that the

Young’s modulus is higher for experimental fibrosis mice (E = 18.2 ± 3.7 kPa) than for untreated mice (E = 6.8 ± 2.2 kPa) and control mice (E = 3.6 ± 1.2 kPa) (Figure 3). The median value of the CCl4 injected group is clearly higher than the median of the two other groups. The age factor can explain the difference between the values obtained for the untreated mice and the control mice. Indeed, the control group mice were 4 month-old compared with the 16 month-old mice of the oil-injected group. Another explanation might be that oil injections increase liver stiffness by inducing a thickening of the Glisson capsule surrounding the liver. The TME measurements are also in good accordance with histology (Figure 4): A correlation performed between the picrosirius red staining extent and the Young’s modulus gives a Spearman’s coefficient equal to 0.88 (p-value < 0.01) suggesting that transient elastography permits a clear detection of fibrosis at an early stage.

C H CCl4

5

10

15

20

Live

r st

iffne

ss (

kPa)

Group

Figure 3. Relationship between liver stiffness and CCl4 induced fibrosis. Liver stiffness was measured in control mice (C), oil injected mice (H) and CCl4 induced fibrosis mice (CCl4). The top and bottom of the boxes are the

25th and 75th percentiles. The length of the box is the interquartile range (IQR).

The line through the middle of the box represents the median (50th percentile).

The upper adjacent value is the largest observation that is less than or equal to the 75th percentile plus 1.5 IQR. The lower adjacent value is the smallest

observation that is greater than or equal to the 25th percentile minus 1.5 IQR. The crosses are outliers, that is to say data with values lower than the 25th percentile minus 1.5 IQR or greater than the 75th percentile plus 1.5 IQR.

Figure 4. Histological sections for control mice (1-3), oil injected mice (4-6) and CCl4 injected mice (7-9). After picrosirius red staining, the samples were stained with haematoxylin and eosin for light microscopy

quantitative evaluation of the fibrosis content. The figure displays the median and the IQR of the LSM measurements, the results of ALT testing and the percentage of picrosirius red staining (SR).

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IV. DISCUSSION AND CONCLUSION

The values of elasticity obtained for control mice are close to those reported in literature. Using magnetic resonance elastography (MRE), Yin et al. [7] measured a shear modulus of 1.38 ± 0.20 kPa that is to say a Young’s modulus of 4.14 ± 0.6 kPa in wild type mice. However, the experiment proposed was invasive since it required the insertion of a needle to transmit a 120 Hz vibration in the liver. Salameh et al. [8] also reported elasticity scores in the same range with a shear modulus of 1.76 ± 0.37 kPa for normal rats (5.28 ± 1.11 kPa for the Young’s modulus) using MRE with a shear wave frequency of 200 Hz. The method we propose here has the advantage of being fast, totally non-invasive and low-cost compared with MRE. In oncology, the use of ultrasonic static elastography on small animal models has been previously reported (Bilgen et al. [9]) but the method was only applied in vitro or in situ in mice whose bodies had to be embedded in gelatin blocks. Furthermore, the necessity to apply a uniform compression on the measured sample appears as a major drawback for the application of static elastography in vivo. This technique was applied to investigate Crohn’s disease in rats [10] but the results were solely qualitative which is unsuitable for longitudinal studies.

Transient microelastography should make it possible to measure the evolution of pathologies such as fibrosis in the same animal during longitudinal studies. Thus, TME could be a valuable non-invasive tool to assess the evolution of disease as a function of time and the response to treatment of fibrosis in murine models in vivo without proceeding to euthanasia.

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