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Ex vivo study of acoustic radiation force impulse imaging elastography for evaluation of rat liver with steatosis
Accepted Manuscript Ex vivo study of acoustic radiation force impulse imaging elastography for evaluation of rat liver with steatosis Yanrong Guo, Changfeng Dong, Haoming Lin, Xinyu Zhang, Huiying Wen, Yuanyuan Shen, Tianfu Wang, Siping Chen, Yingxia Liu, Xin Chen PII: DOI: Reference:
S0041-624X(16)30224-4 http://dx.doi.org/10.1016/j.ultras.2016.10.009 ULTRAS 5397
To appear in:
Ultrasonics
Received Date: Revised Date: Accepted Date:
6 April 2016 29 September 2016 16 October 2016
Please cite this article as: Y. Guo, C. Dong, H. Lin, X. Zhang, H. Wen, Y. Shen, T. Wang, S. Chen, Y. Liu, X. Chen, Ex vivo study of acoustic radiation force impulse imaging elastography for evaluation of rat liver with steatosis, Ultrasonics (2016), doi: http://dx.doi.org/10.1016/j.ultras.2016.10.009
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Ex vivo study of acoustic radiation force impulse imaging elastography for evaluation of rat liver with steatosis Yanrong Guo1, Changfeng Dong2, Haoming Lin1, Xinyu Zhang1, Huiying Wen1, Yuanyuan Shen1, Tianfu Wang1, Siping Chen1, Yingxia Liu2, Xin Chen1* 1
School of Biomedical Engineering, Shenzhen University, Shenzhen, China, National-Regional Key Technology Engineering Laboratory for Medical Ultrasound, Guangdong Key Laboratory for Biomedical Measurements and Ultrasound Imaging;
2
Shenzhen Institute of Hepatology, The Third People’s Hospital of Shenzhen, Shenzhen, China;
* Corresponding Author: Prof. Xin Chen School of Medicine, Shenzhen University Email: [email protected]
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Abstract Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases in developed countries. Accurate, noninvasive tests for diagnosing NAFLD are urgently needed. The goals of this study were to evaluate the utility of acoustic radiation force impulse (ARFI) elastography for determining the severity grade of steatosis in rat livers, and to investigate the changes in various histologic and biochemical characteristics. Steatosis was induced in the livers of 57 rats by gavage feeding of a high fat emulsion; 12 rats received a standard diet only and served as controls. Liver mechanics were measured ex vivo using shear wave velocity (SWV) induced by acoustic radiation force. The measured mean values of liver SWV ranged from 1.33 to 3.85 m/s for different grades of steatosis. The area under the receiver operative characteristic curve (≥S1) was equal to 0.82 (95% CI = 0.69, 0.96) between the steatosis group and the normal group, and the optimal cutoff value was 2.59 with sensitivity of 88% and specificity of 76%. However, there are no significant differences in SWV measurements between the steatosis grades. SWV values did not correlate with the early grade of inflammation. In conclusion, ARFI elastography is a promising method for differentiating normal rat liver from rat liver with steatosis, but it cannot reliably predict the grade of steatosis in rat livers. The early grade of inflammation activity did not significantly affect the SWV measurements.
Keywords: nonalcoholic fatty liver disease; acoustic radiation force impulse elastography; shear wave velocity; steatosis; inflammation.
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Introduction Nonalcoholic fatty-liver disease (NAFLD) is an important cause of chronic liver disease [1]. At present, the prevalence of this condition is approximately 20% to 30% of the general population in affluent countries, and it threatens to become a serious public health problem [2, 3]. NAFLD is defined as the accumulation of lipid deposits in hepatocytes that are not due to excessive alcohol use [4]. It represents a spectrum of diseases ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), which can progress to liver fibrosis, cirrhosis, and hepatocellular carcinoma [3, 4]. In clinical, the non-invasive assessment of steatosis level is of great interest besides the staging of hepatic fibrosis and distinguishing NASH from simple steatosis. The hepatic steatosis is linked to an increase in cardiovascular and diabetic patients’ risk of death; and it is associated with adverse perioperative outcomes following partial hepatectomy. Moreover, it is also the threshold criteria for the diagnosis of NAFLD [5-8].
Liver biopsy is currently considered the gold standard for the diagnosis steatosis. However, biopsy is an invasive procedure associated with severe complications in 0.3% to 3.0% of cases, and leads to death in 0.01% of cases [9]. Furthermore, it is highly dependent on the experience of the operator, and subject to sampling error because of the small size of biopsy samples [9, 10]. Therefore, a simple and noninvasive method of detecting steatosis severity in patients is of major clinical interest.
Many different MRI techniques have been developed to improve the performance of 3
this modality in the diagnostic spectrum of NAFLD. Magnetic resonance elastography (MRE) uses a modified phase-contrast method to image the propagation characteristics of the shear wave in the liver. Early studies of MRE suggest that it could accurately differentiate simple steatosis from NASH with or without fibrosis and has good accuracy for diagnosing NASH [11-13]. Magnetic resonance spectroscopy (MRS) that directly measures the proton signals from acyl groups in hepatocyte triglyceride stores has shown incredible accuracy for diagnosing and quantifying steatosis[14, 15]. However, MRI is costly and time-consuming for use in routine clinical practice.
Ultrasonography (US) is a non-invasive, cost effective, and readily available screening tool for NAFLD, but it has some limitations, such as dependence on the reliability of the operator and equipment, limited use in obese patients, and low sensitivity when the degree of steatosis is less than 20% to 30% [16-19]. Research over the past 2 decades has led to significant new developments in ultrasound elastographic methods, including transient elastography (TE) and acoustic radiation force impulse (ARFI) elastography. These techniques were initially applied to evaluate viral hepatitis induced liver fibrosis by measuring liver stiffness [20-24]. The TE technique has been developed into a commercial ultrasound device called Fibroscan (EchoSens, Paris, France). Clinical trials using Fibroscan have indicated that its stiffness measurements are significantly correlated with the stage of fibrosis, and the areas under the receiver operating characteristic curves (AUROCs) were close 4
to those achieved by biopsy [25, 26]. ARFI elastography is a technique that measures shear wave velocity (SWV) using high-intensity, short-duration acoustic pulses to generate localized displacements in tissue, so as to provide information on the mechanical properties of soft tissue [27-29]. It can be useful for the real-time evaluation of stiffness during standard ultrasonography examinations of the liver, and the acoustic radiation force can be transmitted through fat layers or ascites. It also shows good results for assessing liver fibrosis in chronic viral hepatitis [30-32].
Ultrasound elastography has recently been used for the diagnosis of patients with NAFLD . However, obesity, a major risk factor for NAFLD; it has been associated with TE failure in up to 25% of patients [33]. To improve the diagnostic accuracy of FibroScan in patients with NAFLD, some researchers have applied a new probe, which uses a lower frequency than the standard M probe, to provide a higher rate of measurement in patients with an increased body mass index (BMI) [34-36]. In contrast, ARFI can transmit acoustic radiation force through fat layers or ascites; it more suitable for patients with NAFLD. Many studies have shown that ARFI elastography is a promising method for differentiating patients with nonalcoholic steatohepatitis from patients with simple steatosis [37, 38]. ARFI has also been found useful in predicting significant fibrosis in patients with NAFLD [34, 39, 40].
Hepatic steatosis presents with histological signs of fat accumulation in hepatocytes, and almost no clinical manifestations [23]. Because of the invasive nature of liver 5
biopsy, for both ethical and practical reason, has limited use in patients with steatosis. Therefore, clinical research on steatosis by ARFI technology has mainly focused on steatosis with fibrosis or NASH [34, 37-41], while steatosis without fibrosis has been poorly studied. A few studies have used animal model to investigate the correlation between elastographic measurement and steatosis grade [42, 43]. For example, Aroca et al. showed an increase in ARFI results in an experimental fatty liver model using chickens [43]; Kang et al. found no significant differences in the elasticity of rat livers between the normal group and the group with simple steatosis [42]. However, the reasons for the differences in ARFI results within the steatosis grades are not clear.
The purpose of the present study was to develop a rat model with different severity grade of steatosis, use the ARFI elastography method to measure the SWV of rat livers, and determine the performance of SWV measurements for evaluating steatosis.
Materials and methods Animal model Steatosis of the liver was induced in male Sprague-Dawley (SD) rats (Guangdong Medical Laboratory Animal Center, Guangdong, China) weighing about 180 to 200 g. They were housed in sterile isolated cages with a 10/14-hour light/dark cycle at constant temperature (20° - 26°C) and humidity (50% - 70%). Sixty-nine SD rats were randomly divided into the control group (57 rats) and the fatty liver group (12 rats). The control group was provided with a standard diet with sterilized food and 6
water. The fatty liver group had been fed on a high fat emulsion (20% lard, 10% cholesterol, 20% sodium cholate, 0.5% propylthiouracil, and 30% fructose), once a day at 1 mL/100 g rat weight, for different numbers of days to induce different severity grade of steatosis. Two, 4, 6, and 8 weeks later, several rats (see Table 1 for the exact number each week) were humanely killed, the right lateral lobe of the liver was harvested for SWV measurements, and the other lobes were used for histologic assessment. The steatosis grade was ultimately determined by the histologic results, not by the length of time the rats were gavaged with high fat emulsion. All procedures in these studies were approved by the Animal Care Committee of Shenzhen University and the Guangdong Medical Laboratory Animal Center.
Blood test Blood samples (1.5 mL) were extracted from the orbital venous sinus into tubes after a 12-hour fasting period. Plasma was separated and analyzed for total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) using an automatic biochemical analyzer (Type 7020, Hitachi High-Tech Companies, Tokyo, Japan).
Histologic assessment The excised liver tissues were fixed in 10% formalin solution for at least 24 hours. After being washed and dehydrated, they were embedded in paraffin and sliced to a 7
thickness of 7 um. The paraffin slices were stained with Oil Red O, hematoxylin, and Masson’s trichrome by histopathology technicians, and analyzed using an Olympus BX41 microscope by an expert pathologist (20 years of experience) blinded to the results of the ultrasound measurements. The severity grade of steatosis was assessed on the basis of the percentage of hepatocytes containing macrovesicular fat droplets as follows: S0, no steatosis; S1, less than 25% of the hepatocytes contain macrovacuoles of fat; S2, 25% to 50% of the hepatocytes contain macrovacuoles of fat; S3, 50% to 75% of the hepatocytes contain macrovacuoles of fat; S4, more than 75% of the hepatocytes contain macrovacuoles of fat. The necroinflammatory severity was assessed and assigned grades as follows: G0, no inflammatory cells; G1, a small numbers of inflammatory cells are gathered around the portal area or central vein; G2, the inflammatory cells are gathered around the portal area, central vein, and hepatic sinus or lobular bridge; G3, there is infiltration of focal inflammatory cells, with liver cell denaturation and necrosis; G4, there is diffuse infiltration of inflammatory cells and degeneration/necrosis of a large number of liver cells. The stage of fibrosis was evaluated according to the METAVIR scoring system [44]: F0, no fibrosis; F1, portal fibrosis without septa; F2, portal fibrosis with a few septa; F3, numerous septa without cirrhosis; and F4, cirrhosis.
ARFI Elastography ARFI imaging is an elastographic technology that targets an anatomic region to be examined for its elastic properties using a region of interest (ROI) cursor while 8
performing real-time B-mode imaging. Tissue in the region of interest is mechanically stimulated using short-duration acoustic pulses that are automatically produced by an ultrasound probe. The acoustic push pulse transmitted by the transducer toward the tissue induces elastic shear waves that propagate through the tissue. The velocity, measured directly in meters per second (m/s), is displayed on the screen.
In an ex vivo experiment, the right liver lobe was processed and embedded in a fabricated gelatin solution (gelatin from porcine skin, Sigma–Aldrich, St. Louis, MO) in a container (11 cm × 11 cm × 7 cm). The ARFI imaging was performed by a sonographer (with 8 years of experience) using the Virtual Touch Tissue Quantification mode implemented on the Siemens Acuson S2000 ultrasound system with an 8-MHz linear ultrasound transducer. As shown in Figure 1, an area was chosen where the liver tissue was free of large blood vessels. The measurements were made at a 1.5 to 2 cm depth below the gelatin simulation. All the ultrasound studies were performed blind to the results of histologic assessment by the sonographer. Ten successful acquisitions were performed in each liver. The measurement was considered invalid due to motion artifact when the screen displayed “xxx”. The final results were expressed as the median value of the total measurements.
Statistical methods A total of 69 rats, which were grouped on the basis of histopathologic grade, were processed in this study. For each steatosis grade, the SWV values of all the rats at that 9
grade were grouped. The mean SWV of each rat was then used to estimate the population mean of each steatosis group. A Tukey test, in conjunction with analysis of variance (ANOVA), was performed to compare SWV measurements of the inflammation and steatosis severity grades. The diagnostic accuracy of the SWV parameters in discriminating between the grades of steatosis was studied using nonparametric AUROCs. Cutoff values were chosen by maximizing the Youden index [45]. Sensitivity and specificity values were computed with exact 95% confidence intervals. The correlations between SWV values and biological characteristics, and SWV values and inflammation activity, were tested using Spearman’s correlation coefficient. For all tests, significance was achieved at P < 0.05.
RESULTS Table 1 lists the distribution of rats with NAFLD according to their histologic characteristics at each sacrifice date. Figure 2 shows box plots of SWV measurements for each grade of steatosis. The median SWV was 2.25 ± 0.52 m/s (range 1.33 3.26m/s) in the S0 group, 2.66 ± 0.40 m/s (range 1.53 - 3.04 m/s) in the S1 group, 2.90 ± 0.40 m/s (range 1.93 - 3.85 m/s) in the S2 group, 2.93 ± 0.27 m/s (range 2.62 3.52 m/s) in the S3 group, and 2.82 ± 0.28 (range 2.36 - 3.18 m/s) m/s in the S4 group. The analysis of variance results show significant differences between the normal group and the steatosis group (P < 0.05). There were no significant differences with increasing histologic steatosis severity. As shown in Figure 3, SWV measurements have a good accuracy in predicting steatosis grade (S ≥ 1), with an AUROC of 0.82 10
(95% CI = 0.69, 0.96) and the optimal cutoff value was 2.59 with a sensitivity of 88% and specificity of 76%. Spearman’s correlation coefficient between SWV measurements and histologically determined steatosis grades showed a moderately significant correlation (r = 0.458, P < 0.05). As shown in Figure 4, the mean inflammation results were 2.70 ± 0.42 m/s (range 1.53 - 3.52 m/s) in the G0 group, 2.63 ± 0.59 m/s (range 1.53 - 3.52 m/s) in the G1 group, and 3.01 ± 0.18 m/s (range 2.80 - 3.16 m/s) in the G2 group. The 1-way ANOVA results showed no significant differences between the grades of inflammation. SWV values did not correlate with the grade of inflammation. Table 2 shows the clinical and biologic parameters observed in the rats. There was an increase in all lipid parameters measured in the plasma of rats fed a special diet, when compared to the control rats fed the standard diet. SWV measurements correlated significantly with ALT (r = 0.50, P < 0.05), AST (r = 0.42, P < 0.05), TC (r = 0.47, P < 0.05), and LDL (r = 0.45, P < 0.05) but no significant correlation was found between SWV and either TG or HDL. No fibrosis was observed on Masson staining.
DISCUSSION NAFLD has become a focus of increasing medical attention because obesity, hyperlipidemia, and disturbances related to excessive or unbalanced food ingestion have reached epidemic proportions throughout the world over the past decade [1, 3]. It is, moreover, a complex disorder involving genetic predisposition and environmental factors, and much is still unknown about its pathophysiology in humans. Animal models of the spectrum of NAFLD provide the necessary tools to 11
overcome confounding variables, such as genetic heterogeneity, and environmental factors, including diet and lifestyle [46-48]. Given the difficulty of studying all the factors involved in steatosis in human populations, the animal model may have helped to improve our understanding of the physiopathology of the disease.
NAFLD accompanies various histologic abnormalities, including hepatic steatosis, inflammation, and fibrosis, which may potentially affect ultrasound transmission and shear wave propagation [22, 23, 39, 42, 49]. For the clinical studies, it was found that that SWV values were significantly correlated with the stages of liver fibrosis in patients with NAFLD, and could distinguish patients with NASH from those with simple steatosis [30, 34, 37]. In clinical situations, all these abnormalities may combine with each other and contribute to the change in SWV values. Therefore, it is difficult to estimate the discrete effect of each disease on the SWV measurements.
As shown in Table 1, there was mild inflammation (G1, n = 25; G2, n = 3; G3, n = 0; G4, n = 0) without fibrosis in our rat livers. The 1-way ANOVA results showed that there was no significant difference between G0, G1, and G2 (P > 0.05) in the group with inflammation. The 2-way ANOVA indicated that there was no significant interaction between the early grade of inflammation and steatosis. Therefore, the mild inflammation in our animal model did not affect the SWV measurements.
In our study, the results of ROC analysis for the diagnostic performance of SWV in 12
grading hepatic steatosis severity can be seen in Figure 2. The mean SWV measured with the ARFI method was 2.25 ± 0.52 m/s in the normal group (range 1.33 - 3.26 m/s ) and 2.83 ± 0.37 m/s in the fatty liver group (range 1.53 - 3.85 m/s), with statistically significant differences observed between the 2 groups (P < 0.05). This finding is consistent with another animal study, which showed that the median velocity measured by the ARFI method was 0.9 ± 0.1 m/s in the control group and 1.9 ± 0.2 m/s in the hyperlipidemic diet group with statistically significant differences between the 2 groups (P < 0.001) [43]. However, the finding of this study is in conflict with some clinical studies. The study of Nightingale et al. found that there was no correlation with steatosis and SWS measurements in patients with NAFLD[41]. Another 2 studies showed a stepwise decrease in ARFI velocity with increasing histologic hepatic steatosis in patients with NAFLD [34, 37]. The discrepancy between our study and these clinical studies may be due to the differences between these conditions in animal models and in clinical patients. As mentioned above, clinical patients with NAFLD usually have several combined diseases—such as steatosis, inflammation, and fibrosis—which may contribute to the difference in ARFI velocity. On the other hand, the disease in the animal model is relatively simple. In the rat model of our study, the effects of steatosis is the dominant cause of the change in ARFI velocity. In the rat model, Kang et al. found no significant differences in the elasticity of rat livers between the normal group and the group with simple steatosis. The discrepancy may be due to the different method of developing animal model and measuring technique. 13
Although significant difference was found between the SWV values of normal group and steatosis group, the box plot and the ROC analysis show that the SWV values have no significant difference between the grades of steatosis. As shown in Fig. 2, the SWV values increase rapidly with the fat accumulation less than 25 percent (first steatosis grade); and this increase trend slows down with further fat accumulation (second to fourth steatosis grades). The possible explanation for this finding is the nature of liver steatosis which is caused by intra-cellular fat accumulation. Compared with inter-cellular change of collagen in liver fibrosis, this micro-level change may have less impact on shear wave propagation, especially in high steatosis grades [41].
One limitation of our study is that it would have been ideal to have rat model in which a single steatosis feature was expressed, but this was not possible. That is, inflammation may also precede or develop in parallel with hepatic steatosis owing to the increasing duration of feeding [42, 50]. Although mild inflammation had been found in the control group and fatty liver group, as shown in Table 1, mild inflammation did not affect the SWV by the 2-way ANOVA. Another limitation of our study is that liver stiffness was examined in ex vivo samples. This condition may overcome rat confounding environmental factors, such as different body boundary conditions and different thickness of the fat in our experiment. But mechanical and physiological characteristics may possibly change from in vivo to ex vivo. Meanwhile, the SWV value used to analyze the relationship between the ex vivo liver and lab 14
parameters may also have been limited.
In conclusion, our results indicate that ARFI elastography offers good diagnostic accuracy in evaluating the normal liver and hepatic steatosis, but no significant differences are observed between the grade of steatosis. Furthermore, the early grade of inflammation activity does not significantly affect the SWV measurements.
Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 81471735, 81570552, 61427806, 61101025, 61201041), National Natural Science Foundation of Guangdong Province (Grant No. 2016A030310047), and the National Science & Technology Pillar Program (2015BAI01B02)
References [1]
B.Q. Starley, C.J. Calcagno, and S.A. Harrison, Nonalcoholic fatty liver disease and hepatocellular carcinoma: a weighty connection, Hepatology, 51 (2010) 1820-1832.
[2]
A. Imhof, W. Kratzer, B. Boehm, K. Meitinger, G. Trischler, G. Steinbach, I. Piechotowski, and W. Koenig, Prevalence of non-alcoholic fatty liver and characteristics in overweight adolescents in the general population, European journal of epidemiology, 22 (2007) 889-897.
[3]
P. Angulo, Nonalcoholic fatty liver disease, New England Journal of Medicine, 346 (2002) 1221-1231.
[4]
I. Liou and K.V. Kowdley, Natural history of nonalcoholic steatohepatitis, Journal of clinical gastroenterology, 40 (2006) S11-S16.
[5]
Z.M. Younossi, T. Gramlich, C.A. Matteoni, N. Boparai, and A.J. Mccullough, Nonalcoholic fatty liver disease in patients with type 2 diabetes, Clinical Gastroenterology & Hepatology, 2 (2004) 262-265.
[6]
G. Targher, C.P. Day, and E. Bonora, Risk of cardiovascular disease in patients with nonalcoholic fatty liver disease, New England Journal of Medicine, 363 (2010) 1341-50.
[7]
L. Mccormack, P. Dutkowski, A.M. El-Badry, and P.A. Clavien, Liver transplantation using fatty livers: Always feasible?, Journal of hepatology, 54 (2011) 1055-62.
15
[8]
M.V.E. De, B.T. Kalish, M. Puder, and J.N.M. Ijzermans, Systematic review and meta-analysis of steatosis as a risk factor in major hepatic resection, British Journal of Surgery, 97 (2010) 1331-9.
[9]
M.R. Pagadala and A.J. McCullough, The relevance of liver histology to predicting clinically meaningful outcomes in nonalcoholic steatohepatitis, Clinics in liver disease, 16 (2012) 487-504.
[10]
G.A. Grandison and P. Angulo, Can NASH be diagnosed, graded, and staged noninvasively?, Clinics in liver disease, 16 (2012) 567-585.
[11]
L. Huwart, C. Sempoux, E. Vicaut, N. Salameh, L. Annet, E. Danse, F. Peeters, L.C.T. Beek, J. Rahier, and R. Sinkus, Magnetic Resonance Elastography for the Noninvasive Staging of Liver Fibrosis, Gastroenterology, 135 (2008) 32-40.
[12]
J. Chen, J.A. Talwalkar, M. Yin, K.J. Glaser, S.O. Sanderson, and R.L. Ehman, Early detection of nonalcoholic steatohepatitis in patients with nonalcoholic fatty liver disease by using MR elastography, Radiology, 259 (2011) 749-56.
[13]
S.K. Venkatesh and R.L. Ehman, Magnetic resonance elastography of liver, Magnetic Resonance Imaging Clinics of North America, 22 (2014) 433-46.
[14]
S. McPherson, J.R. Jonsson, G.J. Cowin, P.O. Rourke, A.D. Clouston, A. Volp, L. Horsfall, D. Jothimani, J. Fawcett, G.J. Galloway, M. Benson, and E.E. Powell, Magnetic resonance imaging and spectroscopy accurately estimate the severity of steatosis provided the stage of fibrosis is considered, Journal of hepatology, 51 (2009) 389–397.
[15]
G.J. Cowin, J.R. Jonsson, J.D. Bauer, S. Ash, A.A. Bhsc, E.J.O. Bhsc, D.M. Purdie, and G.J. Galloway, Magnetic resonance imaging and spectroscopy for monitoring liver steatosis, Journal of Magnetic Resonance Imaging, 28 (2008) 937–945.
[16]
S. Dasarathy, J. Dasarathy, A. Khiyami, R. Joseph, R. Lopez, and A.J. McCullough, Validity of real time ultrasound in the diagnosis of hepatic steatosis: a prospective study, Journal of hepatology, 51 (2009) 1061-1067.
[17]
C.C. Mottin, M. Moretto, A.V. Padoin, A.M. Swarowsky, M.G. Toneto, L. Glock, and G. Repetto, The role of ultrasound in the diagnosis of hepatic steatosis in morbidly obese patients, Obesity surgery, 14 (2004) 635-637.
[18]
C.K. Ryan, L.A. Johnson, B.I. Germin, and A. Marcos, One hundred consecutive hepatic biopsies in the workup of living donors for right lobe liver transplantation, Liver transplantation, 8 (2002) 1114-1122.
[19]
R. Bouzitoune, M. Meziri, C.B. Machado, F. Padilla, and W.C. Pereira, Can early hepatic fibrosis stages be discriminated by combining ultrasonic parameters?, Ultrasonics, 68 (2016) 120-126.
[20]
G. Ferraioli, C. Filice, L. Castera, B.I. Choi, I. Sporea, S.R. Wilson, D. Cosgrove, C.F. Dietrich, D. Amy, and J.C. Bamber, WFUMB Guidelines and Recommendations for Clinical Use of Ultrasound Elastography: Part 3: Liver, Ultrasound in medicine & biology, 41 (2015) 1161-1179.
[21]
D. Cosgrove, F. Piscaglia, J. Bamber, J. Bojunga, J.M. Correas, O.H. Gilja, A.S. Klauser, I. Sporea, F. Calliada, and V. Cantisani, EFSUMB guidelines and recommendations on the clinical use of ultrasound elastography. Part 2: Clinical applications, Ultraschall in Der Medizin, 34 (2013) 238-253.
[22]
W.K. Jeong, H.K. Lim, H.K. Lee, J.M. Jo, and Y. Kim, Principles and clinical application of
16
ultrasound elastography for diffuse liver disease, Ultrasonography, 33 (2014) 149-160. [23]
M.V. Machado and H. Cortez-Pinto, Non-invasive diagnosis of non-alcoholic fatty liver disease. A critical appraisal, Journal of hepatology, 58 (2013) 1007-19.
[24]
X. Chen, Y. Shen, Y. Zheng, H. Lin, Y. Guo, Y. Zhu, X. Zhang, T. Wang, and S. Chen, Quantification of liver viscoelasticity with acoustic radiation force: a study of hepatic fibrosis in a rat model, Ultrasound in medicine & biology, 39 (2013) 2091-102.
[25]
S.M. Martinez, G. Crespo, M. Navasa, and X. Forns, Noninvasive assessment of liver fibrosis, Hepatology, 53 (2011) 325-335.
[26]
P. Marcellin, M. Ziol, P. Bedossa, C. Douvin, R. Poupon, V. De Lédinghen, and M. Beaugrand, Non-invasive assessment of liver fibrosis by stiffness measurement in patients with chronic hepatitis B, Liver international, 29 (2009) 242-247.
[27]
A.P. Sarvazyan, O.V. Rudenko, S.D. Swanson, J.B. Fowlkes, and S.Y. Emelianov, Shear wave elasticity imaging: a new ultrasonic technology of medical diagnostics, Ultrasound in medicine & biology, 24 (1998) 1419-1435.
[28]
K. Nightingale, M.S. Soo, R. Nightingale, and G. Trahey, Acoustic radiation force impulse imaging: in vivo demonstration of clinical feasibility, Ultrasound in medicine & biology, 28 (2002) 227-235.
[29]
L. Zhai, M.L. Palmeri, R.R. Bouchard, R.W. Nightingale, and K.R. Nightingale, An integrated indenter-ARFI imaging system for tissue stiffness quantification, Ultrasonic imaging, 30 (2008) 95-111.
[30]
H. Ebinuma, H. Saito, M. Komuta, K. Ojiro, K. Wakabayashi, S. Usui, P.-s. Chu, R. Umeda, Y. Ishibashi, and T. Takayama, Evaluation of liver fibrosis by transient elastography using acoustic radiation force impulse: comparison with Fibroscan®, Journal of gastroenterology, 46 (2011) 1238-1248.
[31]
M. Friedrich-Rust, K. Wunder, S. Kriener, F. Sotoudeh, S. Richter, J. Bojunga, E. Herrmann, T. Poynard, C.F. Dietrich, and J. Vermehren, Liver Fibrosis in Viral Hepatitis: Noninvasive Assessment with Acoustic Radiation Force Impulse Imaging versus Transient Elastography 1, Radiology, 252 (2009) 595-604.
[32]
R. Goertz, Y. Zopf, V. Jugl, R. Heide, C. Janson, D. Strobel, T. Bernatik, and T. Haendl, Measurement of liver elasticity with acoustic radiation force impulse (ARFI) technology: an alternative noninvasive method for staging liver fibrosis in viral hepatitis, Ultraschall in der Medizin (Stuttgart, Germany: 1980), 31 (2010) 151-155.
[33]
R. Kwok, Y.K. Tse, G.H. Wong, Y. Ha, A. Lee, M. Ngu, H.Y. Chan, and V.S. Wong, Systematic review with meta-analysis: non-invasive assessment of non-alcoholic fatty liver disease-the role of transient elastography and plasma cytokeratin-18 fragments, Alimentary pharmacology & therapeutics, 39 (2014) 254-269.
[34]
M. Yoneda, K. Suzuki, S. Kato, K. Fujita, Y. Nozaki, K. Hosono, S. Saito, and A. Nakajima, Nonalcoholic fatty liver disease: US-based acoustic radiation force impulse elastography 1, Radiology, 256 (2010) 640-647.
[35]
M. Sasso, S. Audière, A. Kemgang, F. Gaouar, C. Corpechot, O. Chazouillères, C. Fournier, O. Golsztejn, S. Prince, and Y. Menu, Liver Steatosis Assessed by Controlled Attenuation Parameter (CAP) Measured with the XL Probe of the FibroScan: A Pilot Study Assessing Diagnostic Accuracy, Ultrasound in medicine & biology, 42 (2016) 92-103.
[36]
M. Friedrich-Rust, H. Hadji-Hosseini, S. Kriener, E. Herrmann, I. Sircar, A. Kau, S. Zeuzem,
17
and J. Bojunga, Transient elastography with a new probe for obese patients for non-invasive staging of non-alcoholic steatohepatitis, European radiology, 20 (2010) 2390-2396. [37]
C.F. Braticevici, I. Sporea, E. Panaitescu, and L. Tribus, Value of acoustic radiation force impulse imaging elastography for non-invasive evaluation of patients with nonalcoholic fatty liver disease, Ultrasound in medicine & biology, 39 (2013) 1942-1950.
[38]
F. Aroca, M. Frutos-Bernal, A. Bas, J. Luján-Mompeán, M. Reus, J. de Dios Berná-Serna, and P. Parrilla, Detection of non-alcoholic steatohepatitis in patients with morbid obesity before bariatric surgery: preliminary evaluation with acoustic radiation force impulse imaging, European radiology, 22 (2012) 2525-2532.
[39]
T. Deffieux, J.L. Gennisson, L. Bousquet, M. Corouge, S. Cosconea, D. Amroun, S. Tripon, B. Terris, V. Mallet, and P. Sogni, Investigating liver stiffness and viscosity for fibrosis, steatosis and activity staging using shear wave elastography, Journal of hepatology, 62 (2015) 317-24.
[40]
V.W.S. Wong, J. Vergniol, G.L.H. Wong, J. Foucher, H.L.Y. Chan, B. Le Bail, P.C.L. Choi, M. Kowo, A.W.H. Chan, and W. Merrouche, Diagnosis of fibrosis and cirrhosis using liver stiffness measurement in nonalcoholic fatty liver disease, Hepatology, 51 (2010) 454-462.
[41]
K.R. Nightingale, N.C. Rouze, S.J. Rosenzweig, M.H. Wang, M.F. Abdelmalek, C.D. Guy, and M.L. Palmeri, Derivation and analysis of viscoelastic properties in human liver: impact of frequency on fibrosis and steatosis staging, IEEE Transactions on Ultrasonics Ferroelectrics & Frequency Control, 62 (2015) 165-75.
[42]
B.K. Kang, S.S. Lee, H. Cheong, S.M. Hong, K. Jang, and M.G. Lee, Shear Wave Elastography for Assessment of Steatohepatitis and Hepatic Fibrosis in Rat Models of Non-Alcoholic Fatty Liver Disease, Ultrasound in medicine & biology, 41 (2015) 3205-15.
[43]
F.G. Aroca, I. Ayala, L. Serrano, J.D. Berná-Serna, M.T. Castell, B. García-Pérez, and M. Reus, Assessment of liver steatosis in chicken by using acoustic radiation force impulse imaging: preliminary results, European radiology, 20 (2010) 2367-2371.
[44]
E.M. Brunt, Nonalcoholic steatohepatitis: definition and pathology, Seminars in Liver Disease, 21 (2001) 3-16.
[45]
W.J. Youden, Index for rating diagnostic tests, Cancer, 3 (1950) 32-35.
[46]
R.M. London and J. George, Pathogenesis of NASH: animal models, Clinics in liver disease, 11 (2007) 55-74.
[47]
N.F. Schwenzer, F. Springer, C. Schraml, N. Stefan, J. Machann, and F. Schick, Non-invasive assessment and quantification of liver steatosis by ultrasound, computed tomography and magnetic resonance, Journal of hepatology, 51 (2009) 433-445.
[48]
I. Ayala, A.M. Castillo, G. Adanez, A. Fernandez-Rufete, B.G. Pérez, and M.T. Castells, Hyperlipidemic chicken as a model of non-alcoholic steatohepatitis, Experimental biology and medicine, 234 (2009) 10-16.
[49]
F. Piscaglia, S. Marinelli, S. Bota, C. Serra, L. Venerandi, S. Leoni, and V. Salvatore, The role of ultrasound elastographic techniques in chronic liver disease: current status and future perspectives, European Journal of Radiology, 83 (2013) 450-5.
[50]
N. Chalasani, L. Wilson, D.E. Kleiner, O.W. Cummings, E.M. Brunt, and A. Unalp, Relationship of steatosis grade and zonal location to histological features of steatohepatitis in adult patients with non-alcoholic fatty liver disease, Journal of hepatology, 48 (2008) 829-34.
18
Figure Captions Fig. 1. B-mode image from one of the isolated rat liver phantoms. The green box represents the ROI for reconstructing the shear velocity.
19
Fig. 2. Box plots of SWV measurements in each steatosis grade. Boundaries of the boxes indicate the 25th and 5th percentiles; lines within the boxes indicate medians; and error bars indicate ranges for each grade.
20
Fig. 3. Receiver operating characteristic curves for differentiation different steatosis grades.
21
Fig. 4.
Box plots of SWV measurements in each inflammation grade.
22
Table 1. Number of rats and histopathologic results at each sacrifice date Duration of fat emulsion administration
Number of rats
Steatosis group (0/1/2/3/4)
Inflamation group (0/1/2/3/4)
Fibrosis group (0/1/2/3/4)
Control
12
12/0/0/0/0
8/4/0/0/0
12/0/0/0/0
2 weeks
15
3/7/4/1/0
7/7/1/0/0
15/0/0/0/0
4 weeks
13
1/3/5/4/0
3/8/2/0/0
13/0/0/0/0
6 weeks
14
0/1/8/5/0
11/3/0/0/0
14/0/0/0/0
8 weeks
15
1/3/2/2/7
12/3/0/0/0
15/0/0/0/0
Table 2. Results of liver enzymes and plasma lipids from rats. Steatosis Group
ALT (U/L)
AST (U/L)
TC (mmol/L)
TG (mmol/L)
HDL-C (mmol/L)
LDL-C (mmol/L)
S0
45.59±9. 25* 73.08±3 0.14 59.59±1 7.61 71.42±1 7.47 103.57± 19.31
119.19±17 .39 153.10±31 .75 144.82±25 .43 139.40±24 .69 321.00±79 .20
4.50±3.07
1.59±0.62
0.97±0.40
0.72±0.89
9.08±2.87
1.51±0.65
1.34±0.36
2.66±1.94
19.74±12. 28 14.09±4.2 3 13.27±2.1 1
1.67±0.72
1.47±0.38
6.89±6.00
1.84±1.06
1.54±0.39
5.63±3.30
3.23±0.80
1.88±0.42
5.65±1.30
S1 S2 S3 S4
ALT = alanine aminotransferase; AST = aspartate aminotransferase; TC = total cholesterol; TG = Triglyceride; HDL-C = high density lipoprotein cholesterol; LDL-C = low density lipoprotein cholesterol *The values of biochemical characteristics are expressed as the mean ± standard deviation.
23
24
Highlights
The research by ARFI technology is focused on steatosis without fibrosis. ARFI elastography offers good diagnostic accuracy in evaluating the normal rat liver and rat liver with steatosis. The SWV measurements have no significant difference between the grades of steatosis. The early grade of inflammation activity does not significantly affect the SWV measurements.
25
S0041-624X(16)30224-4 http://dx.doi.org/10.1016/j.ultras.2016.10.009 ULTRAS 5397
To appear in:
Ultrasonics
Received Date: Revised Date: Accepted Date:
6 April 2016 29 September 2016 16 October 2016
Please cite this article as: Y. Guo, C. Dong, H. Lin, X. Zhang, H. Wen, Y. Shen, T. Wang, S. Chen, Y. Liu, X. Chen, Ex vivo study of acoustic radiation force impulse imaging elastography for evaluation of rat liver with steatosis, Ultrasonics (2016), doi: http://dx.doi.org/10.1016/j.ultras.2016.10.009
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Ex vivo study of acoustic radiation force impulse imaging elastography for evaluation of rat liver with steatosis Yanrong Guo1, Changfeng Dong2, Haoming Lin1, Xinyu Zhang1, Huiying Wen1, Yuanyuan Shen1, Tianfu Wang1, Siping Chen1, Yingxia Liu2, Xin Chen1* 1
School of Biomedical Engineering, Shenzhen University, Shenzhen, China, National-Regional Key Technology Engineering Laboratory for Medical Ultrasound, Guangdong Key Laboratory for Biomedical Measurements and Ultrasound Imaging;
2
Shenzhen Institute of Hepatology, The Third People’s Hospital of Shenzhen, Shenzhen, China;
* Corresponding Author: Prof. Xin Chen School of Medicine, Shenzhen University Email: [email protected]
1
Abstract Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases in developed countries. Accurate, noninvasive tests for diagnosing NAFLD are urgently needed. The goals of this study were to evaluate the utility of acoustic radiation force impulse (ARFI) elastography for determining the severity grade of steatosis in rat livers, and to investigate the changes in various histologic and biochemical characteristics. Steatosis was induced in the livers of 57 rats by gavage feeding of a high fat emulsion; 12 rats received a standard diet only and served as controls. Liver mechanics were measured ex vivo using shear wave velocity (SWV) induced by acoustic radiation force. The measured mean values of liver SWV ranged from 1.33 to 3.85 m/s for different grades of steatosis. The area under the receiver operative characteristic curve (≥S1) was equal to 0.82 (95% CI = 0.69, 0.96) between the steatosis group and the normal group, and the optimal cutoff value was 2.59 with sensitivity of 88% and specificity of 76%. However, there are no significant differences in SWV measurements between the steatosis grades. SWV values did not correlate with the early grade of inflammation. In conclusion, ARFI elastography is a promising method for differentiating normal rat liver from rat liver with steatosis, but it cannot reliably predict the grade of steatosis in rat livers. The early grade of inflammation activity did not significantly affect the SWV measurements.
Keywords: nonalcoholic fatty liver disease; acoustic radiation force impulse elastography; shear wave velocity; steatosis; inflammation.
2
Introduction Nonalcoholic fatty-liver disease (NAFLD) is an important cause of chronic liver disease [1]. At present, the prevalence of this condition is approximately 20% to 30% of the general population in affluent countries, and it threatens to become a serious public health problem [2, 3]. NAFLD is defined as the accumulation of lipid deposits in hepatocytes that are not due to excessive alcohol use [4]. It represents a spectrum of diseases ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), which can progress to liver fibrosis, cirrhosis, and hepatocellular carcinoma [3, 4]. In clinical, the non-invasive assessment of steatosis level is of great interest besides the staging of hepatic fibrosis and distinguishing NASH from simple steatosis. The hepatic steatosis is linked to an increase in cardiovascular and diabetic patients’ risk of death; and it is associated with adverse perioperative outcomes following partial hepatectomy. Moreover, it is also the threshold criteria for the diagnosis of NAFLD [5-8].
Liver biopsy is currently considered the gold standard for the diagnosis steatosis. However, biopsy is an invasive procedure associated with severe complications in 0.3% to 3.0% of cases, and leads to death in 0.01% of cases [9]. Furthermore, it is highly dependent on the experience of the operator, and subject to sampling error because of the small size of biopsy samples [9, 10]. Therefore, a simple and noninvasive method of detecting steatosis severity in patients is of major clinical interest.
Many different MRI techniques have been developed to improve the performance of 3
this modality in the diagnostic spectrum of NAFLD. Magnetic resonance elastography (MRE) uses a modified phase-contrast method to image the propagation characteristics of the shear wave in the liver. Early studies of MRE suggest that it could accurately differentiate simple steatosis from NASH with or without fibrosis and has good accuracy for diagnosing NASH [11-13]. Magnetic resonance spectroscopy (MRS) that directly measures the proton signals from acyl groups in hepatocyte triglyceride stores has shown incredible accuracy for diagnosing and quantifying steatosis[14, 15]. However, MRI is costly and time-consuming for use in routine clinical practice.
Ultrasonography (US) is a non-invasive, cost effective, and readily available screening tool for NAFLD, but it has some limitations, such as dependence on the reliability of the operator and equipment, limited use in obese patients, and low sensitivity when the degree of steatosis is less than 20% to 30% [16-19]. Research over the past 2 decades has led to significant new developments in ultrasound elastographic methods, including transient elastography (TE) and acoustic radiation force impulse (ARFI) elastography. These techniques were initially applied to evaluate viral hepatitis induced liver fibrosis by measuring liver stiffness [20-24]. The TE technique has been developed into a commercial ultrasound device called Fibroscan (EchoSens, Paris, France). Clinical trials using Fibroscan have indicated that its stiffness measurements are significantly correlated with the stage of fibrosis, and the areas under the receiver operating characteristic curves (AUROCs) were close 4
to those achieved by biopsy [25, 26]. ARFI elastography is a technique that measures shear wave velocity (SWV) using high-intensity, short-duration acoustic pulses to generate localized displacements in tissue, so as to provide information on the mechanical properties of soft tissue [27-29]. It can be useful for the real-time evaluation of stiffness during standard ultrasonography examinations of the liver, and the acoustic radiation force can be transmitted through fat layers or ascites. It also shows good results for assessing liver fibrosis in chronic viral hepatitis [30-32].
Ultrasound elastography has recently been used for the diagnosis of patients with NAFLD . However, obesity, a major risk factor for NAFLD; it has been associated with TE failure in up to 25% of patients [33]. To improve the diagnostic accuracy of FibroScan in patients with NAFLD, some researchers have applied a new probe, which uses a lower frequency than the standard M probe, to provide a higher rate of measurement in patients with an increased body mass index (BMI) [34-36]. In contrast, ARFI can transmit acoustic radiation force through fat layers or ascites; it more suitable for patients with NAFLD. Many studies have shown that ARFI elastography is a promising method for differentiating patients with nonalcoholic steatohepatitis from patients with simple steatosis [37, 38]. ARFI has also been found useful in predicting significant fibrosis in patients with NAFLD [34, 39, 40].
Hepatic steatosis presents with histological signs of fat accumulation in hepatocytes, and almost no clinical manifestations [23]. Because of the invasive nature of liver 5
biopsy, for both ethical and practical reason, has limited use in patients with steatosis. Therefore, clinical research on steatosis by ARFI technology has mainly focused on steatosis with fibrosis or NASH [34, 37-41], while steatosis without fibrosis has been poorly studied. A few studies have used animal model to investigate the correlation between elastographic measurement and steatosis grade [42, 43]. For example, Aroca et al. showed an increase in ARFI results in an experimental fatty liver model using chickens [43]; Kang et al. found no significant differences in the elasticity of rat livers between the normal group and the group with simple steatosis [42]. However, the reasons for the differences in ARFI results within the steatosis grades are not clear.
The purpose of the present study was to develop a rat model with different severity grade of steatosis, use the ARFI elastography method to measure the SWV of rat livers, and determine the performance of SWV measurements for evaluating steatosis.
Materials and methods Animal model Steatosis of the liver was induced in male Sprague-Dawley (SD) rats (Guangdong Medical Laboratory Animal Center, Guangdong, China) weighing about 180 to 200 g. They were housed in sterile isolated cages with a 10/14-hour light/dark cycle at constant temperature (20° - 26°C) and humidity (50% - 70%). Sixty-nine SD rats were randomly divided into the control group (57 rats) and the fatty liver group (12 rats). The control group was provided with a standard diet with sterilized food and 6
water. The fatty liver group had been fed on a high fat emulsion (20% lard, 10% cholesterol, 20% sodium cholate, 0.5% propylthiouracil, and 30% fructose), once a day at 1 mL/100 g rat weight, for different numbers of days to induce different severity grade of steatosis. Two, 4, 6, and 8 weeks later, several rats (see Table 1 for the exact number each week) were humanely killed, the right lateral lobe of the liver was harvested for SWV measurements, and the other lobes were used for histologic assessment. The steatosis grade was ultimately determined by the histologic results, not by the length of time the rats were gavaged with high fat emulsion. All procedures in these studies were approved by the Animal Care Committee of Shenzhen University and the Guangdong Medical Laboratory Animal Center.
Blood test Blood samples (1.5 mL) were extracted from the orbital venous sinus into tubes after a 12-hour fasting period. Plasma was separated and analyzed for total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) using an automatic biochemical analyzer (Type 7020, Hitachi High-Tech Companies, Tokyo, Japan).
Histologic assessment The excised liver tissues were fixed in 10% formalin solution for at least 24 hours. After being washed and dehydrated, they were embedded in paraffin and sliced to a 7
thickness of 7 um. The paraffin slices were stained with Oil Red O, hematoxylin, and Masson’s trichrome by histopathology technicians, and analyzed using an Olympus BX41 microscope by an expert pathologist (20 years of experience) blinded to the results of the ultrasound measurements. The severity grade of steatosis was assessed on the basis of the percentage of hepatocytes containing macrovesicular fat droplets as follows: S0, no steatosis; S1, less than 25% of the hepatocytes contain macrovacuoles of fat; S2, 25% to 50% of the hepatocytes contain macrovacuoles of fat; S3, 50% to 75% of the hepatocytes contain macrovacuoles of fat; S4, more than 75% of the hepatocytes contain macrovacuoles of fat. The necroinflammatory severity was assessed and assigned grades as follows: G0, no inflammatory cells; G1, a small numbers of inflammatory cells are gathered around the portal area or central vein; G2, the inflammatory cells are gathered around the portal area, central vein, and hepatic sinus or lobular bridge; G3, there is infiltration of focal inflammatory cells, with liver cell denaturation and necrosis; G4, there is diffuse infiltration of inflammatory cells and degeneration/necrosis of a large number of liver cells. The stage of fibrosis was evaluated according to the METAVIR scoring system [44]: F0, no fibrosis; F1, portal fibrosis without septa; F2, portal fibrosis with a few septa; F3, numerous septa without cirrhosis; and F4, cirrhosis.
ARFI Elastography ARFI imaging is an elastographic technology that targets an anatomic region to be examined for its elastic properties using a region of interest (ROI) cursor while 8
performing real-time B-mode imaging. Tissue in the region of interest is mechanically stimulated using short-duration acoustic pulses that are automatically produced by an ultrasound probe. The acoustic push pulse transmitted by the transducer toward the tissue induces elastic shear waves that propagate through the tissue. The velocity, measured directly in meters per second (m/s), is displayed on the screen.
In an ex vivo experiment, the right liver lobe was processed and embedded in a fabricated gelatin solution (gelatin from porcine skin, Sigma–Aldrich, St. Louis, MO) in a container (11 cm × 11 cm × 7 cm). The ARFI imaging was performed by a sonographer (with 8 years of experience) using the Virtual Touch Tissue Quantification mode implemented on the Siemens Acuson S2000 ultrasound system with an 8-MHz linear ultrasound transducer. As shown in Figure 1, an area was chosen where the liver tissue was free of large blood vessels. The measurements were made at a 1.5 to 2 cm depth below the gelatin simulation. All the ultrasound studies were performed blind to the results of histologic assessment by the sonographer. Ten successful acquisitions were performed in each liver. The measurement was considered invalid due to motion artifact when the screen displayed “xxx”. The final results were expressed as the median value of the total measurements.
Statistical methods A total of 69 rats, which were grouped on the basis of histopathologic grade, were processed in this study. For each steatosis grade, the SWV values of all the rats at that 9
grade were grouped. The mean SWV of each rat was then used to estimate the population mean of each steatosis group. A Tukey test, in conjunction with analysis of variance (ANOVA), was performed to compare SWV measurements of the inflammation and steatosis severity grades. The diagnostic accuracy of the SWV parameters in discriminating between the grades of steatosis was studied using nonparametric AUROCs. Cutoff values were chosen by maximizing the Youden index [45]. Sensitivity and specificity values were computed with exact 95% confidence intervals. The correlations between SWV values and biological characteristics, and SWV values and inflammation activity, were tested using Spearman’s correlation coefficient. For all tests, significance was achieved at P < 0.05.
RESULTS Table 1 lists the distribution of rats with NAFLD according to their histologic characteristics at each sacrifice date. Figure 2 shows box plots of SWV measurements for each grade of steatosis. The median SWV was 2.25 ± 0.52 m/s (range 1.33 3.26m/s) in the S0 group, 2.66 ± 0.40 m/s (range 1.53 - 3.04 m/s) in the S1 group, 2.90 ± 0.40 m/s (range 1.93 - 3.85 m/s) in the S2 group, 2.93 ± 0.27 m/s (range 2.62 3.52 m/s) in the S3 group, and 2.82 ± 0.28 (range 2.36 - 3.18 m/s) m/s in the S4 group. The analysis of variance results show significant differences between the normal group and the steatosis group (P < 0.05). There were no significant differences with increasing histologic steatosis severity. As shown in Figure 3, SWV measurements have a good accuracy in predicting steatosis grade (S ≥ 1), with an AUROC of 0.82 10
(95% CI = 0.69, 0.96) and the optimal cutoff value was 2.59 with a sensitivity of 88% and specificity of 76%. Spearman’s correlation coefficient between SWV measurements and histologically determined steatosis grades showed a moderately significant correlation (r = 0.458, P < 0.05). As shown in Figure 4, the mean inflammation results were 2.70 ± 0.42 m/s (range 1.53 - 3.52 m/s) in the G0 group, 2.63 ± 0.59 m/s (range 1.53 - 3.52 m/s) in the G1 group, and 3.01 ± 0.18 m/s (range 2.80 - 3.16 m/s) in the G2 group. The 1-way ANOVA results showed no significant differences between the grades of inflammation. SWV values did not correlate with the grade of inflammation. Table 2 shows the clinical and biologic parameters observed in the rats. There was an increase in all lipid parameters measured in the plasma of rats fed a special diet, when compared to the control rats fed the standard diet. SWV measurements correlated significantly with ALT (r = 0.50, P < 0.05), AST (r = 0.42, P < 0.05), TC (r = 0.47, P < 0.05), and LDL (r = 0.45, P < 0.05) but no significant correlation was found between SWV and either TG or HDL. No fibrosis was observed on Masson staining.
DISCUSSION NAFLD has become a focus of increasing medical attention because obesity, hyperlipidemia, and disturbances related to excessive or unbalanced food ingestion have reached epidemic proportions throughout the world over the past decade [1, 3]. It is, moreover, a complex disorder involving genetic predisposition and environmental factors, and much is still unknown about its pathophysiology in humans. Animal models of the spectrum of NAFLD provide the necessary tools to 11
overcome confounding variables, such as genetic heterogeneity, and environmental factors, including diet and lifestyle [46-48]. Given the difficulty of studying all the factors involved in steatosis in human populations, the animal model may have helped to improve our understanding of the physiopathology of the disease.
NAFLD accompanies various histologic abnormalities, including hepatic steatosis, inflammation, and fibrosis, which may potentially affect ultrasound transmission and shear wave propagation [22, 23, 39, 42, 49]. For the clinical studies, it was found that that SWV values were significantly correlated with the stages of liver fibrosis in patients with NAFLD, and could distinguish patients with NASH from those with simple steatosis [30, 34, 37]. In clinical situations, all these abnormalities may combine with each other and contribute to the change in SWV values. Therefore, it is difficult to estimate the discrete effect of each disease on the SWV measurements.
As shown in Table 1, there was mild inflammation (G1, n = 25; G2, n = 3; G3, n = 0; G4, n = 0) without fibrosis in our rat livers. The 1-way ANOVA results showed that there was no significant difference between G0, G1, and G2 (P > 0.05) in the group with inflammation. The 2-way ANOVA indicated that there was no significant interaction between the early grade of inflammation and steatosis. Therefore, the mild inflammation in our animal model did not affect the SWV measurements.
In our study, the results of ROC analysis for the diagnostic performance of SWV in 12
grading hepatic steatosis severity can be seen in Figure 2. The mean SWV measured with the ARFI method was 2.25 ± 0.52 m/s in the normal group (range 1.33 - 3.26 m/s ) and 2.83 ± 0.37 m/s in the fatty liver group (range 1.53 - 3.85 m/s), with statistically significant differences observed between the 2 groups (P < 0.05). This finding is consistent with another animal study, which showed that the median velocity measured by the ARFI method was 0.9 ± 0.1 m/s in the control group and 1.9 ± 0.2 m/s in the hyperlipidemic diet group with statistically significant differences between the 2 groups (P < 0.001) [43]. However, the finding of this study is in conflict with some clinical studies. The study of Nightingale et al. found that there was no correlation with steatosis and SWS measurements in patients with NAFLD[41]. Another 2 studies showed a stepwise decrease in ARFI velocity with increasing histologic hepatic steatosis in patients with NAFLD [34, 37]. The discrepancy between our study and these clinical studies may be due to the differences between these conditions in animal models and in clinical patients. As mentioned above, clinical patients with NAFLD usually have several combined diseases—such as steatosis, inflammation, and fibrosis—which may contribute to the difference in ARFI velocity. On the other hand, the disease in the animal model is relatively simple. In the rat model of our study, the effects of steatosis is the dominant cause of the change in ARFI velocity. In the rat model, Kang et al. found no significant differences in the elasticity of rat livers between the normal group and the group with simple steatosis. The discrepancy may be due to the different method of developing animal model and measuring technique. 13
Although significant difference was found between the SWV values of normal group and steatosis group, the box plot and the ROC analysis show that the SWV values have no significant difference between the grades of steatosis. As shown in Fig. 2, the SWV values increase rapidly with the fat accumulation less than 25 percent (first steatosis grade); and this increase trend slows down with further fat accumulation (second to fourth steatosis grades). The possible explanation for this finding is the nature of liver steatosis which is caused by intra-cellular fat accumulation. Compared with inter-cellular change of collagen in liver fibrosis, this micro-level change may have less impact on shear wave propagation, especially in high steatosis grades [41].
One limitation of our study is that it would have been ideal to have rat model in which a single steatosis feature was expressed, but this was not possible. That is, inflammation may also precede or develop in parallel with hepatic steatosis owing to the increasing duration of feeding [42, 50]. Although mild inflammation had been found in the control group and fatty liver group, as shown in Table 1, mild inflammation did not affect the SWV by the 2-way ANOVA. Another limitation of our study is that liver stiffness was examined in ex vivo samples. This condition may overcome rat confounding environmental factors, such as different body boundary conditions and different thickness of the fat in our experiment. But mechanical and physiological characteristics may possibly change from in vivo to ex vivo. Meanwhile, the SWV value used to analyze the relationship between the ex vivo liver and lab 14
parameters may also have been limited.
In conclusion, our results indicate that ARFI elastography offers good diagnostic accuracy in evaluating the normal liver and hepatic steatosis, but no significant differences are observed between the grade of steatosis. Furthermore, the early grade of inflammation activity does not significantly affect the SWV measurements.
Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 81471735, 81570552, 61427806, 61101025, 61201041), National Natural Science Foundation of Guangdong Province (Grant No. 2016A030310047), and the National Science & Technology Pillar Program (2015BAI01B02)
References [1]
B.Q. Starley, C.J. Calcagno, and S.A. Harrison, Nonalcoholic fatty liver disease and hepatocellular carcinoma: a weighty connection, Hepatology, 51 (2010) 1820-1832.
[2]
A. Imhof, W. Kratzer, B. Boehm, K. Meitinger, G. Trischler, G. Steinbach, I. Piechotowski, and W. Koenig, Prevalence of non-alcoholic fatty liver and characteristics in overweight adolescents in the general population, European journal of epidemiology, 22 (2007) 889-897.
[3]
P. Angulo, Nonalcoholic fatty liver disease, New England Journal of Medicine, 346 (2002) 1221-1231.
[4]
I. Liou and K.V. Kowdley, Natural history of nonalcoholic steatohepatitis, Journal of clinical gastroenterology, 40 (2006) S11-S16.
[5]
Z.M. Younossi, T. Gramlich, C.A. Matteoni, N. Boparai, and A.J. Mccullough, Nonalcoholic fatty liver disease in patients with type 2 diabetes, Clinical Gastroenterology & Hepatology, 2 (2004) 262-265.
[6]
G. Targher, C.P. Day, and E. Bonora, Risk of cardiovascular disease in patients with nonalcoholic fatty liver disease, New England Journal of Medicine, 363 (2010) 1341-50.
[7]
L. Mccormack, P. Dutkowski, A.M. El-Badry, and P.A. Clavien, Liver transplantation using fatty livers: Always feasible?, Journal of hepatology, 54 (2011) 1055-62.
15
[8]
M.V.E. De, B.T. Kalish, M. Puder, and J.N.M. Ijzermans, Systematic review and meta-analysis of steatosis as a risk factor in major hepatic resection, British Journal of Surgery, 97 (2010) 1331-9.
[9]
M.R. Pagadala and A.J. McCullough, The relevance of liver histology to predicting clinically meaningful outcomes in nonalcoholic steatohepatitis, Clinics in liver disease, 16 (2012) 487-504.
[10]
G.A. Grandison and P. Angulo, Can NASH be diagnosed, graded, and staged noninvasively?, Clinics in liver disease, 16 (2012) 567-585.
[11]
L. Huwart, C. Sempoux, E. Vicaut, N. Salameh, L. Annet, E. Danse, F. Peeters, L.C.T. Beek, J. Rahier, and R. Sinkus, Magnetic Resonance Elastography for the Noninvasive Staging of Liver Fibrosis, Gastroenterology, 135 (2008) 32-40.
[12]
J. Chen, J.A. Talwalkar, M. Yin, K.J. Glaser, S.O. Sanderson, and R.L. Ehman, Early detection of nonalcoholic steatohepatitis in patients with nonalcoholic fatty liver disease by using MR elastography, Radiology, 259 (2011) 749-56.
[13]
S.K. Venkatesh and R.L. Ehman, Magnetic resonance elastography of liver, Magnetic Resonance Imaging Clinics of North America, 22 (2014) 433-46.
[14]
S. McPherson, J.R. Jonsson, G.J. Cowin, P.O. Rourke, A.D. Clouston, A. Volp, L. Horsfall, D. Jothimani, J. Fawcett, G.J. Galloway, M. Benson, and E.E. Powell, Magnetic resonance imaging and spectroscopy accurately estimate the severity of steatosis provided the stage of fibrosis is considered, Journal of hepatology, 51 (2009) 389–397.
[15]
G.J. Cowin, J.R. Jonsson, J.D. Bauer, S. Ash, A.A. Bhsc, E.J.O. Bhsc, D.M. Purdie, and G.J. Galloway, Magnetic resonance imaging and spectroscopy for monitoring liver steatosis, Journal of Magnetic Resonance Imaging, 28 (2008) 937–945.
[16]
S. Dasarathy, J. Dasarathy, A. Khiyami, R. Joseph, R. Lopez, and A.J. McCullough, Validity of real time ultrasound in the diagnosis of hepatic steatosis: a prospective study, Journal of hepatology, 51 (2009) 1061-1067.
[17]
C.C. Mottin, M. Moretto, A.V. Padoin, A.M. Swarowsky, M.G. Toneto, L. Glock, and G. Repetto, The role of ultrasound in the diagnosis of hepatic steatosis in morbidly obese patients, Obesity surgery, 14 (2004) 635-637.
[18]
C.K. Ryan, L.A. Johnson, B.I. Germin, and A. Marcos, One hundred consecutive hepatic biopsies in the workup of living donors for right lobe liver transplantation, Liver transplantation, 8 (2002) 1114-1122.
[19]
R. Bouzitoune, M. Meziri, C.B. Machado, F. Padilla, and W.C. Pereira, Can early hepatic fibrosis stages be discriminated by combining ultrasonic parameters?, Ultrasonics, 68 (2016) 120-126.
[20]
G. Ferraioli, C. Filice, L. Castera, B.I. Choi, I. Sporea, S.R. Wilson, D. Cosgrove, C.F. Dietrich, D. Amy, and J.C. Bamber, WFUMB Guidelines and Recommendations for Clinical Use of Ultrasound Elastography: Part 3: Liver, Ultrasound in medicine & biology, 41 (2015) 1161-1179.
[21]
D. Cosgrove, F. Piscaglia, J. Bamber, J. Bojunga, J.M. Correas, O.H. Gilja, A.S. Klauser, I. Sporea, F. Calliada, and V. Cantisani, EFSUMB guidelines and recommendations on the clinical use of ultrasound elastography. Part 2: Clinical applications, Ultraschall in Der Medizin, 34 (2013) 238-253.
[22]
W.K. Jeong, H.K. Lim, H.K. Lee, J.M. Jo, and Y. Kim, Principles and clinical application of
16
ultrasound elastography for diffuse liver disease, Ultrasonography, 33 (2014) 149-160. [23]
M.V. Machado and H. Cortez-Pinto, Non-invasive diagnosis of non-alcoholic fatty liver disease. A critical appraisal, Journal of hepatology, 58 (2013) 1007-19.
[24]
X. Chen, Y. Shen, Y. Zheng, H. Lin, Y. Guo, Y. Zhu, X. Zhang, T. Wang, and S. Chen, Quantification of liver viscoelasticity with acoustic radiation force: a study of hepatic fibrosis in a rat model, Ultrasound in medicine & biology, 39 (2013) 2091-102.
[25]
S.M. Martinez, G. Crespo, M. Navasa, and X. Forns, Noninvasive assessment of liver fibrosis, Hepatology, 53 (2011) 325-335.
[26]
P. Marcellin, M. Ziol, P. Bedossa, C. Douvin, R. Poupon, V. De Lédinghen, and M. Beaugrand, Non-invasive assessment of liver fibrosis by stiffness measurement in patients with chronic hepatitis B, Liver international, 29 (2009) 242-247.
[27]
A.P. Sarvazyan, O.V. Rudenko, S.D. Swanson, J.B. Fowlkes, and S.Y. Emelianov, Shear wave elasticity imaging: a new ultrasonic technology of medical diagnostics, Ultrasound in medicine & biology, 24 (1998) 1419-1435.
[28]
K. Nightingale, M.S. Soo, R. Nightingale, and G. Trahey, Acoustic radiation force impulse imaging: in vivo demonstration of clinical feasibility, Ultrasound in medicine & biology, 28 (2002) 227-235.
[29]
L. Zhai, M.L. Palmeri, R.R. Bouchard, R.W. Nightingale, and K.R. Nightingale, An integrated indenter-ARFI imaging system for tissue stiffness quantification, Ultrasonic imaging, 30 (2008) 95-111.
[30]
H. Ebinuma, H. Saito, M. Komuta, K. Ojiro, K. Wakabayashi, S. Usui, P.-s. Chu, R. Umeda, Y. Ishibashi, and T. Takayama, Evaluation of liver fibrosis by transient elastography using acoustic radiation force impulse: comparison with Fibroscan®, Journal of gastroenterology, 46 (2011) 1238-1248.
[31]
M. Friedrich-Rust, K. Wunder, S. Kriener, F. Sotoudeh, S. Richter, J. Bojunga, E. Herrmann, T. Poynard, C.F. Dietrich, and J. Vermehren, Liver Fibrosis in Viral Hepatitis: Noninvasive Assessment with Acoustic Radiation Force Impulse Imaging versus Transient Elastography 1, Radiology, 252 (2009) 595-604.
[32]
R. Goertz, Y. Zopf, V. Jugl, R. Heide, C. Janson, D. Strobel, T. Bernatik, and T. Haendl, Measurement of liver elasticity with acoustic radiation force impulse (ARFI) technology: an alternative noninvasive method for staging liver fibrosis in viral hepatitis, Ultraschall in der Medizin (Stuttgart, Germany: 1980), 31 (2010) 151-155.
[33]
R. Kwok, Y.K. Tse, G.H. Wong, Y. Ha, A. Lee, M. Ngu, H.Y. Chan, and V.S. Wong, Systematic review with meta-analysis: non-invasive assessment of non-alcoholic fatty liver disease-the role of transient elastography and plasma cytokeratin-18 fragments, Alimentary pharmacology & therapeutics, 39 (2014) 254-269.
[34]
M. Yoneda, K. Suzuki, S. Kato, K. Fujita, Y. Nozaki, K. Hosono, S. Saito, and A. Nakajima, Nonalcoholic fatty liver disease: US-based acoustic radiation force impulse elastography 1, Radiology, 256 (2010) 640-647.
[35]
M. Sasso, S. Audière, A. Kemgang, F. Gaouar, C. Corpechot, O. Chazouillères, C. Fournier, O. Golsztejn, S. Prince, and Y. Menu, Liver Steatosis Assessed by Controlled Attenuation Parameter (CAP) Measured with the XL Probe of the FibroScan: A Pilot Study Assessing Diagnostic Accuracy, Ultrasound in medicine & biology, 42 (2016) 92-103.
[36]
M. Friedrich-Rust, H. Hadji-Hosseini, S. Kriener, E. Herrmann, I. Sircar, A. Kau, S. Zeuzem,
17
and J. Bojunga, Transient elastography with a new probe for obese patients for non-invasive staging of non-alcoholic steatohepatitis, European radiology, 20 (2010) 2390-2396. [37]
C.F. Braticevici, I. Sporea, E. Panaitescu, and L. Tribus, Value of acoustic radiation force impulse imaging elastography for non-invasive evaluation of patients with nonalcoholic fatty liver disease, Ultrasound in medicine & biology, 39 (2013) 1942-1950.
[38]
F. Aroca, M. Frutos-Bernal, A. Bas, J. Luján-Mompeán, M. Reus, J. de Dios Berná-Serna, and P. Parrilla, Detection of non-alcoholic steatohepatitis in patients with morbid obesity before bariatric surgery: preliminary evaluation with acoustic radiation force impulse imaging, European radiology, 22 (2012) 2525-2532.
[39]
T. Deffieux, J.L. Gennisson, L. Bousquet, M. Corouge, S. Cosconea, D. Amroun, S. Tripon, B. Terris, V. Mallet, and P. Sogni, Investigating liver stiffness and viscosity for fibrosis, steatosis and activity staging using shear wave elastography, Journal of hepatology, 62 (2015) 317-24.
[40]
V.W.S. Wong, J. Vergniol, G.L.H. Wong, J. Foucher, H.L.Y. Chan, B. Le Bail, P.C.L. Choi, M. Kowo, A.W.H. Chan, and W. Merrouche, Diagnosis of fibrosis and cirrhosis using liver stiffness measurement in nonalcoholic fatty liver disease, Hepatology, 51 (2010) 454-462.
[41]
K.R. Nightingale, N.C. Rouze, S.J. Rosenzweig, M.H. Wang, M.F. Abdelmalek, C.D. Guy, and M.L. Palmeri, Derivation and analysis of viscoelastic properties in human liver: impact of frequency on fibrosis and steatosis staging, IEEE Transactions on Ultrasonics Ferroelectrics & Frequency Control, 62 (2015) 165-75.
[42]
B.K. Kang, S.S. Lee, H. Cheong, S.M. Hong, K. Jang, and M.G. Lee, Shear Wave Elastography for Assessment of Steatohepatitis and Hepatic Fibrosis in Rat Models of Non-Alcoholic Fatty Liver Disease, Ultrasound in medicine & biology, 41 (2015) 3205-15.
[43]
F.G. Aroca, I. Ayala, L. Serrano, J.D. Berná-Serna, M.T. Castell, B. García-Pérez, and M. Reus, Assessment of liver steatosis in chicken by using acoustic radiation force impulse imaging: preliminary results, European radiology, 20 (2010) 2367-2371.
[44]
E.M. Brunt, Nonalcoholic steatohepatitis: definition and pathology, Seminars in Liver Disease, 21 (2001) 3-16.
[45]
W.J. Youden, Index for rating diagnostic tests, Cancer, 3 (1950) 32-35.
[46]
R.M. London and J. George, Pathogenesis of NASH: animal models, Clinics in liver disease, 11 (2007) 55-74.
[47]
N.F. Schwenzer, F. Springer, C. Schraml, N. Stefan, J. Machann, and F. Schick, Non-invasive assessment and quantification of liver steatosis by ultrasound, computed tomography and magnetic resonance, Journal of hepatology, 51 (2009) 433-445.
[48]
I. Ayala, A.M. Castillo, G. Adanez, A. Fernandez-Rufete, B.G. Pérez, and M.T. Castells, Hyperlipidemic chicken as a model of non-alcoholic steatohepatitis, Experimental biology and medicine, 234 (2009) 10-16.
[49]
F. Piscaglia, S. Marinelli, S. Bota, C. Serra, L. Venerandi, S. Leoni, and V. Salvatore, The role of ultrasound elastographic techniques in chronic liver disease: current status and future perspectives, European Journal of Radiology, 83 (2013) 450-5.
[50]
N. Chalasani, L. Wilson, D.E. Kleiner, O.W. Cummings, E.M. Brunt, and A. Unalp, Relationship of steatosis grade and zonal location to histological features of steatohepatitis in adult patients with non-alcoholic fatty liver disease, Journal of hepatology, 48 (2008) 829-34.
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Figure Captions Fig. 1. B-mode image from one of the isolated rat liver phantoms. The green box represents the ROI for reconstructing the shear velocity.
19
Fig. 2. Box plots of SWV measurements in each steatosis grade. Boundaries of the boxes indicate the 25th and 5th percentiles; lines within the boxes indicate medians; and error bars indicate ranges for each grade.
20
Fig. 3. Receiver operating characteristic curves for differentiation different steatosis grades.
21
Fig. 4.
Box plots of SWV measurements in each inflammation grade.
22
Table 1. Number of rats and histopathologic results at each sacrifice date Duration of fat emulsion administration
Number of rats
Steatosis group (0/1/2/3/4)
Inflamation group (0/1/2/3/4)
Fibrosis group (0/1/2/3/4)
Control
12
12/0/0/0/0
8/4/0/0/0
12/0/0/0/0
2 weeks
15
3/7/4/1/0
7/7/1/0/0
15/0/0/0/0
4 weeks
13
1/3/5/4/0
3/8/2/0/0
13/0/0/0/0
6 weeks
14
0/1/8/5/0
11/3/0/0/0
14/0/0/0/0
8 weeks
15
1/3/2/2/7
12/3/0/0/0
15/0/0/0/0
Table 2. Results of liver enzymes and plasma lipids from rats. Steatosis Group
ALT (U/L)
AST (U/L)
TC (mmol/L)
TG (mmol/L)
HDL-C (mmol/L)
LDL-C (mmol/L)
S0
45.59±9. 25* 73.08±3 0.14 59.59±1 7.61 71.42±1 7.47 103.57± 19.31
119.19±17 .39 153.10±31 .75 144.82±25 .43 139.40±24 .69 321.00±79 .20
4.50±3.07
1.59±0.62
0.97±0.40
0.72±0.89
9.08±2.87
1.51±0.65
1.34±0.36
2.66±1.94
19.74±12. 28 14.09±4.2 3 13.27±2.1 1
1.67±0.72
1.47±0.38
6.89±6.00
1.84±1.06
1.54±0.39
5.63±3.30
3.23±0.80
1.88±0.42
5.65±1.30
S1 S2 S3 S4
ALT = alanine aminotransferase; AST = aspartate aminotransferase; TC = total cholesterol; TG = Triglyceride; HDL-C = high density lipoprotein cholesterol; LDL-C = low density lipoprotein cholesterol *The values of biochemical characteristics are expressed as the mean ± standard deviation.
23
24
Highlights
The research by ARFI technology is focused on steatosis without fibrosis. ARFI elastography offers good diagnostic accuracy in evaluating the normal rat liver and rat liver with steatosis. The SWV measurements have no significant difference between the grades of steatosis. The early grade of inflammation activity does not significantly affect the SWV measurements.
25