ORIGINAL RESEARCH
Intravoxel Incoherent Motion MRI Evaluation for the Staging of Liver Fibrosis in a Rat Model Genwen Hu, MD,1,2 Queenie Chan, PhD,3 Xianyue Quan, MD,1* Xuhui Zhang, MD,1 Yufa Li, MD,4 Xing Zhong, MD,5 and Xiaoying Lin, MD2 Purpose: To explore the characteristics of intravoxel incoherent motion (IVIM) in various stages of liver fibrosis, and their relationships with fibrotic stages in rats. Materials and Methods: Fifty rats were given various doses of carbon tetrachloride (CCl4) to induce various fibrotic stages in rats; 15 untreated rats served as controls. Diffusion-weighted magnetic resonance imaging (MRI) was performed and eight b-values (0–800 s/mm2) were applied to obtain IVIM parameters (D, pure molecular diffusion; f, perfusion fraction; D*, pseudodiffusion). The stages of liver fibrosis (stages F0–F4) were evaluated histologically using METAVIR scores. Fifty-seven rats (15 controls and 42 with fibrosis) were analyzed by nonparametric methods and receiver operating characteristic curves to determine diagnostic accuracy. Results: Significant differences (P < 0.001) were found between stages (stages F0–F4) by D, f, D*, and apparent diffusion coefficient (ADC). There were inverse correlations between fibrosis stages and D, f, D*, ADC (r 5 –0.657, r 5 –0.631, r 5 –0.711 r 5 –0.719, respectively). Multivariate analysis showed that the combination models (D, f, D*) were better than the individual parameter (ADC) for the evaluation fibrosis stages (area under the curve [AUC]: 0.821–1.000 vs. AUC: 0.753–0.918) Conclusion: IVIM-derived parameters showed significant correlations with stages of liver fibrosis in a rat model. J. MAGN. RESON. IMAGING 2015;42:331–339.
L
IVER FIBROSIS is a consequence of sustained prolonged injury from a variety of causes including hepatitis, autoimmune disorders, biliary atresia, and metabolic diseases. Progression of early fibrosis can be reversed by treatment with specific antifibrotic therapy or removal of the cause.1–3 Therefore, early and accurate diagnosis of liver fibrosis is important in the prevention of morbidity and mortality. Liver biopsy is the reference standard for the assessment of chronic liver disease including liver fibrosis. However, liver biopsy is an invasive technique, and may have complications including pain and bleeding.4 Furthermore, liver biopsy may also result in sampling error.5,6
Thus, noninvasive tools assessing the degree of injury and fibrosis of the whole liver are needed. Liver fibrosis results in extracellular accumulation of collagen, glycosaminoglycans, and proteoglycans that may restrict the molecular diffusion of water,7 thus suggesting that magnetic resonance imaging (MRI) diffusion-weighted imaging (DWI) may be useful for assessing fibrosis. In addition, studies have shown that the apparent diffusion coefficients (ADCs) from a monoexponential model in cirrhotic livers were significantly lower than those in nonfibrotic livers.7–9 However, ADC in the living tissue can be affected not only by the molecular diffusion of water, but also by the microcirculation blood capillary perfusion.
View this article online at wileyonlinelibrary.com. DOI: 10.1002/jmri.24796 Received Sep 10, 2014, Accepted for publication Oct 23, 2014. *Address reprint requests to: X.Q., Department of Medical Image Center, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, P.R. China. E-mail: [email protected] From the 1Department of Medical Image Center, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong Province, P.R. China; Department of Medical Image Center, Shenzhen Bao’an Maternal and Child Health Hospital, Shenzhen, Guangdong Province, P.R. China; 3Philips Healthcare, Hong Kong, P.R. China; 4Department of Pathology, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong Province, P.R. China; and 5Department of Medical Image Center, First Affiliated Hospital, Jinan University, Guangzhou, Guangdong Province, P.R. China
2
Potential conflict of interest: Nothing to report. The first two authors contributed equally to this work.
C 2014 Wiley Periodicals, Inc. V 331
Journal of Magnetic Resonance Imaging
FIGURE 1: A rat with fibrosis stage F2; (a) white circles denote ROIs manually placed over the liver parenchyma; (b) axial T2weighted image; (c) ADC map showing mean values (0.92 3 1023 mm2/s); (d) pure molecular diffusion (D) map showing mean values (0.80 3 1023 mm2/s); (e) perfusion fraction (f) map showing mean values (0.17); (f) pseudodiffusion (D*) map showing mean values (29.13 3 1023 mm2/s).
Recently, intravoxel incoherent motion (IVIM), a DWI technique that is able to measure both diffusion and perfusion parameters, has been found to be superior to ADC in diagnosing liver fibrosis.10,11 IVIM reflects the random microscopic motion that occurs in voxels on MRI of intracellular or extracellular water molecules and the microcirculation of blood. At present, only a few studies using IVIM in liver fibrosis have been reported,10,12–15 and fewer on the assessment of staging in liver fibrosis.16 The aim of the current study was to investigate the relationship between liver IVIM measurements and various stages of carbon tetrachloride (CCl4)-induced liver fibrosis in a rat model, and to assess the application value of IVIM in the staging of liver fibrosis.
Materials and methods Animals and Treatment A total of 65 adult male Sprague-Dawley (SD) rats (200 6 20 g) (specific pathogen-free, Certificate No. 44002100001642) from the
Southern Medical University Laboratory Animal (Guangzhou, China) were used. The rats were provided with standard feed and water ad libitum and individually housed at a constant temperature (18–20 C) and humidity (60–70%) with a 12-hour light/dark cycle. All experimental procedures were approved by the Institutional Animal Ethics Committee of Southern Medical University. Rats were randomly divided into two groups: liver fibrosis model group (n 5 50) and control group (n 5 15). We used the liver fibrosis modeling method as described previously.17,18 After a week of acclimatization to the standard conditions, the liver fibrosis model group rats were administered 50% (v/v) CCl4: olive oil, subcutaneously at a dose of 0.3 mL/100 g of body weight, twice a week for 10 weeks. During the procedure, rats were weighed twice a week to adjust the dose of drug. The control group, 15 rats, were scanned by MRI after a week of acclimatization. Fifty rats in the liver fibrosis model group were given the first injection of CCl4 on the same day. After the first injection, five rats from the liver fibrosis model group were randomly selected for MRI at each of nine timepoints of 14, 21, 28, 35, 42, 49, 56, 63, and 70 days. Five rats in the model group died in the process of intervention.
Conventional MRI Techniques Abbreviations
CCl4 D D* f IVIM TFE TSE
332
carbon tetrachloride pure molecular diffusion pseudodiffusion perfusion fraction intravoxel incoherent motion turbo-field-echo turbo-spin-echo
All MRIs were performed using a 3.0T MRI (Achieva 3.0T TX, Philips Healthcare, Best, Netherlands), using an animal coil. MRI scans were performed after the rats were anesthetized (3% pentobarbital [w/v] at 0.2 mL/100 g of body weight by intraperitoneal injection). The rats were placed in a prone position, head forward. The abdomen of each rat was fixed with a belt to decrease respiratory motion. The standard sequences were performed: 1) axial Volume 42, No. 2
Hu et al.: IVIM in Various Stages of Liver Fibrosis
FIGURE 2: Examples of typical H&E staining and Masson’s trichrome staining. a–e: Liver fibrosis stages 0–4 (H&E stain; 10 3 20). f–j: Liver fibrosis stages 0–4 (Masson stain; 10 3 40).
T2-weighted fast field echo (2D-FFE) (repetition time / echo time [TR/TE] 5 206/9.2 msec; field of view [FOV] 5 60 3 60 mm; matrix 5 100 3 100; slice thickness 5 3 mm); 2) axial T1-weighted turbo-spin-echo (TSE) (TR/TE 5 400/10ms; FOV 5 60 3 60 mm; matrix 5 120 3 93; slice thickness 5 3 mm); and 4) axial T2-weighted TSE (TR/TE 5 1080/120 msec; FOV 5 60 3 60 mm; matrix 5 120 3 88; slice thickness 5 3 mm). All routine scans had 13 slices.
Diffusion-Weighted MRI Diffusion-weighted MRI was performed using a single-shot spinecho echo-planar imaging sequence, with the following parameters: TR/TE 5 2000/55 msec, echo-planar imaging factor 5 63, FOV 5 50 3 50 mm, slice thickness 5 3 mm, matrix 5 64 3 63, receiver bandwidth 5 2,735.7 Hz per pixel, motion probing gradients in three orthogonal axes, number of signal averages 5 3, parallel imaging (sensitivity encoding [SENSE]) factor 5 3, and spectral presaturation inversion recovery fat suppression. Nine axial slices were selected to cut through the liver. A total of eight bvalues were used: 0, 25, 50, 100, 150, 300, 500, and 800 s/mm2.
Image Analysis The biexponential model from an IVIM sequence was described by Le Bihan et al.19 as:
Sb =S0 5ð12f Þ exp ð2bDÞ1f :½2bðD1D Þ where Sb is the signal intensity in the pixel with diffusion gradient b, S0 is the signal intensity in the pixel without a diffusion gradient, D is the true diffusion as reflected by pure molecular diffusion, f is the fractional perfusion related to microcirculation, and D* is the pseudodiffusion coefficient related to perfusion. August 2015
In this study ADC was calculated assuming monoexponential decay using b values of 0 and 800. Image analysis was performed with software supplied by the manufacturer (PRIDE DWI Tool, v. 1.5, Philips Healthcare) and fitted on a pixel-by-pixel basis by the Levenberg–Marquardt algorithm, as described before.12,13,20 D was obtained by a simplified linear fit equation using b values greater than 200 s/mm2. f and D* were calculated by a nonlinear regression algorithm using all b values. The IVIM parameters obtained in each slice were calculated on a pixel-by-pixel basis and expressed as mean values of all pixels within the volumes of interest. Five regions of interest (ROIs) of 3–4 mm2 were manually drawn to avoid the inclusion of vessels/bile ducts and the margin of the liver. Subsequent analysis was performed using ImageJ (NIH, Bethesda, MD). The mean values of these five ROIs were considered to be the parameter values (Fig. 1). Images of three rats could not be evaluated (n 5 3, F3 5 1, F4 5 2), because of unacceptable image quality.
Histopathological Analysis Five to 15 hours after MR scanning, rats were sacrificed by an overdose of 3% pentobarbital by intraperitoneal injection. Rat livers were excised and fixed in 10% formalin for histological examination of 5-lm sections stained with hematoxylin and eosin (H&E) for routine histology, and Masson’s trichrome stained to detect collagen. Serial sections were examined under a microscope (Leica DM2000) and photographed. The stages of liver fibrosis were scored by an experienced histopathologist (L.Y.F., 9 years of liver pathology) using the METAVIR classification system,21,22 and blinded to the results of the MR, in which F0 5 no fibrosis, F1 5 portal fibrosis without septa, F2 5 portal fibrosis and a few septa, F3 5 numerous septa without cirrhosis, and F4 5 cirrhosis. 333
Journal of Magnetic Resonance Imaging
TABLE 1. Correlation and 95% CI Between Fibrosis Stage and Parameters
Model Biexponential
Monoexponential
Parameters
Fibrosis stage
P
D
20.657 (20.774, 20.467)
Intravoxel Incoherent Motion MRI Evaluation for the Staging of Liver Fibrosis in a Rat Model Genwen Hu, MD,1,2 Queenie Chan, PhD,3 Xianyue Quan, MD,1* Xuhui Zhang, MD,1 Yufa Li, MD,4 Xing Zhong, MD,5 and Xiaoying Lin, MD2 Purpose: To explore the characteristics of intravoxel incoherent motion (IVIM) in various stages of liver fibrosis, and their relationships with fibrotic stages in rats. Materials and Methods: Fifty rats were given various doses of carbon tetrachloride (CCl4) to induce various fibrotic stages in rats; 15 untreated rats served as controls. Diffusion-weighted magnetic resonance imaging (MRI) was performed and eight b-values (0–800 s/mm2) were applied to obtain IVIM parameters (D, pure molecular diffusion; f, perfusion fraction; D*, pseudodiffusion). The stages of liver fibrosis (stages F0–F4) were evaluated histologically using METAVIR scores. Fifty-seven rats (15 controls and 42 with fibrosis) were analyzed by nonparametric methods and receiver operating characteristic curves to determine diagnostic accuracy. Results: Significant differences (P < 0.001) were found between stages (stages F0–F4) by D, f, D*, and apparent diffusion coefficient (ADC). There were inverse correlations between fibrosis stages and D, f, D*, ADC (r 5 –0.657, r 5 –0.631, r 5 –0.711 r 5 –0.719, respectively). Multivariate analysis showed that the combination models (D, f, D*) were better than the individual parameter (ADC) for the evaluation fibrosis stages (area under the curve [AUC]: 0.821–1.000 vs. AUC: 0.753–0.918) Conclusion: IVIM-derived parameters showed significant correlations with stages of liver fibrosis in a rat model. J. MAGN. RESON. IMAGING 2015;42:331–339.
L
IVER FIBROSIS is a consequence of sustained prolonged injury from a variety of causes including hepatitis, autoimmune disorders, biliary atresia, and metabolic diseases. Progression of early fibrosis can be reversed by treatment with specific antifibrotic therapy or removal of the cause.1–3 Therefore, early and accurate diagnosis of liver fibrosis is important in the prevention of morbidity and mortality. Liver biopsy is the reference standard for the assessment of chronic liver disease including liver fibrosis. However, liver biopsy is an invasive technique, and may have complications including pain and bleeding.4 Furthermore, liver biopsy may also result in sampling error.5,6
Thus, noninvasive tools assessing the degree of injury and fibrosis of the whole liver are needed. Liver fibrosis results in extracellular accumulation of collagen, glycosaminoglycans, and proteoglycans that may restrict the molecular diffusion of water,7 thus suggesting that magnetic resonance imaging (MRI) diffusion-weighted imaging (DWI) may be useful for assessing fibrosis. In addition, studies have shown that the apparent diffusion coefficients (ADCs) from a monoexponential model in cirrhotic livers were significantly lower than those in nonfibrotic livers.7–9 However, ADC in the living tissue can be affected not only by the molecular diffusion of water, but also by the microcirculation blood capillary perfusion.
View this article online at wileyonlinelibrary.com. DOI: 10.1002/jmri.24796 Received Sep 10, 2014, Accepted for publication Oct 23, 2014. *Address reprint requests to: X.Q., Department of Medical Image Center, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, P.R. China. E-mail: [email protected] From the 1Department of Medical Image Center, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong Province, P.R. China; Department of Medical Image Center, Shenzhen Bao’an Maternal and Child Health Hospital, Shenzhen, Guangdong Province, P.R. China; 3Philips Healthcare, Hong Kong, P.R. China; 4Department of Pathology, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong Province, P.R. China; and 5Department of Medical Image Center, First Affiliated Hospital, Jinan University, Guangzhou, Guangdong Province, P.R. China
2
Potential conflict of interest: Nothing to report. The first two authors contributed equally to this work.
C 2014 Wiley Periodicals, Inc. V 331
Journal of Magnetic Resonance Imaging
FIGURE 1: A rat with fibrosis stage F2; (a) white circles denote ROIs manually placed over the liver parenchyma; (b) axial T2weighted image; (c) ADC map showing mean values (0.92 3 1023 mm2/s); (d) pure molecular diffusion (D) map showing mean values (0.80 3 1023 mm2/s); (e) perfusion fraction (f) map showing mean values (0.17); (f) pseudodiffusion (D*) map showing mean values (29.13 3 1023 mm2/s).
Recently, intravoxel incoherent motion (IVIM), a DWI technique that is able to measure both diffusion and perfusion parameters, has been found to be superior to ADC in diagnosing liver fibrosis.10,11 IVIM reflects the random microscopic motion that occurs in voxels on MRI of intracellular or extracellular water molecules and the microcirculation of blood. At present, only a few studies using IVIM in liver fibrosis have been reported,10,12–15 and fewer on the assessment of staging in liver fibrosis.16 The aim of the current study was to investigate the relationship between liver IVIM measurements and various stages of carbon tetrachloride (CCl4)-induced liver fibrosis in a rat model, and to assess the application value of IVIM in the staging of liver fibrosis.
Materials and methods Animals and Treatment A total of 65 adult male Sprague-Dawley (SD) rats (200 6 20 g) (specific pathogen-free, Certificate No. 44002100001642) from the
Southern Medical University Laboratory Animal (Guangzhou, China) were used. The rats were provided with standard feed and water ad libitum and individually housed at a constant temperature (18–20 C) and humidity (60–70%) with a 12-hour light/dark cycle. All experimental procedures were approved by the Institutional Animal Ethics Committee of Southern Medical University. Rats were randomly divided into two groups: liver fibrosis model group (n 5 50) and control group (n 5 15). We used the liver fibrosis modeling method as described previously.17,18 After a week of acclimatization to the standard conditions, the liver fibrosis model group rats were administered 50% (v/v) CCl4: olive oil, subcutaneously at a dose of 0.3 mL/100 g of body weight, twice a week for 10 weeks. During the procedure, rats were weighed twice a week to adjust the dose of drug. The control group, 15 rats, were scanned by MRI after a week of acclimatization. Fifty rats in the liver fibrosis model group were given the first injection of CCl4 on the same day. After the first injection, five rats from the liver fibrosis model group were randomly selected for MRI at each of nine timepoints of 14, 21, 28, 35, 42, 49, 56, 63, and 70 days. Five rats in the model group died in the process of intervention.
Conventional MRI Techniques Abbreviations
CCl4 D D* f IVIM TFE TSE
332
carbon tetrachloride pure molecular diffusion pseudodiffusion perfusion fraction intravoxel incoherent motion turbo-field-echo turbo-spin-echo
All MRIs were performed using a 3.0T MRI (Achieva 3.0T TX, Philips Healthcare, Best, Netherlands), using an animal coil. MRI scans were performed after the rats were anesthetized (3% pentobarbital [w/v] at 0.2 mL/100 g of body weight by intraperitoneal injection). The rats were placed in a prone position, head forward. The abdomen of each rat was fixed with a belt to decrease respiratory motion. The standard sequences were performed: 1) axial Volume 42, No. 2
Hu et al.: IVIM in Various Stages of Liver Fibrosis
FIGURE 2: Examples of typical H&E staining and Masson’s trichrome staining. a–e: Liver fibrosis stages 0–4 (H&E stain; 10 3 20). f–j: Liver fibrosis stages 0–4 (Masson stain; 10 3 40).
T2-weighted fast field echo (2D-FFE) (repetition time / echo time [TR/TE] 5 206/9.2 msec; field of view [FOV] 5 60 3 60 mm; matrix 5 100 3 100; slice thickness 5 3 mm); 2) axial T1-weighted turbo-spin-echo (TSE) (TR/TE 5 400/10ms; FOV 5 60 3 60 mm; matrix 5 120 3 93; slice thickness 5 3 mm); and 4) axial T2-weighted TSE (TR/TE 5 1080/120 msec; FOV 5 60 3 60 mm; matrix 5 120 3 88; slice thickness 5 3 mm). All routine scans had 13 slices.
Diffusion-Weighted MRI Diffusion-weighted MRI was performed using a single-shot spinecho echo-planar imaging sequence, with the following parameters: TR/TE 5 2000/55 msec, echo-planar imaging factor 5 63, FOV 5 50 3 50 mm, slice thickness 5 3 mm, matrix 5 64 3 63, receiver bandwidth 5 2,735.7 Hz per pixel, motion probing gradients in three orthogonal axes, number of signal averages 5 3, parallel imaging (sensitivity encoding [SENSE]) factor 5 3, and spectral presaturation inversion recovery fat suppression. Nine axial slices were selected to cut through the liver. A total of eight bvalues were used: 0, 25, 50, 100, 150, 300, 500, and 800 s/mm2.
Image Analysis The biexponential model from an IVIM sequence was described by Le Bihan et al.19 as:
Sb =S0 5ð12f Þ exp ð2bDÞ1f :½2bðD1D Þ where Sb is the signal intensity in the pixel with diffusion gradient b, S0 is the signal intensity in the pixel without a diffusion gradient, D is the true diffusion as reflected by pure molecular diffusion, f is the fractional perfusion related to microcirculation, and D* is the pseudodiffusion coefficient related to perfusion. August 2015
In this study ADC was calculated assuming monoexponential decay using b values of 0 and 800. Image analysis was performed with software supplied by the manufacturer (PRIDE DWI Tool, v. 1.5, Philips Healthcare) and fitted on a pixel-by-pixel basis by the Levenberg–Marquardt algorithm, as described before.12,13,20 D was obtained by a simplified linear fit equation using b values greater than 200 s/mm2. f and D* were calculated by a nonlinear regression algorithm using all b values. The IVIM parameters obtained in each slice were calculated on a pixel-by-pixel basis and expressed as mean values of all pixels within the volumes of interest. Five regions of interest (ROIs) of 3–4 mm2 were manually drawn to avoid the inclusion of vessels/bile ducts and the margin of the liver. Subsequent analysis was performed using ImageJ (NIH, Bethesda, MD). The mean values of these five ROIs were considered to be the parameter values (Fig. 1). Images of three rats could not be evaluated (n 5 3, F3 5 1, F4 5 2), because of unacceptable image quality.
Histopathological Analysis Five to 15 hours after MR scanning, rats were sacrificed by an overdose of 3% pentobarbital by intraperitoneal injection. Rat livers were excised and fixed in 10% formalin for histological examination of 5-lm sections stained with hematoxylin and eosin (H&E) for routine histology, and Masson’s trichrome stained to detect collagen. Serial sections were examined under a microscope (Leica DM2000) and photographed. The stages of liver fibrosis were scored by an experienced histopathologist (L.Y.F., 9 years of liver pathology) using the METAVIR classification system,21,22 and blinded to the results of the MR, in which F0 5 no fibrosis, F1 5 portal fibrosis without septa, F2 5 portal fibrosis and a few septa, F3 5 numerous septa without cirrhosis, and F4 5 cirrhosis. 333
Journal of Magnetic Resonance Imaging
TABLE 1. Correlation and 95% CI Between Fibrosis Stage and Parameters
Model Biexponential
Monoexponential
Parameters
Fibrosis stage
P
D
20.657 (20.774, 20.467)
