Application of Diffusion Weighted Proton MRI for Evaluating Molecular Diffusion of Water and Tissue Perfusion in Diethylnitrosamine Induced Rat Liver Fibrosis

Introduction Chronic liver disease is a major public health problem worldwide (1). Early and accurate diagnosis of liver cirrhosis is crucial for prognostic assessment of the course of the disease. Liver biopsy, the standard for diagnosing liver fibrosis has many limitations, including invasiveness, sampling error, observer variability, and the use of categorical scoring system. Measurement of water apparent diffusion coefficient (ADC) by diffusion weighted (DW) H MRI may be useful in diagnosis of cirrhosis, because increased deposition of extracellular matrix components in fibrosis may lead to decreased water ADC (2,3). DW H MRI has potential to provide information about both random molecular diffusion and tissue perfusion by carefully selecting a range of low (<100 mm/s) and high (> 100 mm/s) b-factors (4). The main goal of this research was to determine the effects of hepatic fibrosis in an animal model of cirrhosis produced by diethyl nitrosamine (DEN) on molecular diffusion of water and tissue perfusion, using DW H MRI. Methods Liver fibrosis was induced in male Wistar rats by continuous administration of 0.01% DEN via drinking water for 6 weeks (DEN group, n = 10) (5). The control (CON) animals received normal water over the same period (CON group, n = 6). MR experiments were conducted on a Varian 9.4 T, 31 cm horizontal bore system using a 6.3 cm diameter quadrature birdcage coil after 6 weeks of treatment. A 10 mm diameter tube filled with 54 mM NaCl was placed inside the coil and used as a H signal intensity (SI) reference. Total, water and fat H MRI were obtained with a spin-echo sequence (TR/TE = 1100/12 ms) without and with fat or water suppression using chemical shift selective (CHESS) technique (3). DW H MRI was collected using a modified spin-echo sequence (TR/TE = 1100/25 ms) and 10 b-factors (b = 0, 10, 20, 30, 100, 220, 350, 600, 1000, 1600 s/mm). Twelve transaxial slices with 1.5 mm slice thickness and 0.5 mm slice gap and 128 × 128 data points over 64 × 64 mm FOV were collected for all MRI. Respiratory gating was used to minimize the effects of motion on water ADC measurements. Mortal ischemia was produced by increasing the isoflurane to 4.5 % in medical air at 4 L/min. Respiration was monitored until it stopped, then again DWI H MRI were collected for the 10 b-factors. After the MRI experiments, the liver was fixed for histological analysis. DW MRI SI versus b-factor data were fit to the following biexponential equation: 1 , where SI is signal intensity for a given b-factor, ADCfast and ADCslow are the fast and slow ADC component which are related to tissue perfusion and random molecular diffusion of water, respectively, and Af is the relative contribution of ADCfast which is related to the relative vascular volume. Results Fig. 1 shows representative transaxial sections of total, water, and fat images from a DEN and CON treated rat. As shown in Table 1, water SI relative to the reference was significantly higher in DEN group (0.52 ± 0.05) compared to CON group (0.38 ± 0.07). However, fat SI relative to the reference was not significantly different in the two groups. The increase in water MRI SI with DEN treatment was consistent with a lower dry-to-wet weight ratio in DEN group compared to CON group. DW H MRI data for rats in both CON and DEN groups showed biexponential plots for live animals and monoexponential plots for dead animals. As expected, ADCfast was not detectable in dead animals in either group. In live animals, ADCfast was lower in DEN group compared to CON group (p < 0.05), but Af was higher in DEN group. ADCslow was same in DEN and CON groups for live animals. Upon death ADCslow did not change for DEN group but it decreased for CON group (p < 0.05). This resulted in a higher ADCslow for DEN group compared to the CON group after death. Representative histology slides from CON and DEN treated animals are shown in Fig. 2. DEN caused focal periportal fibrosis, bridging fibrosis, multifocal bile duct proliferation, focal cholangiohepatitis, and regenerative nodules. A scatter plot of ADCfast versus fibrosis score is shown in Fig. 3. ADCfast was lower in the presence of liver fibrosis but a correlation between ADCfast and fibrosis score was not detectable Discussion It is generally believed that ADC of water decreases with liver fibrosis because of increased deposition of extracellular matrix components (3). This idea is supported by data from many previous DW H MRI studies that used low b factors (< 500 mm/s) and analyzed the data using a monoexponential model (3,4). In the present study, contributions of random restricted diffusion of water and tissue perfusion to ADC were separated by carefully selecting ten b-factors and fitting the data to a biexponential model. In contrast to previous studies, the data presented here show that liver fibrosis produced by DEN does not alter the random molecular diffusion of water because ADCslow were identical for live animals in DEN and CON groups. In fact, water diffusion may be increased with fibrosis because ADCslow for dead animals was higher for DEN group compared to CON group. The higher liver water content measured by both H MRI and dry-to-wet weight ratio may be related to the observed increased ADCslow in DEN group. Hepatic fibrosis by DEN also caused a decrease in tissue perfusion because ADCfast was lower in DEN group compared to CON group. The decrease in perfusion appears to be accompanied by an increase in vascular volume because Af was higher in DEN group compared to CON group Similar results have recently been reported in a human study by Luciani et al. (5). Conclusion DW H MRI can be useful in evaluating both restricted molecular diffusion of water and tissue perfusion. The previously reported decrease in ADC with liver fibrosis (3,4) was mainly caused by a decrease in perfusion and not because of a decrease in water diffusion. Thus measurement of perfusion by DW H MRI may be a useful marker for diagnosis of fibrosis. A larger study is need to evaluate the use of DW H MRI for staging of liver fibrosis. References (1) Griffiths et al. Health Statistics Quarterly 2005;28,6-17. (2) Taouli et al. Am J Roentgenol 2007; 189:799-80. (3) Annet et al. J Magn Reson Imaging 2006;25:122-128. (4) Luciani et al. Radiology 2008;249:891-899. (5) Gang et al. Microsc Res Tech 2009;72:672-678. Fig.2. H&E stained histological sections of a CON (top) and DEN (bottom) rat liver with grade 4 fibrosis showing bridging fibrosis, focal cholangiohepatitis, diffuse bile ductproliferation, large dilated cyst , inflammation and regenerative nodules. CON