Experimental Study On Hepatic Fibrosis With MR Diffusion-weighted Imaging And MR Perfusion-weighted Imaging

PrefaceLiver fibrosis is a pathologic state with abnormal synthesis and deposition of extracellular matrix.It is a procedure of cirrhosis resulted from all kinds of chronic liver diseases.Make early diagnosis and assess the severity of liver fibrosis accurately is very important for therapy and prognosis.In recent years the functional imaging techniques of MR including difffusion weighted imaging(DWI)and perfusion-weighted imaging(PWI)have developed fast. DWI sensitizes the MR signal related to the incoherent microscopic motion of water molecules,and PWI evaluates the hemodynamic characteristics of the hepatic lesions. Both of them can assess ultrastructural changes and hemodynamic alterations before morphological alteration in liver fibrosis.In our experimental study,DWI and PWI examinations were underwent in rat model of liver fibrosis.The changes of them in different stages of liver fibrosis were analyzed.DWI and PWI results were compared with histopathological, ultramicrostructural,blood biochemiology,typeâ…£collagen(C-â…£)and laminin(LN) immunohistochemistry expressions to make further interpretations.The diagnosis efficacies of DWI and PWI parameters for liver fibrosis as well as the correlations among them were analyzed,so as to choose noninvasive method for assessing liver fibrosis reasonably.Materials and MethodsSixty Wistar rats were divided into six groups:1 to 5 as experimental group,and 6 as control group with 10 rats each.Mixed resolution(0.2ml/100g)with 60%carbon tetrachloride(CCl₄)and 40%olive oil were hypodermic injected twice a week on rats of experimental group,while in control group were injected 0.2ml/100g saline twice a week.At 4,6,8,10,12 weekends after injection,rats of 1 to 5 experimental groups and two rats of control group were underwent MR examination respectively.All MR examinations were performed on a 1.5 Tesla unit(Philips Intera)with the C3 coil.Three different b values were tested:333,666 and 1000s/mm².Spin echo echo-planar DWI was performed using the following acquisition parameters: TR/TE=1723ms/66ms,slice thickness/slice gap=3mm/0mm,matrix=128×256, NSA=1.The software may provide ADC images automatically after DWI original diffusion images were acquired.Then all images were transferred to Philips workstation.Slice of interesting were selected at hepatic right lobes.Three ROI avoiding large vessels and artifacts were placed over 1-2 representative sections involving hepatic hilar.Signal intensity(SI)and ADC value were recorded and compared among different stages of liver fibrosis.The relationships among DWI parameters,liver fibrosis stage were analyzed.First-pass contrast enhanced perfusion MR imaging was performed on rats in experimental and control groups.A THRIVE(T1-TFE)three dimensional sequence was undertaken with the following acquisition parameters:TR/TE=7.Sms/3.Sms,slice thickness/slice gap=2mm/0mm,block thickness=20mm,matrix=128×128,NSA=1. Sixty phases of dynamic images for ten slices of the whole liver were obtained during 126 seconds after intravenous bolus administration of Gd-DTPA(0.05mmol/kg)via caudal vein.The scan began as soon as bolus administration started.Region-of-interest analyses over the aorta,the portal vein,and liver parenchyma enabled generation of concentration-time curves.Perfusion parameters were recorded as follows:â‘ Time to peak(TP);â‘¡Wash-in rate;â‘¢Wash-out rate.Three parameters of portal vein and liver parenchyma were compared among different stages of liver fibrosis.The relationships among PWI parameters,fibrosis stage were analyzed. Blood samples were obtained in all animals after MR examination to observe serum markers of liver fibrosis including hyaluronic acid(HA),procollagen typeâ…¢(PCâ…¢),typeâ…£collagen(C-â…£)and laminin(LN).The differences of results at various stages of liver fibrosis were compared.Analysis of relationships among serum markers and stage of liver fibrosis were performed.Liver was resected within six hours after MR scanning and fixed in 10%formalin. HE,Masson and reticular fiber staining stained the samples separately.The stage of liver fibrosis was decided by light microscopy.C-â…£and LN immunohistochemistry staining were undertaken by Envision two-step method.A sample with 1×1×3mm volume was obtained and fixed in 2.5%glutaral for making electron microscopic section in 2 rats of control group and of each experimental group.Hepatic ultrastructure was observed by transmission electron microscope.Correlation between ADC and serum marker,perfusion parameter and C-â…£collagen and LN expression were analyzed separately.ResultsTwelve rats of experimental group were eliminated from experiment due to death in the process of breeding.With the prolongation of injection,the severity of hepatic degeneration,necrosis and inflammatory cell infiltration progressed,the amount of collagen and reticular fiber increased.The injury of hepatic cell progressed and the deposition of fibrous tissue increased under electron microscope as the injection time prolonged.Morphology of fat-storing cell changed and hepatic sinusoidal capillarization developed in the process of liver fibrosis.1.Results of serum indices in liver fibrosisAll serum fibrosis indices increased as the stage of liver fibrosis increased,and reached the peak on S4 stage.There was a significantly positive correlation among all serum fibrosis indices and between serum indices and stage of liver fibrosis(P＜0.01).2.Results of C-â…£and LN immunohistochemistry staining We found C-â…£and LN dramatically increased and successionally deposited along the wall of sinusoids and thick fibrous septa.The positive expression of C-â…£and LN increased as the stage of liver fibrosis progressed.There was a significantly positive correlation between C-â…£or LN and stage of liver fibrosis(P＜0.01).The r values amount to 0.900 and 0.912,respectively.3.Results of DWIGrouping according as different stage of fibrosis,we found that SI and ADC values decreased along with b values increasing(P＜0.01).When b values equal to 333, 666 and 1000 s/mm² respectively,SI increased and ADC decreased gradually as the stage of fibrosis progressed(P＜0.01).There was a good relationship between ADC and fibrosis stage,especially when b value was 1000 s/mm².When b values equal to 333 and 666s/mm²,S0-S2 can be distinguished from S3 and S4 stage,and S3 can be distinguished from S4 stage according to SI and ADC values.Moreover b value equal to 1000 s/mm²,S0-S2 can be distinguished from S4 stage.There was excellent negtive correlation between ADC and serum indices(P＜0.05).4.Appearances and results of PWIOn perfusion-weighted image,inferior vena cava,abdominal aorta and portal vein visualized in order.As the stage of fibrosis progressed,TP of portal vein increased progressively,and the wash-in rate and wash-out rate decreased progressively. According to TP of portal vein,S2-S4 stage can be distinguished from S0(P＜0.05,＜0.001,＜0.001).According to wash-in rate and wash-out rate of portal vein,S0-S2 stage can be distinguished from S3 and S4 stage(P＜0.05).As the stage of fibrosis progressed,TP of liver parenchyma increased progressively,and the wash-in rate and wash-out rate decreased progressively.S0-S2 stage can be distinguished from S3 and S4 stage(P＜0.05).Of all parameters,TP of portal vein and liver parenchyma had a positive correlation with the expressions of C-â…£and LN(P＜0.01).The wash-in and wash-out rate of portal vein and liver parenchyma had a negative correlation with the expressions of C-â…£and LN(P＜0.01).Conclusions1.All serum fibrosis indices increased as the stage of liver fibrosis progressed. There was a significantly positive correlation among all indices and between serum indices and stage of liver fibrosis.2.The positive expression of C-â…£and LN increased as the stage of liver fibrosis progressed.There was a significantly positive correlation between C-â…£or LN and stage of liver fibrosis.3.SI increased and ADC value decreased gradually as the stage of fibrosis progressed.There was a good relationship between ADC and stage of fibrosis, especially when b value equal to 1000 s/mm².DWI was an effective method to detect liver fibrosis on S3 and S4 stage.4.There was excellent correlation between ADC and serum indices.It reflected the characteristic pathologic state of abnormal synthesis and deposition of extracellular matrix.5.PWI should be an effective method to quantify severity of liver fibrosis as well as to detect early cirrhosis(S2 stage)and severe fibrosis(S3-S4 stage).MR perfusion parameters were better correlated with fibrosis staging,and were associated with hemodynamic alternations.6.There were excellent correlations between the perfusion parameters and the expressions of C-â…£and LN.These correlations would better interpreted sinusoidal capillarization in liver fibrosis.