Value Of Mri With T₂^* Mapping And Molecular Imaging For Assessing Liver Fibrosis In A Rat Model

PART I ESTABLISHMENT OF LIVER FIBROSIS IN A RAT MODEL EVALUATED WITH MR T2* MAPPINGObjective To establish animal models of liver fibrosis with Sprague-Dawley rats by injecting carbon tetrachloride (CCI4) intraperitoneally, and to evaluate the usefulness of magnetic resonance imaging (MRI) with T2* mapping for determining the stage of liver fibrosis.Methods Thirty-five male Sprague-Dawley rats were randomly assigned to the experimental group (n=30) and the control group (n=5). Among them, thirty rats in experiment group were injected intraperitoneally with a 0.1 mL/100 g mixture of CC14 and oil (1:1 v/v) twice a week for 12 weeks. For the quantification of liver R2* values, we used T2* mapping sequence with a multi-echo fast gradient echo sequence (TR=160 ms, TE= 2.7-22.3 ms, FOV=10×10 cm). The liver iron content (LIC) was evaluated by a flame atomic absorption spectrophotometer. For the pathological study, hematoxylin-eosin (HE) staining and Masson trichrome staining were performed to assess the severity of liver fibrosis. Statistical analyses were performed by using SPSS 17.0 software and the performances of R2* values and LIC for each fibrosis stage were assessed. The optimal cutoff values for fibrosis stage were determined by receiver operating characteristic (ROC) curve analysis.Results Twenty-three rats in the experimental group survived and 7 died after the experiment. The mortality is 23.3%. The histological staging showed 1 stage 4 rats,2 stage 8 rats,3 stage 7 rats, and 4 stage 4 rats in the experimental group and 0 stage 5 rats in the control group. R2* values and LIC gradually increased during the progression of the liver fibrosis, and were positively correlated with the stage of liver fibrosis. There were significant differences in R2* values and LIC among the stages of liver fibrosis except for SO versus S1 and S1 versus S2. The most discriminating cutoff values of R2* were 46.84 Hz for greater than or equal to S1,55.30 Hz for greater than or equal to S2,68.06 Hz for greater than or equal to S3, and 78.79 Hz for S4. The most discriminating cutoff values of LIC were 131.29 μg/g for greater than or equal to S1,155.39 μg/g for greater than or equal to S2,187.09 μg/g for greater than or equal to S3, and 222.95 μg/g for S4.Conclusion Long-term intraperitoneal injection of mixture of CCl4 and oil (1:1 v/v) at 0.1 mL/100 g, twice a week is a suitable method to establish rat model of liver fibrosis with high success rate and fast processes. R2* values can be used for prediction of the presence of moderate and advanced liver fibrosis and the quantitative evaluation of LIC.PART Ⅱ PREPARATION AND PERFORMANCE TESTING OF AN ASIALOGLYCOPROTEIN RECEPTOR-TARGETED SUPERPARAMAGNETIC PERFLUOROOCTYLBROMIDE NANOPARTICLESObjective To prepare an asialoglycoprotein receptor (ASGPR)-targeted superparamagnetic perfluorooctylbromide nanoparticles (M-PFOBNP) and investigate its characterization and safety in vitro and vivo for magnetic resonance imaging (MRI).Methods GalPLL, a ligand of ASGPR, was synthesized by reductive amination. ASGPR-targeted M-PFOBNP was prepared by a film hydration method coupled with sonication. The morphological characterization was observed by a light microscope. The concentration of Fe3O4 inclusion in the emulsion was estimated by a flame atomic absorption spectrophotometer. The mean size and Zeta potential of ASGPR-targeted M-PFOBNP were obtained by means of dynamic light scattering. The targeting effect in vitro was observed by a fluorescence microscopy. The influence of ASGPR-targeted M-PFOBNP uptake on the cell viability was estimated by using the methyl thiazolyl tetrazolium (MTT) assay. The in-vivo MR T2* mapping was performed to evaluate of the enhancement effect in rat liver.Results The prepared ASGPR-targeted M-PFOBNP was uniform and well dispersed. The concentration of Fe3O4 nanoparticles inclusion in the emulsion was about 52.79 μg/mL, and the mean size was 285.6±4.6 nm with a Zeta potential that was+15.8 mV. Many ASGPR-targeted M-PFOBNP combined with BRL cells were observed after 60 min in vitro. The MTT test showed that there was no significant difference in the absorption values of cells incubated with all ASGPR-targeted M-PFOBNP concentrations. Compared with M-PFOBNP and superparamagnetic Fe3O4 nanoparticles, the decrease in T2* relaxation time of ASGPR-targeted M-PFOBNP is larger and the increase in R2* value is higher.Conclusion ASGPR-targeted M-PFOBNP may potentially serve as a liver-targeted contrast agent for MR receptor imaging.PART Ⅲ MR MOLECULAR IMAGING OF EARLY LIVER FIBROSIS IN A RAT MODEL WITH ASGPR-TARGETED SUPERPARAMAGNETIC PERFLUOROOCTYLBROMIDE NANOPARTICLESObjective To evaluate the usefulness of MR molecular imaging with ASGPR-targeted superparamagnetic perfluorooctylbromide nanoparticles (M-PFOBNP) for determining the early liver fibrosis.Methods Twenty male Sprague-Dawley rats were injected intraperitoneally with a 0.1 mL/100 g mixture of carbon tetrachloride (CCl4) and olive oil (1:1 v/v) twice a week for 6 weeks. The other five rats injected the normal saline were also selected to perform the experiment as a control group. All rats were treated with ASGPR-targeted M-PFOBNP by injecting via tail vein at a dose of 0.2 mL/100 g. MR images were obtained before and 2 h after the injection of ASGPR-targeted M-PFOBNP. The R2* value changes before and after ASGPR-targeted M-PFOBNP injection were analyzed. After the MR examinations, the rats were sacrificed with deep anesthesia (3% pentobarbital sodium, intraperitoneally). The liver harvested from the rats was divided into two parts for histopathological examinations and detection of the content of liver ASGPR, respectively.Results Fourteen rats in the experimental group survived and 6 died after the experiment. The histological staging of liver fibrosis included stage 1 in 9 cases, stage 2 in 5 cases in the experimental group and stage 0 in 5 cases in the control group. The mean R2* values before ASGPR-targeted M-PFOBNP injection for fibrosis pathologically staged as SO, S1, and S2 were 43.42±3.39 Hz,49.02±5.82 Hz,53.50±4.72 Hz, respectively. There was only significant difference in R2* values between stage 0 and stage 2 fibrosis. The mean R2 values after ASGPR-targeted M-PFOBNP injection for fibrosis pathologically staged as S0, S1, and S2 were 422.92±6.59 Hz,406.57±9.39 Hz,392.21±8.38 Hz, respectively. There was a significant difference in the R2* value changes before and after ASGPR-targeted M-PFOBNP injection. There were also significant differences between stage 0 and stage 1 fibrosis, stage 0 and stage 2 fibrosis, and stage 1 and stage 2 fibrosis. The most discriminating cutoff values of R2* were 414.16 Hz for stage 1 or greater and 390.71 Hz for stage 2 or greater with areas under the curve of 0.957 and 0.914, respectively. There was a trend toward a decrease in hepatic R2* value and the content of liver ASGPR with increasing degree of fibrosis.Conclusion MR molecular imaging with ASGPR-targeted M-PFOBNP may be used for prediction of the presence of the early liver fibrosis in a rat model.