The Establishment Of Rat Model Of Early Stage Hepatic Fibrosis And Experimental Study On MR Imaging Of Activated Hepatic Stellate Cells

Objective: To establish SD rat model of early stage hepatic fibrosis by injectingcarbon tetrachloride (CCl4) subcutaneously to provide reliable animal model forexperimental study on hepatic fibrosis. Then, to investigate the feasibility of the ultrasmallsuperparamagnetic iron oxide (USPIO) modified by anti-integrin α5β1monoclonalantibody for targeting hepatic stellate cells (HSCs) in early stage fibrosis rats in vivo usinga clinical1.5T MRI scanner.Material and Methods:(1) Establishment of rat model:30rats were injectedsubcutaneously with a3ml/kg body weight mixture of CCl4and oil twice a week for3weeks as experimental group, the initial dose is5ml/kg body weight and purified water isthe drinking water. Another10normal rats were selected to perform the experiment ascontrol group by injecting physiologic saline subcutaneously and the dosage and usage issame with the experimental group.(2) Construction of molecular probe: α5β1-USPIOspecific probe was prepared by conjugating anti-integrin α5β1monoclonal antibody withUSPIO coated with carboxyl through carbodiimide method.(3) Grouping experiment ofrats:12rats and4rats were selected randomly from experimental group and control grouprespectively for MR liver scanning at3days after the final CCl4injection. The remainingrats were sacrificed immediately and the liver samples were collected for histopathologicalexamination (HE、Masson and reticular fiber staining) with the routine staining protocoland transmission electron microscope(TEM) examination. The twelve fibrosis rats weredivided randomly into3groups, so that four rats in each group were given α5β1-USPIO、naked USPIO at an iron dose of100μmol/kg body weight and anti-integrin α5β1monoclonal antibody respectively via femoral vein catheter. Four normal rats were givenα5β1-USPIO at the same iron dose.(4) MR imaging and analysis: abdominal MR imagingwas performed for all the rats using a clinical1.5T MRI scanner before and4h after the injection of α5β1-USPIO、naked USPIO or anti-integrin α5β1monoclonal antibody. Theliver: muscle contrast-to-noise ratio (CNR) of the rats in all4groups before and4h afterinjection was calculated and analyzed respectively.(5) Histopathological examination ofrat liver: after MR scanning, all the rats were sacrificed with deep anesthesia and the liverharvest from the rats was divided into several lumps for histopathological examination(HE、Masson、reticular fiber and Prussian blue staining) and TEM examination.Results:(1) The results of the rat model:24rats of experimental group weresuccessful and6were dead. The mortality was20%. All the rats of experimental grouppresented the symptoms of nutritional disturbance and chronic liver disease. Thehistopathologic examination of liver showed that there was hepatic cell degeneration andnecrosis, collagen and reticular fiber hyperplasia, et al. The rats of experimental groupwere all in the early stage of hepatic fibrosis according to Ishak fibrosis scoring. The rats ofcontrol group were survival totally and had no corresponding symptoms or fibrosis.(2)MR signal changes of rat liver: the MR imaging4h after injection showed that the signalintensity of liver decreased in different degree in fibrosis rats given by α5β1-USPIO orUSPIO and normal rats given by α5β1-USPIO, while the signal intensity of liver in fibrosisrats given by anti-integrin α5β1monoclonal antibody didn’t change at all.(3) Analysis ofthe liver: muscle CNR: the liver: muscle CNR of rats before and4h after injection were44.63±1.61and255.74±18.3(fibrosis rats given by α5β1-USPIO),45.37±1.87and175.51±12.49(normal rats given by α5β1-USPIO),44.56±2.08and157.96±13.72(fibrosisrats given by USPIO),44.82±1.39and45.46±8.24(fibrosis rats given by anti-integrinα5β1monoclonal antibody) respectively. There were significant differences between theliver: muscle CNR before injection and that4h after injection in fibrosis rats given byα5β1-USPIO, normal rats given by α5β1-USPIO and fibrosis rats given by USPIO(P＜0.01), but there was no significant difference in fibrosis rats given by anti-integrin α5β1monoclonal antibody(t=1.45, P=0.24). The liver: muscle CNR is the highest in fibrosis ratsgiven by α5β1-USPIO4h after injection and the liver: muscle CNR decreased gradually innormal rats given by α5β1-USPIO, fibrosis rats given by USPIO and fibrosis rats given byanti-integrin α5β1monoclonal antibody. There was difference among all four groups.Further comparision between any two groups showed that the liver: muscle CNR had nosignificant difference between normal rats given by α5β1-USPIO and fibrosis rats given byUSPIO (P=0.094), while there were significant differences among all the other groups(P＜ 0.01).(4) The results of histopathological examination of rat liver: the Prussian bluestaining of liver of fibrosis rats given by α5β1-USPIO showed that stained blue ironparticles mainly distributed in the perisinusoidal space of Disse where was consistent inlocation with the HSCs. TEM showed that there were some lysosomes embodying a fewiron particles in the HSC of the fibrosis rat liver treated with α5β1-USPIO, but no particleswas found in HSC of normal rats given by α5β1-USPIO and fibrosis rats given by USPIO.Conclusion: The rat model of early stage hepatic fibrosis could be establishedsuccessfully by injecting CCl4subcutaneously. The activated HSCs in early stage fibrosisrat liver could engulf α5β1-USPIO specifically. In vivo targeted imaging of activatedhepatic cells in early stage liver fibrosis using α5β1-USPIO targeting integrin α5β1wasfeasible using a clinical1.5T MRI scanner.