Non-alcoholic Steatohepatitis Kupffer Cells In Mr Imaging Of Experimental Research

This study used self-developed the Fe₃O₄ nanoparticles modified with chitosan-graft-poly (acryl acid) (CS-g-PAA) as a magnetic resonance imaging contrast agent, taking rat as experimental materials, to establish the animal model of non-alcoholic steatohepatitis (NASH) , to perform liver 1.5T MRI, were imaged before and after CS-g-PAA / Fe₃O₄ nanoparticles from the tail vein infusion, assessing rat NASH Kupffer cells (KC) phagocytic function, Which was divided into three parts.Part I: The preparation of Fe₃O₄ nanoparticles modified with chitosan-graft-poly (acryl acid) (CS-g-PAA)Objective: The well-dispersed Fe₃O₄ nanoparticles suspension was successfully prepared through surface modification with chitosan–poly (acrylic acid) (CS–PAA) copolymer by graft copolymerization technique.Methods: The graft polymerization was prepared by Chitosan–poly (acrylic acid) (CS–PAA) surface modification of Fe₃O₄ and get their water dispersed stable system. The structure of synthesized nanoparticles was characterized by Fourier transform infrared spectrometer (FTIR). The size and morphology of the nanoparticles was investigated by field emission scanning electron microscopy (FE-SEM) and Transmission electron microscope (TEM). The crystallization and superparamagnetism of the nanoparticles were characterized by X-ray diffraction (XRD) and vibrating sample magnetometer (VSM) respectively. The hydrodynamic radius of the modified particles was characterized by Dynamic light scattering (DLS). The results indicated that the hydrodynamic radius was dependant on PH of the system.Results:The Fe₃O₄ nanoparticles successfully modified with chitosan-g-polyacrylic acid (CS-g-PAA). CS-g-PAA/Fe₃O₄ nanoparticles were in water (PH=7.4)dispersion stability on globose or ellipse, diameter 15-60nm, uniform size distribution, and its crystal structure has not changed, with the stability of superparamagnetism.Conclusions: The well-dispersed Fe₃O₄ nanoparticle suspension was successfully prepared on PH=7.4 through surface modification with Chitosan–poly (acrylic acid) (CS–PAA) copolymer, the stability of its suspension mechanism is electrostatic repulsion and steric effects of synergy. CS-g-PAA/Fe₃O₄ nanoparticle size distribution of the 15.0-60.0nm, Which has the spinel structure, superparamagnetism slightly lower, but still meet the requirements of MRI contrast agent.Part II: MR imaging of CS-g-PAA/Fe₃O₄ nanoparticles in vitroObjective: To study the stability and the changing patterns of MR signals in differrnt concentration of CS-g-PAA/Fe₃O₄ nanoparticles (PH=7.4) in vitro magnetic resonance imaging.Methods: The CS-g-PAA/Fe₃O₄ nanoparticles phosphate solution were disposed by density, to observe the stability of CS-g-PAA/Fe₃O₄ and regular of MR signal; MR imaging was performed as following sequences: FSE T1WI, FRFSE T2WI and FIESTA T2*WI.Results: The CS-g-PAA/Fe₃O₄ nanoparticles were homogeneous MR signal (P>0.05). The signal intensity of CS-g-PAA/Fe₃O₄ nanoparticles on T1WI was firstly increased, but decreased following the increase of the concentration of Fe₃O₄. The signal on T2WI and T2*WI decreased as the increase of the concentration of Fe₃O₄. The lower the concentration, the more sensitive on T1WI and T2*WI when compared with that of T2WI, with T2*WI the most significant (P<0.05). The changes of signal intensity in all sequences were more distinct as the iron concentration≤20mg/l.Conclusion: The stability of CS-g-PAA/Fe₃O₄ nanoparticles could be examined by 1.5T MR scan using in clinical practice, the changes of signal intensity were remarked when iron concentrations≤20mg/l. T2*WI was the most sensitive sequence for detecting the CS-g-PAA/Fe₃O₄ nanoparticles in vitro.Part III: A preliminary study on the phagocytic activity of Kupffer cells with CS-g-PAA/Fe₃O₄ nanoparticles enhanced MR imaging in a rat nonalcoholic steatohepatitis modelObjective: To investigate the feasibility of using CS-g-PAA/Fe₃O₄ nanoparticles as MRI contrast agent to assess rat nonalcoholic steatohepatitis Kupffer cells (KC) function.Methods: Twenty male SD rats were randomly divided into experimental and control group, 10 rats in each group, experimental group was fed high fat diet, control group fed normal diet. After eight weeks, plain MR and CS-g-PAA/Fe₃O₄ enhanced were performed in all the rats; Blood lipids were measured, and HE and Perl's blue staining in all livers specimen was done. The related result of the staining were analyzed.Results: Experimental group TC and TG levels [(6.58±1.25) mmol/L and (1.53±0.23) mmol/L respectively] were significantly higher than control group [(1.64±0.22) mmol/L and (0.55±0.14) mmol/L respectively] (t = 11.716 and 11.558, P <0. 01); Experimental and control group hepatic signal intensity decreased in all sequences after CS-g-PAA/Fe₃O₄ enhanced, but in experimental group the level of decline [(34.78±4.51)% and (60.38±3.49%) respectively] was less than control group [(64.96±2.42)% and (81.08±1.66)% respectively] on PDWI and T1WI, and statistically significant difference (t = -18.415 and -16.240, P <0.01) were found. In experimental group the ratio of signal intensity of liver to spleen(1.002±0.141, 5.000±0.516, 20.004±1.490 and 2.601±0.077)was more than control group(0.400±0.102, 1.500±0.115, -0.3±0.058 and 0.503±0.105)before and after contrast enhancement on PDWI, T2WI, T2*WI and T1WI (t = 10.745, 19.800, 39.168 and 92.785, P <0.01). Typical histological hepatic lesions of NASH were observed in experimental group. Perl's staining-positive particles in experimental group(2.33±0.50)were fewer than in control group (4) (t= -10.000, P <0.01).Conclusion: The high-fat diet-induced model of SD rats close to the human NASH and was easy to establish. Clinical application of CS-g-PAA/Fe₃O₄ enhanced MR successfully assessed the phagocytic activity of Kupffer cells in the study, and it suggested that the pathogenesis of NASH related to the decreased phagocytic activity of Kupffer cells.