Establishment Of An Hepatic Injury Model Induced By Hydrodynamic Injection In Mice And Detection Of The Factors Involved In Its Liver Repair

Background and objectiveHydrodynamic injection is a method that allows the efficient transfer of genesto mammalian tissue, especially to the liver. It is the simplest and most effectivenon-viral gene delivery technique. While hydrodynamic injection has caused liverinjury as well. In this paper, we establish an hepatic injury model by hydrodynamicinjection and detect the factors involved in the liver repair to provide basic data forfurther study of the repair mechanisms of hydrodynamic induced liver injury.MethodsHydrodynamic injections were performed by intravenous injection of largevolume of saline (1.8-2ml) to Balb/c mice in3s. Blood samples were collected forALT assay at30min,8h,1d,3d,5d and7d after injection. Animals were sacrifcedand the livers were harvested for macroscopic observation and H&E staining wasperformed for histopathological analysis. The factors invovled were detected byReal-time PCR, such as TNF-α, IL-6, HGF, TGF-α, EGF, HNF4α, Notch2, VEGF,Cyclin D1, Bax and Bcl-2. Immunohistochemical staining of PCNA was performedto examine the proliferative activity of hepatocytes and cholangiocytes. Non-injectedmice were used as controls.Results1. Levels of serum ALTSerum ALT levels began to rise at8h, reached a peak at1d, and returned tonormal levels by7d.2. Macroscopic changes of the liversMice were sacrificed at different times after hydrodynamic injection. The liverswere weighed and fixed in phosphate-buffered10%formaldehyde for macroscopic observation. At30min after injection, the livers looked grey-red, then turned to lightgrey-red at8h. The liver basically return the same color to control until7d.While at30min after injection, the ratio of liver weight and body weight had nosignificant changes, decreased lower than the30min and control at8h, thengradually increased, and recovered to the normal until7d.3. Morphological examination of the liversH&E staining showed that the injury were mainly located in the junction areabetween Zone1and Zone2&3. At30min after hydrodynamic injection, hepatocyteslocated in Zone2&3were swollen and their cytoplasm was vacuolized and stainedless with eosin.Then the swollen hepatocytes decreased and some hepatocytes withdifferent degrees of damage showed cytoplasmic inclusions of RBCs at8h, and thenumber of hepatocytes increased,but the volume decreased. Double-nuclear andmotic hepatocytes appeared at this time as well.Hemorrhagic necrosis was significantly reduced and the motic hepatocytes wereincreased at1d. At3d after injection, hemorrhagic necrosis almost disappeared andthe number of hepatocytes, double-nuclear and motic hepatocytes decreased. Whileat5d-7d, the liver structure basically returned to normal.4. Immunohistochemical staining of PCNAImmunohistochemical staining showed that, PCNA-positive rate of hepatocytesin Zone2&3began to increase from30min after injection, reached a peak at1d, thengradually reduced, basically returned to control group by7d. While the trend ofPCNA-positive rate of hepatocytes in Zone1was similar as Zone2&3. However,cholangiocytes PCNA-positive rate reached the highest level at30min after injection,obviously higher than control group, then gradually decreased to control group by7d.5. Factors expression in the liverReal-time PCR results demonstrated that, at30min after injection, TNF-α, IL-6,EGF, HNF4α and VEGF mRNA levels were significantly higher than the other timepoints, then declined. While HGF, TGF-α mRNA levels began to rise at30min after injection, to the maximum at3d, then decreased. CyclinD1beggan to rise at30minas well, but to the maximum at1d. Notch2mRNA level was no significant differeceat each time point. However, Bax and Bcl-2mRNA levels were higher than othertime points at8h after injection. Bax/Bcl-2was highest at1d after injection.Conclusions1. Liver injury induced by hydrodynamic injection mainly caused the cellularswelling of hepatocytes and necrosis foci in Zone2&3and leaded to elevation ofserum ALT levels.2. Liver injury induced by hydrodynamic injection could be repaired naturally in aweek.First, non-parenchymal cells participated in liver repair by secreting TNF-α,IL-6, EGF, VEGF, Bcl-2and Bax.And then HGF and TGF-α promoted hepatocytes proliferation by regulatingCyclinD1.Notch2mihgt not involed in the repairment of liver injury induced byhydrodynamic injection.