| Objective:To observe the change of TGF-β1、MMP9and iron metabolism in rats secondaryto intraventricular hemorrhage and to investigate the mechanism of the ventriculardamage and apoptosis after IVH.Methods:(1)84male Wistar rats were randomly divided into9groups:normal、shamsurgery（1d,3d,7d,28d after saline injection）and IVH groups(1d,3d,7d,28d afteroperation).Total130ul autologous tail arterial blood was injected into right ventriclefor two times to establish animal models. And sham groups animals were injected thesame amount of saline.Brain specimen, electron microscopy scanning and HE stainingwers used to evaluate ventricle damage at the different time points after sugery.(2)108male Wistar rats were randomly assigned to: normal、sham surger（y1d,3d,7d,28d after saline injection） and IVH groups（1d,3d,7d,28d after operation）. Wedetected the TGF-β1mRNA level with real-time PCR and the expression of MMP9with Western blot.(3)25male Wistar rats were randomly divided into sham surgery and IVH groups(1d,3d,7d,28d after operation).Bathophenanthroline was used to measure the serumand brain nonheme iron content.(4)162male Wistar rats were randomly assigned to normal、sham surgery andIVH groups (1d,3d,7d,28d after operation).Real-time PCR, Western Blot andimmunofluorescence staining were used to detect ferritin (Fn), transferrin (Tf),transferrin receptor1(TfR1), divalent metal ions transporter1(DMT1), ironregulatory protein2(IRP2), heme oxygenase1(HO1) expression in tissues aroundthe ventricle. immunofluorescence staining for8-OHdG and Tunel staining wereused for apoptosis detection.(5)18Wistar rats were randomly divided into normal、deferoxamine treatmentand placebo groups after IVH. We observed ventricles under electron microscope andapoptosis was detected by Tunel. Results:(1)Ventricular wall and its surrounding tissues of control groups were normalwith ependymal cells in alignment and structure organized. In IVH groups it’sshowed that the ventricle full of blood.We can also observed white matter edemaaround ventricle and disordered arrangement of ependymal cells1d after operation.3days after surgery lateral ventricle hematocele begin to scatter.ependymal fracture andependyma cells hyperplasia can also be found. glial cell hyperplasia and ependymalfracture were even more apparent at day28.Under the electron microscope,cilia wasdense、 regular and orderly and basement membrane was intact in controlgroups.While in IVH groups cilia was disorder and fractured and the basementmembrane was bare.(2)Postoperative1day TGF-β1mRNA rise obviously and significantly higherthan that of saline group (P <0.01).Then it significantly decreased (P <0.001)3dayslater, but still higher than the normal(P <0.05).7d after surgery TGF-β1mRNA riseagain to form the second peak and fell at days28.But it’s still higher than salinegroup.The differences between levels on28d and control groups were statisticallysignificant (P <0.05). MMP9protein rised from day1with a significant differencecompared to saline group (P <0.05).Then with the time gone its expression continueincrease and peaked at day28.(3)Serum iron concentration between groups were significantly differente afterthe IVH (P <0.01),while each group was undifferentiated compared to control group.1day postoperative brain iron content increased and peaked at7day. Then thequantity of iron in brain was high continuously even at day28.(4) Tf mRNA increased over time after operation. It’s significantly higher on day1than that of control group. Tf mRNA reach its peak at3day and then graduallydecline While obviously lower than that of the control group.Tf protein wasincreased at day1and peaked at day3.It declined from day7and the quantity wasobviously lower compared to the control group (P <0.01).TfR1decreased aftersurgery.It underwent the minimum at day3and then upregulated with time go on.There’sno difference between day7and day28but they were all higher than the control group.DMT1mRNA continued to fall with the time go on. It was significantly lowerthan the control group at day1(P <0.01) and got minimum at day7.It seemd to getrecovery at day28with no significant change compared to the control group.DMT1expression has no obvious change compared with controls at day1and thendecreased.Its value was minimum at day7and significantly lower than the controlgroup. It seemed to be normal at day28with no statistical difference compared withcontrol group.IRP2mRNA was also decreased after IVH.It fell to the lowest7d aftersurgery.The level of mRNA recover to some extent, but still significantly lower thanthe control group (P <0.001). A same trend of IRP protein was appeared. HO1increased significantly than the control group1day after IVH while fell slightly atday3with no significant difference compared to the level of day1.It was still higherthan normal group28days later.(5)8-OHdG positive cells in the control group were very little. Then the positiverate increased continuously. And there was no significant difference between day7and28.The trends of Tunel staining for hippocampal and ventricle surrounding tissueswere consistent. The low positive rate was very low in the control group thenincreased significantly and peaked at day28.（6）When observed under electron microscope7d after IVH and salinetreatment. It’s found that ventricular wall cilia was attenuated and fractured withbasal membrane peeling. Change in DFO treatment group was less obvious; Thepositive rate of tunel staining in DFO treatment group was significantly declineingcompared with the saline group.Conclusions:(1)The early pathological change of ventricle surrounding tissues after IVH isedema and hyperplasia was obvious gradually with time go on;(2)The ventricular injury caused by IVH may be associated with the imbalanceof TGF-β1and MMP9;(3)Iron metabolism disorder and iron overload plays an important role on theventriclar damage and apoptosis secondary to IVH;(4)Deferoxamine treatment can reduce the brain ventricular wall injury and apoptosis after IVH. |
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