The Effects Of Salvia Miltiorrhiza On Cell Apoptosis And The Expression Of Fas Protein And Fas Ligand In Cochlear Of Guinea Pigs After Ischemia-reperfusion Injury

Objective: To establish the animal model of ischemia reperfusion injury in cochlea of guinea pigs:1. To observe the change of morphology in cochlear after ischemia reperfusion in different time.2. To analysis the expression of Fas protein and Fas ligand in cochlear after ischemia reperfusion in different time.3. To observe the cell apoptosis in cochlear after ischemia reperfusion in different time.4. To analysis the relationship between the expression of Fas protein/ Fas ligand and the cell apoptosis in cochlear after ischemia reperfusion.5. To investigate the application of salvia miltiorrhiza.Methods: 72 healthy guinea pigs were devided into the normal group, negative control group, negative control group after the application of salvia miltiorrhiza, model group and drug treated group given salvia miltiorrhiza 150mg/kg everyday by intraperitoneal injection for 7 consective days before onset of ischemia. Every groups have 8 guinea pigs and we will take samples 30 minutes after ischemia,6 hours and 24 hours after reperfusion in the last two groups.The method to establish the model of ischemia reperfusion in cochlear of guinea pig: making a median incision in the cervical part of guinea pig anesthetized by chlora hydrate, one side of common carotid artery was occluded by specialized microvascular clamp and two sides of vertebral arterys were scorched,so the animal model of ischemia was finished;then the clamp was removed and the blood flow restored at the time point (6hour,24hour),so the model of reperfusion. The negative control group was just opened and exposed the arterys. The hismorphology of cochlear was observed with hematoxylineosin (HE) staining. The Fas protein/ Fas ligand immunoactivity in cochlear was studied by immunohistochemisty and labeling ratio of Fas protein/ Fas ligand expression was studied by image analysis. The cell apoptosis was studied by TUNEL (terminal-deoxynucleotidy transferase mediated d-UTP nick-end labeling). Statistical analysis was performed with one-way ANOVA of SPSS 13.0 statistical package and statistical significance was defined as P<0.05.Result:There were no lesions of hair cells ,stria vascularis and the spiral ganglion in the normal group and the control group wherever the application of salvia miltiorrhiza or not. Lesions were detected in the hair cells,the stria vascularis and the spiral ganglion in model group. Compared with the ischemia group, the lesions were more obvious in the reperfusion groups, especially in reperfusion 24 hours group. But the morphological changes of the cochlea in the groups that had been applicated salvia miltiorrhiza were more subtle than the groups in the same time points without drugs. In the model groups, labeling ratio of Fas protein/ Fas ligand expression increased significantly compared with the normal group and the control group ( P<0.05). The labeling ratio of Fas protein / Fas ligand expression in every reperfusion group increased gradually compared with the ischemia group (P<0.05). However, compared with the model groups, the labeling ratio of Fas protein / Fas ligand expression in cochlear decreased in drug groups. There were almost no cell apoptosis in the normal group and the control groups. Compared with the three groups before, there were more cell apoptosis in the ischemia group and the reperfused groups (P<0.05). The numbers of apotosis cells in the drug groups were smaller than the groups without drug.Conclusion:We can establish the model of ischemia reperfusion in cochleas of guinea pigs by specialized microvascular clamp and scorched two sides of vertebral arterys. Lesions were detected in the cochleas in ischemia group and reperfusion groups, and it is better in the drug groups. There is slight positive expression of Fas protein / Fas ligand in the cochleas of normal group and control groups. In the ischemia group and reperfusion groups, labeling ratio of Fas system expression increased significantly compared with the normal group and the control groups. Compared with the model groups, the labeling ratio of Fas system expression in cochlear decreased in drug groups. There were almost no cell apoptosis in the normal group and the control group. But there were more cell apoptosis in the ischemia group and the reperfusion groups (P<0.05). The numbers of apotosis cells in the drug groups were smaller than the groups without drug. All of above indicates that Fas protein / Fas ligand may get involve in the cochlear injury caused by the ischemia reperfusion injury. The ischemia reperfusion injury can cause the cell apoptosis in cochlear. The therapy of salvia miltiorrhiza may preserve the cochlear in the ischemia reperfusion injury through blocking the expression of Fas protein / Fas ligand and inhibitting the cell apoptosis .