| INTRODUCTIONCerebral ischemia-reperfusion injury (CIRI) is a common pathological process in clinical practice, which seriously threatens the patient's life and blights the prognosis. It happens, for example, after the treatment of shock, cardiopulmonary-cerebral resuscitation, cerebral vascular thrombosis reperfusion and releasing from the pressure of brain tumors, and so on. At present, there are still no precise methods for preventing and treating CIRI.Neuron-specific enolase (NSE) is a specific enzyme that participates in glycolysis in neurons and neuroendocrine cells. The NSE content increased in serum and cerebrospinal fluid after brain injury and the positive correlation between the increased NSE level and the extent of brain damage makes NSE as a sensitive biochemistry indicator for the central nervous system damage.In recent years, the study on pharmacological function of Salvia demonstrated that Salvia protects brain against CIRI. Tanshinone IIA (Tan IIA) is the most important fat-soluble component of Salvia. At the present time, there is no study on the proper time and dose of Tan IIA in preventing and treating CIRI. In this study, different doses of Tan IIA were given before ischemia or immediately after reperfusion. We measured the serum NSE level and observed on the morphological change of brain tissue to search a more effective method for preventing and treating CIRI and to provide an experimental basis for clinical treatment.MATERIALS AND METHODS一,Animals and Surgery Healthy male Sprague-Dawley rats (275 to 325g) were subjected to CIRI. All experimental animals were caged with natural lighting, drinking and eating at will, and were left in the lab for 3 days to adapt themselves to the new environment.The animals were anesthetized with aether during surgical preparation. First, the animals were moored on the operating table, lying prone, with their heads made an angle of 30 degrees with their trunks. A median incision was made on dorsal cervix, approximately 1-2cm length. A small opening was made by blunt dissection performed with a pair of hemostatic forceps; the transverse foramens on both sides of the first cervical vertebra came into view. The pinhead No. 6 1/2 without pinpoint was heated on the spirit lamp till it gets dark red heat and the two vertebral arteries, going through the transverse foramens were cauterized to break by the pinhead. After that, the wound was sutured. Second, the animals were moored on the operating table in the supine position. A median incision was made on ventral cervix, approximately 1-2cm length. Bilateral carotis communis were separated, exposed, and blockaded with microvascular clamps after sternocleidomastoids were retracted towards both sides in carotid triangle. After 20 minutes of 4-vascular occlusion (4-VO), cerebral blood flow was restored by removal of the clamps.Acceptance criterion of model: After removal of ether anesthesia, the animals were still unconsciousness; the righting reflex disappeared; labium oris and eyes were pale; corneal reflex disappeared; mydriasis; no gnawing while metal tweezers stimulating lips, but those symptoms mentioned above faded 1-2 minutes after removing the clamps. The animals fell into disuse when jerk and massive haemorrhage happened in the course of surgical procedure.二,Serum NSE Concentration AssayThe animals were killed 24 hours after reperfusion. About 2ml blood was taken out of heart, laid steadily, centrifuged. The supernatants were analyzed at 450nm using a microtiter reader. Determine the corresponding concentration of in from the standard curve. The assay was performed in strict accordance with the instructions of goat anti-Rat NSE ELISA Kit.三,Histological ObservationCoronal sections were cut into slices, which were immersed in 40 g/L Polyformaldehyde (pH 7.4), then the slices were dehydrated, embed, HE stained; in the end, the morphological changes of brain tissue can be seen through light microscopy.四,Statistical AnalysisThe data are expressed as mean±SD(x±s). The factorial experiment of random effect model was achieved using Two-way classification ANOVA with Sigma Stat software. Significance was accepted with P<0.05. Comparison between two groups was achieved using Dunnett test. Significance was accepted with P<0.05.RESULTS1. No difference in the level of serum NSE was observed between the 0mg/kg dose group and the model group. Serum NSE levels were significantly lower in each group that given effective drugs compared with the 0mg/kg dose group and the model group.2. In different timing of medicine treatment groups with 3mg/kg dose or 6mg/kg dose, serum NSE levels were significantly lower in the rats given drugs immediately after reperfusion and in the rats given drugs both 30 minutes before ischemia and immediately after reperfusion compared with the levels in the rats given drugs 30 minutes before ischemia. No difference in the level of serum NSE was observed in different timing of medicine treatment groups with 12mg/kg dose.3. Serum NSE levels were significantly lower in the rats given 12mg/kg dose compared with the levels in the rats given 3mg/kg dose or 6mg/kg dose when the drugs were given 30 minutes before ischemia. No difference in the level of serum NSE was observed in the rats given different dose whether the drugs were given immediately after reperfusion or both 30 minutes before ischemia and immediately after reperfusion.4. Watched through the optical microscope, the neural cells in hippocampal CA1 section of the 0mg/kg dose group and the model group were swelling obviously. Chromatin was fine granulated or dyed dark diffusely, the nucleolus and the chromatin fell into confusion, the membrane dissolved, cytoplasm leaked out, cells became incomplete, the cell number decreased and arrayed loosenly, and other nerve cell injury changes, which also appeared in the nerve cells in hippocampal CA1 section of each group that given effective drugs, but the damage degrade was lighter. DISCUSSIONIn this experiment, we copied the CIRI model, and given 3mg/kg, 6mg/kg and 12mg/kg different doses of Tan IIA sodium injection in different timing of medicine treatment such as 30 minutes before ischemia, immediately after reperfusion and both 30 minutes before ischemia and immediately after reperfusion to observe the influence of the timing and dose on CIRI protective effect. The results shows that it could reduce the serum NSE concentration after CIRI in rat by given different doses of Tan IIA in the set time, that is, reducing brain injury, which proved the protection of Tan IIA on CIRI. And it becomes more apparent that the effect of medicine treatment given immediately after reperfusion and both 30 minutes before ischemia and immediately after reperfusion.There is no difference in the effect of medicine treatment between given immediately after reperfusion and given both 30 minutes before ischemia and immediately after reperfusion, accordingly, there is no need to combine medicine treatment 30 minutes before ischemia with medicine treatment immediately after reperfusion, when the Tan IIA is in application to protect brain tissue and reduce ischemia/reperfusion injury. The medicine treatment immediately after reperfusion will be OK. When given different doses of tanshinone II A, we found that the effect of low-dose and high-dose is similar in medicine treatment immediately after reperfusion or combined with 30 minutes before ischemia. For the reduction of drug side effects, in the application of Tan IIA to protect brain tissue and reduce ischemia/reperfusion injury, the use of low-dose will be proper.CONCLUSION1. Tan IIA can protect brain tissue against CIRI.2. The protective effect against CIRI by giving Tan IIA immediately after reperfusion is better than before ischemia .3. The effect of low-dose and high-dose is similar by giving Tan II A immediately after reperfusion. |
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