| Objective:We established the mice model of cerebral ischemia reperfusion injury(CIRI) with monofilament, using Huazhuo Jiedu Huoxue Tongluo prescription to intervene it, by observing the neurological function deficits scales、the volume of cerebral infarction、the pathological type of the brain tissue、the expression of Vascular endothelial growth factor(VEGF) and Microvessel density(MVD). from the perspective of therapeutic angiogenesis to discuss the brain protective mechanism of Huazhuo Jiedu Huoxue Tongluo prescription on mice with CIRI.Methods:Fifty-four healthy male SD mice were randomly divided into six groups: the sham operation group, the model group, the aspirin group, the low dose of Huazhuo Jiedu Huoxue Tongluo prescription(the low dose group for short), the middle dose of Huazhuo Jiedu Huoxue Tongluo prescription(the middle dose group for short) and the high dose of Huazhuo Jiedu Huoxue Tongluo prescription(the high dose group for short), nine mice in each group. Mice modes of CIRI were established through improved thread embolism method. The eligible mice were brought into the later experiment according to the Neurologic deficits symptoms score. All mice were returned to their cages after the operation, ate food and water freely after models were successfully established, and the drugs were given by gastric perfusion the next day. Physiological saline were given to the mice in the sham operation group and the model group. The aspirin group were given the same amount of aspirin solution and the respective dose of Huazhuo Jiedu Huoxue Tongluo prescription were given to the low dose group、the middle group and the high dose group. The drugs were given once a day and for 14 days continuously. Neurologic deficits symptoms score was graded again after the treatment. Then the mice were sacrificed and the brains were collected for the tests. Three of them were stained by TTC to measure the volume of cerebral infarction. The others of each group were taken to observe the pathological type of the brain tissue by using HE dyeing、the expression of Vascular endothelial growth factor(VEGF) by using RT-PCR and the Microvessel density(MVD) by immunohistochemistry.Results:1 The Neurologic deficits symptoms score of each group: Compared with the sham operation group, the scores of the model group increase obviously(P<0.05); compared with the model group, the scores of the middle dose group、 the high dose group and the aspirin group decrease(P<0.05), but the low dose group had no significant difference with the model group(P>0.05); the score of the high dose group has no significant difference with the aspirin group(P>0.05).2 The pathological morphological changes of the right cerebral tissues in mice: tissues on the right side of the cerebral cortex were arranged regularly and tightly, the form of cellswerenormal, and there had little dead cells. The ischemic regions were obviously on the cerebral cortex of the model group, tissues were arranged irregularly, the extracellular space of cells was broadening significantly, a large number of necrotic neuron cells exist; cells on the right side of the cerebral cortex from the middle dose group、the high dose group and the aspirin group were increased significantly than the model group. There was little difference between the high dose group and the aspirin group, but still existed cells of degeneration and necrosis. The form of organization on the low dose group was similar to the model group.3 the volume of cerebral infarction on each group stained by TTC: Compared with the sham operation group, distinct infarctions were visible in the model group and the treatmentgroups. Compared with the model group, the volume of infarction significantly reduced in the middle dose group、 the high dose group and the aspirin group decrease(P<0.05), but the low dosegroup had no significant difference with the model group(P>0.05); the volume of infarction in the aspirin group was less than the the low dose group and the middle dose group(P<0.05), but there was little difference between the high dose group and the aspirin group(P>0.05).4 The expression of Vascular endothelial growth factor(VEGF) by RT-PCR: The expression of VEGF in the model group was obviously increased than the sham operation group(P<0.05); compared with the model group, the expression of VEGF was increased in the middle dose group、 the high dose group and the aspirin group decrease(P<0.05), and the high dose group was better than the middle dose group(P<0.05), but the low dose group had little difference with the model group(P>0.05); the high dose group had no difference on the expression of VEGF Compared with the aspirin group(P>0.05).5 the Microvessel density(MVD) by immunohistochemistry :Under optical microscope, most of the positive cells could be seen around ischemia area in infarct side, and the positive cells immunohistochemisty staining were sandy, the nucleus of the positive endothelial cells were round or oval. By the count of the MVD in each group, we can see that: The MVD in the model group was obviously increased compared with the sham operation group(P<0.05); and the MVD in the middle dose group、the high dose group and the aspirin group were higher than the model group(P<0.05); but the low dose group increased little than the model group(P>0.05); compared with the aspirin group, the MVD of the middle dose group was less than the aspirin group(P<0.05), but the high dose group had no difference with the aspirin group(P>0.05).Conclusion:1 Huazhuo Jiedu Huoxue Tongluo prescription can significantly reduce the neurologic deficits symptoms score in CIRI model rats, and improve the pathological morphological damage in the ischemic regions, promote the recovery of neural function.2 Huazhuo Jiedu Huoxue Tongluo prescription can significantly reduce the volume of cerebral infarction.3 Huazhuo Jiedu Huoxue Tongluo prescription can promote the expression of VEGF after CTIR, promote angiogenesis and increase the MVD, establish effective collateral circulation to protect the ischemic penumbra.4 the mechanism of Huazhuo Jiedu Huoxue Tongluo prescription on mice with CIRI may be related to the promotion of the expression of VEGF 、angiogenesis, and helped to establish effective collateral circulation. The brain protection has an dose-dependent. |
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