| Stroke includes ischemic stroke and hemorrhagic stroke and the former one accounts for 70%-80%. Cerebral middle artery,which is the vulnerable area of ischemic stroke. The embolism of cerebral middle artery produces the focal cerebral ischemia. Clinical manifestation of cerebral ischemia is hemiplegia,language disorder, hypesthesia and so on. Cerebral ischemia,which behaves high morbidity rates, high mortality rates, high disability rates,high relapse rates,has already become the one of principal diseases threatening people's health,which brings the socity and families big spiritual pressure and heavy economic burden. Therefor,developing new therapeutic medicine as soon as possible has a huge medical value.Tetramethylpyrazine is an alkaloid from Szechuan Lovage Rhizome. From the modern pharmacology study,we could find that TMP posses antioxidant effect, anticalcuim effect, boosting neurogenesis and so on.TMP is often used on myocardial ischemia and cerebral ischemia. We used combination principles to synthetize a series of new medicine with TMP and other medical compounds.Using primary neurons, we got a new compound called TMP-Vanillic acid. The effect and mechanism was not explored. To address this issue,a variety of multidisciplinary approaches including Ethology Application, Light microscope, enzyme-linked immuno sorbent assay(ELISA) and immunohistochemical staining were used.Part 1 Effect of TMP-Xs on primary neurons growthObjective:to screen the compound that promotes the neurons growth.Method:Rats with pregnant for 16-18d was used to plant primary neurons,culutured for 48h,and then incubation with medice.The medicine and their concentration were as following: 26-2PH (TMP-Vanillic acid:60μM,30μM,15μM),10PH (100μM,50μM,10μM) 18PH (60μM,40μM,20μM),23PH (60μM,40μM,20μM),25PH (60μM,40μM,20μM),26-1PH (50μM,40μM,30μM,20μM),34-2PH (50μM,40μM,30μM,20μM),35-2PH (60μM,40μM,20μM),38-2PH (60μM,40μM,20μM),40PH (60μM,40μM,20μM),43PH (60μM,40μM,20μM). Cell viability was measured by MTT test after incubation with medice for 12h,24h,36h or 48h. All of the experiments above were repeated for 3 times.Results:10PH,18PH,23PH,25PH,26-1PH,34-2PH,35-2PH,38-2PH.40PH,43PH had a toxic effect on neurons,however, TMP-Vanillic acid promoted the primary neurons growth.Conclusion:TMP-Vanillic acid showed some potential on promoting neurons growth.Part 2 Effect of TMP-Vanillic acid on cerebral ischemia in mice and rats2.1 Effect of TMP-Vanillic acid on the gasping duration after decapitation in miceObjective:To study the effect of global cerebral ischemia in mice.Method:After oral adimistration of medicine for 10d,cut the head of mice after swimming for 3min in the 4℃cold water,then recorded the gasping duration after decapitation.Results:Compared to control, With 10d treat of TMP-Vanillic acid (200mg/kg,100mg/kg,50 mg/kg), gasping duration after decapitation was prolonged.Following with the increase of the dosage of TMP-Vanillic acid,the effect got better.There was no significant difference between TMP-Vanillic acid and TMP+Vanillic acid at the same amount of substance.Conclusion:TMP-Vanillic acid could prolong gasping duration after decapitation in mice,effective on global cerebral ischemia in mice.2.2 Effect of TMP-Vanillic acid on the infarct area in rats with MCAOObjective:To study the effect of TMP-Vanillic acid on the infarct area in rats with MCAO.Method:The model made basing on the surgical insertion of a nylon monofilament (diameter:0.25mm) through the right internal carotid artery. This resulted in occlusion of the ostium of the ipsilateral MCA.After oral adimistration of medicine for 10d, rats were killed then brain slices were immersed in a 1.2% solution of triphenyl-tetrazolium chloride(TTC) in normal saline at 37℃for 30 min,after which sections were fixed in 10% phosphate-buffered formalin for testing.The weight of infarct area accounting for the whole brain was assessed.Results:No infarct in sham.The infarct area of model rats accounted for 18.86% of the whole brain,however, TMP-Vanillic acid 120mg/kg and TMP-Vanillic acid 60mg/kg could diminish the infarct area to 14.14% and 12.51% respectively.Conclusion:TMP-Vanillic acid could protect the rats from MCAO.2.3 Effect of TMP-Vanillic acid on impaired functional recovery in rats with MCAOObjective:To study the effect of TMP-Vanillic acid on impaired functional recovery in rats with MCAO.Method:Repeated the model as the method above. Ischemic medication rats received intragastric administration once a day at 3-10day post-ischemia. Animals were evaluated with the neurological score, beam-walking tests, adhesivetape-exposing tests and bar-grasping tests at 6d and 9d post-ischemia. Results:Compared with sham-operated group, model rats showed significantly severe neurological deficit at 6d post-ischemia,however,got better at 9d post-ischemia.Rats receiving TMP-Vanillic acid had a little more rapid recover obeam-walkingtests,adhesivetape-exposing tests and bar-grasping tests than model rats. Especially,the TMP-Vanillic acid at the doseage of 60mg/kg showed a significant improvement on beam-walking test than other groups.Conclusion:TMP-Vanillic acid could improve the impaired functional recovery in rats with MCAO.2.4 Effect of TMP-Vanillic acid on Histopathological morphological changes in rats with MCAOObjective:To study the effect of TMP-Vanillic acid on cortical neurons in rats with MCAO.Method:Repeated the model as the method above. At 12d after MCAO, animals were transcardially perfused with 0.9% physiological saline, followed by 4% paraformaldehyde (PH=7.2). Brains were embedded in paraffin wax and 6-μm sections were cut at 8 predetermi-ned coronal levels throughout the middle cerebral artery territory.Then we observed the morp-hological changes of cortical neurons through light microscope after hematoxylin-eosin staini-ng and Nissl staining.Results:Most neurons in the brain sections from sham-operated group of animals were of the intact type. Acute ischemic changes (shrinkage/scalloping, swelling and vacuolizing) of neuronal perikarya were visible in increasing numbers as a function of time elapsed after MCA occlusion. Nissl body with light colour was in the model rats,and also there was less Nissl bodies than sham-operated group. There were more intact neural cells and Nissl bodies in the groups treated with TMP-Vanillic acid.Conclusion:TMP-Vanillic acid could reduce the magnitude of cerebral ischemic neural cells damage.Part 3 Probe into the molecular mechanism of TMP-Vanillic acid involved with its prohibitive effects on cerebral ischemiaObjective:To study the molecular mechanism of TMP-Vanillic acid involved with its prohibitive effects on cerebral ischemia based on activity of SOD and Ca2+-Mg2+ ATP enzyme,the amount of MDA and VEGF.Method:Repeated the modle as the method above.We detected activity of SOD and Ca2+-Mg2+ ATP enzyme,the amount of MDA by Chemical Colorimetric Analysis. VEGF was quantitatively dected by ELISA and was locatingly examined by immunohistochemistry. There was an extra experiment on chicken chorioallantoic membrane(CAM) done by my cooperater. Results:The activities of SOD has been reduced in the rats with MCAO compared with shme-operated.However, Ca2+-Mg2+ ATP enzyme was advanced by feedback adjusting.The magnitude of MDA ascended in model rats.On the contrary,the amount of VEGF dropped in rats treated with vehicle.In the groups treated with TMP-Vanillic acid, SOD and Ca2+-Mg2+ ATP enzyme were more activated,less MDA,more VEGF.Conclusion:The contribution of TMP-Vanillic acid to cerebral ischemiac damage maybe was associated with activating SOD and Ca2+-Mg2+ ATP enzyme,and down-regulation of MDA,and advancing VEGF.Ending NotesOur data showed that TMP-Vanillic acid had remarkable effects on restraining cerebral ischemia.TMP-Vanillic acid could prolong the gasping duration after decapitation in mice, diminish the infarct area, improve the impaired functional recovery, reduce the magnitude of cerebral ischemic neural cells damage.The mechianism of TMP-Vanillic acid was linked to antioxidant,anticalcium and advancing VEGF and so on. |
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