The Influence Of Musk With Borneol On The Rats With Focal Cerebral Ischemia/Reperfusion Injury

Stroke is one of the most important fatal diseases all over the world. The percentage ofattack rate and death rate of stroke in the general death case among the Chinese peopletakes 20% in the urban area and 19% in the rural area. Ischemic stroke takes more than80% among all the types of stroke. The principle of treating ischemic injury clinically is torestore reperfusion of blood ASAP, but the recent researches shows ischemic injurybecomes aggravated after restoring blood supply in the ischemic area and the quantity ofbrain cell death increased. This secondary injury after reperfusion of blood is calledcerebral ischemia reperfusion injury(CIRI). CIRI causes the cerebral functional impairmentand structure damage become worse and death rate higher, degrades therapeutic efficacygreatly. CIRI draws the people attention extensively globally and becomes one of thehottest focuses in cerebral ischemia researches in recent years.CIRI has complicated patho-phisiological process and the specific mechanism are stillunclear. Recent studies shows the injury of brain cell caused by cerebral blood flowinterruption and reperfusion is a rapid cascade reaction which includes many links such asenergy obstacle, generous release of oxygen free radical, cell acidosis, increased release ofexcitatory amino acids, damage of intracellular cacium homeostasis, production of freeradicals and apoptosis gene activation. All these links interacted, inter-overlapped andintercommunicated and caused vicious circle and leads to cell apoptosis and necrosisfinally.These is no ideal drugs to treat CIRI although CIR1 increases greatly after restorationof blood circulaton nowdays. Based on long term of clinical practice, Pro. Pen Shenquanraises that the brain is the the house of mentality and place of the upper orifice whichcontrols the mental activities. The main pathogenesis of the clinical stroke is blockage ofthe upper orifice and disturbance of Qi and blood, the principle of early treatment on stokeshould be resuscitation-inducing method and musk and borneol are the main herbs when giving prescription. Musk is one of the comman herbs used on brain diseases and otherserious diseases which can dredging the blocks of all orifices, meridians and collaterals.Borneol is also the commonly used herb on nervous system diseases such as stroke, conaetc, borneol can enhance therapeutic effect when using with musk together.Based on the past clinical and empirical study achievements, this study willapproach the mechanism of anti-CIRI by musk with borneol in the whole and molecularlevel. This study will widen the thoughts of preventing and treating CIRI by Chinese herbsand provide scientific evidence for improving therapeutic effect of herbs clinically.1 Observation of Therapeutic Effect on CIRI using Musk with Borneol1.1 Duplication and evaluation of rats model with cerebral focal ischemia with reperfusion.Objective: Duplicating SD rat model of cerebral focal ischemia with reperfusion andevaluatng the result. Methods: The model of MCAO-reperfusion was induced by themethod of nylon monofilament via the internal carotid artery. Observing and evaluating theearly neural function and brain histological changes on the model. Results: Rats displayedfocal nerves' functional impairment and brain tissue get swelling in the ischemic side. lightMost nerve cells have Karyopyknosis, interstitial substance rarefaction and vacant spacearound the cell under light microscope. Conclusion: Successfully duplicatedMCAO/Reperfusion model.1.2 The influence of musk with borneol on the cerebral infarction area and neural functionin rats with focal cerebral ischemia/reperfusionObjective:To investigate the effect of musk with borneol on the volume of cerebralinfarction and the Neural function in rats with cerebral focal ischemia with reperfusion.Methods: The models of MCAO-reperfusion was induced by the method of nylonmonofilament via the internal carotid artery. Technique of image analysis was used tocalculate the volume of cerebral infarction and scores of neural function were evaluated.Results: After cerebral ischemia for 2 hours and reperfusion for 24 hours, the volume ofcerebral infarction in the model group increased significantly(P＜0.01), the scores of theneural function increased(P＜0.01). The volume of cerebral infarction in musk withborneol group was significantly lower than that in the model group (P＜0.05). The scoresof the neural function in musk with borneol group, musk group were significantly lowerthan that in the model group (P＜0.05). Conclusion: Musk with borneol could decrease thevolume of cerebral infarction and improve the Neural function after focal cerebral ischemiawith reperfusion.1.3 Effect of musk with borneol on brain water content and blood-brain barrier permeability in the rat model of cerebral focal ischemia with reperfusionObjective: To investigate the effect of Musk with Borneol on Brain water content and BBBpermeability in the rat model of cerebral focal ischemia with reperfusion. Methods: Themodel of MCAO-reperfusion was induced by the method of nylon monofilament via theinternal carotid artery. Method of dry and wet weight was used to observe water contentand Evans blue(EB) was used to observe the changes of BBB permeability. Results: Aftercerebral ischemia for 2 hours and reperfusion for 24 hours, the content of water and EB inthe model group increased significantly compared with sham operation group(P＜0.01),which indicated the BBB was destroyed; The water content in Musk with Borneol groupwas significantly lower than that of the model group (P＜0.05) and the content of BBB inMusk with Borneol group and Borneol group was significantly lower than that of the modelgroup (P＜0.01). Conclusion: Musk with Borneol can decrease the water content andpermeability of BBB, therefore protect the structure of BBB after focal cerebral ischemiawith reperfusion.1.4 Effect of musk with Borneol on nerve cell and synapse ultrastructure in the rat model ofcerebral focal ischemia with reperfusionObjective: To investigate the effect of Musk with Borneol on nerve cell and Synapseultrastructure in the rat model of cerebral focal ischemia with reperfusion. Methods: Themodel of MCAO-reperfusion was induced by the method of nylon monofilament via theinternal carotid artery, electronic microscope was used to observe the changes of nerve cellultrastructure and synapse in Cortex of parietal lobe. Results: After cerebral ischemia for 2hours and reperfusion for 24 hours, the ultrastructure of the nerve cell has damaged: nervecell contracted, cytochondriome get swelling and rough endoplasmic reticulum getdistending et. And Musk with Borneol has protecting and repairing effect on lobe nerve cell.The quantity of the synapses in neuropil of the model group decreasedsignificantly(P＜0.01), and the typical synapse structure was destroyed; the quantity of thesynapses in neuropil of Musk with Borneol group was significantly higher than that of themodel group (P＜0.05) and the synapse form was almost normal. Conclusion: Musk withBorneol can increase the quantity of the synapses in neuropil and protect the Synapseultrastructure after focal cerebral ischemia with reperfusion.2 The molecule mechanism study of musk with borneol on the Rats with FocalCerebral Ischemia/Reperfnsion Injury2.1 Study of AQP-4 mRNA expression on the rats with focal cerebral ischemia/reperfusionInjury with musk and borneol Objective: To investigate the AQP-4 mRNA expression on the Rats with Focal CerebralIschemia/Reperfusion Injury with musk and borneol. Methods: Determination is made byutilizing Real-time PCR via preparation of TaqMan probe. Results: AQP-4 mRNAexpression in the ischemic brain tissue of the model group increased significantly comparedwith sham operation group(P＜0.01). AQP-4 mRNA expression decreased significantly ingroups of musk with borneol and borneol compared with model group(P＜0.05).Conclusion: musk with borneol and bornel can restrain AQP-4 mRNA expressionsignificantly.2.2 Study of ICAM-1 mRNA expression on the rats with focal cerebral ischemia/reperfusion injury with musk and borneolObjective: To investigate the ICAM-1 mRNA expression on the Rats with Focal CerebralIschemia/Reperfusion Injury with musk and borneol. Methods: Determination is made byutilizing Real-time PCR via preparation of TaqMan probe. Results: ICAM-1 mRNAexpression in the ischemic brain tissue of the model group increased significantly comparedwith sham operation group(P＜0.01). ICAM-1 mRNA expression decreased significantly inall medicine groups (P＜0.05～0.01), and musk with borneol group decrease mostly.Conclusion: musk with borneol can restrain ICAM-1 mRNA expression significantly andso can restrain adhesion of white cell with endothelial cell in brain ischemia withreperfusion, alleviate ischemic brain injury.3 Peculiarities and new ideas in this studyThere are affluent data globally of musk or bornel fundatrnental and clinical researches, andwe are the first one to use animal experiment to evaluate anti-CIRI using musk withborneol systematically from therapeutic effect and molecule mechanism. As for the animalmodel, we duplicated MCAO/reperfusion model on SD rats via reformed nylonmonofilament method and did obsevation and evaluation on early ethological andhistological changes, the constancy and reproducibility are satisfactory. The traditionaldetecting method on AQP-4 and ICAM-1 in the formal researches were mostly RT-PCRmethod or immunohistochemical method, we are the first one to use Real-time PCRdectecting method by preparing probe of AQP-4 and ICAM-1 genes, degree of accuracyand reliability are increased.