| Cerebrovascular disease (CVD) is a kind of disease with high morbidity,mortality and disability rate globally, it is ranked second only to heart systemdisease and cancer in mortality rate. While the ischemic cerebrovasculardisease(ICVD) become the highest incidence among the cerebrovasculardiseases. Studies have found that a number of patients with vascular occlusioncan recanalized naturally by hemolysis after recanalization from cerebralischemia, but the brain functional recovery from cerebral ischemia reperfusioninjury is quit slow in some patients, even the patient’s condition exacerbated.As the ischemic brain tissue restored by reperfusion, but the function ofischemia brain tissue did not recovery, the phenomenon on aggravating theinjury and dysfunction is known as cerebral ischemia-reperfusion (CIR) injury.Therefore recanalization is an important procedure for preventing the ischemiccerebrovascular disease. The N-butyl phthalide (NBP) is a bio-organic activeelement extracted from celery seed, more and more studies have demonstratedthat the NBP plays a protective role in protecting cardiovascular andcerebrovascular system effectively. NBP pretreatment is refers to use NBP tocarry out prior protecting to the tissues of organs which is subjected ischemia,so as to Mobilize endogenous protective mechanisms for reducing thesubsequent ischemia-reperfusion injury to the tissues. In recent years, studieshave shown that NBP ischemic pretreatment can significantly reduce the braincell apoptosis, promote the neuron functional recovery and brain protection,but its mechanism is remained unclear. The aim of our experiment is toinvestigate the protective effects about NBP on the model hippocampalneurons prepared by using local cerebral ischemia reperfusion and to exploreintrinsic or potential mechanism of protection functions. Objective:The experiment by using N-butyl phthalide (NBP) pretreatment in rats,Preparing the focal cerebral ischemia reperfusion model by ligating rightmiddle cerebral atery with Improved Zea Longa wire, comparing the nervefunctional assessment score in the different groups; counting the percentage ofinfarction volume of hippocampus, hereby to study the effective actions byobserving the changes of the neurons and the the expression of5-hydroxytryptamine in related neurons in hippocampus, for the purpose ofrevealing the potential protection mechanism of NBP pretreatment.Methods:160male Sprague Dawley (SD) rats were randomly divided into5groups(n=32): sham group (Sham), ischemia-reperfusion group (IR), NBP low dosegroup (NBPⅠ g roup), NBP middle dose group (NBP Ⅱ group), NBP highdosegroup (NBP Ⅲg roup).Each group of the NBP pretreatment were given20mgkg-1,40mg kg-1,80mg kg-1respectively,1times each day within1week;Sham and IR group were only given normal saline solution, after the lastdosing until24hours of surgery. The model of ischemia reperfusion in ratswas established with the suture occluded method invented by Zea Longa, butthis method was properly improved in our lab. Two hours later, the suture wirewas cut off to recover the perfusion. The neurological deficit score of rats ineach group was calculated after24h ischemia reperfusion. The rats are broughtinto the experiment group strictly by the criterion of requirement, after thespecimen cut into slices, then staining them with2%TTC for expressing thebrain infarct portions and volume; HE staining for observing histopathologicalchanges of hippocampal neurons; the ultrastructural and pathological changesin hippocampus neurons were observed by EM; Immunohistochemistry wasused to observe the expression of5-HT，and using Western blotting to identifythe protein expression of the5-HT. Using the SPSS17.0software for statisticalanalysis, All data were processed and analysis with one-way ANOVA andLSD-t test by SPSS17.0statistic software and presented withx±s, P<0.01was regarded to have statistical significant difference. Results:1Neurological evaluation of focal cerebral ischemia reperfusion in ratsAfter2h arterial occlusion and subsequent24h of reperfusion, theneurological syndromes were shown up, Rats in the Sham group emerged noany abnormal activity. IR group animals displayed paralysis in the left limbs,independently automatical activity tend to the left side, even dumped. WhenHung up the rat by the tail, the rat was unable to fully stretch out itself, and theleft rear limb rotated to the contralateral side centered on the left hind legas.The neurological score of NBP pretreatment in low, middle and high dosegroups was significantly lower than the IR group, it have a significantdifference (P<0.01, P<0.05)statistically. There is no significantly different(P>0.05) between the NBP middle and high dose groups.2The measurement of infarcted area of hippocampusThe infarction size was measured by2,3,5-Triphenyltetrazolium chloride(TTC) staining technique. The right side of the brain tissue of the sham groupmanifest uniform red, Hippocampus shows no local infarction, IR groupshows pale infarcts. The calculated infarction volume in NBP low, NBPmiddle and NBP high dose groups were obviously reduced than IR group(P<0.01, P<0.05). there is no significantly different between NBP middleand high dose group (P>0.05).3The changes of neurons in hippocampus observed by microscopeThe neurons displayed normal shape in clear margin, clear nucleolus inthe Sham group, the nuclear membrane and nucleolus is clearly visible,nucleolus were enlarge and circle; the pyramidal cells arranged disorderly inthe Ischemia-reperfusion group, the mumber of pyramidal cell reduced, thebroadened gaps appeared; some severe ischemic changes occurred inpyramidal cells, the cell body swelling into a polygon, nuclear becomekaryopyknosis. The hippocampal pyramidal cells in NBP medium dose groupand NBP in high dose group were arranged in more neat rows than low dosegroup, most of the cellular structure is in structurally completely completelyintegrity.the small nerve cell shows abnormalities morphologically.4The ultrastructural changes of hippocampal neurons observed by transmission electron microscopy(TEM)In the Sham group, the hippocampal neuron’s membrane was intactcondition, clear boundary and nucleolus is located in the center of the cell,chromatin were uniformly distributed, abundant mitochondrions, organelles,such as golgi apparatus and endoplasmic reticulums were integrity, themorphology of endotheliocyte cells was normal, basement membranes wereintegrated. In the IR group, the swelling and necrosis of the hippocampalneurons, pyknosis and dispersion of chromatin can be seen. Cell membraneruptured, dissolved and vacuolization of cytoplasm appeared significantly, thenumber of mitochondria reduced. The morphology of endotheliocyte cells andmembranes were irregular, basement merebranes were intact incompletely.NBP pretreatment group compared with IR group showed some improvements,which the NBPⅡ、Ⅲ group effect is more obvious than NBPⅠ group. Mostmorphology of cells shows normal, nuclear membrane and basementmembranes were almost integrity, amount of swelling of the neurons reduced,mitochondrial is almost completelly intact, evenly distributed of chromatin,Golgi apparatus and Lysosomal tended to be in normal state.5Effect of NBP pretreatment on5-HT immunoreactive cells in hippocampalneurons following ischemia reperfusion in rats.Sham group shows that the5-HT positive cells are widely distributed inthe surrounding area of the hippocampus. IR group was visible,5-HT positivecells was significantly reduced (P<0.01, P<0.05). The5-HT positive neuronsin NBP low, in middle and high dose group were significantly increasedcompared with IR group(P<0.01, P<0.05); Moreover, NBP medium dosegroup compared with the NBP high dose group have no significant difference(P>0.05).6The protein expression of the5-HT was measured by western blotting.Compared with the sham group, the5-HT protein expression inhippocampus in IR group was significantly reduced (P<0.01, P<0.05). Theexpression in NBP low, middle and high dose group compare with IR groupwas obviously increased (P<0.01, P<0.05), NBP middle and high dose group shown not significantly different (P>0.05).Conclusions:1N-buty l phthalide (NBP) pretreatment act as a significant role in brainprotection.2Different doses of N-butyl phthalide (NBP) pretreatment with differentprotection, the medium doses group, high doses group of NBP pretreatmenteffects on neurons can exert significantly protective function, it is much betterthan the low dose group. But there was no significant difference between themiddle doses group and high doses group.3The neuroprotection of3-n-butylphthalide may have close relaton to theupregulated the expression of5-HT in focal cerebral ischemia rats. |
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