The Effect Of Dlbutyphthalide On Expression Of Nogo-a And Igf-1 Flowing Chronic Cerebral Hypoperfusion In Rats

BackgroundCerebral vascular disease is one of the most important senile diseases. high incidence of a disease,the disabled and recrudescence is its main feature. Cerebral vascular disease,cardiac disease and malignant tumor consitute the main reason which to cause human death. Ischemic cerebrovascular disease accounts for 70% cerebral vascular disease .Chronic cerebral ischemia is a disease that can cause brain functional disturbance,which initiates the brain ischemic and anoxemic. it was introduced by japanese in 1990.As the medical treatment levels are improved, the death rate of cerebrovascular disease has decreased, but the high mutilation rate gave the person,the fimily and the country the heavy burden. thus, the research in the cerebral fuction has become a hot spot .the factor of the fuction,which influences the remodeling after cerebrovascular disease, is very complex, that includes roughly promoting agent and inhibitory factor . Cerebral ischemia induces neuronal injury or death,whereas the endogenous neuroprotective mechanismsare triggered to resist these changes. The balance of the ischemic injury and the neuroprotective mechanisms determines the outcome of the ischemic neuronal cells.It is well established that insulin-like growth factor (IGF-1) has potent neuroprotective effects on cerebral ischemia in the rat and sheep model. it is a small peptide (7.5kDa) structurally related to pro-insulin.Insulin-like growth factor (IGF-1) belongs to the family of growth factors required by developing neurons for proliferation,differentiation and survival.Insulin-like growth factor is a neurotrophic factor that plays important roles in promoting cell proliferation and differentiation during normal brain development and maturation.It attenuates apoptosis in hippocampus neurons caused by cerebral ischemia and reperfusion in stroke-prone spontaneously hypertensive rats. Administration of IGF-1 shortly after injury has been shown to be neuroprotective.IGF-1 also protects cortical neurons from apoptosis induced by serum deprivation and motor neurons from glutamate-induced death in vitro. The adult mammalian CNS has limited neuroregenerative capacity in response to injury. The nerve growth inhibitory factor is possibably the main reason which includes Nogo-A,Nogo-B and Nogo-C, which can cause disaggregation of the nervous process growing tip. One such protein is the recently cloned myelin-associated neurite inhibitory factor Nogo-A. They are the important reason for neural regeneration after the mammal's central nerve injury. Neutralization of Nogo-A results in anatomical plasticity and behavioural recovery after ischemic brain lesions. Similarly, inactivation of the Nogo-A gene produces a robust protection from photothrombotic stroke in mice. Some researches reveals that Nogo-A intensity expression can inhibite neogenesis granula cell axonal growth as ischemia stimulus following chronic cerebral ischemia ,which can cause interneuronal fiber connection damage and induce cognitive ability's decrease.D1-3-n-Butylphthalide(NBP) was a new drug that isolated from the seeds of celery, then was synthesized artificially.By occluding bilateral carotid artery, 2VO model of chronic cerebral ischemia was established. Cognitive ability of all the rats was evaluated by Morris water maze three month after 2V0. The expressions of Nogo-A and IGF-1 in different brain field were observed immunohistochemically.The aim of this study is to explore the mechanism of the protective effect of NBP on learning and memory after chronic cerebral ischemia.Materials and methods1. Experiment animal and grouping Selected 80 healthy Wistar rats and distributed to 4 groups randomly, 20 rats each group.Group A:control group(sham operation plus); Group B:ischemia group; Group C:ischemia and low dose treatment group; Group D:ischemia and high dose treatment group.The rats of groups C and D began to receive daily oral dose of 60mg/kg and 120mg/kg NBP (dissolved in 2ml oil) form the 61^st day after the operation, The rats of groups A and B received the same volume of oil, which all will last for a month.2. Producing the modelChronic cerebral ischemic rat model was established by permanent occlusion and snip of bilateral common carotid arteries.Rats undergoing permanent bilateral occlusion and snip of the common carotid arteries were distributed into groupB,C,D.Rats with bilateral common carotid arteries only isolated but not ligated and snipped were into group A.3. Producing the specimenAfter three months, all rats will be be-headed after the heart irrigation and the brains will be fixed in the paraffin section after adding 4% paraformaldehyde for HE and immunohistochemistry staining.4. The content of observation4.1 Cognitive ability of all the rats was evaluated by Morris water maze three month after 2VO.4.2 The changes of neuron in temporal cortex and hippocampus were observed by HE.4.3 The expressions of Nogo-A and IGF-1 were observed by immunohistochemically.5. Statistical methodThe data was handled with SPSS12.0 static software.The difference of every two groups was compared with one-way analysis of variance and LSD method, significant level is a=0.05.Result1. The conditions of experiment animal After operation all the rats were suppressed,behavior disorder,ate little.The next day,the rats of group A recovered significantly, the rats of other groups did not recover obviously. The operation groups recovered slowly until 4-6 days.A week later, they were normal.2. The results of cognitive abilityAfter 2VO three months the difference between group B,C,D and group A was significant (p<0.05). cognitive ability were improved for group C and group D comparing with group B.the difference was significant (p<0.05). The difference between group C and group D about cognitive ability was significant too. (p<0.05)3. The results of pathological section with HE stainingIn hippocampus, neurons were normal in group A.The number of degenerated and dead neuron significantly increased in group B compared with group A.The number of degenerated and dead neurons was less in group C comparing with group B, the normal neuron cells in group C are more than group B.The number of degenerated and dead neurons was less in group D compared with group C, the normal neuron cells in group D are more than group C.4. The results of immunohistochemistry for IGF-1The expression of IGF-1 positive protein mainly in temporal cortex and hippocampus,corpus striatum,the positive cells display buffy or isabelline.There were small IGF-1 immunopositive cells in group A .The number of IGF-1 immunopositive cells was not remakably increased in group B. The difference between group B and group A was not significant (p>0.05).The number of IGF-1 immunopositive cells was remarkably increased in group C and group D comparing with group B.The difference between group C,D and group B was signifiant (p<0.05).The number of IGF-1 immunopositive cells was remarkably increased in group D comparing with group C. The difference between group D and group C was significant (p<0.05).5. The results of immunohistochemistry for Nogo-AThe Nogo-A immunopositive cells were mainly in glial cell,neuron,fiber endochylema and apophysis. the positive cells display buffy or isabelline. There were small Nogo-A immunopositive cells in group A. The number of Nogo-A immunopositive cells was remakably increased in group B. The difference between group B and group A was significant (p<0.05). The number of Nogo-A immunopositive cells was remarkably decreased in group C and group D comparing with group B.The difference between group C,D and group B was signifiant (p<0.05). The number of Nogo-A immunopositive cells was remarkably decreased in group D comparing with group C.The difference between group D and group C was significant (p<0.05).Conclusion1. Three months after chronic cerebral ischemia,the number of IGF-1 immunopositive cells was not remakably increased and the number of Nogo-A immunopositive cells was remakably increased.2. NBP can also promote the expression of IGF-1 and degrade the expression of Nogo-A in temporal cortex and hippocampus after chronic cerebral ischemia.high dose group is more significant.