| Background and AimThe chronic cerebral hypoperfusion is the important foundation of vascular dementia (VD)and Alzheimer's disease(AD).In the recent years,many research has proved that egardless of vascular dementia (VD) or Alzheimer's disease(AD) has the obvious blood vessel factor. The role of brain hypoperfusion brings to the attention day by day in the process of demented advancement , and proposed the vascular cognitive impairment (VCI) this new concept. Therefore, it is important to study pathogenesis for preventing and treating chronic cerebral hypoperfusion.The chronic cerebral hypoperfusion can cause nerve injury and its main performance is cognitive disorder such as study, memory, and so on. The chronic cerebral hypoperfusion cause the hippocampal change to contain two principal aspects : Nerve cell's damage and the retardant function impairment of cholinergic nerve. Some researchs report that the impairment of frontal lobe and the cholinergic neurons possibly be the morphological foundation which can cause cognitive disorder,and the cognitive impaired mechanism involves changes of the cholinergic acceptor, and so on . Chat is a rate-limiting enzyme,which control the synthesis of acetycholine( Ach).So it is always regarded as the special signal of cholinergic neurons . D1-3-n-Butylphthalide (NBP) was shown to improve brain energy metabolism in mice with complete cerebral ischemic, to enhance the rCBF of rats with regional ischemia,and to improve impairment of learning and memory of rat with MCAO, and so on. Present study aimed to determine the change of Chat in hippocampus in rats brain by the methods of reverse transcription PCR and Immunohistochemical staining and dot immunobinding assay. By occlusiving bilateral carotid artery, 2VO model of chronic cerebral ischemia was established. The expression of Chat in hippocampus was observed through three methods.The aim of this study is to explore that mechanism of the protective effect of NBP on learning and memory after chronic cerebra ischemia.Materials and methods1. Subjects were divided into 5 groups 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; group E: ischemia and middle dose treatment. In front of operation ,the rats of groupE give NBP according to 80mg/kg fill the stomach every day, and after the operation , continues to fill the stomach for two months according to this dosage.The rats of groups C and D began to receive daily oral dose of 60mg/kg and 120mg/kg NBP (dissolved into 2ml oil) from the 61st day after the operation, which will last a month. The rats of groups A and B received the same volume of oil, which will last the same time.2. Chronic 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 ischemia group or ischemia treatment group. Rats with bilateral common carotid arteries only isolated but not ligated and snipped were into control group.3. After three months , all rats after the heart irrigation, behead take the left brains, fixed makes the paraffin section after 4% paraformaldehyde, for immunohistochemistry staining; The right brains were put in the supercold refrigerator, for RT-PCR and Dot Immunobinding Assay.4. Used the immunohistochemical staining to observe the distribution of Chat immunopositive cells in hippocampus in rats brain ; Used the RT-PCR method to examine ChatmRNA transcription level in hippocampus in rats brain; Used the dot immunobinding assay method to study the expression of Chat protein in hippocampus in rats brain. 5. The data was handled with SPSS13.0(Inc). The diference of each group was compared with one-way analysis of variance,and only values < 0.05 were considered statistically significant.Results1. The conditions of animal All the rats were depressed, reacted slowly, ate less after operation. The next day, sham groups recovered significantly, however the operation groups recovered slowly until 3-5 days. A week later, they were normal.2. The results of immunohistochemical staining for CHAT The number of Chat immunopositive cells in hippocampus was significant decreased in group B comparing with group A. Significant differences were observed between group B and group A. The number of Chat immunopositive cells was markly increased in group C , group D and group E comparing with B group. Significant differences were observed between group C, group D and group E. The number of Chat immunopositive cells was markly increased in group D comparing with group C and group E. Significant differences were observed between group C, group D and group E.No significant differences were observed between group C and group E.3. The results of RT-PCR for CHAT Significant differences were observed between group B and group A.The optical density value was significant increased in group C , group D and group E comparing with B group. Significant differences were also observed between group C, group D and group B. The number was significant increased in group D comparing with group C and group E. And significant differences were observed between group C, group D and group E. No significant differences were observed between group C and group E.4. The results of Chat Dot Immunobinding Assay The grey number was significant decreased in group B comparing with group A. Significant differences were observed between group B and group A.The grey number was markly increased in group C , group D and group E comparing with B group. Significant differences were observed between group C, group D and group B. The grey number was significant increased in group D comparing with group C and group E. Significant differences were observed between group C, group D and group E .However, No significant differences were observed between group C and group E.Conclusions1. Chronic cerebral ischemia rat model was established by permanent occlusion and snip of bilateral common carotid arteries.The progress of the model and chronic cerebral ischemia was almost same. Its operation is simple and repeat well.2. Three months after, significantly functional impairment appeared in hippocampus in rats brain.3. NBP can suppress the diminution of immunopositive cells in hippocampus in rats brain which chronic cerebral hypoperfusion caused, enhance ChatmRNA the transcription level, increase the Chat protein the expression.
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