| Objective:To establish model of stroke by the middle cerebral artery occlusion, after that the rats were divided into experimental and control groups. Experimental group were application of chronic unpredictable mild stimulus joint single housed to established post stroke depression model. After that the rats were sacrificed to get frontal lobe and hippocampus. In order to study changes of 5-HT、DA、NE and the variation of the fibroblast growth factor (FGF-2) in the PSD rats brain and to explore the relationship between all of them and post stroke depression, HOLC was used to determination of 5-HT、DA、NE; western blot were used to determination of the fgf-2 protein expression level of frontal lobe and hippocampus, real-time PCR to test the expression levels of fgf-2 mRNA in the frontal lobes of both groups.Methods:1. Reference improved Koizumi method, a mouse model of post stroke depression was established by means of middle cerebral artery occlusion (MCAO). Success criteria for judging the Model of rats:This method of scoring is a reference to nerve function:Score of 1 to 3 points is a sign of successful operation, the socre of 0,4 are regarded as model-making failure. Then the rats were randomly divided into two groups:control and modification groups.2. After 7 days of surgery period of 21 days, reference Willner’s improved CMS method, prepare left internal carotid artery stroke middle cerebral artery model. After 7 days of surgery period of 21 days, reference Willner’s improved CMS method, the CMS includes:①fasting for 20 hours;②water deprivation 17 hours;③cage titled (45°) 17h;④constant light 17 h;⑤wet cage(100g sawdust+200 mLwater)21h;⑥4℃swim 5 min;⑦horizontal shaking 5 min;⑧behavior restrictions 2 h;⑨tail clamp 1 min。with isolate raised, one kind of those stimulations was taken randomly everyday to established PSD model. Open-box test was conduct after the PSD model was successfully established. The number of animals across the bottom lattice as the level activity score, generally animals step aside walk, across 1 lattice was 1 time. If the rats walk in the diameter direction, generally every 10cm recorded as 1 times, the number of vertical activities as score. The rats leave the ground regard as 1 time no matter how long the rats stand up.Thoroughly clean the boxes before the next open-box test, each measurement time are 5 min.3. Application HPLC to analysis the levels of 5-HT、NE、DA, which in the frontal and hippocampus tissues4. Application western blot to analysis the protein expression levels of fgf-2 which in the frontal and hippocampus tissues. Extract protein of rat frontal cortex and hippocampus with protein extraction(1 mL extraction reagent mixture added 5 u L protease inhibitors,5μL phenylmethanesulfonyl fluoride, PMSF). Total proteins were prepared by the addition of 500μL of lysis buffer. Plates were incubated at 4℃for 30 minutes, scraped and the protein concentration determined with a Bio—Rad protein assay kit. Total protein(25μg)in each lane was separated by SDS/PAGE on 10% acrylamide precast gels. The electrophoresed proteins were transferred to a nitrocellulose membrane. After blocking with 3% BSA, a panel of monoclonal antibodies against sm—α--actin (1:5000), sm-MHC(1:200)andβ-actin(1:5000)were applied. Signals were visualized using a horseradish peroxidase—conjugated goat antirabbit IgG(Jackson, USA; 1:5000)and enhanced chemiluminescent reagent (ECL Amersham-Pharmacia) and were quantified on auto radiographs that depicted bands within a linear range of exposures.5. Then further tests, which are to test the expression levels of fgf-2 mRNA in the rat frontal lobe tissues, were conducted by the real time quantitative PCR(RT-PCR). Extraction of total RNA by the methods of Triziol after crushing the rat frontal cortex tissue, Then using reverse transcriptase reverse transcriptase mRNA into cDNA. Establish polymerase system(Table 1) The gene was amplified using the FGF-2 primers with the following PCR condition:95℃for 5min; 35 cycles of 95℃for 30s,55℃for 30 s,72℃for lmin; 72℃for 10 min. The SYBR fluorescence real-time quantitative PCR reactions, which to detect FGF-2 mRNA expression levels, between the target gene and housekeeping gene was made using real-time quantitative PCR. Specificity for realtime PCR reactions was tested by electrophoresis through a 1.5% agarose gel and melting curve analyses. A standard curve that describes the relationship between experimental and control group copy numbers and cycle threshold (CT) values was generated using serial dilutions of a known copy number of the FGF-2 rRNA. We calculated the copy numbers directly from the concentration of extracted plasmid DNA by spectrophotometry (Nanodrop Technologies, Rockland, Del, USA). Melting curve analysis was performed from 55℃to 95℃with a reading made every 1℃and the samples held for 1 s between readings.Results:1. In this study 60 rats were used to established the stroke model,45 rats were modeling success, the rate of this methods is 75.0%2.23 rats were used to established the psd model by the CMS and isolate raised,20 rats were modeling success, the rate of this methods is 86.9%3. The levels of 5-HT、NE and DA, which in the frontal and hippocampus, was significantly lower than the control group(P<0.01, t test)4. Western blot:The modification groups expression levels of fgf-2 in the frontal was significantly lower than the control group(P<0.01, t test), the protein expression of fgf-2 in the hippocampus was not significant(P>0.05,t test).5. Real-time PCR:The modification groups expression levels of fgf-2 mRNA in the frontal was significantly lower than the control group(P<0.01, ttest)Conclusion:1.Decreased levels of monoamine neurotransmitters in the frontal cortex and hippocampus have a certain connection in the incidence of PSD2 Decreased expression levels of fgf-2 protein in PSD rats frontal lobe is one of the reasons that caused PSD.3. The reason that the fgf-2 protein express levels decline due to the decreased express levels of fgf-2 mRNA4. Frontal lobe play a major role in the incidence of post-stroke depression... |
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