| Objective: Based on the previous studies of correlation between endostatin(ES) and vascular endothelial growth factor(VEGF) in blood serum of rats induced by spinal cord ischemia-reperfusion injury, we detect the expression and associativity between ES and VEGF in rats subjected to cerebral ischemia-reperfusion injury. Methods: The models of focal brain reperfusion injury by middle cerebral artery occlusion (MCAO) were established in wistar rats according to the advanced Zea Longa's method. Rats were randomly divided into two groups: sham- operated group(group A) and cerebral ischemia-reperfusion injury group ( group B). Group A (n=8) underwent the same surgical procedure but without the introduction of the filament into the MCA. Group B(n=40) was divided into 5 subgroup(n=8 for each) and the subgroups were anesthetized and euthanized by decapitation at 6h, 1d, 2d, 4d, and 7d after reperfusion respectively.1. Neurologic examination was Clinically performed on each rat after 6h, 1d, 2d, 4d, 7d reperfusion according to the Zea Longa's scoring method.2. At the given end point,rats were sacrificed and brain tissue biopsy was immediately taken. Cerebral slices were stained with triphenyl tetrazolium choride (TTC) and observed under light microscope;3. Expression levels of VEGF mRNA and ES mRNA in brain tissue were quantified with real time Polymerase chain reaction (RT-PCR) and ratios of ES/VEGF were calculated.Measurement data was presented as mean ± SD. Data analysis was performed by one way ANOVA followings t test and q test,and analysis of linear correlation was used to evaluate the correlation between neurological function score and the ratio ofE/V with SPSS 13.0. P < 0.05 was considered statistically significant.Results :1 .Neurological deficits of the rats:Compared with group A, the neurological deficits were significant in group B at each time point. In group B1, seven rats dextrad circled during crawl and one tumbled rightward, and score was 2.125 ± 0. 354. Group B2 exhibited slightly difference to group B1.In group B3, five rats lost autonomic activities and three tumbled rightward, and score was 3.625±0. 518. Most rats in group B4 and B5 scored 2 points, and scores was 2.375 ± 0. 518 and 2.250±0.463.2. TTC staining of cerebral slices:Cerebral slices of rats in group A showed no infarct; so did the right hemispheres of rats in group B .Six hours after reperfusion, left cerebrums of group B began to swell and showed small infarction. On 1 day post-reperfusion, infarction area appeared more obvious and most infarction were in the cortical substance. And with the deterioration of ischemia, larger infarction area appeared at day 2.Then the encephaledema relieved, while the infarction were still obvious after 4 days.3. Expression of VEGF mRNA and ES mRNA in brain tissues with RT-PCR.The VEGF mRNA expression quantity of rats in group A was set as 1. Compared with those in group A, VEGF mRNA expression in brain tissues of group B elevated significantly: the quantities upregulated as early as 6 hours after reperfusion (2.499±0.399, P<0.05 ) and peaked at 1d point (4.243±0.352, P<0.01) followed by a noticeable decrease at 2d point (1.321±0.215) .Interestingly, the level of VEGF mRNA elevated at 4d after reperfusion (1.647±0.264) and kept a higher level at 7d point (1.920±0.253, P<0.05). The quantity level of those in subset B2 was distinguished contrast to those of other subsets (P<0.05) .There were no statistical difference observed among subgroup B1, B3, B4, and B5.Compared to group A who's ES mRNA was set as 1, the expression of ES mRNA remained a normal level (0.989±0.308,P>0.05) at 6h point. The level went up dramaticlly after 1 d post reperfusion (3.229±0.247, P<0.01), and the peak value was distinguished at 2d point (7.301±0.242,P<0.01) .From 4d to 7d after reperfusion, the ES mRNA expression still kept a higher level compared with those of group A (P<0.01). Quantity of subset B3 exhibited a statistically significant compared with those of other subsets (P<0.01).In group B, values of ES/VEGF decreased at both 6h and 1d points, but increased gradually until 2d after reperfusion (5.588±0.786, P<0.01).But it is still at high levels at 4d point (2.850±0.377,P<0.05) and 7d point (2.323±0.257,P<0.05) .Valueof subgroup B3 was distinguished compared with those of other subgroups (P<0.01) and values of subgroup B4 and B5 was significantly higher contrast to those of group B1 and B2(P<0.05).The correlation analysis showed a strong relationship between neurological function scores of the rats subjected to ischemic reperfusion injury and their E/V values (r=0.811) . ConclusionSubjected to reperfusion injury following 90 minutes of cerebral ischemia, rats central nervous systems begin to work abnormally, especially 2 days after reperfusion when neurological function scores rise to the peak; left cerebrums showed various signs of ischemia and large area of infarction could be observed. Expressions of VEGF mRNA expression in brain tissues increase as early as 6 hours after reperfusion and peak at 1d point followed by a noticeable down regulation at 2d point however still higher than the sham group. Expressions of ES mRNA don't upregulate until 1d after reperfusion and reach the peak at 2d point. Ratio of E/V shows a falling trend during the early time of reperfusion, but it goes up significantly at day 2 and remains a high level later. Neurological function scores shows closely related to the ratio of E/V.In conclusion, during the procedure of brain ischemia reperfusion of rats, the balance of ES and VEGF, which may be reflected from the value of E/V, is tightly correlated with the neurological dysfunctions. Balance of enhancing and inhibiting factors of angiogenesis may be the key factor affecting severity and prognosis of injury after cerebral ischemic reperfusion. Since there are noticeable upregulation of VEGF mRNA expression and no significant elevation of ES during the early period and especially before 2 days after reperfusion, this period may be of crucial importance to the rebuilding of microcirculation. |
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