| Objectives: Chronic cerebral hypoperfusion is a common pathological course of the developing of vascular dementia (VD), Alzheimer's disease (AD) and Bingswanger's disease, et al. In our country, VD is one of major senile dementia, which not only impairs patients'physical and mental health badly, but also brings about a heavy load to the society and family. At present, the pathogenesis of VD has not been illustrated clearly, and dealed with effectively, either. Therefore, it's an important subject in medicine field to explore the pathogenesis of VD and make a reasonable therapeutic regimen.Chronic cerebral hypoperfusion may be one of the major causes for VD. Some studies show that cognitive function can be impaired under hypoperfusion and hypometabolism. And the disorder of free radical metabolism in brain may be one of the major possible mechanism, which results in impairment of cerebral texture and microvascular system. SOD as a specific enzyme of scavenging superoxide anion in vivo, its change responds the vivo's ability to scavenge free radical indirectly. MDA is the metabolic end product of lipid peroxidatic reaction, which responds its intensity indirectly caused by free radical in vivo. After cerebral ischemia, lipid peroxidatic reaction is active, which results in producing more MDA and consuming more SOD. Hypoxia-inducible factor-1α(HIF-1α) found in recent years is a core regulatory factor in participating in regulating unbalance of oxygen homeostasis. It is induced to express in hypoxia and play versatile effects. Some studies investigate that HIF-1αmay take part in the compensation and accomodation mechanism of chronic cerebral hypoperfusion in rats.Edaravone as an oxygen free radical scavenger has been attentioned by more and more people for its neuroprotective effect in acute cerebral ischemia. But most studies have only focused on acute cerebral ischemia model at home and abroad, while the researches about its effect on chronic cerebral ischemia model have still lacked. This experiment mimicried human chronic cerebral hypoperfusion by the ligation of bilateral carotid arteries in Sprague-Dawley male rats, to observe their changes of learning and memory, SOD activity and MDA content in forebrain and the expression of HIF-1αat hippocampal CA1 area, and at the same time to explore the effect of edaravone, a new type of free radical scavenger, on them. We provided a new evidence for clinic treating vascular cognitive disorder as well as revealed the pathogenesis of VD.Methods: 84 Sprague-Dawlay rats, 3~4 months old, 280~340g in weight, were randomly divided into sham group(n=20), saline group(n=32) and edaravone group(n=32). Each group was randomly divided into 6 weeks group and 8 weeks group again. Saline group and edaravone group were followed permanent occlusion of bilateral common carotid arteries (2-VO) accoding to de la Torre's method. Sham group was handled as Saline and edaravone group except not ligating bilateral common carotid arteries. After 4 weeks following the operation, edaravone group were injected edaravone (5mg·kg-1·d-1) through abdomen cavity for 2 weeks in 6 weeks group and 4 weeks in 8 weeks group. Saline group were injected isodose saline. Each group rats were executed at the corresponding time after water maze test. We dissected rat brain at ice disk, removed brainstem and cerebellum, rinsed in 0~4℃physiological saline(PS) to get rid of blood and then dried it by fiter-paper. At post-chiasm opticum 1mm and 4mm, we cut off the brain coronally and took the middle tissue which was hippocampus to fix, dewater, immerse wax, embedded and made into paraffin section. Morphological change was examined by HE staining. The expression of HIF-1αwas assayed by immunohistochemistry analysis. We respectively measured integrated optic density (IOD) of HIF-1αpositive neurons in the same area of hippocampal CA1 area. SOD activity and MDA content of anterior-chiasm opticum brain tissue were assayed. One-way analysis of variance was carried out with SPSS 10.0 statistics software, LSD-t test was used to compare the differences among the groups. P<0.05 meant statistical significance. Results: (1) The survival condition of each group: 0 died in the sham group (including 6 weeks and 8 weeks groups). 6 and 8 rats died respectively in the saline 6 weeks group and 8 weeks group. The death rates were 31.3% and 37.5% respectively. 5 and 4 rats died respectively in the edaravone 6 weeks group and edaravone 8 groups. The death rates were 31.3% and 25% respectively. All survival rats of each group were as study objects. (2) Water maze test: Learning and memory performances of rats decreased obviously in saline 6 weeks group and 8 weeks group, showing escape latency(EL) extended which were clearly higher than sham group (P<0.05). Compared between saline 6 weeks group and 8 weeks group, the latter behaved worse(P<0.05). Learning and memory performances in edaravone 6 weeks group and 8 weeks group improved significantly(P<0.05), compared with saline 6 weeks group and 8 weeks group. The performances in edaravone 6 weeks group still decreased, if compared with sham group(P<0.05). While performances of edaravone 8 weeks group had no obvious distinction, compared with sham group(P>0.05). (3) HE stainning in hippocampus: The arrangement of pyramydal neurons at hippocampus CA1 in sham group was tight and in order, nucleus were large and round, and the chromatospherites were evident; In saline group, the arrangement of pyramydal neurons was loose and not in order, pyramydal neurons lacked and inflammatory cells infiltrated, cell bodies shrinked, karyopycnosis and cytoplast dyed densely; The arrangement of pyramidal neurons was better than saline group in edaravone group, the phenomenon of karyopycnosis and inflammatory cells infiltrate was less. (4) The expression of HIF-1αat hippocampal CA1 area: only few positive neurons were observed at hippocampal CA1 area in sham group; There were a few of positive neurons expressed in saline 6 weeks group and saline 8 weeks group, which aligned loosely, cell bodys shrinked and karyopycnosis. The integrated optic density (IOD) of positive neurons was higher obviously than that in sham group. Compared IOD between saline 6 weeks group and saline 8 weeks group, there was no statistical significance(P<0.05). A great quantity of HIF-1αpositive neurons distributed at hippocampal CA1 area in the edaravone 6 weeks group and edaravone 8 weeks group. The positive substance located mainly in intracytoplasm, and nucleuses also expressed a little. The positive neurons ranged tightly, brown and yellow, the cell bodies were round or ellipse, and prominences were observed in some bodies. We discovered that the IOD of edaravone 6 weeks group increased significantly compared with sham group and saline 6 weeks group(P<0.01). Similarly, the positive neuron IOD of edaravone 8 weeks group was clearly higher than that of sham group and saline 8 weeks group(p<0.01), and also higher than that of edaravone 6 weeks(P<0.05)(.5)The results of SOD and MDA : Compared with sham group, SOD activity of forebrain tissue decreased in saline 6 weeks group and saline 8 weeks group, and MDA content increased(P<0.05). Compared with saline 6 weeks group, the SOD activity of saline 8 weeks group decreased more, and MDA content increased more (P>0.05). After edaravone treatment, the SOD activity increased and MDA content decreased(P<0.05). And if compared with edaravone 6 weeks group, SOD activty of edaravone 8 weeks group increased more, and MDA decreased more(P>0.05).Conclusions: (1) This experiment successfully established chronic cerebral hypoperfusion model by ligation of bilateral common carotid arteries in rats. Water maze test verified that model rats existed learning and memory disorder, and HE stain in hippocampus showed pyramidal cells were damaged, which could imitate the clinical intelligence impairment of VD patients. At the same time, the effect of edaravone was proved in the study. (2) With immunohistochemistry, this study confirmed that the expression of HIF-1αincreased at hippocampus of rats under chronic cerebral hypoperfusion, which may participate the compensation and accomodation mechanism of chronic cerebral hypoperfusion. We adopted xanthine-oxydation and thio-barbituric acid to measure SOD activty and MDA content respectivly, and proved that SOD activity of forebrain decreased while MDA content increased after 2-VO. After edaravone treatment the expression of HIF-1αat hippocampus increased obviously, SOD activty of forebrain strengthened clearly and MDA content decreased significantly. All of those illuminated that edaravone could improve vascular cognitive impairment by multi-ways such as promoting HIF-1αexpression, inhibiting the activity of xanthine oxidase and hypoxanthine oxidase, lowering hydroxy radical concentration and so on. (3) Furthermore, this study proved that learning and memory ability could improve lastly by extending treatment time which suggested that the treatment of vascular cognitive impairment (VCI) need long time maintenance. |
