| Background Ischemic cerebrovascular disease is a kind of common disease in clinic,which has high incidence rate and high mortality rate, it threatens people's health and life seriously. This disease named "Stroke" in Traditional Chinese Medicine. In clinic, ischemic cerebrovascular disease account for the stroke of 70～80%, cerebral thrombosis, cerebral embolism is the directly cause of cerebral infarction. Middle cerebral artery occlusion is the most common disease of focal cerebral ischemia. Therefore, it is very important to study the regeneration and repair mechanism in focal cerebral ischemia/reperfusion. As a compensatory response to ischemia, the regeneration of nerves and blood vessels is very important.Revascularization can happen in angiogenesis and vasculogenesis. Vasculogenesis, a nove process when vessels are formed by endothelial progenitor cells (EPCs), which occurs mostly during embryogenesis. Angiogenesis, which invoves vascular sprouting from mature of pre-existing blood vessel. The emergence of EPCs, makes us realize another mechanism of neovascularization which updated the traditional theory of revascularization after vascular injury. That is EPCs participate in neovascularization after ischemia, both of angiogenesis and vasculogenesis are playing a very important role in the vascular repair process.EPCs mainly come from the bone marrow after cerebral ischemia. The cerebral ischemic region release some factors, which is one of the conditions mobilizes bone marrow EPCs homing. Among these factors, SDF-1αis the most representative one which promote vasculogenesis. SDF-1αand CXCR4 binding help EPCs homing.The mechanism of neovascularization is activated immediately after cerebral ischemia, including vasculogenesis and angiogenesis, but the body's ability of repairing respond is very weak. It isn't sufficient to improve the situation of tissue perfusion and ability of vascular network reconstruction. Through acupuncture, which from the surface points, stimulating the endogenous protection mechanism of the body is an effective method of non-drug treatment way after cerebral ischemia. Electroacupuncture(EA) is widly used in clinic, it can improve neurological deficit scores in the treatment of stroke. Previous studies of our experimental group showed that EA can upregulate vascular endothelial growth factor and angiopoietin, downregulate endostatin, promote angiogenesis after cerebral ischemia. And VEGF also plays an important role in vasculogenesis after focal cerebral ischemia/reperfusion, both of VEGF and SDF-1αpromote vasculogenesi. For these reasons, our study focuses on the changes of the expression of SDF-1α/CXCR4 axis and angiogenesis in rat brain under intervention of electroacupuncture and AMD3100 (CXCR4 inhibitor) after focal cerebral ischemia/reperfusion. To investigate the mechanism of electroacupuncture promoting revasculariza- tion in the rat brain of focal cerebral ischemia/reperfusion.Objective (1) To investigate the expression pattern of SDF-1α/ CXCR4 axis in rats brain after focal cerebral ischemia/reperfusion and the effect of electroacupuncture and AMD3100;(2) To investigate the expression of microvessel in rats brain after focal cerebral ischemia/reperfusion and the effect of electroacupuncture and AMD3100;(3) To investigate the effect of SDF-1α/CXCR4 axis on revasculariza- tion after focal cerebral ischemia/reperfusion, and the status of vasculogen- esis in vascular repair;(4) To investigate the mechanism of electroacupuncture in vasculoge- nesis for focal cerebral ischemia/reperfusion.Methods Male SD (Sprague-Dawley) rats were randomly divided into control group(NC group), model group(I/R group), electroacupuncture group (I/RE group), drug group(I/RA group), saline group(I/RN group). The model of focal cerebral ischemia /reperfusion was made by filament occlusion, I/R group and I/RE group were divided into 5 subgroups according to accept reperfusion 1d,3d, 7d,14d, 21d after 1h ischemia. Each subgroup has 5 rats for immunohistochemistry and RT-PCR. EA was given at bilateral"Hegu"poin(tLI 4)in I/RE group. These rats were treated with continuous stimulation for 15min each day up to 7 days. 7d time point was selected to observe the I/RA group and I/RN group. From the first to fourth day, AMD3100 was injected at 1.25mg/Kg once daily in I/RA group, and the same volume of saline was injected in I/RN group. Each of NC group, I/RN group and I/RA group has 4 rats.To investigate the pathological difference of brain tissue by HE staining. To evaluate the neurological function impairment of focal cerebral ischemia/reperfusion by neurological symptom score. The expression of SDF-1αmRNA was detected with reverse transcription -polymerase chain reaction(RT-PCR). The immunohistochemical method was employed to detect the expression of SDF-1α, CXCR4 proteins and microvessel count.Results 1. Neurological symptom scoreEA can effectively improve the neurological function deficit, and promote recovery of neurological function after focal cerebral ischemia/reperfusion injury. At the time points of reperfusion 2h,1d,3d, score gradually reduced and the difference between I/R group and I/RE group was not statistically significant (p>0.05). With prolonged reperfusion, the rats have a recovery of neurological function, at the time points of 7d, 14d, the difference between I/R group and I/RE group was significantly different (p<0.05).2. The expression of SDF-1αmRNANC group has a small amount of SDF-1αmRNA expression on the right cerebral cortex. In I/R group, SDF-1αmRNA expression has a single peak-like increase, compared with NC group, increased strikingly at 1d (P< 0.01), reached a peak value at 7d point, then decreased gradually, but still higher than the NC group (P<0.01). At the time points of 3d,7d,14d, I/RE group has a higher expression than I/R group, at 3d and 7d points, the difference of SDF-1αmRNA expression between them was significant(P<0.01), at 14d, the difference was still statistically significant (P<0.05).3. The results of immunohistochemistry(1) The expression of SDF-1αproteinNC group: a small amount of SDF-1αprotein expression in the cerebral cortex, hippocampus, paraventricular and other places around the brain. I/R group: ischemic cerebral cortex shows a large number of brown cells, bilateral brain contrasted, ischemic cerebral hemisphere has deepen color. In I/R group, SDF-1αprotein expression began to increase at 1d(P<0.05), peaked at 7d(P<0.01), it is still higher than NC group at 21d(P<0.01). I/RE group and I/R group has the same law, but the former has a higher level. At 3d, the difference of SDF-1αprotein expression between them was statistically significant(P<0.05). At 7d, 14d, 21d, the difference was very significant (P<0.01). Compared with I/R group and I/RN group, the I/RA group has a higher level(P<0.01), but compared with I/RE group, I/RA group is lower, the difference was significant(P<0.01).(2) CXCR4-positive cell countNC group: in cerebral cortex, there has very limited number of CXCR4 -labeled cells, some sections don't discover CXCR4-positive cells, but we find that CXCR4 protein expresses around some small blood vessel. I/R group: cerebral cortex, hippocampus, lateral ventricles show a large number of CXCR4 positive cells. The CXCR4-labeled cells expression has a single peak-like increase, reach a peak value at 7d. Compared with I/R group ,the I/RE group has the same law, but the later has a higher level. At 3d, 7d, the difference of CXCR4-labeled cells expression between them is statistically significant(P<0.05), at 14d, the expression decrease gradually. but still higher than the I/R group, the difference is significant (P<0.01). The number of CXCR4-positive cells in I/RA group has a higher level than I/R group (P<0.01), I/RE group has a higher level than I/RA group,but the difference was not statistically significant(P>0.05).(3) Microvessel countI/R group: some clustering microvessel can be seen in ischemic cerebral cortex. In the first day to 14th day, microvessel count increased gradually and peaked at 14d, at 21d, it decreased slightly, but remained at a high level. I/RE group has the same law with I/R group, the expression of the peak is still at 14d, but the I/RE group has a higher level than I/R group in each point, at 3d, 7d, 14d, 21d point, the difference between I/R group and I/RE group was significant difference (P<0.01). Microvessel count in I/RA group is higher than I/R group, the difference was statistically significant (P<0.05), but compared with I/RE group, the difference was not statistically significant (P>0.05).Conclusion (1) EA can effectively promote recovery of neurological function after focal cerebral ischemia/reperfusion;(2) EA could promote SDF-1αmRNA expression in ischemic cortex after focal cerebral ischemia/reperfusion;(3) EA could stimulate the SDF-1α/CXCR4 axis in ischemic cortex after focal cerebral ischemia/reperfusion;(4) EA could promote revascularization in ischemic cortex after focal cerebral ischemia/reperfusion;(5) EA may promote revascularization by increaseing the expression of SDF-1α/CXCR4 in the rats brain of focal cerebral ischemia/reperfusion.
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