| Objective:1. To observe whether the bone-marrow endothelial progenitor cells(EPCs) could be mobilized to peripheral blood, and homed to the ischemic brain regions in focal cerebral ischemia/reperfusion rat;2. To investigate the effect of electroacupuncture on the number of VEGFR2+EPCs and CD34+EPCs in rat bone marrow and peripheral blood after focal cerebral ischemia/reperfusion;3. To investigate the effect of electroacupuncture on the angiogenesis in ischemic cortex after focal cerebral ischemia/reperfusion in rat;4. To investigate the effect of electroacupuncture on the number of VEGFR2+EPCs and CD34+EPCs via eNOS in rat bone marrow and peripheral blood after focal cerebral ischemia/reperfusion;5. To investigate the effect of electroacupuncture on the angiogenesis via eNOS in ischemic cortex after focal cerebral ischemia/reperfusion.Method:The rats received filament occlusion of the right middle cerebral artery for 1.5 hours followed by reperfusion. A total of 90 healthy male adult Sprague-Dawley (SD) rats were randomly divided into I/R+NS group, I/R+DIL-acLDL group. Immunofluorescence were used to test bone marrow DIL-acLDL+cells, bone marrow and peripheral blood CD34+DIL-acLDL+cells, and DIL-acLDL+cells in ischemic brain regions of these rats. A total of 160 healthy male adult Sprague-Dawley (SD) rats were randomly divided into normal group(N), sham group(S), model group(I/R), electroacupuncture group(I/RE) and I/RE plus L-NAME (a specific antagonist of eNOS) group (I/REL), and were further divided into 1d,2d,3d,7d subgroups after reperfusion,10 rats each group, in addition to the N group. Electroacupuncture was applied to " Baihui" (GV 20)/ " Siguan" (Hegu LI 4/Taichong LR 3) acupoints for 20 min, once a day. Flow cytometer was used to detect the percentage of CD34+EPCs, VEGFR2+EPCs in bone marrow and peripheral blood. ELISA was used to detect the expression of eNOS protein in peripheral blood. The expression of VEGFR2 mRNA in ischemic cortex was tested by fluorogenic quantitative PCR. Immunohistochemical method was selected to detect the expression of VEGFR2 protein, CD34+ microvessels and eNOS protein in ischemic cortex.Results:1. The results of I/R+NS group were negative. DIL-acLDL+cells were found after 1d of intra-bone marrow injection in bone marrow, and CD34+DIL-acLDL+cells were found after 1d,2d,3d and 7d of intra-bone marrow injection respectively in bone marrow and peripheral blood. DIL-acLDL+cells were not found after intra-bone marrow injection in ischemic brain regions.2. The changes of the number of CD34+EPCs, VEGFR2+EPCs in rat bone marrowCompared with sham group, the number of CD34+EPCs in bone marrow was higher at 1d,2d,3d after reperfusion in I/R group(P<0.01), in which the increase were more obvious in I/RE group at each time point(P<0.01), but it was decreased by the inhibitor of eNOS(P<0.01). Compared with I/R group, there was a significant up-regulation of the percentage of VEGFR2+EPCs in bone marrow by electroacupuncture (P<0.01, P<0.05), but it was decreased by the inhibitor of eNOS(P<0.01).3. The changes of the number of CD34+EPCs, VEGFR2+EPCs in rat peripheral bloodCompared with sham group,the number of CD34+EPCs was significantly increased at 1d,2d,3d after reperfusion in I/R group(P<0.01). In I/RE group, the number of CD34+EPCs was obviously increased at 1d, 2d,3d(P<0.01) compared with I/R group, but it was decreased by the inhibitor of eNOS(P<0.01). Compared with I/R group, there was a significant up-regulation of the percentage of VEGFR2+EPCs in peripheral blood by electroacupuncture(P<0.01, P<0.05), but it was decreased by the inhibitor of eNOS(P<0.01).4. The expression of eNOS protein in rat peripheral bloodCompared with sham group, the expression of eNOS protein was significantly increased at 1d,2d,3d after reperfusion(P<0.01). In I/RE group, the expression of eNOS protein was obviously increased at 1d,2d, 3d(P<0.01) compared with I/R group, but it was decreased by the inhibitor of eNOS(P<0.01).5. The expression of CD34+vessels in cerebral ischemic cortexCompared with I/R group, the expression of CD34+vessels was highly increased at each group in I/RE group(P<0.01), but it was decreased by the inhibitor of eNOS(P<0.01).6. The expression of VEGFR2 mRNA and VEGFR2+cells in rat cerebral ischemic cortexThe expression of VEGFR2 mRNA and VEGFR2 positive cells were increased gradually along with the ischemic time. Compared with N group, the expression of VEGFR2+cells was highly increased at each group in I/R group(P<0.01). In I/RE group, the expression of VEGFR2 mRNA and VEGFR2 positive cells were obviously increased at each group(P<0.01) compared with I/R group, while both of them were decreased by the inhibitor of eNOS(P<0.01).7. The expression of eNOS protein in rat cerebral ischemic cortexCompared with sham group, the expression of eNOS protein was significantly increased at 1d,2d,3d after reperfusion(P<0.01). In I/RE group, the expression of eNOS protien was obviously increased at Id,2d, 3d(P<0.01) compared with I/R group, but it was decreased by the inhibitor of eNOS(P<0.01).Conclusions:1. In vivo, bone-marrow cells can be tagged by DIL-acLDL in focal cerebral ischemia/reperfusion rat. We found that bone-marrow CD34+DIL-acLDL+EPCs can also be tested in peripheral blood, demonstrating the moving trajectory of CD34+DIL-acLDL+EPCs directly;2. Electroacupuncture can effectively mobilize EPCs to promote the revascularization in focal cerebral ischemia/reperfusion rat, which will be impaired by the inhibitor of eNOS. So we speculated that EPCs could be mobilized by electroacupuncture via activating the expression of eNOS to promote the revascularization. |
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