Résumé
Objective To investigate whether silencing repulsive guidance molecule A (KGMa) shows protective effects on blood brain barrier (BBB) and tight junction protein after the injury of cerebral ischemia and reperfusion (I/H) in rats. Methods Middle cerebral artery occlusion (MCAO)reperfusion was employed to establish the models in the male adult rats. Fourty male Sprague-Dawley rats were divided into blank control group (sh-con) and RGMa interference group (sh-RGMa). The effects of adenovirus on RGMa were observed at day 1 and day 3 after injection. The remaining 120 SD rats were randomly divided into sham group, I/R group, I/'R+sh-con group and I/R+sh-RGMa group. After reperfusion for 72 hours, the neurological recovery was evaluated in rats by neurological deficit score, the infarcted volume was measured by 2,3,5-triphenyl tetrazolium chloride (TTC) staining and the permeability of BBB was performed through evans blue by tail vein injection. The expression of RGMa was detected by Western blotting and immunohistochemistry assay. The expression of claudin-5, matrix metalloprotein 9 (MMP-9)and zonula occludin l(ZO-l)were detected by Western blotting. Results Silencing RGMa could improve the permeability of BBB, the infarcted volume, down-regulate the expression of MMP-9, and up-regulate the expression of claudin-5 and Z0-1. Conclusion Silencing RGMa shows protective effects on BBB after I/R injury in rats.
Résumé
Objective: To investigate the expression of repulsive guidance molecule A (RGMa) in polarized microglia after rat focal cerebral ischemia/reperfusion injury. Methods: Adult male Sprague-Dawley (SD) rats were randomly divided into control group, 7 days cerebral ischemia/reperfusion injury group (I/R), and 14 days I/R group. The transient middle cerebral occlusion (tMCAO) model was induced by ligation with nylon monofilament. Real-time PCR was used to test the mRNA expression of Ml and M2 microglia marker interleukin-1Î2 (IL-1Î2), inducible nitric oxide synthase (iNOS), arginase 1 (Arg-1) and CD206 in ipsilateral cortex at day 7 and day 14. Double immunofluorescence staining was performed to investigate the co-expression of RGMa and Ibal in microglia. The microglia was incubated with lipopolysaccharides (LPS)or IL-4 in vitro. The mRNA expression of Ml and M2 microglia marker (IL-1Î2, iNOS, Arg-1, CD206) was tested by Real-time PCR. Western blotting was used to assess the proteins level of RGMa in Ml and M2 microglia. Results: M1 and M2 microglia marker mRNA level were increased in ipsilateral cortex at day 7 and day 14. RGMa was strongly expressed in reactive microglia at day 7 and day 14. LPS or IL-4 treatment polarized microglia to Ml or M2 in vitro. The expression of RGMa protein in Ml and M2 microglia was increased. Conclusion: RGMa was strongly expressed in reactive Ml and M2 microglia after ischemia/reperfusion injury. RGMa may play an important role in the process of microglia activation and polarization. RGMa may be a novel therapeutic target for stroke.
Résumé
Objective To investigate the effect of RNA interference targeting repulsive guidance molecule a (RGMa) on angiogenesis in rat cerebral cortex after focal cerebral ischemia/reperfusion (I/R) injury and its potential mechanism. Methods The experimental animals were randomly divided into sham operated group(S), cerebral I/R group, I/R plus RGMa-specific recombinant adenovirus (I/R+rAd-shRGMa) and I/R plus empty carrier recombinant adenovirus group (I/R+rAd-HK). Adenovirus were injected into the right cortex and hippocampus in rats before undergoing I/R surgery. The transient middle cerebral occlusion (tMCAO) model was induced by ligation with nylon monofilament. Location of RGMa and Neogenin was assessed by Laser confocal microscope 2 days after tMCAO. Seven days after adenovirus injection, the expression of CD31 was detected by immunohistochemistry and the expression of vascular endothelial growth factor a (VEGFa) was detected by Western blotting analysis. Neurological score was evaluated before animals were sacrificed. Results Both RGMa and Neogenin were expressed in CD31+ cells after focal cerebral I/R injury as shown by double staining. Suppression of RGMa via RNA interference increased the number of CD31+ cells and the expression of VEGFa, and improved the neurological deficits. ConclusionRGMa-targeted RNA interference can promote angiogenesis in peri-infarction cortex after cerebral I/R injury, which is partly related to the increase of VEGFa expression in the cerebral tissues.