| Background: Stroke is a health destroyer of human beings for itshigh morbidity, mortality and disability rates. The population is aging inChina, the patients of stroke are increasing, which influences life quality ofelder population. Animal research found that blood brain barrier leakageafter cerebral ischemia is the leading cause of brain edema andhemorrhagic transformation, so attenuating blood brain barrier leakageafter I/R can alleviate brain injury, delay ischemia process and improve theprognosis. Most current studies concerned with blood brain barrier after I/Rlargely focused on the role of endothelial cells. However, the importance ofsupporting cells was ignored. In fact, endothelial cells with extra cellmatrix, pericytes, astrocytes and neurons formed Neurovascular Unite,which plays significant role in the management of neurovascular function.ECM occupy an pivotal place in NVU, because it not only supportsendothelial cells, pericytes and astrocytes anatomically, but also mediatessignaling transduction (e.g. Integrin signaling). Fibulin-5is a new memberof Fibulin family, Fibulin-5can enhance endothelial cells attachment to ECM, which contribute to maintain the integrity of blood brain barrier.Furthermore, endothelial cell-ECM interactions via integrin directly affectinterendothelial cell tight junction expression and brain microvascularpermeability. Fibulin-5binding to α5β1integrin leads to inhibition offibronectin-mediated downstream signaling and then suppress theproduction of ROS. Meanwhile, interaction between Fibulin-5andextracellular superoxide dismutase SOD is required for ecSOD binding tovascular tissues, thereby Fibulin-5could regulate vascular O–2levels.Endothelial cells stimulated by ROS would contract, leading to increasedgaps between cells and leakage of BBB finally. ROS can also augment theactivation of tyrosine kinase and matrix metalloproteinase after I/R,which can degradate ECM and then increase BBB damage. Fibulin-5wasalso shown to inhibit angiogenesis by antagonise the expression of VEGF, awell-known vascular permeability factor that increases microvascularpermeability. Based on the BBB protection mechanisms of Fibulin-5what we have mentioned above and the fact that the expression of Fibulin-5is up-regulated after cerebral ischemia, we hypothesized that Fibulin-5is aself-repair factor after I/R. Methods to augment functions of Fibulin-5afterI/R might be an new way to attenuate BBB compromise.Objective: In this study, adenoviruses carrying the Fibulin-5gene orcontrol gene were injected into ipsilateral cortex and hippocampus of ratsto investigate the effect of over-expression Fibulin-5mediated by adenoviruses on blood brain barrier leakage after cerebralischemia/reperfusion injury in rats and its potential mechanisms.Methods:1.32adult male Sprague-Dawly rats were randomly divided intoshame operation group, low adenovirus titer group, medium adenovirustiter group and high adenovirus titer group. The same amount of saline ordifferent titer of adenovirus were injected stereotactically into the rightcortex and hippocampus.7days after injection, rats were decapitated andfrozen sections around the injection site were applied to detect greenfluorescent protein with laser confocal microscopy (n=4). Inflammation,provocated by brain adenovirus injection, was presented by detection ofinterleukin-10using ELISA kit(n=4).2.112Adult male Sprague-Dawly rats were randomly divided intosham operation group, cerebral ischemia/reperfusion group, I/R plusAD-Fibulin-5group, I/R plus AD-HK group. Adenovirus carrying theFibulin-5gene or control gene were injected into right cortex andhippocampus in rats. After7-day gene transfer, rats underwent90min ofischemia and then were reperfused for24h. The expression of Fibulin-5,occludin, MMP-9and VEGF were detected by western-blot andrt-PCR(n=8for WB, n=4for rt-PCR). Dual immunofluorescent withRECA1(endothelial cells marker) and occludin revealed expression andlocation of occludin(n=4), Evan-blue extravasation(n=4) and brain water content(n=4) were applied to evaluate BBB leakage. TTC staining(n=4)and behavior assessment(n=4) were investigate to evaluate brain infarctvolume and neurological deficit recovery.Results:1.Laser confocal microscopy showed that there were no emission ofgreen fluorescent on frozen sections of shame operate group rats, whilelittle, medium and large amount of green fluorescent were detected onfrozen sections of low adenovirus titer group, medium adenovirus titergroup and high adenovirus titer group respectively.2.Fibulin-5protein and mRNA were significantly increased in ratstransfected with AD-Fibulin-5compared to other groups (P<0.01) bywestern-blot and rt-PCR found that. The amount of Evans blue dyeextravasation in the right cerebral hemisphere was significantly increased at24h after reperfusion compared with the other groups, whileover-expression of Fibulin-5can decrease Evan-blue extravasation andbrain water content(P<0.01). Meanwhile, Evan-blue extravasation andbrain water content were no difference between I/R group and I/R+AD-HKgroup(P>0.05). Over-expression of Fibulin-5decreased TTC staininginfarct volume of rats and improve neurological deficit24h afterreperfusion(p<0.01) compared to I/R and I/R+AD-HK group.3.Increased occludin expression and down regulation of MMP-9and VEGF were detected in I/R+AD-FBLN5goup rats(P<0.01) by western-blot and rt-PCR. Confocal microscopy found that occludinlocalized at the peripheral of endothelial cells under normalconditions,while delocalization and degradation of occludin was detectedat24h after reperfusion. The disruption of occludin was markedlyalleviated in adenovirus injection group.Conclusions:1.All three titers of adenovirus were able to transfect the right cortexand hippocampus in rats successfully.2.Adenovirus obviously up-regulated Fibulin-5gene transcription inischemia cortex and ipsilateral hippocampus. Over-expresstion of Fibulin-5attenuated BBB leakage after cerebral ischemia.3.Fibulin-5increased the expression of occludin, suppressed MMP-9activation and VEGF releasing of brain tissue in rats. These may be themechanisms of Fibulin-5protection on BBB. |
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