The Response Of Microglia And Astrocytes After Blood Brain Barrier Opening And The Regulation Mechanism Of Injuried Neuron On Blood Brain Barrier

The blood-brain barrier (BBB) is a barrier between blood and brain perikaria, consisting of microvascular endothelial cells, basement membrane, pericytes, astrocytes and neuronal process. Under normal condition, BBB may filter nutrients including glucose and oxygen into the brain tissue. At the same time, BBB is also a special barrier that shields the brain from toxic substances in the blood, filters harmful compounds from the brain back to the bloodstream supplies, sustains environment steability, and ensures proper function of the central nervous system (CNS). Lots of reports have suggested the BBB permeability damage are closely related to CNS diseases, including stroke, brain tumor, Alzheimer's disease (AD), Parkinson's disease (PD). Under pathological condition, the increased BBB permibility will lead large molecules extruding into brain tissue, which correspondly induce immue responses. In our previous study, we have successfully established a rat model of BBB opening induced by injection of adrenalin (AD). We found that large molecules such as IgG extruded to brain parenchyma after BBB opening. But little is know on the details of immumological reaction that occurs under BBB opening. One the other hand, the damage of brain can affect BBB permeability, and finally affect the brain function. But the mechanisms of these processes still remain unknown. Therefore, there is great significance to study on the immune responses after BBB opening, and the regulation mechanism of brain damage on BBB.The reseach included two parts.ⅠThe immune response of microglia and astrocytes after BBB opening.In the present study, we used the established rat BBB opeing model induced by AD. We observed the immune response pathways of Fcγreceptor I (FcγRI) and Toll-like receptor 4 (TLR4) on microglia and astrocytes by PCR, Western blotting, immunohistochemistry, double immunofluorescence staining, and discussed the possible immune mechanism of microglia and astrocytes following BBB opening.The results showed that the arterial blood pressure was greatly increased and IgG extruded to brain parenchyma after the injection of AD, demonstrating the success of the BBB opening model. PCR, Western blotting and double immunofluorescence staining results showed that both FcγR? mRNA level and its protein expression were significantly increased. Spleen tyrosine kinase (Syk) is the key molecule in the downstream of FcγR? pathway, and cytokines interleukin (IL)-10, IL-4 are important effectors of anti-inflamation. Real time PCR results showed that all of these three FcγR? pathway related molecules increased significantly. For TLR4 pathway, even the molecule TLR4 expression were greatly increased, the expressions of the key molecule MyD88 and the effectors IL-12a, IL-1βof downstream of TLR4 pathway were greatly decreased.These results demonstrated after AD-induced BBB opening, FcγR? mediated anti-inflammatory responses were activated, while TLR4-mediated pro-inflammatory responses were inhibited. These indicated that microglia and astrocytes may serve a house-keeping function to limit excessive inflammatory reactions when large circulating molecules enter brain parenchyma after BBB opening.ⅡThe regulation of oxygen glucose deprivation (OGD)-treated neuron on the permeability of monolayer microvessel endothelial cell BBB model.We employed an in vitro BBB model of the monolayer microvessels endothelial cells co-cultured with astrocytes. OGD was used as a method to induce the damage to the neuron. We first co-cultured OGD-treated neuron with astrocytes. Then the astrocytes were co-cultured with endothelial cells. We tested the permeability of monolayer endothelial cells using fluorescence labeled large molecule, and tight junction proteins by Western blotting. As VEGF is a key molecule to regulate the endothelial cell permeability, we used VEGF siRNA to knockdown VEGF expression to observe its effect on the permeability of endothelial cells and the tight junction proteins induced by OGD-treated neuron.The results showed that the astrocytes co-cultured with OGD-treated neuron increased the permeability of monolayer endothelial cells, but the astrocytes co-cultured with normal neuron did not. Occludin and Claudin-5, two important tight junction proteins, were greatly decreased in OGD-treated neuron group, suggesting the proteins may play important roles in the OGD-treated neuron increased permeability of monolayer endothelial cell. If we removed the astrocytes, the increased OGD-neuron induced permeability of monolayer endothelial cell disappeared. And accordingly, the protein levels of Occludin and Claudin-5 did not change either. This result suggested that astrocytes were involved in the process of increasing permeability of monolayer endothelial induced by OGD-treated neuron. VEGF expression on astrocytes was found to be highly increased by OGD-treated neuron using Western blotting and ELISA. If the VEGF expression was knockdowned by VEGF siRNA in astrocytes before they were co-cultured with OGD-treated neuron, the increased permeability of monolayer endothelial cells induced by OGD-treated neuron was greatly inhibited, as well as the decreased expression of tight junction proteins, Occludin and Claudin-5.These results demonstrated that OGD-damaged neuron could increase the permeability of monolayer endothelial cells, which process was mediated by astrocytes. The OGD-treated neuron stimulated the astrocytes to secrete VEGF to down regulate Occludin and Claudin-5 expression, finally leading to promote the permeability of monolayer endothelial cells.