| Objective: Vascular endothelial cells and astrocytes are important components of the BBB.In the pathogenic cause of the disease of the pathogenic bacteria,crossing BBB is the key link,but the mechanism is unknown.It is indicated that cPLA2 may be related to the invasion of pathogenic microorganisms to the CNS,but the role of cPLA2 in the invasion of brain tissue through the invasion of BBB is not clear.This study aims to explore astrocytes in brain tissue distribution and degree of brain endothelial cells closely linked,then study the expression of cPLA2 in brain tissue especially in the two types of cells,and through innovative approaches to solve positioning H99 invades brain tissue location problem,the establishment of experimental murine cryptococcal meningitis model,use of specific drugs that block,finally confirmed the important role of cPLA2 in H99 invades brain tissue.Methods: Immunohistochemistry and immunofluorescence method first detect the distribution of astrocytes(marker GFAP)in cerebral cortex and hippocampus around vascular endothelial cell(marker PECAM-1),then detect the expression of cPLA2 in cerebral cortex and hippocampus of mice peripheral vascular endothelial cells and astrocytes in the experiment of cryptococcal meningitis;model of mouse invasion of H99,using H99-GFP with high expression of GFP features,combined with frozen sections and immunofluorescence,location H99 in invasion of brain tissue and the expression of cPLA2 in vascular endothelial cells;by tail vein injection of cPLA2 inhibitor(AACOCF3)were observed that can inhibite H99 invasive brain tissue;in isolated mouse brain capillaries,and the separation culture and identification of astrocytes,by immunofluorescence and Western blot method,detection the expression of vascular endothelial cells or astrocytes in cPLA2.Results: In the cortex and hippocampus of perivascular astrocytes located rich astrocytes and has following relationship: either cortex or hippocampus,diameter of different vascular(4-8μm,8-12μm and >12μm),with increase of vessel diameter and its distribution of astrocytes around were significant increased(t test,n = 8,P < 0.001);cPLA2 is expressed in cortex and hippocampus of vascular endothelial cells and perivascular astrocytes;In 7 days after H99-GFP invasion,H99-GFP invaded the l brain tissue with the following characteristics: in cortex(about Bregma0.74mm)M1-M2 and cortex(about Bregma-2.18mm)RSA-V2 L,hippocampus(about Bregma-2.18mm)in the CA1,SLu,DLG-LPLR-LPMR,LMol-Mol and striatum CPU position were found Cryptococcus H99-GFP and around the organization appear different degree of damage;In the same local area of two vessels,a high expression of c PLA2 by H99-GFP in invasion of vascular endothelial cells,but other vessels not invaded by H99-GFP was not detected,indicating the expression of cPLA2 in endothelial cells may plays an important role in H99 invade the brain tissue;then through the experiment of tail intravenous injection of cPLA2 inhibitor AACOCF3,H99 invades brain tissue was significantly inhibited(t test,n=6,P<0.05),indicating that cPLA2 plays an important role in H99 invasion of brain;in isolated mouse brain capillary and cultured astrocyte,observed that cPLA2 and p-cPLA2 was expression,and are consistent with the whole animal,visible,cultured brain capillary and separation has been successfully isolated astrocytes can be used to build the next related cell model in vitro.Conclusions: Cortex and hippocampus of mouse vascular surrounding astrocyte distribution has the following characteristics: with the increase of vessel diameter,the richness of astrocytes end feet around the blood vessels is increased;cPLA2 expression in the cortex,hippocampus vascular endothelial cells and astrocyte and plays an important role in H99 invasion of brain tissue;successfully established the experimental cryptococcal meningitis Cryptococcus and positioning H99 location of brain tissue;successfully separated cultured astrocytes,initially established the in vitro experimental model. |
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