| [Objective] The incidence of stroke is increasing year by year, There is a growing concern about the high morbidity of stroke. The destruction of BBB plays an important role in the progression of stroke. BBB is a dynamic interface which is between the blood and brain. it can prevent macromolecules through it and maintain the central nervous system homeostasis. In clinical practice, we found that stroke patients often accompanied by fever. Fever will add to the destruction of the BBB, further aggravate brain damage, affect the prognosis. However, the exact mechanism fever aggravated BBB damage has not been fully elucidated. Studies have found that matrix metal loproteinases, in particular MMP-9, may lead to BBB damage by disrupting the tight junction which is between cells. Our preliminary animal experiments also found that fever increased MMP-9activity, degraded claudin-1protein, resulting in the increasing of BBB permeability. However, at normal temperature, MMP-9whether have the same effect to destruct the claudin-1protein, and whether fever increased MMP-9to destruct the claudin-1protein? It is still not entirely clear. So in the present study, we used primary cultured SD rat brain microvascular endothelial cells and astrocytes to co-culture by Transwell chamber, Construction of in vitro BBB model. Treated the model with adding MMP-9and heating, then observed the changes of the tight junction protein claudin-1, to investigate the impact of MMP-9and fever to claudin-1protein.[Methods]First we use enzymatic digestion technology culture cells, then identified the cultured cells was the SD rat brain microvascular endothelial cells and astrocytes which were we needed. We used the two cells to co-culture by Transwell chamber, building in vitro BBB model. By leakage experiments and Lucifer yellow permeability measurement to make sure we have succeeded. The models were randomly divided into37℃,39℃fever,37℃+MMP-9,39℃fever+MMP-9four groups, the LY permeability was measured in30min,60min,90min, Each group in vitro BBB model’s permeability change was observed. Finally, Using immunohistochemistry and western blot technology observed the change of tight junction protein claudin-1.[Results]1.The cultured cells were brain microvascular endothelial cells and astrocytes of SD rat by HE staining, immunofluorescenceas technology identified. The co-culture BBB model’s leakage test was positive. Models with a low permeability coefficient (Permeability coefficient, Pe,1.08±0.16×10-3cm/min).2.Each group internal compared at different time points, model’s permeability experiments show,37℃and37℃+MMP-9group, in vitro BBB model’s permeability change has no significant difference (P>0.05).39℃fever group and39"Cfever+MMP-9group, in vitro BBB model’s permeability change gradually increased with time, the difference was statistically significant (P>0.05).3.Comparison between two groups at the same time points, model’s permeability experiments show,37℃+MMP-9group compared with37℃group, in vitro BBB model’s permeability change has no significant difference (P>0.05).But39℃fever group compared with37℃group,39℃fever+MMP-9group compared with39℃fever group,39℃fever+MMP-9group compared with37℃+MMP-9group, in vitro BBB model’s permeability change were significantly increased (P<0.05).4.Each group internal compared at different time points, immunohistochemistry technology show, in the four group the number of claudin-1positive cells have no significant difference (P>0.05). Western blot technology show,37℃and37℃+MMP-9group the number of claudin-1positive cells have no significant difference (P>0.05), but39℃fever and39℃fever+MMP-9group, the number of claudin-1positive cells gradually decreased with time, the difference was statistically significant (P>0.05).5.Comparison between two groups at the same time points, immunohistochemistry technology and western blot technology both show,37℃+MMP-9group compared with37℃group, the expression of claudin-1protein have no significant difference (P>0.05), but39℃fever group compared with37℃group,39℃fever+MMP-9group compared with37℃+MMP-9group, the expression of claudin-1protein were significantly decreased (P<0.05).6.Comparison between two groups at the same time points, immunohistochemistry technology show,39℃fever+MMP-9group compared with39℃fever group, only at90min, the expression of claudin-1protein were significantly decreased (P<0.05).7.Comparison between two groups at the same time points, western blot technology show,39℃fever+MMP-9group compared with39℃fever group, the expression of claudin-1protein were significantly decreased (P<0.05).[Conclusions]1. We primary cultured brain microvascular endothelial cells and astrocytes of SD rat and successfully established in vitro BBB model.2. Under the experimental conditions, adding MMP-9alone can not change the permeability of in vitro BBB model, also can not destroy the tight junction protein claudin-1.3.39℃fever can significantly increase the permeability of the in vitro BBB models, adding MMP-9can further increase the damage of fever to the integrity of the in vitro BBB model.4.39℃fever will cause in vitro BBB model tight junction protein claudin-1expression decreased; adding MMP-9can further decrease the expression of tight junction protein claudin-1. |
PDF Full Text Request