Tumor necrosis factor-alpha increases in vitro blood brain barrier permeability / 中国组织工程研究

ABSTRACT

BACKGROUND:The levels of interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) are increased during infectious brain edema, and are positively relevant to the degree of brain damage. However, whether TNF-α can enhance blood brain barrier (BBB) permeability remains unclear, especially in vitro.OBJECTIVE: To understand the changes and possible mechanism of the BBB permeability induced by TNF-α in vitro.DESIGN: Randomized controlled cell model study in vitro.SETTING:Department of Pediatrics, Xiangya Hospital, Central South University; Department of Biochemistry, Xiangya Medical College, Central South University.MATERIALS: Twenty 7-day-old healthy Sprague-Dawley rats, of clean grade and either gender, were provided by the Animal Center, Xiangya Hospital, Central South University. TNF-α was purchased from sigma Company; DMEM fluid medium and fetal bovine serum were purchased from Hyclone Company; Y-27632 was purchased from Alexis Company,and rabbit anti-human factor Ⅷ -related antigen was purchased from Zymed Company; Mouse anti-rat glial fibrillary acidic protein (GFAP) was purchased from Neomarkers. Other biochemical reagents were imported (Sigma Company).METHODS: This experiment was carried out in the Xiangya Hospital, Central South University between March 2004 and April 2005. Brain microvascular endothelial cells and astrocytes were co-cultured 10 days to set up rat models of BBB in vitro. Then, the cells were divided into 4 groups: model group(BBB models were prepared), TNF-α group ( BBB model incubated with 0.01 g/L TNF-α for 5 hours), Y-27632 pretreated group ( BBB model incubated with 30 μmol/L Y-27632 for 1 hour before 0.01g/L TNF-α challenge ) and Y-27632 control group (BBB models only incubated with Y-27632 as those in the Y-27632 pretreated group). The effect of TNF-α on BBB permeability was observed by detecting the 125 I -BSA, which passed through the inserts at each time point (30,60,120 and 240 minutes) using .γradioimmunoassay counter.MAIN OUTCOME MEASURES: The 125 I -BSA, which passed through the inserts in rat models of BBB at different time points after intervention.RESULTS: The 125 I -BSA, which passed through the inserts in rat models of BBB, was all significantly higher in the TNF-α group than in the other groups at 30, 60, 120 and 240 minutes after intervention, respectively (P ＜ 0.01), and reached the peak at 240 minutes; The 125 I -BSA, which passed through the inserts, was lower in the Y-27632 pre-treated group than in the TNF-α group at 30 and 60 minutes after intervention (P＜ 0.01). There was also significant difference in 125 I -BSA permeation between Y-27632 pretreated group and Y-27632 control group after 120 minutes (P ＜ 0.05).CONCLUSION: TNF-α can increase BBB permeability, and Y-27632 pretreatment can early reverse the effect of TNF-α on BBB permeability.