Cucullo Luca, Couraud Pierre-Olivier, Weksler Babette, Romero Ignacio-Andres, Hossain Mohammed, Rapp Edward, Janigro Damir
Division of Cerebrovascular Research, Cleveland Clinic Lerner College of Medicine, Cleveland, Ohio 44195, USA.
J Cereb Blood Flow Metab. 2008 Feb;28(2):312-28. doi: 10.1038/sj.jcbfm.9600525. Epub 2007 Jul 4.
In evaluating drugs that enter or are excluded from the brain, novel pharmaceutical strategies are needed. For this reason, we have developed a humanized Dynamic In vitro Blood-Brain Barrier model (hDIV-BBB) based on a novel human brain vascular endothelial cell line (HCMEC/D3), which closely mimics the BBB in vivo. In this system, HCMEC/D3 was grown in the lumen of hollow microporous fibers and exposed to a physiological pulsatile flow. Comparison with well-established humanized DIV-BBB models (based on human brain and non-brain vascular endothelial cells co-cultured with abluminal astrocytes) demonstrated that HCMEC/D3 cells cultured under flow conditions maintain in vitro physiological permeability barrier properties of the BBB in situ even in the absence of abluminal astrocytes. Measurements of glucose metabolism demonstrated that HCMEC/D3 cells retain an aerobic metabolic pathway. Permeability to sucrose and two relevant central nervous system drugs showed that the HCMEC/D3 cells grown under dynamic conditions closely mimic the physiological permeability properties of the BBB in situ (slope=0.93). Osmotic disruption of the BBB was also successfully achieved. Peak BBB opening in the DIV-BBB lasted from 20 to 30 mins and was completely reversible. Furthermore, the sequence of flow cessation/reperfusion in the presence of leukocytes led to BBB failure as demonstrated by a biphasic decrease in transendothelial electrical resistance. Additionally, BBB failure was paralleled by the intraluminal release of proinflammatory factors (interleukin-6 and interleukin-1beta) and matrix metalloproteinase-9 (MMP-9). Pretreatment with ibuprofen (0.125 mmol/L) prevented BBB failure by decreasing the inflammatory response after flow cessation/reperfusion.
在评估进入或被排除在脑外的药物时，需要新的制药策略。因此，我们基于一种新型的人脑血管内皮细胞系（HCMEC/D3）开发了一种人源化动态体外血脑屏障模型（hDIV-BBB），该模型能紧密模拟体内的血脑屏障。在这个系统中，HCMEC/D3细胞生长在中空微孔纤维的管腔内，并暴露于生理性搏动流中。与成熟的人源化DIV-BBB模型（基于与人脑和非脑血管内皮细胞与腔外星形胶质细胞共培养）比较表明，即使在没有腔外星形胶质细胞的情况下，在流动条件下培养的HCMEC/D3细胞仍能在体外维持血脑屏障原位的生理通透性屏障特性。葡萄糖代谢测量表明，HCMEC/D3细胞保留有氧代谢途径。对蔗糖和两种相关中枢神经系统药物的通透性表明，在动态条件下生长的HCMEC/D3细胞紧密模拟血脑屏障原位的生理通透性特性（斜率=0.93）。血脑屏障的渗透性破坏也成功实现。DIV-BBB中血脑屏障开放的峰值持续20至30分钟，且完全可逆。此外，在存在白细胞的情况下停止流动/再灌注的顺序导致血脑屏障功能障碍，这通过跨内皮电阻的双相下降得以证明。此外，血脑屏障功能障碍与腔内促炎因子（白细胞介素-6和白细胞介素-1β）和基质金属蛋白酶-9（MMP-9）的释放平行。用布洛芬（0.125 mmol/L）预处理通过减少停止流动/再灌注后的炎症反应来防止血脑屏障功能障碍。
