Establishment of an in vitro blood-cerebrospinal fluid barrier model to study the mechanism of lead-induced brain barrier damage.

摘要

The brain barrier separating the circulation between the blood and cerebrospinal fluid (CSF) is named the blood-cerebrospinal fluid barrier (BCB) and is primarily located in the choroid plexus. The choroid plexus plays essential roles in the early stages of brain development, maturation and neuroendocrine regulation by actively producing and secreting the CSF and a variety of useful materials such as transthyretin, vasopressin, and transferrin into the brain. Unlike the blood-brain barrier (BBB), which consists mainly of cerebral endothelial cells, the BCB is composed of an epithelial cell monolayer. Nonetheless, both barriers share the same structural basis for their barrier function, i.e., tight junctions. Typical tight junctional proteins include ZO-1, occludin, and claudin-1. Establishment of an in vitro BCB model based on an immortal cell line is necessary due to the limitations of in vivo studies and in vitro primary cell-based studies, such as high animal and labor costs, time-consuming experimentation, and the relative short lifespan of primary cultures. Recently, this laboratory developed the immortalized rat choroidal epithelial Z310 cell line (Zheng and Zhao, 2002a). We hypothesized that Z310 cells could be developed, through chemical modulations, into a useful in vitro BCB model for toxicological investigations, for instance, Pb-induced leakage of brain barriers. In summary, this project (i) characterized the presence and location of typical tight junctional proteins in 2310 cells (Chapter 2), (ii) identified that dexamethasone was an effective factor in improving the tightness of the 2310 cell-based in vitro BCB model (Chapter 3), (iii) validated that the 2310 cell-based in vitro BCB model was physiologically comparable to the benchmark of an in vitro primary cell-based BCB model and that the presence of dexamethasone in culture media increased the tightness by significantly up-regulating the expression of claudin-1 (mRNA and protein level) and down-regulating the expression of claudin-2 (mRNA level) (Chapter 4), and (iv) found that early exposure to Pb prior to the formation of tight junctions decreased the tightness of BCB by selective inhibition of claudin-1 expression (Chapter 5). The results conclude that the in vitro BCB model established in this study is suitable for practical applications in toxicological and pharmacological studies (Chapter 6).