Characterisation of an in vitro Blood-Brain Barrier Model and Analysis of the Effects of Inflammatory Modulators on the model.

Abstract

The blood-brain barrier (BBB) acts as an interface between the periphery of the body and the central nervous system (CNS). The function of the BBB as a physical, molecular and chemical barrier is critical for CNS homeostasis and functioning. Recent studies have reported its disruption and dysfunction, in a number of neurological disorders during neuroinflammation. Neuroinflammation is a chronic inflammation response that includes production of pro-inflammatory markers, including IL-1β and TNF-α, and invasion of activated immune cells into the CNS. This is reported to be a part of the pathology of neurological conditions, including traumatic brain injuries, Parkinson’s disease and Alzheimer’s disease. Understanding the role of the BBB in such conditions, has begun to receive considerable attention within the research field. However, the availability of reliable human in vitro BBB models that are suitable for such studies is limited, and therefore hindering the progression of such research. The present study aims to characterise the use of a newly commercially available immortalised human cerebral microvasculature endothelial cells (hCMVECs), as an in vitro BBB model and its use in the analysis of neuroinflammation. The main objectives of this study include; structural and molecular characterisation of the hCMVEC in vitro BBB model; assessment of the effects of known pro-inflammatory modulators IL-1β and TNF-α on the BBB model; and assessment of novel neuroinflammatory cytokines S1P, IL-17A and IL-25 on the in vitro BBB model. hCMVECs exhibited expression of endothelial cell type-specific junction proteins VEcadherin and ZO1, as well as endothelial markers PECAM1 (CD31) and JAM1 (CD321). hCMVECs exhibited good TEER values and high adhesion analysed using the xCelligence biosensor, which supports use of hCMVECs as an in vitro BBB model. Upon treatment of IL- 1β, TNF-α and S1P, hCMVECs exhibited increased expression of ICAM-1 andVCAM-1, required for immune cell attachment to the BBB. hCMVECs exhibited increased secretion of pro-inflammatory markers including; IL-6, IL-8 and MCP-1, consistent with a proinflammatory response. Comparison of xCelligence, CBA and flow cytometry data, revealed the differential temporal profile of the effects of IL-1β, TNF-α and S1P. Despite the ability of all three to evoke a pro-inflammatory response, IL-17A and IL-25 appeared to have no effect on hCMVECs.