Communication from the periphery to the hypothalamus through the blood-brain barrier: An in vitro platform

Abstract

One of the major routes of communication from the peripheral systems to the hypothalamus, the core structure of body homeostasis, is the humoral transmission through the blood-brain barrier (BBB). The BBB cultures are the in vitro model of choice to depict the mechanisms behind blood-brain interplay. Still, this strategy excludes the integration of the brain tissue response and, therefore, the resulting output might be limited. In this study, two in vitro assays were established: BBB coculture model and hypothalamic organotypic cultures. The combination of these two assays was used as a platform to address the two critical steps in the humoral transmission through the BBB to the brain: blood-BBB/BBB-brain. The in vitro model of the BBB was performed according to a coculture system using a brain microvascular endothelial cell line (bEnd.3) and primary astrocytes. The expression of junctional molecules as claudin-5, ZO-1, occludin and VE-cadherin was observed in the bEnd.3 cell-cell contact, confirming the BBB phenotype of these endothelial cells. Moreover, the transendothelial electrical resistance (TEER) values (71.1±9.4Ω× cm(2)) and the permeability coefficients (Pe) obtained in the transendothelial flux test (3.3±0.11×10(-6)cm/sec) support high integrity of the established barrier. The hypothalamic organotypic cultures were prepared from 8-days-old C57Bl/6 mice brains, based on the air-medium interface culture method. High cell viability (82±9.6%) and a dense neuronal network were achieved. The stimulation with dexamethasone resulted in an increased neuropeptide (NPY) expression, confirming the responsiveness of the neuronal system of these organotypic cultures. After optimization and characterization of each assay, the functionality of the platform was validated through the evaluation of the hypothalamic response to deep wound encompassing skin and muscle in mice. Results allowed to identify increased NPY activity in hypothalamic slices in response to peripheral signals within the plasma from wounded animals when compared with non-injured animals after surpassing and/or interacting with the BBB. This differential NPY response between the different animal conditions validated the functionality of the in vitro platform. In conclusion, this approach can be greatly anticipated as a useful tool for studying biologic or pharmacological circulating molecules and their impact on the hypothalamic activity.

Keywords: Blood–brain barrier; Hypothalamus; In vitro tools; Organotypic cultures; Periphery-to-brain communication.