Abstract
BackgroundIt is now recognized that the neuro-vascular unit (NVU) plays a key role in several neurological diseases including epilepsy, stroke, Alzheimer’s disease, multiple sclerosis and the development of gliomas. Most of these disorders are associated with NVU dysfunction, due to overexpression of inflammatory factors such as vascular endothelial growth factor (VEGF). Various in vitro models have been developed previously to study the micro-environment of the blood–brain barrier (BBB). However none of these in vitro models contained a complete complement of NVU cells, nor maintained their interactions, thus minimizing the influence of the surrounding tissue on the BBB development and function. The organotypic hippocampal culture (OHC) is an integrative in vitro model that allows repeated manipulations over time to further understand the development of cell circuits or the mechanisms of brain diseases.Methods/designOHCs were cultured from hippocampi of 6–7 day-old Sprague Dawley rats. After 2 weeks in culture, seizures were induced by application of kainate or bicuculline into culture medium. The regulation of BBB integrity under physiological and pathological conditions was evaluated by immunostaining of the main tight junction (TJ) proteins and of the basal membrane of microvessels. To mimic or prevent BBB disassembly, we used diverse pro- or anti-angiogenic treatments.DiscussionThis study demonstrates that NVU regulation can be investigated using OHCs. We observed in this model system an increase in vascularization and a down-regulation of TJ proteins, similar to the vascular changes described in a chronic focus of epileptic patients, and in rodent models of epilepsy or inflammation. We observed that Zonula occludens-1 (ZO-1) protein disappeared after seizures associated with neuronal damage. In these conditions, the angiopoeitin-1 system was down-regulated, and the application of r-angiopoeitin-1 allowed TJ re-assembly. This article demonstrates that organotypic culture is a useful model to decipher the links between epileptic activity and vascular damage, and also to investigate NVU regulation in diverse neurological disorders.
Highlights
It is recognized that the neuro-vascular unit (NVU) plays a key role in several neurological diseases including epilepsy, stroke, Alzheimer’s disease, multiple sclerosis and the development of gliomas
We observed in this model system an increase in vascularization and a down-regulation of tight junctions (TJs) proteins, similar to the vascular changes described in a chronic focus of epileptic patients, and in rodent models of epilepsy or inflammation
Organotypic cultures: a tool to decipher the mechanisms of blood–brain barrier (BBB) failure in epileptic disease Since empty vessels respond to angiogenic factors, we studied the vascular remodelling after epileptic seizures using Organotypic hippocampal slices (OHCs) [20,25,26]
Summary
Due to the presence of all cell types and their interactions, preservation of TJs between endothelial cells, as well as BBB carriers and transporters, brain slices provide a complete ex vivo model of the NVU, in the absence of blood flow. The presence of transporters such as glucose transporter−1 and P-glycoprotein on brain endothelial cells in organotypic slices has been previously documented. Staining for serum protein leakage or the measurement of TEER in vitro [75] is not possible in this model To counteract this issue, a co-culture model of endothelial cells and brain slices has been developed in which Duport and colleagues showed that the BBB permeability can be evaluated by microdialysis [76]. Using organotypic slice cultures to study NVU embedded in the microenvironment of anatomically organized parenchymal cells and maintaining many important physiological functions, will undoubtedly facilitate future studies on mechanisms and impact of pathological conditions on NVU remodeling as well as its role in disease processes.
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