Multicellular 3D Neurovascular Unit Model for Assessing Hypoxia and Neuroinflammation Induced Blood-Brain Barrier Dysfunction

Goodwell Nzou,Vishruti S Makani,Nicole R Vanostrand,Anthony J Atala,Elizabeth E Wicks,M C Seeds,Carl M Nechtman,John D Jackson,Robert T Wicks,Sean V Murphy,Stephanie A Seale,Aya El-Taibany,Eric J Marrotte,Gehad A Mekky

doi:10.1038/s41598-020-66487-8

Abstract

The blood-brain barrier (BBB) is a dynamic component of the brain-vascular interface that maintains brain homeostasis and regulates solute permeability into brain tissue. The expression of tight junction proteins between adjacent endothelial cells and the presence of efflux proteins prevents entry of foreign substances into the brain parenchyma. BBB dysfunction, however, is evident in many neurological disorders including ischemic stroke, trauma, and chronic neurodegenerative diseases. Currently, major contributors to BBB dysfunction are not well understood. Here, we employed a multicellular 3D neurovascular unit organoid containing human brain microvascular endothelial cells, pericytes, astrocytes, microglia, oligodendrocytes and neurons to model the effects of hypoxia and neuroinflammation on BBB function. Organoids were cultured in hypoxic chamber with 0.1% O2 for 24 hours. Organoids cultured under this hypoxic condition showed increased permeability, pro-inflammatory cytokine production, and increased oxidative stress. The anti-inflammatory agents, secoisolariciresinol diglucoside and 2-arachidonoyl glycerol, demonstrated protection by reducing inflammatory cytokine levels in the organoids under hypoxic conditions. Through the assessment of a free radical scavenger and an anti-inflammatory endocannabinoid, we hereby report the utility of the model in drug development for drug candidates that may reduce the effects of ROS and inflammation under disease conditions. This 3D organoid model recapitulates characteristics of BBB dysfunction under hypoxic physiological conditions and when exposed to exogenous neuroinflammatory mediators and hence may have potential in disease modeling and therapeutic development.

Highlights

Major hurdles to the development of novel molecular therapies for ischemic stroke include, but are not limited to; the lack of an agreed upon ischemic stroke in vitro model for pre-clinical drug screening, and the fundamental differences in blood-brain barrier (BBB) organization and architecture between humans and the most common animal models
We have shown that these endothelial cells form a functional BBB12
The cell count varied within groups (Fig. 1C), organoids from the same batch under normoxic and hypoxic conditions were qualitatively similar in size (Fig. 1B,E) and cell lysates had approximately equal total protein levels from both groups (Fig. 1D)

Summary

Introduction

Major hurdles to the development of novel molecular therapies for ischemic stroke include, but are not limited to; the lack of an agreed upon ischemic stroke in vitro model for pre-clinical drug screening, and the fundamental differences in BBB organization and architecture between humans and the most common animal models. We evaluated and observed disrupted tight junction markers in organoids cultured under hypoxic condition[12] We utilized this human cell-based 3D in vitro model to measure the effects of hypoxia on BBB structure and function. Through the assessment of a free radical scavenger and an anti-inflammatory endocannabinoid, we hereby report the utility of the model in drug development for drug candidates that may reduce the effects of ROS and inflammation under disease conditions This human cortex organoid placed within a hypoxic environment mimics normal physiologic response and forms the basis for a promising disease model that could potentially be implemented as an initial in vitro drug screening tool in the evaluation of novel therapeutics

Methods

Results

Conclusion