Gut microbiota is able to communicate with the brain through complex bidirectional routes constituting the so-called microbiota-gut-brain axis (MGBA). Gut microbial dysbiosis increases local inflammation also thanks to the secretion of bacterial lipopolysaccharides (LPS), which disturbs the gastrointestinal and also the blood-brain barrier (BBB) permeability thus propagating inflammation to the brain 1. A fluid-clearance pathway in the brain, the glymphatic system (GS), was recently discovered with increasing evidence of its role in neurodegeneration by controlling the clearance of neurotoxic proteins through convective exchange of flows 2. An in vitro model of GS in the scenario of neuroinflammation is missing. Here, we developed a GS model based on tunable levels of brain fluids clearance in a 3D brainlike environment, to be integrated in a MGBA engineered multiorgan-on-a-chip platform developed within the ERC project “MINERVA”, which aims at studying the intestinal microflora impact on brain functionality 3. The presence of a GS model in the brain unit of the multiorgan platform, combined with gut epithelial and BBB on-a-chip systems will allow to recapitulate LPS-mediated neuroinflammation in the presence of pathophysiological brain fluids drainage representing an innovative tool to study MGBA and GS role in dementia.
The hydrogel based 3D brain model was engineered starting from a previously validated formulation 4.
Interstitial flow levels and molecules transport within the 3D brain model were predicted by multiphysics computational analysis and subsequently validated by measuring dextran molecules transport at different molecular weights (4, 40 and 70 kDa). The biological validation was performed by measuring cell viability for the cell models used to model the gut and BBB barriers as well as brain inflammation: intestinal epithelial cells (CaCo2); endothelial cells (bEND.3); astrocytes (C8D1A); neuroglioma cells (H4). Transepithelial Electrical Reistance (TEER) was measured to evaluate gut epithelial and BBB barriers integrity. Fluorescent LPS was tracked along the platform by fluorescence intensity detection. Interleukin 6 (IL-6) production by H4 cells at different fluid clearance levels was measured by ELISA.
Computational analysis predicted flow velocities profile inside the 3D matrix and a physiological range of 1-10 uL/min was selected with clearance velocities corresponding to 0.14-1 um/s. Solutes clearance was validated with higher molecular wheight molecules being entrapped inside the hydrogel at low level of flows. The platform sustained the long-term culture of all cells models.
After LPS stimulation of the barrier models, TEER values decreased and IL-6 detectable levels inside the 3D brain model were measured. Preliminary results showed IL-6 production being modulated at different clearance flows levels suggesting an active role of fluid drainage in controlling neuroinflammatory response.
The integration of GS in a MGBA engineered multiorgan platform represents a suitable tool for modelling in vitro both physiological and pathological drainage of fluids inside the brain by recreating tuned GS interstitial flows within MGBA inflammation models.
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