Extracellular environment contribution to astrogliosis—lessons learned from a tissue engineered 3D model of the glial scar

Abstract

Glial scars are widely seen as a (bio)mechanical barrier to central nervous system regeneration. Due to the lack of a screening platform, which could allow in-vitro testing of several variables simultaneously, up to now no comprehensive study has addressed and clarified how different lesion microenvironment properties affect astrogliosis. Using astrocytes cultured in alginate gels and meningeal fibroblast conditioned medium, we have built a simple and reproducible 3D culture system of astrogliosis mimicking many features of the glial scar. Cells in this 3D culture model behave similarly to scar astrocytes, showing changes in gene expression (e.g., GFAP) and increased extra-cellular matrix production (chondroitin 4 sulfate and collagen), inhibiting neuronal outgrowth. This behavior being influenced by the hydrogel network properties. Astrocytic reactivity was found to be dependent on RhoA activity, and targeting RhoA using shRNA-mediated lentivirus reduced astrocytic reactivity. Further, we have shown that chemical inhibition of RhoA with ibuprofen or indirectly targeting RhoA by the induction of extracellular matrix composition modification with chondroitinase ABC, can diminish astrogliosis. Besides presenting the extracellular matrix as a key modulator of astrogliosis, this simple, controlled and reproducible 3D culture system constitutes a good scar-like system and offers great potential in future neurodegenerative mechanism studies, as well as in drug screenings envisaging the development of new therapeutic approaches to minimize the effects of the glial scar in the context of central nervous system disease.

Piezo1 regulates calcium oscillations and cytokine release from astrocytes

“…Differences were analyzed using a two-way ANOVA with the Holm-Sidak posthoc test and *p < .05 [Color figure can be viewed at wileyonlinelibrary.com] and A2 phenotypes recently described (Liddelow et al, 2017). Astrocytes sense and react to the local biochemical milieu of the brain parenchyma, as well as to the mechanical properties of the extracellular matrix and neighboring cells, particularly after CNS injury (Bowers, Fiori, Khadela, Janmey, & Galie, 2019;Rocha, Ferraz-Nogueira, Barrias, Relvas, & Pego, 2015). Here, primary mouse astrocyte cultures were used to investigate several potential functions of astroglial Piezo1 channels in the cerebral cortex during a neuroinflammatory stimulus.…”

High-throughput platforms for the screening of new therapeutic targets for neurodegenerative diseases

“…We have developed a 3D in vitro tissue engineered glial scar [101] by culturing primary astrocytes within alginate hydrogels, in the presence of meningeal-fibroblast-conditioned medium. Hydrogel mechanical properties were shown to influence cell behaviour significantly with intermediate stiffness gels promoting astrogliosis.…”

“…Ultimately, this will translate into new drug targets, biomarkers, biomolecule-based therapies and novel and moreefficient disease treatment or management approaches. [101] Abbreviations: BBB, blood-brain barrier; CNS, central nervous system; LPS, lipopolysaccharide; NSCs, neural stem cells.…”

Biomaterial Approaches to Modulate Reactive Astroglial Response

“…Since astrocytes are not as reactive in 3D and appear to more accurately mimic a healthy tissue phenotype, others have studied how astrocytes impact neurite outgrowth. Cortical rat astrocytes and cortical neurons cultured in 3D alginate hydrogels with fibroblast conditioned medium induced a reactive astrocyte phenotype, altering their gene and protein expression towards a more reactive state while inhibiting neurite outgrowth [Rocha et al, 2015].…”