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.
Highlights
Astrocytes are the most abundant cells in the central nervous system (CNS) (Lu et al, 2006) and are known to play a pivotal role in glial scar formation
Astrocytes cultured in the presence of conditioned medium (CM) showed increased expression of astrogliosis hallmark genes as Gfap and Vimentin (Figure 1B) and proteins as glial fibrillary acidic protein (GFAP) and C4S at 3 days when compared to controls (Figures 1C,D)
Astrocytes cultured with CM exhibited significantly increased levels of excreted active MMP2 upon 3 days of culture compared with controls (Figure 1F)
Summary
Astrocytes are the most abundant cells in the central nervous system (CNS) (Lu et al, 2006) and are known to play a pivotal role in glial scar formation. The regeneration failure in the adult CNS is multi-factorial but the glial scar has been ascribed has a highly inhibitory environment While it has been a widely explored therapeutic target (Jones et al, 2003; Koechling et al, 2011), very little is known about the causes and mechanisms underlying astrocyte activation. Several animal models have been developed to study the processes of CNS degeneration and regeneration These are time consuming, costly, and raise technical and ethical issues when one intends to perform routine assays to elucidate molecular mechanisms or screening for potential therapeutics. Mechanical properties of CNS tissue are known to be altered when a glial scar is formed (Bonneh-Barkay and Wiley, 2009; Freimann et al, 2011; Murphy et al, 2012) and ECM components are thought to play a pivotal role on the mechanotransduction processes in healthy and diseased tissues. We show that the Rho-ROCK signaling pathway can regulate astrogliosis constituting a possible therapeutic target
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