Cell Growth in Response to Mechanical Stiffness is Affected by Neuron- Astroglia Interactions

Abstract

Cell adhesion and morphology are affected by the mechanical properties of the extracellular matrix. Using polyacrylamide gels as cell substrates, the cellular response to substrate compliance was investigated in pure neuronal, pure astroglial, or mixed co-cultures. Substrates used spanned a large range of stiffnesses including that of brain tissue. In both pure and mixed cultures, immature (vimentin+) astroglia adhered best to stiffest gels. Mature (GFAP+) astrocyte adhesion peaked on intermediate stiffness, while pure GFAP+ astroglial adhesion displayed no intermediate preference and increased with stiffness. Neurite length was constant with stiffness; however, primary dendrite number was lowest on intermediate gels. Pure neuronal cultures were more adherent to hard gels, while mixed cultures had no stiffness preference. Furthermore, we investigated the role of stiffness in the modulation of the neurotoxic effect of glutamate. Exposure to two glutamate concentrations (500 and 1000 M) of cultured spinal cord neurons induced cell death. The damage elicited by 500 m glutamate to neurons in a mixed culture of spinal cord cells is most severe on soft 300 Pa gels. The neurotoxic effect of glutamate on neurons cultured on hard gels where astrocytes are present was strongly attenuated compared with that observed on soft gels, where there is a relatively low number of astrocytes. Our data suggest that mechanical stiffness of the substrate affects the response of both neurons and astroglia, and this response is varied by interaction between the two cell types.

Regulation of Dendrite Arborization by Substrate Stiffness is Mediated by Glutamate Receptors

“…Our laboratory has shown that astrocytes influence neuronal growth on compliant substrates. 24 Mixed hippocampal cultures were treated with Ara-C to obtain a pure neuronal population. 24 Neurons were immunostained for MAP-2 for dendrite visualization.…”

“…24 Mixed hippocampal cultures were treated with Ara-C to obtain a pure neuronal population. 24 Neurons were immunostained for MAP-2 for dendrite visualization. 24 We found that as substrate stiffness increased, primary and secondary dendrite number increased for neurons grown in pure neuronal cultures, i.e., neurons grown on gels of 3000 Pa stiffness had more primary and secondary branches than neurons grown on gels of 300 Pa stiffness (Figs.…”

“…24 Since the physical interaction between these two cell types may affect neuronal morphology, we asked whether secreted astroglial factors affect dendrite branching. To determine this, we treated pure neuronal cultures with media conditioned from pure astrocyte cultures.…”

“…17,19,[24][25][26]31,38,42 Some of these studies have determined mechanisms for mechanosensing. For example, the cytoskeleton and cytoskeletal signaling proteins, such as focal adhesion kinases, change behavior on compliant substrates.…”

“…Our group has determined that the interaction between neurons and astrocytes affects neuronal growth on compliant substrates. 24,38 We also determined that glutamate concentration affects neuronal growth on compliant substrates, possibly through glutamate released by astrocytes. 24,38 In this study, we examined how factors secreted by astrocytes affect dendrite outgrowth on compliant substrates.…”