Physical cues of the scaffolds, elasticity, and stiffness significantly guide adhesion, proliferation, and differentiation of stem cells. In addressable microenvironments constructed by three-dimensional graphene foams (3D-GFs), neural stem cells (NSCs) interact with and respond to the structural geometry and mechanical properties of porous scaffolds. Our studies aim to investigate NSC behavior on the various stiffness of 3D-GFs. Two kinds of 3D-GFs scaffolds present soft and stiff properties with elasticity moduli of 30 and 64 kPa, respectively. Stiff scaffold enhanced NSC attachment and proliferation with vinculin and integrin gene expression were up-regulated by 2.3 and 1.5 folds, respectively, compared with the soft one. Meanwhile, up-regulated Ki67 expression and almost no variation of nestin expression in a group of the stiff scaffold were observed, implying that the stiff substrate fosters NSC growth and keeps the cells in an active stem state. Furthermore, NSCs grown on stiff scaffold exhibited enhanced differentiation to astrocytes. Interestingly, differentiated neurons on stiff scaffold are suppressed since growth associated protein-43 expression was significantly improved by 5.5 folds.
BackgroundSpinal cord astrocyte swelling is an important component to spinal cord edema and is associated with poor functional recovery as well as therapeutic resistance after spinal cord injury (SCI). High mobility group box-1 (HMGB1) is a mediator of inflammatory responses in the central nervous system and plays a critical role after SCI. Given this, we sought to identify both the role and underlying mechanisms of HMGB1 in cellular swelling and aquaporin 4 (AQP4) expression in cultured rat spinal cord astrocytes after oxygen-glucose deprivation/reoxygenation (OGD/R).MethodsThe post-natal day 1–2 Sprague-Dawley rat spinal cord astrocytes were cultured in vitro, and the OGD/R model was induced. We first investigated the effects of OGD/R on spinal cord astrocytic swelling and HMGB1 and AQP4 expression, as well as HMGB1 release. We then studied the effects of HMGB1 inhibition on cellular swelling, HMGB1 and AQP4 expression, and HMGB1 release. The roles of both toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) signaling pathway and interleukin-6 (IL-6) in reducing cellular swelling resulting from HMGB1 inhibition in spinal cord astrocytes after OGD/R were studied. Intergroup data were compared using one-way analysis of variance (ANOVA) followed by Dunnett’s test.ResultsThe OGD/R increased spinal cord astrocytic swelling and HMGB1 and AQP4 expression, as well as HMGB1 release. Inhibition of HMGB1 using either HMGB1 shRNA or ethyl pyruvate resulted in reduced cellular volume, mitochondrial and endoplasmic reticulum swelling, and lysosome number and decreased upregulation of both HMGB1 and AQP4 in spinal cord astrocytes, as well as HMGB1 release. The HMGB1 effects on spinal cord astrocytic swelling and AQP4 upregulation after OGD/R were mediated—at least in part—via activation of TLR4, myeloid differentiation primary response gene 88 (MyD88), and NF-κB. These activation effects can be repressed by TLR4 inhibition using CLI-095 or C34, or by NF-κB inhibition using BAY 11-7082. Furthermore, either OGD/R or HMGB1 inhibition resulted in changes in IL-6 release. IL-6 was also shown to mediate AQP4 expression in spinal cord astrocytes.ConclusionsHMGB1 upregulates AQP4 expression and promotes cell swelling in cultured spinal cord astrocytes after OGD/R, which is mediated through HMGB1/TLR4/MyD88/NF-κB signaling and in an IL-6-dependent manner.
High mobility group box-1 (HMGB1) could function as an early trigger for pro-inflammatory activation after spinal cord injury (SCI). Spinal cord edema contributes to inflammatory response mechanisms and a poor clinical prognosis after SCI, for which efficient therapies targeting the specific molecules involved remain limited. This study was designed to evaluate the roles of HMGB1 on the regulation of early spinal cord edema, astrocyte activation, and aquaporin-4 (AQP4) expression in a rat SCI model. Adult female Sprague-Dawley rats underwent laminectomy at T10, and the SCI model was induced by a heavy falling object. After SCI, rats received ethyl pyruvate (EP) or glycyrrhizin (GL) via an intraperitoneal injection to inhibit HMGB1. The effects of HMGB1 inhibition on the early spinal cord edema, astrocyte activation (glial fibrillary acidic protein [GFAP] expression), and AQP4 expression after SCI (12 h-3 days) were analyzed. The results showed that EP or GL effectively inhibited HMGB1 expression in the spinal cord and HMGB1 levels in the serum of SCI rats. HMGB1 inhibition improved motor function, reduced spinal cord water content, and attenuated spinal cord edema in SCI rats. HMGB1 inhibition decreased SCI-associated GFAP and AQP4 overexpression in the spinal cord. Further, HMGB1 inhibition also repressed the activation of the toll-like receptor 4/myeloid differentiation primary response gene 88/nuclear factor-kappa B signaling pathway. These results implicate that HMGB1 inhibition improved locomotor function and reduced early spinal cord edema, which was associated with a downregulation of astrocyte activation (GFAP expression) and AQP4 expression in SCI rats.
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