Softening of the chronic hemi-section spinal cord injury scar parallels dysregulation of cellular and extracellular matrix content.

Regeneration following spinal cord injury (SCI) is challenging in part due to the modified tissue composition and organization of the resulting glial and fibrotic scar regions. Inhibitory cell types and biochemical cues present in the scar have received attention as therapeutic targets to promote regeneration. However, altered Young's modulus of the scar as a readout for potential impeding factors for regeneration are not as well-defined, especially in vivo. Although the decreased Young's modulus of surrounding tissue at acute stages post-injury is known, the causation and outcomes at chronic time points remain largely understudied and controversial, which motivates this work. This study assessed the glial and fibrotic scar tissue's Young's modulus and composition (scar morphometry, cell identity, extracellular matrix (ECM) makeup) that contribute to the tissue's stiffness. The spatial Young's modulus of a chronic (~18-wks, post-injury) hemi-section, including the glial and fibrotic regions, were significantly less than naïve tissue (~200 Pa; p < 0.0001). The chronic scar contained cystic cavities dispersed in areas of dense nuclei packing. Abundant CNS cell types such as astrocytes, oligodendrocytes, and neurons were dysregulated in the scar, while epithelial markers such as vimentin were upregulated. The key ECM components in the CNS, namely sulfated proteoglycans (sPGs), were significantly downregulated following injury with concomitant upregulation of unsulfated glycosaminoglycans (GAGs) and hyaluronic acid (HA), likely altering the foundational ECM network that contributes to tissue stiffness. Our results reveal the Young's modulus of the chronic SCI scar as well as quantification of contributing elastic components that can provide a foundation for future study into their role in tissue repair and regeneration.

Metabolomic and Signaling Programs Induced by Immobilized versus Soluble IFN γ in Neural Stem Cells.

Neural stem cells (NSCs) provide a strategy to replace damaged neurons following traumatic central nervous system injuries. A major hurdle to translation of this therapy is that direct application of NSCs to CNS injury does not support sufficient neurogenesis due to lack of proper cues. To provide prolonged spatial cues to NSCs IFN-γ was immobilized to biomimetic hydrogel substrate to supply physical and biochemical signals to instruct the encapsulated NSCs to be neurogenic. However, the immobilization of factors, including IFN-γ, versus soluble delivery of the same factor, has been incompletely characterized especially with respect to activation of signaling and metabolism in cells over longer time points. In this study, protein and metabolite changes in NSCs induced by immobilized versus soluble IFN-γ at 7 days were evaluated. Soluble IFN-γ, refreshed daily over 7 days, elicited stronger responses in NSCs compared to immobilized IFN-γ, indicating that immobilization may not sustain signaling or has altered ligand/receptor interaction and integrity. However, both IFN-γ delivery types supported increased ßIII tubulin expression in parallel with canonical and noncanonical receptor-signaling compared to no IFN-γ. Global metabolomics and pathway analysis revealed that soluble and immobilized IFN-γ altered metabolic pathway activities including energy, lipid, and amino acid synthesis, with soluble IFN-γ having the greatest metabolic impact overall. Finally, soluble and immobilized IFN-γ support mitochondrial voltage-dependent anion channel (VDAC) expression that correlates to differentiated NSCs. This work utilizes new methods to evaluate cell responses to protein delivery and provides insight into mode of action that can be harnessed to improve regenerative medicine-based strategies.