Chondroitin sulfate proteoglycans (CSPGs) act as potent inhibitors of axonal growth and neuroplasticity after spinal cord injury (SCI). Here we reveal that CSPGs also play a critical role in preventing inflammation resolution by blocking the conversion of pro-inflammatory immune cells to a pro-repair phenotype in rodent models of SCI. We demonstrate that enzymatic digestion of CSPG glycosaminoglycans enhances immune cell clearance and reduces pro-inflammatory protein and gene expression profiles at key resolution time points. Analysis of phenotypically distinct immune cell clusters revealed CSPG-mediated modulation of macrophage and microglial subtypes which, together with T lymphocyte infiltration and composition changes, suggests a role for CSPGs in modulating both innate and adaptive immune responses after SCI. Mechanistically, CSPG activation of a pro-inflammatory phenotype in pro-repair immune cells was found to be TLR4-dependent, identifying TLR4 signalling as a key driver of CSPG-mediated immune modulation. These findings establish CSPGs as critical mediators of inflammation resolution failure after SCI in rodents, which leads to prolonged inflammatory pathology and irreversible tissue destruction.
Spinal cord injuries (SCIs) result in the loss of sensory and motor function with massive cell death and axon degeneration. We have previously shown that transplantation of spinal cord-derived ependymal progenitor cells (epSPC) significantly improves functional recovery after acute and chronic SCI in experimental models, via neuronal differentiation and trophic glial cell support. Here, we propose an improved procedure based on transplantation of epSPC in a tubular conduit of hyaluronic acid containing poly (lactic acid) fibres creating a biohybrid scaffold. In vitro analysis showed that the poly (lactic acid) fibres included in the conduit induce a preferential neuronal fate of the epSPC rather than glial differentiation, favouring elongation of cellular processes. The safety and efficacy of the biohybrid implantation was evaluated in a complete SCI rat model. The conduits allowed efficient epSPC transfer into the spinal cord, improving the preservation of the neuronal tissue by increasing the presence of neuronal fibres at the injury site and by reducing cavities and cyst formation. The biohybrid-implanted animals presented diminished astrocytic reactivity surrounding the scar area, an increased number of preserved neuronal fibres with a horizontal directional pattern, and enhanced coexpression of the growth cone marker GAP43. The biohybrids offer an improved method for cell transplantation with potential capabilities for neuronal tissue regeneration, opening a promising avenue for cell therapies and SCI treatment.
Background Toll-like receptors (TLRs) represent critical effectors in the host defense response against various pathogens; however, their known function during development has also highlighted a potential role in cell fate determination and neural differentiation. While glial cells and neural precursor cells (NPCs) of the spinal cord express both TLR2 and TLR4, their influence on self-renewal and cell differentiation remains incompletely described. Methods TLR2, TLR4 knock-out and the wild type mice were employed for spinal cord tissue analysis and NPCs isolation at early post-natal stage. Sox2, FoxJ1 and Ki67 expression among others served to identify the undifferentiated and proliferative NPCs; GFAP, Olig2 and β-III-tubulin markers served to identify astrocytes, oligodendrocytes and neurons respectively after NPC spontaneous differentiation. Multiple comparisons were analyzed using one-way ANOVA, with appropriate corrections such as Tukey's post hoc tests used for comparisons. Results We discovered that the deletion of TLR2 or TLR4 significantly reduced the number of Sox2-expressing NPCs in the neonatal mouse spinal cord. While TLR2-knockout NPCs displayed enhanced self-renewal, increased proliferation and apoptosis, and delayed neural differentiation, the absence of TLR4 promoted the neural differentiation of NPCs without affecting proliferation, producing long projecting neurons. TLR4 knock-out NPCs showed significantly higher expression of Neurogenin1, that would be involved in the activation of this neurogenic program by a ligand and microenvironment-independent mechanism. Interestingly, the absence of both TLR2 and TLR4, which induces also a significant reduction in the expression of TLR1, in NPCs impeded oligodendrocyte precursor cell maturation to a similar degree. Conclusions Our data suggest that Toll-like receptors are needed to maintain Sox2 positive neural progenitors in the spinal cord, however possess distinct regulatory roles in mouse neonatal spinal cord NPCs—while TLR2 and TLR4 play a similar role in oligodendrocytic differentiation, they differentially influence neural differentiation.
Background: Toll-Like Receptors (TLRs) represent critical effectors in the host defense response against various pathogens; however, their known function during development has also highlighted a potential role in cell fate determination and neural differentiation. While glial cells and neural precursor cells (NPCs) of the spinal cord express both TLR2 and TLR4, their influence on self-renewal and cell differentiation remains incompletely described. Methods: TLR2, TLR4 knock-out and the wild type mice were employed for spinal cord tissue analysis and NPCs isolation at early post-natal stage. Sox2, FoxJ1 and Ki67 expression among others served to identify the undifferentiated and proliferative NPCs; GFAP, Olig2 and b-III-tubulin markers served to identify astrocytes, oligodendrocytes and neurons respectively after NPC spontaneous differentiation. Multiple comparisons were analyzed using one-way ANOVA, with appropriate corrections such as Tukey's post hoc tests used for comparisons. Results: We discovered that the deletion of TLR2 or TLR4 significantly reduced the number of Sox2-expressing NPCs in the neonatal mouse spinal cord. While TLR2-knockout NPCs displayed enhanced self-renewal, increased proliferation and apoptosis, and delayed neural differentiation, the absence of TLR4 promoted the neural differentiation of NPCs without affecting proliferation, producing long projecting neurons. TLR4 knock-out NPCs showed significantly higher expression of Neurogenin1, that would be involved in the activation of this neurogenic program by a ligand and microenvironment-independent mechanism. Interestingly, the absence of both TLR2 and TLR4 in NPCs impeded oligodendrocyte precursor cell maturation to a similar degree. Conclusions: Our data suggest that Toll-Like receptors are needed to maintain Sox2 positive neural progenitors in the spinal cord, however possess distinct regulatory roles in mouse neonatal spinal cord NPCs - while TLR2 and TLR4 play a similar role in oligodendrocytic differentiation, they differentially influence neural differentiation.
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