Rho/ROCK signaling induced after spinal cord injury (SCI) contributes to secondary damage by promoting apoptosis, inflammation, and axon growth inhibition. The specific Rho-kinase inhibitor fasudil can contribute to functional regeneration after SCI, although inherent low stability has hampered its use. To improve the therapeutic potential of fasudil, we now describe a family of rationally-designed bioresponsive polymer-fasudil conjugates based on an understanding of the conditions after SCI, such as low pH, enhanced expression of specific proteases, and a reductive environment. Fasudil conjugated to poly-l-glutamate via a self-immolative redox-sensitive linker (PGA-SS-F) displays optimal release kinetics and, consequently, treatment with PGA-SS-F significantly induces neurite elongation and axon growth in dorsal root ganglia explants, spinal cord organotypic cultures, and neural precursor cells (NPCs). The intrathecal administration of PGA-SS-F after SCI in a rat model prevents early apoptosis and induces the expression of axonal growth- and neuroplasticity-associated markers to a higher extent than the free form of fasudil. Moreover, a combination treatment comprising the acute transplantation of NPCs pre-treated with PGA-SS-F leads to enhanced cell engraftment and reduced cyst formation after SCI. In chronic SCI, combinatory treatment increases the preservation of neuronal fibers. Overall, this synergistic combinatorial strategy may represent a potentially efficient clinical approach to SCI treatment.

After spinal cord injury (SCI), cyclic adenosine monophosphate (cAMP) levels drop in the spinal cord, cortex and brainstem, unlike in regenerating peripheral neurons. To address SCI recovery, we expressed photoactivatable adenylate cyclase (bPAC) in corticospinal neurons of female rats with dorsal hemisection for on-demand cAMP inductions. bPAC stimulation restored passive and firing properties of corticospinal neurons, promoted early and sustained locomotor recovery and increased corticospinal tract plasticity. Additionally, bPAC enhanced sparing of lumbar-projecting brainstem neurons after SCI, accompanied by activation of cAMP signaling in the raphe-reticular formation and increased excitatory/inhibitory neurotransmitter balance. Accordingly, augmented density of serotonergic tracts was found caudal to the injury in bPAC rats, correlating with enhanced functional performance. Serotonergic implication in motor recovery was further evidenced by selective depletion, resulting in the abrogation of bPAC-mediated recovery. Overall, our findings underscore that cAMP induction in corticospinal neurons enhances locomotion after SCI, through a cortical rerouting pathway via the serotonergic descending tract.

Cell-free therapies based on the mesenchymal stem cells (MSCs) secretome are being widely explored for spinal cord injury (SCI). Induced pluripotent stem cells (iPSCs) provide a scalable source for generating mesenchymal stem cells (iMSCs), but limited studies have evaluated the modulatory capacity of iMSCs for CNS applications. Due to the importance of the glial and vascular responses to SCI progression, this study evaluated the activity of the iMSCs secretome on glial and endothelial cells in SCI-relevant paradigms. In primary mixed spinal cord cultures, the secretome was tested under supplement withdrawal and hyperosmotic injury conditions, while endothelial network formation was assessed in a morphogenesis assay. In vitro, the iMSCs secretome promoted a modest protective effect on cell survival, modulated astrocyte morphology and preserved myelin basic protein expression within the oligodendroglial lineage, without clear alterations in microglial morphometric readouts. Secretome treatment also increased Arg1 expression in mixed spinal cord cultures. In endothelial cultures, the secretome promoted a denser and more interconnected endothelial network. In a mouse thoracic SCI model, the secretome was administered intravenously, and tissue was analyzed one week after injury. In vivo, the secretome reduced perilesional GFAP coverage and increased selected vascular morphometric parameters without altering lesion size or microglia morphological readouts. Overall, these findings indicate that the iMSCs secretome produced context-dependent changes in glial and vascular cells, mainly regarding morphological parameters, supporting further studies of iPSC-derived MSCs as a scalable source for secretome-based strategies.