Tissue engineering, including cell transplantation and the application of biomaterials and bioactive molecules, represents a promising approach for regeneration following spinal cord injury (SCI). We designed a combinatorial tissue-engineered approach for the minimally invasive treatment of SCI—a hyaluronic acid (HA)-based scaffold containing polypyrrole-coated fibers (PPY) combined with the RAD16-I self-assembling peptide hydrogel (Corning^® PuraMatrix™ peptide hydrogel (PM)), human induced neural progenitor cells (iNPCs), and a nanoconjugated form of curcumin (CURC). In vitro cultures demonstrated that PM preserves iNPC viability and the addition of CURC reduces apoptosis and enhances the outgrowth of Nestin-positive neurites from iNPCs, compared to non-embedded iNPCs. The treatment of spinal cord organotypic cultures also demonstrated that CURC enhances cell migration and prompts a neuron-like morphology of embedded iNPCs implanted over the tissue slices. Following sub-acute SCI by traumatic contusion in rats, the implantation of PM-embedded iNPCs and CURC with PPY fibers supported a significant increase in neuro-preservation (as measured by greater βIII-tubulin staining of neuronal fibers) and decrease in the injured area (as measured by the lack of GFAP staining). This combination therapy also restricted platelet-derived growth factor expression, indicating a reduction in fibrotic pericyte invasion. Overall, these findings support PM-embedded iNPCs with CURC placed within an HA demilune scaffold containing PPY fibers as a minimally invasive combination-based alternative to cell transplantation alone.

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.

Optogenetics is an emerging discipline with multiple applications in neuroscience, allowing to study neuronal pathways or serving for therapeutic applications such as in the treatment of anxiety disorder, autism spectrum disorders (ASDs), or Parkinson's disease. More recently optogenetics is opening its way also to stem cell-based therapeutic applications for neuronal regeneration after stroke or spinal cord injury. The results of optogenetic stimulation are usually evaluated by immunofluorescence or flow cytometry, and the observation of transient responses after stimulation, as in cardiac electrophysiology studies, by optical microscopy. However, certain phenomena, such as the ultra-fast calcium waves acquisition upon simultaneous optogenetics, are beyond the scope of current instrumentation, since they require higher image resolution in real-time, employing for instance time-lapse confocal microscopy. Therefore, in this work, an optogenetic stimulation matrix controllable from a graphical user interface has been developed for its use with a standard 24-well plate for an inverted confocal microscope use and validated by using a photoactivable adenyl cyclase (bPAC) overexpressed in rat fetal cortical neurons and the consequent calcium waves propagation upon 100 ms pulsed blue light stimulation.

Effective spinal cord injury (SCI) treatment remains a significant challenge, given the complex nature of the primary injury and associated devastating loss of neural activity. Neural progenitor cell (NPC)-based therapy has emerged as a potent strategy for the treatment of SCI. However, the invasive nature of direct cell transplantation and the need to enhance graft integration into host tissue remain critical issues. We implemented an improved combinatorial approach to SCI treatment by functionalizing electrospun poly-lactic acid (PLA) membranes that support the sustained delivery of curcumin (PLA-curcumin) and act as a carrier for NPC for local transplantation. In vitro experiments demonstrate that curcumin prevents harmful oxidative and inflammatory stress by preventing death and inhibiting NF-κB activation (mimicked by treatment with hydrogen peroxide or lipopolysaccharide acid). Curcumin also enhances neurite-like outgrowth in NPC and cortical neurons in culture, which may enhance neural connectivity. In vivo transplantation of NPC on a PLA-curcumin electrospun membrane enables cell migration, reduces injured area size, and increases neuronal fiber preservation to induce a slowing of acute neural damage.