Cervical spinal cord injury (SCI) remains a devastating event without adequate treatment options despite decades of research. In this context, the usefulness of common preclinical SCI models has been criticized. We, therefore, aimed to use a clinically relevant animal model of severe cervical SCI to assess the long-term effects of neural precursor cell (NPC) transplantation on secondary injury processes and functional recovery. To this end, we performed a clip contusion-compression injury at the C6 level in 40 female Wistar rats and a sham surgery in 10 female Wistar rats. NPCs, isolated from the subventricular zone of green fluorescent protein (GFP) expressing transgenic rat embryos, were transplanted ten days after the injury. Functional recovery was assessed weekly, and FluoroGold (FG) retrograde fiber-labeling, as well as manganese-enhanced magnetic resonance imaging (MEMRI), were performed prior to the sacrifice of the animals eight weeks after SCI. After cryosectioning of the spinal cords, immunofluorescence staining was conducted. Results were compared between the treatment groups (NPC, Vehicle, Sham) and statistically analyzed (p < 0.05 was considered significant). Despite the severity of the injury, leading to substantial morbidity and mortality during the experiment, long-term survival of the engrafted NPCs with a predominant differentiation into oligodendrocytes could be observed after eight weeks. While myelination of the injured spinal cord was not significantly improved, NPC treated animals showed a significant increase of intact perilesional motor neurons and preserved spinal tracts compared to untreated Vehicle animals. These findings were associated with enhanced preservation of intact spinal cord tissue. However, reactive astrogliosis and inflammation where not significantly reduced by the NPC-treatment. While differences in the Basso–Beattie–Bresnahan (BBB) score and the Gridwalk test remained insignificant, animals in the NPC group performed significantly better in the more objective CatWalk XT gait analysis, suggesting some beneficial effects of the engrafted NPCs on the functional recovery after severe cervical SCI.

Objective and consistent assessment of locomotion recovery remains challenging in rodent spinal cord injury (SCI). We, therefore, studied the validity and relevance of the CatWalk XT^® gait analysis as a tool for assessing functional outcome in a clinically relevant cervical SCI model in rats. In total, 20 Wistar rats were randomly assigned to either a C6 clip compression/contusion SCI or a sham laminectomy. Locomotion recovery was assessed weekly using the CatWalk XT^® gait analysis and the BBB open field score. Six weeks after SCI, the percentage of preserved spinal cord tissue was measured by glial fibrillary acidic protein (GFAP) immunohistochemistry (IHC) staining. Statistical analyses were performed to assess the correlation of the BBB and the percentage of preserved tissue with 30 different CatWalk XT^® parameters. SCI caused a bilateral and significant functional impairment in all studied CatWalk XT^® parameters. Similar to the BBB, a significant spontaneous recovery could be observed in most of the CatWalk XT^® parameters in the following weeks. Correlation between the hindlimb CatWalk XT^® parameters and the BBB was good (53% of r values > 0.6) while the correlation between the forelimb and the hindlimb CatWalk XT^® parameters and the percentage of preserved tissue was even stronger (83% of r values > 0.6). The CatWalk XT^® gait analysis is closely correlated with tissue damage after cervical contusion/compression SCI and can be used as an objective and consistent tool for assessing locomotion recovery.

The Sonic Hedgehog protein (Shh) has been extensively researched since its discovery in 1980. Its crucial role in early neurogenesis and endogenous stem cells of mature brains, as well as its recently described neuroprotective features, implicate further important effects on neuronal homeostasis. Here, we investigate its potential role in the survival, proliferation, and differentiation of neural precursors cells (NPCs) under inflammatory stress as a potential adjunct for NPC-transplantation strategies in spinal cord injury (SCI) treatment. To this end, we simulated an inflammatory environment in vitro using lipopolysaccharide (LPS) and induced the Shh-pathway using recombinant Shh or blocked it using Cyclopamine, a potent Smo inhibitor. We found that Shh mediates the proliferation and neuronal differentiation potential of NPCs in vitro, even in an inflammatory stress environment mimicking the subacute phase after SCI. At the same time, our results indicate that a reduction of the Shh-pathway activation by blockage with Cyclopamine is associated with reduced NPC-survival, reduced neuronal differentiation and increased astroglial differentiation. Shh might thus, play a role in endogenous NPC-mediated neuroregeneration or even be a potent conjunct to NPC-based therapies in the inflammatory environment after SCI.

Astrocytes, which proliferate after brain injury, represent a promising target for cellular reprogramming due to their abundance and ability to support brain repair. In this study, we investigated the in vitro reprogramming of primary cortical astrocytes from neonatal rats into neuronal precursor cells (NPCs) using the transcription factors Oct4, Sox2, and Klf4 (OSK), delivered via lentiviral vectors. We designed a reporter system to trace the conversion of astrocytes to NPCs and neurons by using GFAP-driven iCre and Nestin- or Synapsin1-driven fluorescent reporters. After transduction, we observed morphological changes and the expression of neuronal markers in some cells, while many cells remained in a transitional state, expressing both astrocytic and neuronal features. Importantly, the study was not designed to quantify reprogramming efficiency or demonstrate full astrocyte-to-neuron conversion but rather to establish and evaluate a traceable reporter system. Our data suggest that OSK-mediated reprogramming in this in vitro model can initiate conversion of astrocytes to neuronal precursor-like cells, although the process is complex and incomplete within the one-week timeframe. We also highlight limitations in co-transduction efficiency and potential silencing of the reporter system during reprogramming. These findings provide an initial technical platform to explore astrocyte reprogramming in vitro and inform future studies aiming to refine these methods and apply them in vivo.

Transplanted neural precursor cells (NPCs) can support repair after spinal cord injury (SCI) but often face poor survival and limited differentiation within the hostile post-injury microenvironment. We tested whether subacute activation of Sonic hedgehog (Shh) signaling improves NPC engraftment and functional recovery over the duration of 6 weeks. Female Wistar rats underwent T9/T10 clip-contusion SCI and were assigned to vehicle, Shh-only, NPC-only, Shh + NPC, or sham groups (n = 8 per SCI arm, n = 5 sham, total n = 37). GFP + NPCs (4 × 10⁵) were grafted at 7 days post injury. Recombinant Shh or vehicle was delivered intrathecally via osmotic pumps for 7 days thereafter. Outcome measures included pathway activation (GLI1, SMO), NPC differentiation (NeuN, Olig2, Nestin, GFAP), astrogliosis (GFAP), CSPG deposition, immune cell infiltration (CD3, Iba1), apoptosis (cleaved Caspase-3), and locomotion (BBB, Gridwalk, CatWalk) over 6 weeks. Shh delivery increased GLI1/SMO expression and, when combined with NPCs, significantly elevated NPC-derived neurons and oligodendrocytes while preserving undifferentiated NPCs. Combined therapy reduced astrogliosis, CSPG deposition, T-cell and macrophage/microglial densities, cyst size and apoptotic cells compared with controls. These histological benefits were accompanied by improved locomotor scores in BBB, Gridwalk and selected CatWalk parameters. Our findings highlight the synergistic effects of Shh pathway activation and NPC transplantation in promoting neuroregeneration after SCI. The data support Shh as an adjunct to NPC transplantation for SCI repair.