DISTINCT SPINAL CORD CELLULAR COMPOSITION AND TRANSCRIPTIONAL STATES ASSOCIATED WITH MOTONEURON FATE UNDER NERVE REGENERATIVE AND DEGENERATIVE CONDITIONS IN NEONATAL RATS

Peripheral nerves in adult mammals show considerable regenerative capacity; however, neonatal injuries often result in poor regeneration and motoneuron loss. The mechanisms behind this age-dependent failure remain unclear. To address this problem, we employed a sciatic nerve grafting model in newborn rats (P6 donor nerves to P3 recipients) to promote regeneration by leveraging the developmental advantage of older donor nerves. In contrast, same-age P3/P3 grafts do not support regrowth. This approach enables investigation of age-dependent regenerative mechanisms and associated spinal cord cellular responses, driving regenerative outcomes.
We combined single-nucleus RNA sequencing (snRNA-seq) with immunohistochemistry to characterize the spinal cord cellular responses associated with successful versus failed peripheral nerve regeneration in neonatal rats. Since neonatal motoneurons in rats lack well-defined molecular markers, we performed single-nucleus transcriptome profiling that provides an unbiased strategy to resolve injury-associated transcriptional changes across spinal cord cell types. Enhanced regeneration correlated with increased survival of ChAT⁺ motoneurons, whereas reduced motoneuron number reflected degenerative processes. SnRNA-seq analysis revealed distinct cellular compositions and injury-associated transcriptional states across major spinal cord cell types, indicating divergent cellular responses between regenerative and degenerative conditions. Early after injury, a higher number of Iba1⁺ microglial cells were observed in the regenerative condition. In contrast, microglial activation was delayed and sustained in degenerative cases.
Together, our findings suggest that injury-induced microenvironmental cues shape spinal cord regenerative capacity and highlight a role for neuroimmune dynamics in motoneuron survival following neonatal nerve injury.
Funded by NCN (2020/37/B/NZ4/04065).