Exercise training is a traditional method to maximize remaining function in patients with spinal cord injury (SCI), but the exact mechanism by which exercise promotes recovery after SCI has not been identified; whether exercise truly has a beneficial effect on SCI also remains unclear. Previously, we showed that epigenetic changes in the brain motor cortex occur after SCI and that a treatment leading to epigenetic modulation effectively promotes functional recovery after SCI. We aimed to determine how exercise induces functional improvement in rats subjected to SCI and whether epigenetic changes are engaged in the effects of exercise. A spinal cord contusion model was established in rats, which were then subjected to treadmill exercise for 12 weeks. We found that the size of the lesion cavity and the number of macrophages were decreased more in the exercise group than in the control group after 12 weeks of injury. Immunofluorescence and DNA dot blot analysis revealed that levels of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in the brain motor cortex were increased after exercise. Accordingly, the expression of ten-eleven translocation (Tet) family members (Tet1, Tet2, and Tet3) in the brain motor cortex also elevated. However, no macrophage polarization was induced by exercise. Locomotor function, including Basso, Beattie, and Bresnahan (BBB) and ladder scores, also improved in the exercise group compared to the control group. We concluded that treadmill exercise facilitates functional recovery in rats with SCI, and mechanistically epigenetic changes in the brain motor cortex may contribute to exercise-induced improvements.

Axonal regeneration after spinal cord injury (SCI) is difficult to achieve, and no fundamental treatment can be applied in clinical settings. DNA methylation has been suggested to play a role in regeneration capacity and neuronal growth after SCI by controlling the expression of regeneration-associated genes (RAGs). The aim of this study was to examine changes in neuronal DNA methylation status after SCI and to determine whether modulation of DNA methylation with ascorbic acid can enhance neuronal regeneration or functional restoration after SCI. Changes in epigenetic marks (5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC)); the expression of Ten-eleven translocation (Tet) family genes; and the expression of genes related to inflammation, regeneration, and degeneration in the brain motor cortex were determined following SCI. The 5hmC level within the brain was increased after SCI, especially in the acute and subacute stages, and the mRNA levels of Tet gene family members (Tet1, Tet2, and Tet3) were also increased. Administration of ascorbic acid (100 mg/kg) to SCI rats enhanced 5hmC levels; increased the expression of the Tet1, Tet2, and Tet3 genes within the brain motor cortex; promoted axonal sprouting within the lesion cavity of the spinal cord; and enhanced recovery of locomotor function until 12 weeks. In conclusion, we found that epigenetic status in the brain motor cortex is changed after SCI and that epigenetic modulation using ascorbic acid may contribute to functional recovery after SCI.

Background: The isolation and culture of primary neurons from specific regions of the rat nervous system are fundamental techniques for investigating neuronal function, development, and pathology. These tools allow the exploration of distinct neural populations and their roles in health and disease.Methods: Protocols were optimized for dissection, isolation, and culture of primary neurons from the rat cortex, hippocampus, spinal cord, and dorsal root ganglia. Each methodology was customized to address the unique properties of the respective tissue types, focusing on key steps to enhance neuronal yield and viability whilst minimizing contamination with non-neuronal cells. The protocols incorporate refined enzymatic dissociation techniques, mechanical trituration methods, and specialized culture conditions to support neuronal survival and maturation. Additionally, essential considerations for neuronal culture such as growth medium composition, cell density used for plating, and substrate preparation were addressed.Results: These region-specific methodologies yielded robust and reproducible outcomes, enabling the generation of reliable in vitro models of neurons from both the central and peripheral nervous systems. The optimized procedures effectively increased neuronal viability and purity, making them suitable for a wide range of neuroscience applications.Conclusion: This comprehensive set of protocols represents a valuable resource for researchers working in neuroscience on rats. Practical approaches to isolate and culture neurons from diverse regions of the nervous system in the rat have been described. The methodologies outlined provide a strong foundation for studying neuronal populations and their significance in various physiological and pathological contexts.