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Abstract

To date, there are no specific treatments available that efficiently target the loss of neural connectivity after a spinal cord injury (SCI). Thus patients usually suffer from life-long motor, sensory and autonomic dysfunction. Neuron-intrinsic growth programs are activated after a lesion in the peripheral nervous system (PNS) and can contribute to enhanced regeneration in a subsequent central lesion. Yet this so-called conditioning lesion (CL) holds little translational potential for SCI. Electrical stimulation (ES) can influence various cellular functions, including neuronal growth and could provide a practical approach to enhance regeneration after SCI. However, the mechanisms and a practical means for applying ES as a therapy after SCI are insufficiently understood. I hypothesized that evoked neuronal activity by direct ES of the peripheral nerve can enhance the growth potential of dorsal root ganglia (DRG) neurons in a similar way to CL, supporting the regeneration of the injured central branch ascending in the dorsal column. ES (20Hz, 2*MT, 0.2ms, 1h) was applied in vivo to the sciatic nerve of adult Fischer 344 rats, followed by ex-vivo assessment of the growth potential, showing about 2-fold enhanced neurite growth compared to sham animals. ES increased the percentage of neurons with neurites >100um, but there was no change in the percentage of neurite bearing neurons, indicating that the effect on growth is due to enhanced elongation and not initiation. Longer duration stimulation (7h) also enhances growth by 67 ± 25%, as well as repeated stimulation for 7 days (55 ± 24%). The pattern of growth and timeline is similar to a CL, suggesting a similar or a partial overlap in the mechanism. Growth effects of 1h ES were also assessed in vivo in a model of spinal cord injury, together with cell transplantation of BMSCs (bone marrow stromal cells) at 4 weeks post-injury. Stimulated fibers were labeled by sciatic nerve injection of the transganglionic tracer Cholera toxin B (CTB). Animals with ES for 1h showed significantly increased axonal regeneration into the spinal cell graft within the lesion compared to sham animals. Repeated stimulation with chronic electrodes showed a similar effect, but also a slight influence from chronic electrode implantation in chronic sham animals. Dieback of axons was not modified in any of the conditions. To evaluate possible side effects that may interfere with clinical applicability, I also tested pain-like behavior, showing a lack of allodynia or thermal hyperalgesia after ES. This further highlights the translational potential of this strategy in combinatorial approaches such as cell transplantation. In parallel, I investigated the mechanisms underlying the observed neuronal activity-mediated increases in neurite growth. Using in vitro depolarization of DRG neurons as a model, my data show that neurite growth is influenced depending on the duration of the depolarization and the delay between stimulation and measurement. Since depolarization induces calcium influx, I examined in a separate set of experiments calcium signaling, showing that blocking nuclear calcium signaling with recombinant calmodulin-binding proteins reduces growth in DRG cultures at 72h by 50 ± 10%. However, a cytoplasmic block enhances growth by 35 ± 11%, and has similar effects in vivo after adeno-associated virus gene transfer into lumbar DRGs. This differential effect of nuclear and cytoplasmic calcium signaling provides an explanation for previous reports, which have shown stimulation or reduction of growth following neuronal activity. Furthermore, I investigated HDAC5 (histone deacetylase 5), showing export from the nucleus in DRGs (92 ± 5% nuclear before and 14 ± 1% after depolarization). These in vitro experiments suggest that neuronal activity-mediated effects on axon growth could involve epigenetic mechanisms, dependent on calcium/calmodulin signaling. To follow up on these experiments, RNA sequencing was performed to investigate differential gene expression at 1 day and 7 days after ES, compared to sham animals, naive animals and animals that underwent a peripheral lesion, collecting 30M SE reads/sample on a HiSeq2000. As expected CL induces and represses an extensive number of genes compared to naïve animals. ES induced/reduced expression of a much lower number of genes relative to sham animals with smaller changes in gene expression. Several genes and pathways could be identified that are known to play a role in regeneration, suggesting that ES-mediated effects on axon regeneration are likely a summation of several activated pathways that overlap only partially with CL. Taken together, my results reveal the capacity of neurons to modulate their growth response depending on their activity in vivo. Electrical stimulation is shown to be an effective means to increase axonal regeneration in a central lesion, and could provide a feasible therapeutic approach either alone or in combination with other strategies such as cell transplantation.