Investigating the Mechanism of Conditioning Electrical Stimulation: Identifying Key Parameters and Signaling Pathways
Creators: Hardy, Paige BL
Publisher: name: University of Alberta Library
Year: 2024

<p>Despite timely surgical intervention, regeneration following peripheral nerve injury remains insufficient for adequate functional recovery. Thus, new therapeutic interventions are necessary. Traditionally, electrical stimulation has been delivered postoperatively to promote functional recovery following peripheral nerve injury. Postoperative electrical stimulation (PES) promotes staggered regeneration (increasing axon extension across the injury site), and has been successfully integrated into clinical practice. Conditioning electrical stimulation has recently been shown to accelerate the innate rate of regeneration when administered 7 days prior to a nerve injury. Due to its non-injurious, non-inflammatory nature, CES proves a viable option for clinical translation to enhance recovery in chronic and acute nerve repair, and nerve transfer procedures. However, the parameters of CES have not yet been optimized. The minimum duration of stimulation, period between conditioning and injury (latency period) and neuronal specificity have not been investigated. Likewise, much of the mechanism of CES remains either uninvestigated, or undifferentiated in the literature from PES. However, cAMP and its downstream targets BDNF and pCREB are known to upregulate following CES. cAMP’s downstream effectors PKA and EPAC have not been investigated in the context of CES. <br>
In this study we aim to identify the minimum duration of electrical stimulation, and the minimum latency period required to elicit the pro-regenerative effects of CES. We investigate the neuronal specificity of CES, and identify key signaling pathways involved in the mechanism of CES.<br>
To investigate the minimum duration of CES, CES was administered for 60, 30, 10 or 0 minutes either 3 days prior to DRG harvest to assess regeneration associated gene (RAG) expression, or 7 days prior to nerve repair surgery to assess regeneration. To investigate the latency period of CES, CES was administered 7, 5, 3, or 1 days prior to either neurite extension assays or RAG analysis, and compared to unstimulated DRG controls. Neuronal specificity is investigated through immunohistochemical staining and assessment of regeneration of different neuronal subtypes, including proprioceptors, peptidergic nociceptors, and motor neurons. To investigate the involvement of cAMP and its mediators in CES, CES is administered 3 days prior to DRG harvest and culture, EPAC and PKA pathways are pharmacologically inhibited and neurite extension assessed. <br>
Our data demonstrates that as the duration of stimulation increases, the capacity for regeneration following injury also increases. The most effective regeneration occurred with 1 hour of CES, though 30 minutes also significantly increased regeneration. Latency periods between 3 and 7 days increased neurite extension in vitro, coinciding with the upregulation of RAGs. CES appeared to elicit its effects non-specifically across all neuronal subtypes, despite innate differences in regenerative capacity among subtypes. Pharmacological inhibition of EPAC alone, and EPAC and PKA in combination resulted in the partial ablation of the pro-regenerative effects of CES in vitro, suggesting a compensatory role for both mediators, but a potentially larger role of EPAC in the mechanism of CES. CES administered for 1 hour, with a latency period between 3 and 7 days, will act upon the cAMP pathway to nonspecifically elicit pro-regenerative effects. </p>