Electrical stimulation is emerging as a perioperative strategy to improve peripheral nerve regeneration and enhance functional recovery. Despite decades of research, new insights into the complex multifaceted mechanisms of electrical stimulation continue to emerge, providing greater understanding of the neurophysiology of nerve regeneration. In this study, we summarize what is known about how electrical stimulation modulates the molecular cascades and cellular responses innate to nerve injury and repair, and the consequential effects on axonal growth and plasticity. Further, we discuss how electrical stimulation is delivered in preclinical and clinical studies and identify knowledge gaps that may provide opportunities for optimization.

Peripheral nerve injuries result in the loss of the motor, sensory and autonomic functions of the denervated segments of the body. Neurons can regenerate after peripheral axotomy, but inaccuracy in reinnervation causes a permanent loss of function that impairs complete recovery. Thus, understanding how regenerating axons respond to their environment and direct their growth is essential to improve the functional outcome of patients with nerve lesions. Schwann cells (SCs) play a crucial role in the regeneration process, but little is known about their contribution to specific reinnervation. Here, we review the mechanisms by which SCs can differentially influence the regeneration of motor and sensory axons. Mature SCs express modality-specific phenotypes that have been associated with the promotion of selective regeneration. These include molecular markers, such as L2/HNK-1 carbohydrate, which is differentially expressed in motor and sensory SCs, or the neurotrophic profile after denervation, which differs remarkably between SC modalities. Other important factors include several molecules implicated in axon-SC interaction. This cell-cell communication through adhesion (e.g., polysialic acid) and inhibitory molecules (e.g., MAG) contributes to guiding growing axons to their targets. As many of these factors can be modulated, further research will allow the design of new strategies to improve functional recovery after peripheral nerve injuries.

Brief low-frequency electrical stimulation (ES, 1 h, 20 Hz) of the proximal nerve stump has emerged as a potential adjunct treatment for nerve injury. Despite available experimental and clinical data, the potentials and limitations of the ES therapy still have to be defined using different animal models, types of nerves, and clinical settings. Here, we show that brief ES of the proximal stump of the transected rat femoral nerve causes, as estimated by motion analysis, enhanced functional recovery reaching preoperative levels within 5 months of injury, in contrast to the incomplete restoration in sham-stimulated (SS) animals. The functional advantage seen in ES rats was associated with higher numbers, as compared with SS, of correctly targeted quadriceps motoneurons. In contrast, ES prior to facial nerve suture did not lead to improvement of whisking compared with SS. Lack of functional effects of the treatment was correlated with lack of changes, as compared with SS, in the precision of muscle reinnervation and frequency of abnormally innervated muscle fibers. These results show that ES is an effective therapy in a spinal nerve injury model leading to complete restoration of function. Although this finding and the safety of the procedure are encouraging, the results for the facial nerve model suggest that brief ES may not be a universal treatment for nerve injuries. Anat Rec, 302:1304-1313, 2019. © 2019 Wiley Periodicals, Inc.

The rat femoral nerve is a valuable model allowing studies on specificity of motor axon regeneration. Despite common use of this model, the functional consequences of femoral nerve lesions and their relationship to precision of axonal regeneration have not been evaluated. Here we assessed gait recovery after femoral nerve injuries of varying severity in adult female Wistar rats using a video-based approach, single-frame motion analysis (SFMA). After nerve crush, recovery was complete at 4 weeks after injury (99% of maximum 100% as estimated by a recovery index). Functional restoration after nerve section/suture was much slower and incomplete (84%) even 20 weeks post-surgery. A 5-mm gap between the distal and proximal nerve stumps additionally delayed recovery and worsened the outcome (68% recovery). As assessed by retrograde labeling in the same rats at 20 weeks after injury, the anatomical outcome was also dependent on lesion severity. After nerve crush, 97% of the femoral motoneurons (MNs) had axons correctly projecting only into the distal quadriceps branch of the femoral nerve. The percentage of correctly projecting MNs was only 55% and 15% after nerve suture and gap repair, respectively. As indicated by regression analyses, better functional recovery was associated with higher numbers of correctly projecting MNs and, unexpectedly, lower numbers of MNs projecting to both muscle and skin. The data show that type of nerve injury and repair profoundly influence selectivity of motor reinnervation and, in parallel, functional outcome. The results also suggest that MNs\' projection patterns may influence their contribution to muscle performance. In addition to the experiments described above, we performed repeated measurements and statistical analyses to validate the SFMA. The results revealed high accuracy and reproducibility of the SFMA measurements.

The immune system plays important functional roles in regeneration after injury to the mammalian central and peripheral nervous systems. After damage to the peripheral nerve several types of immune cells, invade the nerve within hours after the injury. To gain insights into the contribution of T- and B-lymphocytes to recovery from injury we used the mouse femoral nerve injury paradigm. RAG2-/- mice lacking mature T- and B-lymphocytes due to deletion of the recombination activating gene 2 were subjected to resection and surgical reconstruction of the femoral nerve, with the wild-type mice of the same inbred genetic background serving as controls. According to single frame motion analyses, RAG2-/- mice showed better motor recovery in comparison to control mice at four and eight weeks after injury. Retrograde tracing of regrown/sprouted axons of spinal motoneurons showed increased numbers of correctly projecting motoneurons in the lumbar spinal cord of RAG2-/- mice compared with controls. Whereas there was no difference in the motoneuron soma size between genotypes, RAG2-/- mice displayed fewer cholinergic and inhibitory synaptic terminals around somata of spinal motoneurons both prior to and after injury, compared with wild-type mice. Extent of myelination of regrown axons in the motor branch of the femoral nerve measured as g-ratio was more extensive in RAG2-/- than in control mice eight weeks after injury. We conclude that activated T- and B-lymphocytes restrict motor recovery after femoral nerve injury, associated with the increased survival of motoneurons and improved remyelination.

Preferential motor reinnervation (PMR) is the tendency for motor axons regenerating after repair of mixed nerve to reinnervate muscle nerve and/or muscle rather than cutaneous nerve or skin. PMR may occur in response to the peripheral nerve pathway alone in juvenile rats (Brushart, 1993; Redett et al., 2005), yet the ability to identify and respond to specific pathway markers is reportedly lost in adults (Uschold et al., 2007). The experiments reported here evaluate the relative roles of pathway and end organ in the genesis of PMR in adult rats. Fresh and 2-week predegenerated femoral nerve grafts were transferred in correct or reversed alignment to replace the femoral nerves of previously unoperated Lewis rats. After 8 weeks of regeneration the motoneurons projecting through the grafts to recipient femoral cutaneous and muscle branches and their adjacent end organs were identified by retrograde labeling. Motoneuron counts were subjected to Poisson regression analysis to determine the relative roles of pathway and end organ identity in generating PMR. Transfer of fresh grafts did not result in PMR, whereas substantial PMR was observed when predegenerated grafts were used. Similarly, the pathway through which motoneurons reached the muscle had a significant impact on PMR when grafts were predegenerated, but not when they were fresh. Comparison of the relative roles of pathway and end organ in generating PMR revealed that neither could be shown to be more important than the other. These experiments demonstrate unequivocally that adult muscle nerve and cutaneous nerve differ in qualities that can be detected by regenerating adult motoneurons and that can modify their subsequent behavior. They also reveal that two weeks of Wallerian degeneration modify the environment in the graft from one that provides no modality-specific cues for motor neurons to one that actively promotes PMR.