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Specificity in regeneration after peripheral nerve injuries is a major determinant of functional recovery. Unfortunately, regenerating motor and sensory axons rarely find their original pathways to reinnervate appropriate target organs. Although a preference of motor axons to regenerate towards the muscle has been described, little is known about the specificity of the heterogeneous sensory populations. Here, we propose the comparative study of regeneration in different neuron subtypes. Using female and male reporter mice, we assessed the regenerative preference of motoneurons (ChAT-Cre/Ai9), proprioceptors (PV-Cre/Ai9), and cutaneous mechanoreceptors (Npy2r-Cre/Ai9). The femoral nerve of these animals was transected above the bifurcation and repaired with fibrin glue. Regeneration was assessed by applying retrograde tracers in the distal branches of the nerve 1 or 8 weeks after the lesion and counting the retrotraced somas and the axons in the branches. We found that cutaneous mechanoreceptors regenerated faster than other populations, followed by motoneurons and, lastly, proprioceptors. All neuron types had an early preference to regenerate into the cutaneous branch whereas, at long term, all neurons regenerated more through their original branch. Finally, we found that myelinated neurons extend more regenerative sprouts in the cutaneous than in the muscle branch of the femoral nerve and, particularly, that motoneurons have more collaterals than proprioceptors. Our findings reveal novel differences in regeneration dynamics and specificity, which indicate distinct regenerative mechanisms between neuron subtypes that can be potentially modulated to improve functional recovery after nerve injury., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Neurons of the peripheral nervous system retain the intrinsic capability of regenerate their axons after injury, by triggering a complex activation response. This genetic switch is dependent of signals from the injured axon. Schwann cells (SCs) in the distal stump of an injured nerve also play an active role in the local regulation of axonal programs, by using cell-to-cell contacts but also secreted signals, the so-called secretome. Secretome contains all the proteins (cytokines, growth factors and others) secreted by the cell and includes extracellular vesicles. The released vesicles can transport signaling proteins and both coding and regulatory RNAs, thus facilitating multilevel communication. It is nowadays clear that secretome of SCs is fundamental to both orchestrate Wallerian degeneration and to sustain axonal regeneration. Therefore, the use of secretome has emerged as an alternative to cell therapy in the field of tissue regeneration. In fact, separate components of SC secretome have been extensively used in experimental models to enhance peripheral nerve regeneration after injury. However, the most used secretome in neural therapies has been the one derived from mesenchymal (MSC) or other derived stem cells. In fact, the effects of cell therapy with MSCs have been mainly associated with the secretion of bioactive molecules and extracellular vesicles, which constitute their secretome. In this review, we first describe the role of SC and macrophage secretomes on Wallerian degeneration and axonal regeneration after peripheral nerve injury. Then, we review the different works reported in the literature that have used secretomes of SCs or MSCs in the treatment of peripheral nerve injuries in experimental models, to highlight the use of secretomes as a promising cell-free therapeutic approach, that reduces some of the risks associated with the use of cells, such as tumor formation or rejection., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Injury of a peripheral nerve not only leads to target denervation, but also induces massive stripping of spinal synapses on axotomized motoneurons, with disruption of spinal circuits. Even when regeneration is successful, unspecific reinnervation and the limited reconnection of the spinal circuits impair functional recovery. The aim of this study was to describe the changes that axotomized motoneurons suffer after peripheral nerve injury and how activity-dependent therapies and neurotrophic factors can modulate these events. We observed a marked decrease in glutamatergic synapses, with a maximum peak at two weeks post-axotomy, which was only partially reversed with time. This decrease was accompanied by an increase in gephyrin immunoreactivity and a disintegration of perineuronal nets (PNNs) surrounding the motoneurons. Direct application of neurotrophins at the proximal stump was not able to reverse these effects. In contrast, activity-dependent treatment, in the form of treadmill running, reduced the observed destructuring of perineuronal nets and the loss of glutamatergic synapses two weeks after injury. These changes were proportional to the intensity of the exercise protocol. Blockade of sensory inputs from the homolateral hindlimb also reduced PNN immunoreactivity around intact motoneurons, and in that case treadmill running did not reverse that loss, suggesting that the effects of exercise on motoneuron PNN depend on increased sensory activity. Preservation of motoneuron PNN and reduction of synaptic stripping by exercise could facilitate the maintenance of the spinal circuitry and benefit functional recovery after peripheral nerve injury., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Bortezomib (BTZ), a proteasome inhibitor, is an effective anti-neoplastic drug used in the treatment of multiple myeloma and mantle cell lymphoma. However, it can induce a reversible peripheral neuropathy that may lead to treatment discontinuation. The mechanism through which BTZ exerts toxic effects in peripheral neurons is not clear. Release of proinflammatory cytokines after nerve damage can induce neurodegeneration, but the effects of BTZ on cytokine expression in neurons are unknown, although BTZ modulates the expression of cytokines, such as TNF-α and IL-6, in tumor cells. The aim of this study was to evaluate the expression and the role of these cytokines on the course of BTZ induced neuropathy in mice. IL-6, TNF-α, TGF-β1 and IL-1β were up-regulated in dorsal root ganglia but TNF-α and IL-6 increased faster and higher. Then, we studied the potential neuroprotective effect of selective antibodies anti-TNF-α and anti-IL-6 on the evolution of the neuropathy. Treatment with anti-TNF-α but not with anti-IL-6 significantly prevented the decrease of sensory nerve action potentials amplitude and the loss of myelinated and unmyelinated fibers. We conclude that monoclonal antibodies directed against TNF-α may be a suitable neuroprotective therapy against the neurotoxicity induced by BTZ., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Injured axons of the peripheral nerve are able to regenerate and, eventually, reinnervate target organs. However, functional recovery is usually poor after severe nerve injuries. The switch of Schwann cells to a proliferative state, secretion of trophic factors, and the presence of extracellular matrix (ECM) molecules (such as collagen, laminin, or fibronectin) in the distal stump are key elements to create a permissive environment for axons to grow. In this review, we focus attention on the ECM components and their tropic role in axonal regeneration. These components can also be used as molecular cues to guide the axons through artificial nerve guides in attempts to better mimic the natural environment found in a degenerating nerve. Most used scaffolds tested are based on natural molecules that form the ECM, but use of synthetic polymers and functionalization of hydrogels are bringing new options. Progress in tissue engineering will eventually lead to the design of composite artificial nerve grafts that may replace the use of autologous nerve grafts to sustain regeneration over long gaps., (© 2013 Elsevier Inc. All rights reserved.)

Bortezomib, a proteasome inhibitor, is an antineoplastic drug to treat multiple myeloma and mantle cell lymphoma. Its most clinically significant adverse event is peripheral sensory neuropathy. Our objective was to characterize the neuropathy induced by bortezomib in a mouse model. Two groups were used; one group received vehicle solution and another bortezomib (1mg/kg/twice/week) for 6weeks (total dose as human schedule). Tests were performed during treatment and for 4weeks post dosing to evaluate electrophysiological, autonomic, pain sensibility and sensory-motor function changes. At the end of treatment and after washout, sciatic and tibial nerves, dorsal ganglia and intraepidermal innervation were analyzed. Bortezomib induced progressive significant decrease of sensory action potential amplitude, mild reduction of sensory velocities without effect in motor conductions. Moreover, it significantly increased pain threshold and sensory-motor impairment at 6weeks. According to these data, histopathological findings shown a mild reduction of myelinated (-10%; p=0.001) and unmyelinated fibers (-27%; p=0.04), mostly involving large and C fibers, with abnormal vesicular inclusion body in unmyelinated axons. Neurons were also involved as shown by immunohistochemical phenotypic switch. After washout, partial recovery was observed in functional, electrophysiological and histological analyses. These results suggest that axon and myelin changes might be secondary to an initial dysfunctional neuronopathy., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

Although injured peripheral axons are able to regenerate, functional recovery is usually poor after nerve transection. In this study we aim to elucidate the role of neuronal activity, induced by nerve electrical stimulation and by exercise, in promoting axonal regeneration and modulating plasticity in the spinal cord after nerve injury. Four groups of adult rats were subjected to sciatic nerve transection and suture repair. Two groups received electrical stimulation (3 V, 0.1 ms at 20 Hz) for 1 h, immediately after injury (ESa) or during 4 weeks (1 h daily; ESc). A third group (ES+TR) received 1 h electrical stimulation and was submitted to treadmill running during 4 weeks (5 m/min, 2 h daily). A fourth group performed only exercise (TR), whereas an untreated group served as control (C). Nerve conduction, H reflex and algesimetry tests were performed at 1, 3, 5, 7 and 9 weeks after surgery, to assess muscle reinnervation and changes in excitability of spinal cord circuitry. Histological analysis was made at the end of the follow-up. Groups that received acute ES and/or were forced to exercise in the treadmill showed higher levels of muscle reinnervation and increased numbers of regenerated myelinated axons when compared to control animals or animals that received chronic ES. Combining ESa with treadmill training significantly improved muscle reinnervation during the initial phase. The facilitation of the monosynaptic H reflex in the injured limb was reduced in all treated groups, suggesting that the maintenance of activity helps to prevent the development of hyperreflexia.

Peripheral nerve injuries usually lead to devastating loss of motor, sensory, and autonomic functions in the patients. Due to the complex requirements for adequate axonal regeneration, functional recovery is often poorly achieved. Experimental models are a useful tool to investigate the mechanisms related to axonal regeneration and reinnervation and to test new strategies to improve functional recovery. Therefore, objective and reliable evaluation methods should be applied for the assessment of regeneration and function restitution after nerve injury in animal models. Electrophysiological tests are commonly used in clinical practice and can be also performed in animal models to determine the nature of peripheral nerve disorders, their severity, and their evolution. These tests provide an integrated approach using sensory and motor nerve conduction studies and electromyography, spinal reflex tests, and motor and sensory evoked potentials if appropriate. The low-invasiveness of several electrophysiological methods allows serial evaluation of sensory and motor reinnervation distal to the injury site at desired intervals without killing the animals or disturbing the regeneration process. This chapter gives a brief review of the most useful electrophysiological methods, their values, and limitations.

We have studied whether electrical stimulation immediately after nerve injury may enhance axonal regeneration and modulate plastic changes at the spinal cord level underlying the appearance of hyperreflexia. Two groups of adult rats were subjected to sciatic nerve section followed by suture repair. One group (ES) received electrical stimulation (3 V, 0.1 ms at 20 Hz) for 1 h after injury. A second group served as control (C). Nerve conduction, H reflex, motor evoked potentials, and algesimetry tests were performed at 1, 3, 5, 7 and 9 weeks after surgery, to assess muscle reinnervation and changes in excitability of spinal cord circuitry. The electrophysiological results showed higher levels of reinnervation, and histological results a significantly higher number of regenerated myelinated fibers in the distal tibial nerve in group ES in comparison with group C. The monosynaptic H reflex was facilitated in the injured limb, to a higher degree in group C than in group ES. The amplitudes of motor evoked potentials were similar in both groups, although the MEP/M ratio was increased in group C compared to group ES, indicating mild central motor hyperexcitability. Immunohistochemical labeling of sensory afferents in the spinal cord dorsal horn showed prevention of the reduction in expression of substance P at one month postlesion in group ES. In conclusion, brief electrical stimulation applied after sciatic nerve injury promotes axonal regeneration over a long distance and reduces facilitation of spinal motor responses.

A lesion of a peripheral nerve before a second injury (conditioning lesion, CL), enhances peripheral and central regeneration of dorsal root ganglion (DRG) axons. This effect is mediated by elevated neuronal cAMP. Here we wanted to investigate whether electrical stimulation (ES) of an intact nerve, which has been shown to accelerate peripheral axon outgrowth, is also effective in promoting axon regeneration of injured DRG axons in vitro and of the central DRG axons in vivo and, whether this effect is mediated by elevation of cAMP. For the in vitro assay, the intact sciatic nerve of adult rats was stimulated at 20 Hz for 1 h, 7 days before harvest and primary culture of DRG neurons on a growth permissive substrate. In the in vivo study, the central axons of the lumbosacral DRGs were cut in the Th8 dorsal column, and the sciatic nerve was either cut or left intact, and subjected to 1 h ES at 20 Hz or 200 Hz. In vitro, ES increased neurite outgrowth 4-fold as compared to non-stimulated DRG neurons. In vivo, ES at 20 Hz significantly increased axon outgrowth into the central lesion site as compared to the Sham control. The 20 Hz ES was as effective as the CL in increasing axon outgrowth into the lesion site but not in promoting axonal elongation even though 20 Hz ES increased intracellular cAMP levels in DRG neurons as effectively as the CL. Thus elevation of cAMP may account for the central axonal outgrowth after ES and a CL.

We examined the effects of FK506 administration on the degree of target reinnervation by regenerating axons (following sciatic nerve crush) and by collateral sprouts of the intact saphenous nerve (after sciatic nerve resection) in the mouse. FK506-treated animals received either 0.2 or 5 mg/kg/day, dosages previously found to maximally increase the rate of axonal regeneration in the mouse. Functional reinnervation of motor, sensory, and sweating activities was assessed by noninvasive methods in the hind paw over a 1-month period following lesion. Morphometric analysis of the regenerated nerves and immunohistochemical labeling of the paw pads were performed at the end of follow-up. In the sciatic nerve crush model, FK506 administration shortened the time until target reinnervation and increased the degree of functional and morphological reinnervation achieved. The recovery achieved by regeneration was greater overall with the 5 mg/kg dose than with the dose of 0.2 mg/kg of FK506. In the collateral sprouting model, reinnervation by nociceptive and sudomotor axons was enhanced by FK506. Here, the field expansion followed a faster course between 4 and 14 days in FK506-treated animals. In regard to dose, while collateral sprouting of nociceptive axons was similarly increased at both dosages (0.2 and 5 mg/kg), sprouting of sympathetic axons was more extensive at the high dose. This suggests that the efficacy of FK506 varies between subtypes of neurons. Taken together, our findings indicate that, in addition to an effect on rate of axonal elongation, FK506 improves functional recovery of denervated targets by increasing both regenerative and collateral reinnervation.