Muscle-Derived Extracellular Vesicles Influence Motor Neuron Regeneration Accuracy.

Extracellular vesicles are lipid bilayer-enclosed extracellular structures. Although the term extracellular vesicles is quite inclusive, it generally refers to exosomes (<200 nm), and microvesicles (~100-1000 nm). Such vesicles are resistant to degradation and can contain proteins, lipids, and nucleic acids. Although it was previously thought that the primary purpose of such vesicles was to rid cells of unwanted components, it is now becoming increasingly clear that they can function as intercellular messengers, sometimes operating over long distances. As such, there is now intense interest in extracellular vesicles in fields as diverse as immunology, cell biology, cancer, and more recently, neuroscience. The influence that such extracellular vesicles might exert on peripheral nerve regeneration is just beginning to be investigated. In the current studies we show that muscle-derived extracellular vesicles significantly influence the anatomical accuracy of motor neuron regeneration in the rat femoral nerve. These findings suggest a basic cellular mechanism by which target end-organs could guide their own reinnervation following nerve injury.

Polyethylene glycol fusion repair prevents reinnervation accuracy in rat peripheral nerve.

Functional recovery following a peripheral nerve injury is made easier when regenerating axons correctly reinnervate their original targets. Polyethylene glycol (PEG) has recently been used in attempts to fuse severed peripheral axons during suture-based repair, but an analysis of target selectivity following such repair has not been undertaken. The rat femoral nerve (in which muscle and cutaneous pathways comingle proximally but segregate distally into separate terminal nerve branches) is a convenient in vivo model for assessing motor neuron regeneration accuracy. The present study uses retrograde labeling of motor neurons to compare reinnervation accuracy after suture-based nerve repair with and without PEG fusion. The results show that adding PEG to the suture repair site blocked the preference of motor neurons to reinnervate correctly the distal terminal nerve branch to muscle that was seen with suture repair. Retrograde transport and diffusion studies also determined that PEG fusion allowed passage of probes across the repair site, as has previously been seen, but did not result in motor neuron labeling in the spinal cord. The results suggest that PEG fusion disrupts the beneficial trophic influence of muscle on motor neuron reinnervation accuracy normally seen after suture repair and that such fusion-based approaches may be best suited to nerve injuries in which accurate target reinnervation at the terminal nerve branch level is not a priority. © 2016 Wiley Periodicals, Inc.

Extracellular vesicles from a muscle cell line (C2C12) enhance cell survival and neurite outgrowth of a motor neuron cell line (NSC-34).

INTRODUCTION: There is renewed interest in extracellular vesicles over the past decade or 2 after initially being thought of as simple cellular garbage cans to rid cells of unwanted components. Although there has been intense research into the role of extracellular vesicles in the fields of tumour and stem cell biology, the possible role of extracellular vesicles in nerve regeneration is just in its infancy. BACKGROUND: When a peripheral nerve is damaged, the communication between spinal cord motor neurons and their target muscles is disrupted and the result can be the loss of coordinated muscle movement. Despite state-of-the-art surgical procedures only approximately 10% of adults will recover full function after peripheral nerve repair. To improve upon such results will require a better understanding of the basic mechanisms that influence axon outgrowth and the interplay between the parent motor neuron and the distal end organ of muscle. It has previously been shown that extracellular vesicles are immunologically tolerated, display targeting ligands on their surface, and can be delivered in vivo to selected cell populations. All of these characteristics suggest that extracellular vesicles could play a significant role in nerve regeneration. METHODS: We have carried out studies using 2 very well characterized cell lines, the C2C12 muscle cell line and the motor neuron cell line NSC-34 to ask the question: Do extracellular vesicles from muscle influence cell survival and/or neurite outgrowth of motor neurons? CONCLUSION: Our results show striking effects of extracellular vesicles derived from the muscle cell line on the motor neuron cell line in terms of neurite outgrowth and survival.

In vitro electrophoresis and in vivo electrophysiology of peripheral nerve using DC field stimulation.

BACKGROUND: Given the movement of molecules within tissue that occurs naturally by endogenous electric fields, we examined the possibility of using a low-voltage DC field to move charged substances in rodent peripheral nerve in vitro. NEW METHOD: Labeled sugar- and protein-based markers were applied to a rodent peroneal nerve and then a 5-10 V/cm field was used to move the molecules within the extra- and intraneural compartments. Physiological and anatomical nerve properties were also assessed using the same stimulation in vivo. RESULTS: We demonstrate in vitro that charged and labeled compounds are capable of moving in a DC field along a nerve, and that the same field applied in vivo changes the excitability of the nerve, but without damage. CONCLUSIONS: The results suggest that low-voltage electrophoresis could be used to move charged molecules, perhaps therapeutically, safely along peripheral nerves.

Motor neuron target selectivity and survival after prolonged axotomy.

PURPOSE: After a cut peripheral nerve is repaired, motor neurons usually regenerate across the lesion site, however they often enter an inappropriate Schwann cell tube and may be directed to an inappropriate target organ such as skin, resulting in continued loss of function. In fact, only about 10% of adults who receive a peripheral nerve repair display full functional recovery. The reasons for this are many and complex, however one aspect is whether the motor neuron has undergone a prolonged period of axotomy prior to nerve repair. Previous studies have suggested a deleterious effect of prolonged axotomy. METHODS: We examined the influence of prolonged axotomy on target selectivity using a cross-reinnervation model of rat obturator motor neurons regrowing into the distal femoral nerve, with its normal bifurcating pathways to muscle and skin. RESULTS: Surprisingly, we found that a prolonged period of axotomy resulted in an increase in motor neuron regeneration accuracy. In addition, we found that regeneration accuracy could be increased even further by a simple surgical manipulation of the distal terminal nerve pathway to skin. CONCLUSIONS: These results suggest that under certain conditions prolonged axotomy may not be detrimental to the final accuracy of motor neuron regeneration and highlight that a simple manipulation of terminal nerve pathways may be one approach to increase such regeneration accuracy.

Polysialic acid expression is not necessary for motor neuron target selectivity.

INTRODUCTION: Recovery after peripheral nerve lesions depends on guiding axons back to their targets. Polysialic acid upregulation by regrowing axons has been proposed recently as necessary for this target selectivity. METHODS: We reexamined this proposition using a cross-reinnervation model whereby axons from obturator motor neurons that do not upregulate polysialic acid regenerated into the distal femoral nerve. Our aim was to assess their target selectivity between pathways to muscle and skin. RESULTS: After simple cross-repair, obturator motor neurons showed no pathway preference, but the same repair with a shortened skin pathway resulted in selective targeting of these motor neurons to muscle by a polysialic acid-independent mechanism. CONCLUSION: The intrinsic molecular differences between motor neuron pools can be overcome by manipulation of their access to different peripheral nerve pathways such that obturator motor neurons preferentially project to a terminal nerve branch to muscle despite not upregulating the expression of polysialic acid.

Influence of terminal nerve branch size on motor neuron regeneration accuracy.

A necessary prerequisite for recovery of motor function following a peripheral nerve injury is the correct choice by regenerating motor neurons to reinnervate the original distal nerve branch to denervated muscle. The present studies use the mouse femoral nerve as a model system to examine factors that influence such motor neuron regeneration accuracy. We examined motor reinnervation accuracy over time in this model under two conditions: 1) when the two terminal nerve branches to either skin (cutaneous) or muscle (quadriceps) were roughly comparable in size, and 2) when the cutaneous branch was larger than the muscle branch. When the terminal nerve branches were similar in size, motor neurons initially projected equally into both branches, but over time favored the terminal muscle branch. When the cutaneous terminal nerve branch was enlarged (via transgenic technology), motor neuron projections significantly favored this inappropriate pathway during early time points of regeneration. These results suggest that regenerating motor neuron projections are not determined by inherent molecular differences between distal terminal nerve branches themselves. Rather, we propose a two-step process that shapes motor neuron reinnervation accuracy. Initial outgrowth choices made by motor axons at the transection site are proportional to the relative amount of target nerve associated with distal nerve axons that previously projected to each of the terminal nerve pathways. Secondly, the likelihood of an axon collateral from a motor neuron remaining in either terminal nerve branch is based upon the relative trophic support provided to the parent motor neuron by the competing terminal pathways and/or end-organs.

Motor neuron regeneration accuracy: balancing trophic influences between pathways and end-organs.

The key to recovery of function following peripheral nerve lesions is guiding axons back to their original target end-organs. The parent femoral nerve splits into two comparable terminal pathways: one to the muscle and the other to the skin. Normally, motor neurons only innervate the pathway to the muscle, but after the parent nerve is repaired regenerating motor neurons are often misrouted to the skin. When the muscle and skin pathways remain connected to their respective targets after the parent nerve is repaired, reinnervation favors the muscle pathway. If contact with the muscle is instead prevented, reinnervation favors the pathway to the skin. Here we examine whether shortening the skin pathway can alter motor reinnervation accuracy when the muscle pathway remains connected to the muscle. We demonstrate that reducing the influence of the skin pathway results in a more rapid and extensive reinnervation of the muscle pathway. These findings suggest that the relative balance of trophic influences from the pathways and their end-organs is an important determinant of motor neuron regeneration accuracy, and that the muscle pathway by itself is not the primary regulator for regeneration accuracy of motor neurons.

Developmentally regulated changes in femoral nerve regeneration in the mouse and rat.

The attractive influence of muscle on regenerating motor neuron axons is well-known. Less is known, however, about the intrinsic abilities of different nerve pathways to support these axons prior to end-organ contact. The age at which a nerve injury is sustained is also known to affect the relationship between regenerating motor axons and muscle. The femoral nerve model, with its distinct muscle and cutaneous pathways, is ideal to study intrinsic pathway properties because the influence of end-organs can easily be removed surgically. However, recent results using this model in adult mice are at odds with the same model in neonatal rats. To reconcile these discrepancies, we used the femoral nerve model to examine possible age differences in intrinsic pathway support for regenerating motor neurons in the mouse and rat. Rat motor neurons showed a preference to regenerate into the muscle pathway after axotomy at 3 weeks of age, but this preference was lost after axotomy at 6 weeks of age. Interestingly, mouse motor neurons showed no pathway preference after axotomy at 3 weeks of age but developed one for the cutaneous pathway after axotomy at 6 weeks of age. These results suggest that in the absence of end-organ contact there is no general preference for motor neurons to project to the muscle pathway.

Manipulations of the mouse femoral nerve influence the accuracy of pathway reinnervation by motor neurons.

Previous studies using the femoral nerve model in both mice and rats have shown that regenerating motor axons prefer to reinnervate the terminal nerve branch to muscle versus a terminal nerve branch to skin, a process that has been termed preferential motor reinnervation (PMR). If end organ contact with muscle and skin is prevented, this preferential motor reinnervation still occurs in the rat. To better understand the process of preferential motor reinnervation in the mouse, we examined motor neuron reinnervation of muscle and cutaneous pathways without any end organ contact as well as with only cutaneous end organ contact. Surprisingly, there was no preferential motor reinnervation: Motor neurons preferred the cutaneous pathway over the muscle pathway when all end organ contact was prevented and showed an even greater preference for the cutaneous pathway when it was attached to skin.

Motor neurons can preferentially reinnervate cutaneous pathways.

Previous work in the rat femoral nerve has shown that regenerating motor neurons preferentially reinnervate a terminal nerve branch to muscle as opposed to skin. This process has been termed preferential motor reinnervation (PMR) and has been interpreted as evidence that regenerating motor axons can differentiate between Schwann cell tubes that reside in muscle versus cutaneous terminal pathways. However, much of this previous work has been confounded by motor axons having access to target muscle during the regeneration period. The present experiments prevented muscle contact by regenerating motor axons. By 8 weeks under these conditions, significantly more motor neurons reinnervated the cutaneous pathway rather than the original muscle pathway. We propose that cutaneous and muscle terminal pathways are not inherently different in terms of their ability to support regeneration of motor neurons. Rather, we suggest that it is the relative level of trophic support provided by each nerve branch that determines whether motor axons will remain in that particular branch. Within the context of the femoral nerve model, our results suggest a hierarchy of trophic support for regenerating motor axons with muscle contact being the highest, followed by the length of the terminal nerve branch and/or contact with skin.

Preferential motor reinnervation in the mouse: comparison of femoral nerve repair using a fibrin sealant or suture.

Previous studies in rat femoral nerve demonstrated that regenerating motor axons preferentially reinnervate a nerve branch to muscle as opposed to skin, a process that has been termed preferential motor reinnervation (PMR). This process has not been previously reported in the mouse, where the use of transgenic animals could be a powerful tool to study the basic mechanisms that determine accuracy of regenerating motor axons. In the mouse, we applied the same nerve repair (suture) and retrograde labeling strategies that successfully demonstrated PMR in the rat femoral nerve but surprisingly were unable to demonstrate PMR. However, if the mouse femoral nerve was repaired with a fibrin sealant, PMR was readily apparent, suggesting that PMR in the mouse is dependent on the method of nerve repair.