Grant A. Robinson

Axotomized mouse retinal ganglion cells containing melanopsin show enhanced survival, but not enhanced axon regrowth into a peripheral nerve graft.

Melanopsin is found in only approximately 2% of mouse retinal ganglion cells (RGCs), making these RGCs uniquely and directly photosensitive. Given that the majority of RGCs die after axotomy and that grafting of a peripheral nerve to the eye provides a permissive environment for axon regrowth, the present study examined the survival and axonal regrowth of melanopsin-containing RGCs in mice. One month after optic nerve transection and grafting, RGCs with regrown axons were labeled from the grafts and retinae were processed to visualize melanopsin and TUJ1. Melanopsin-positive and negative RGCs were counted and compared to axotomized RGCs from ungrafted eyes and uninjured RGCs. Melanopsin-positive RGCs showed a 3-fold increase in survival rate compared to non-melanopsin RGCs. Despite this enhanced survival, melanopsin-containing RGCs did not show increased axon regrowth into nerve grafts.

Netrin-1 and Peripheral Nerve Regeneration in the Adult Rat

Axonal guidance during development of the nervous system is thought to be highly regulated through interactions of axons with attractive, repulsive, and trophic cues. Similar mechanisms regulate axonal regeneration after injury. The netrins have been shown to influence the guidance of several classes of developing axons. Although netrins have been implicated as axonal guidance cues in the developing peripheral nervous system, there has been no direct evidence of netrin-1 expression in either developing or adult peripheral nerve. The present study utilized competitive PCR and immunohistochemistry to demonstrate the localization of netrin-1 within adult rat sciatic nerve. The expression of netrin-1 mRNA and protein was compared for normal or regenerated sciatic nerve 2 weeks following either a crush or a transection and repair injury. The PCR data show that netrin-1 mRNA is normally expressed at low levels in peripheral nerve, and similar low levels are found 2 weeks following a crush injury. However, 2 weeks following nerve transection and repair there is approximately a 40-fold increase in netrin-1 mRNA levels. Immunohistochemistry data show that Schwann cells are the major source of netrin-1 protein in peripheral nerve. Our results suggest that netrin-1 mRNA levels are profoundly affected during peripheral nerve injury and regeneration. The localization of netrin-1 to Schwann cells suggests that this protein is strategically situated to influence axon regeneration in adult peripheral nerve.

The specificity of motor neurone regeneration (preferential reinnervation)

The major determinant of functional recovery after lesions in the peripheral nervous system is the accurate regeneration of axons to their original target end‐organs. Unfortunately, regenerating motor axons are often misrouted to sensory target end‐organs, and sensory axons formerly innervating skin are often misrouted to muscle. As such regeneration is robust, but often inaccurate, a better understanding of how regenerating axons reinnervate terminal pathways would be of fundamental interest to basic and clinical neuroscience. This review will consider the underlying cellular and molecular mechanisms that influence the accuracy of peripheral nerve regeneration, within the context of ‘preferential motor reinnervation’ (PMR). Much previous work with PMR has utilized the rodent femoral nerve and has shown that regenerating motor axons preferentially, albeit incompletely, reinnervate a distal terminal nerve branch to muscle (quadriceps) vs. skin (saphenous). One interpretation of this body of work has been that Schwann cell tubes have a specific identity that can be recognized by regenerating motor axons and that influences their subsequent behaviour. We disagree with that interpretation, and suggest motor and cutaneous pathways are not inherently different in terms of their ability to support regeneration of motor axons. In fact, recent experiments indicate under certain conditions motor axons will preferentially reinnervate the inappropriate terminal cutaneous pathway instead of the appropriate pathway to muscle. 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.

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.

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.

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. Muscle Nerve 28: 227–231, 2003

Schwann cell influence on motor neuron regeneration accuracy.

Extensive peripheral nerve injuries can result in the effective paralysis of the entire limb or distal portions of the limb. The major determinant of functional recovery after lesions in the peripheral nervous system is the accurate regeneration of axons to their original target end-organs. We used the mouse femoral nerve as a model to study motor neuron regeneration accuracy in terms of regenerating motor neurons projecting to their original terminal pathway to quadriceps muscle vs. the inappropriate pathway to skin. Using a variety of surgical manipulations and the selective removal of Schwann cells in the distal nerve via molecular targeting, we have examined the respective roles of end-organ influence (muscle) vs. Schwann cells in this model system. We found evidence of a hierarchy of trophic support that regulates motor neuron regeneration accuracy with muscle contact being the most potent, followed by the number or density of Schwann cells in the distal nerve branches. Manipulating the relative levels of these sources of influence resulted in predictable projection patterns of motor neurons into the terminal pathway either to skin or to muscle.

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.

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.

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.