Sarah K. Walsh

Practical considerations concerning the use of stem cells for peripheral nerve repair

In this review the authors intend to demonstrate the need for supplementing conventional repair of the injured nerve with alternative therapies, namely transplantation of stem or progenitor cells. Although peripheral nerves do exhibit the potential to regenerate axons and reinnervate the end organ, outcome following severe nerve injury, even after repair, remains relatively poor. This is likely because of the extensive injury zone that prevents axon outgrowth. Even if outgrowth does occur, a relatively slow growth rate of regeneration results in prolonged denervation of the distal nerve. Whereas denervated Schwann cells (SCs) are key players in the early regenerative success of peripheral nerves, protracted loss of axonal contact renders Schwann cells unreceptive for axonal regeneration. Given that denervated Schwann cells appear to become effete, one logical approach is to support the distal denervated nerve environment by replacing host cells with those derived exogenously. A number of different sources of stem/precursor cells are being explored for their potential application in the scenario of peripheral nerve injury. The most promising candidate, transplant cells are derived from easily accessible sources such as the skin, bone marrow, or adipose tissue, all of which have demonstrated the capacity to differentiate into Schwann cell-like cells. Although recent studies have shown that stem cells can act as promising and beneficial adjuncts to nerve repair, considerable optimization of these therapies will be required for their potential to be realized in a clinical setting. The authors investigate the relevance of the delivery method (both the number and differentiation state of cells) on experimental outcomes, and seek to clarify whether stem cells must survive and differentiate in the injured nerve to convey a therapeutic effect. As our laboratory uses skin-derived precursor cells (SKPCs) in various nerve injury paradigms, we relate our findings on cell fate to other published studies to demonstrate the need to quantify stem cell survival and differentiation for future studies.

Locally synthesized calcitonin gene-related Peptide has a critical role in peripheral nerve regeneration.

Abstract Regeneration of peripheral nerves involves complex and intimate interactions between axons and Schwann cells. Here, we show that local axon synthesis and action of the neuropeptide calcitonin gene-related peptide (CGRP) is critical for this collaboration. After peripheral sural sensory axon injury in rats, we observed an unexpectedly large proportion of axons that newly expressed CGRP during regeneration. Intense peptide expression accompanied local rises in &agr;CGRP mRNA in the nerve trunk, and there was evidence of transport of &agr;CGRP mRNA into regenerating axons, indicating intra-axonal peptide synthesis. Calcitonin gene-related peptide receptor and its receptor activity modifying protein were expressed onadjacent Schwann cells, where they were available for signaling. Moreover, exogenous CGRP induced proliferation in isolated adult Schwann cells. New axon outgrowth and CGRP expression depended on local peptide synthesis and were inhibited by exposure tolocal translation inhibitors. Local delivery of siRNAs to either &agr;CGRP or receptor activity modifying protein 1 to sites of nerve transection was associated with severe disruption of axon outgrowth.These findings indicate that robust localized intra-axonal translation of the CGRP neuropeptide during regeneration signals Schwann cell proliferation, behavior that is critical for partnering during adult peripheral nerve regrowth.

Collagen Nerve Conduits Promote Enhanced Axonal Regeneration, Schwann Cell Association, and Neovascularization Compared to Silicone Conduits

Peripheral nerve regeneration within guidance conduits involves a critical association between regenerating axons, Schwann cells (SCs), and neovascularization. However, it is currently unknown if there is a greater association between these factors in nonpermeable versus semipermeable nerve guide conduits. We therefore examined this collaboration in both silicone- and collagen-based nerve conduits in both 5- and 10-mm-injury gaps in rat sciatic nerves. Results indicate that collagen conduits promoted enhanced axonal and SC regeneration and association when compared to silicone conduits in the shorter 5-mm-gap model. In addition, collagen tubes displayed enhanced neovascularization over silicone conduits, suggesting that these three factors are intimately related in successful peripheral nerve regeneration. At later time points (1- and 2-month analysis) in a 10-mm-gap model, collagen tubes displayed enhanced axonal regeneration, myelination, and vascularization when compared to silicone-based conduits. Results from these studies suggest that regenerating cables within collagen-based conduits are revascularized earlier and more completely, which in turn enhances peripheral nerve regeneration through these nerve guides as compared to silicone conduits.

Skin-derived precursor cells enhance peripheral nerve regeneration following chronic denervation

While peripheral nerves demonstrate the capacity for axonal regeneration, outcome following injury remains relatively poor, especially following prolonged denervation. Since axon-deprived Schwann cells (SCs) in the distal nerve progressively lose their ability to support axonal growth, we took the approach of using skin-derived precursor cells (SKPs) as an accessible source of replacement SCs that could be transplanted into chronically denervated peripheral nerve. In this study, we employed a delayed cross-reinnervation paradigm to assess regeneration of common peroneal nerve axons into the chronically denervated rodent tibial nerve following delivery of SKP-derived SC (SKP-SCs). SKP-SC treated animals exhibited superior axonal regeneration to media controls, with significantly higher counts of regenerated motorneurons and histological recovery similar to that of immediately repaired nerve. Improved axonal regeneration correlated with superior muscle reinnervation, as measured by compound muscle action potentials and wet muscle weights. We therefore conclude that SKPs represent an easily accessible, autologous source of stem cell-derived Schwann cells that show promise in improving regeneration through chronically injured nerves.

Dose and duration of nerve growth factor (NGF) administration determine the extent of behavioral recovery following peripheral nerve injury in the rat

Nerve growth factor (NGF) has been previously shown to support neuron survival and direct neurite outgrowth in vitro, and to enhance axonal regeneration in vivo. However, a systematic analysis of NGF dose and dose duration on behavioral recovery following peripheral nerve injury in rodents has not been previously investigated. Here, we show that NGF promotes a bell shaped dose-response, with an optimal threshold effect occurring at 800 pg/μl. High dose NGF inhibited regeneration. However, this effect could be reversed through functional blockade of p75 receptors, thus implicating these receptors as mediators of the inhibitory response. Longer term evaluation showed that animals administered NGF at 80 ng/day for 3 weeks had greater sensorimotor recovery compared to all other treatment groups. These animals made significantly fewer errors during skilled locomotion, and displayed both increased vertical and fore-aft ground reaction forces during flat surface locomotion. Furthermore, terminal electrophysiological and myological assessments (EMG, wet gastrocnemius muscle weights) corroborated the behavioral data. Overall, these data support the hypothesis that both appropriate dose and duration of NGF are important determinants of behavioral recovery following nerve injury in the rat.

Use of stem cells to augment nerve injury repair.

OBJECTIVEThe purpose of this review is to summarize the basic science literature related to chronic nerve injuries, and to then use this as the background to provide emerging insights into the promising role of cellular therapy for nerve injury repair. METHODSThe literature pertinent to the experimental and clinical aspects of chronic nerve injury was reviewed, as was emerging literature and our own recent experience in using cellular therapy to repair injured nerves. RESULTSPeripheral nerves have the potential to regenerate axons and reinnervate end organs. Yet, outcome after peripheral nerve injury, even after nerve repair, remains relatively poor. The single most important quantitative contributor to poor motor recovery is chronic denervation of the distal nerve. Chronic denervation is common because of the often extensive injury zone that prevents any axonal outgrowth or (even if outgrowth occurs) the relatively slow rate of regeneration. As a consequence, the distal nerve remains chronically devoid of regrowing axons. In turn, prolonged denervation of Schwann cells (SCs) seems to be the critical factor that makes them unreceptive for axonal regeneration. Regenerative success was demonstrated when denervated SCs were replaced with healthy SCs cultured from a secondary nerve. This cell-replacement strategy is, however, limited in the clinical setting by the inability to obtain sufficient numbers of cells and the requirement for sacrifice of additional nerve tissue. We, along with several other groups, have therefore begun investigating stem cell therapies to improve the regenerative environment. CONCLUSIONThere are several avenues of stem cell-based approaches to peripheral nerve repair. One of these, skin-derived precursor cells, are easily accessible, autologous adult stem cells that can survive and myelinate in the peripheral nerve environment and become SC-like in their apparent differentiation.Injury to the peripheral nerve is common and debilitating. Such injuries can occur in the context of trauma, either blunt or penetrating, or in specific disorders of peripheral nerves known as neuropathies. For example, a severe peripheral nerve injury affects 2.8% of trauma patients (46), and approximately 360 000 people in the United States experience upper extremity peripheral nerve injury annually, resulting in 8 648 000 and 4 916 000 restricted-activity days and bed/disability days, respectively (25). The assumption has been that peripheral nerve injuries recover, as opposed to those of the central nervous system. Although peripheral nerves do regenerate better, recovery is frequently incomplete, misdirected, or associated with debilitating neuropathic pain (64). In particular, nerve transection is associated with notoriously poor outgrowth compared with crush or other injuries, especially when the distance between the injury and target (muscles, skin) is long. Regenerating axons face both a challenging nerve gap and a distal denervated nerve environment whose Schwann cells (SCs) become increasingly less able to support axon regeneration with prolonged denervation. Nerve injury gaps or scar within the nerve prevent regenerating axons from effectively innervating the distal nerve stump. These are currently managed with a repair of the divided nerve or, for the usual scenario of longer gaps or scar segments that need to be resected, placement of interposed nerve grafts (44). Nerve grafts provide a pathway for regenerating axons from the proximal nerve stump to innervate the distal stump (41). Unfortunately, recovery after nerve or nerve graft repair is limited by incomplete regeneration and variable clinical results (10). ABBREVIATIONS: BMSC, bone marrow stromal cell; GFAP, glial fibrillary acidic protein; SC, Schwann cell; SKP, skin-derived precursor; TGF, transforming growth factor USE OF STEM CELLS TO AUGMENT NERVE INJURY REPAIR

Behavioural and anatomical analysis of selective tibial nerve branch transfer to the deep peroneal nerve in the rat

Nerve transfer procedures involving the repair of a distal denervated nerve element with that of a foreign proximal nerve have become increasingly popular for clinical nerve repair as a surgical alternative to autologous nerve grafting. However, the functional outcomes and the central plasticity for these procedures remain poorly defined, particularly for a clinically relevant rodent model of hindlimb nerve transfer. We therefore evaluated the effect of selective tibial branch nerve transfer on behavioural recovery in animals following acute transection of the deep peroneal nerve. The results indicate that not only can hindlimb nerve transfers be successfully accomplished in a rat model but that these animals display a return of skilled locomotor function on a par with animals that underwent direct deep peroneal nerve repair (the current gold standard). At 2 months, ground reaction force analysis demonstrated that partial restoration of braking forces occurred in the nerve transfer group, whereas the direct repair group had fully restored these forces to similar to baseline levels. Ankle kinematic analysis revealed that only animals in the direct repair group significantly recovered flexion during the step cycle, indicating a recovery of surgically induced foot drop. Terminal electrophysiological and myological assessments demonstrated similar levels of reinnervation, whereas retrograde labelling studies confirmed that the peroneal nerve‐innervated muscles were innervated by neurons from the tibial nerve pool in the nerve transfer group. Our results demonstrate a task‐dependent recovery process, where skilled locomotor recovery is similar between nerve transfer and direct repair animals, whereas flat surface locomotion is significantly better in direct repair animals.

Fate of stem cell transplants in peripheral nerves

While damaged peripheral nerves demonstrate some potential to regenerate, complete functional recovery remains infrequent, owing to a functional loss of supportive Schwann cells distal to the injury. An emerging solution to improve upon this intrinsic regenerative capacity is to supplement injured nerves with stem cells derived from various tissues. While many of these strategies have proven successful in animal models, few studies have examined the behavior of transplanted stem cells in vivo, including whether they survive and differentiate. In previous work, we demonstrated that cells derived from neonatal rodent dermis (skin-derived precursor cells, or SKPs) could improve regenerative parameters when transplanted distal to both acute and chronic nerve injuries in Lewis rats. The aim of this work was to track the fate of these cells in various nerve injury paradigms and determine the response of these cells to a known glial growth factor. Here, we report that SKPs survive, respond to local cues, differentiate into myelinating Schwann cells, and avoid complete clearance by the hosts immune defenses for a minimum of 10weeks. Moreover, the ultimate fate of SKPs in vivo depends on the nerve environment into which they are injected and can be modified by inclusion of heregulin-1β.

Motoneuron survival after chronic and sequential peripheral nerve injuries in the rat

OBJECT Surgical repair of peripheral nerves following chronic nerve injury is associated with poor axonal regeneration and outcome. An underlying possibility is that chronic injuries may increase motoneuron cell death. The hypothesis that substantial motoneuron death follows chronic and sequential nerve injuries was tested in adult rats in this study. METHODS Thirty adult male Lewis rats underwent bilateral multistage surgeries. At initial surgery, Fast Blue (FB) tracer was injected at a nerve-crush injury site in the right control femoral motor nerve. The left femoral motor nerve was transected at the same level and either capped to prevent regeneration (Group 1), or repaired to allow axonal regeneration and reinnervation of the target quadriceps muscle (Group 2) (15 rats in each group). After 8 weeks in 6 rats/group, the left femoral nerve was cut and exposed to FB just proximal to prior nerve capping or repair and the rats were evaluated for FB-labeled motoneuron counts bilaterally in the spinal cord (this was considered survival after initial injury). In the remaining 9 animals/group, the left nerve was recut (sequential injury), exposed to FB, and repaired to a fresh distal saphenous nerve stump to permit axonal regeneration. Following another 6 weeks, Fluoro-Gold, a second retrograde tracer, was applied to the cut distal saphenous nerve. This allowed us to evaluate the number of motoneurons that survived (maintained FB labeling) and the number of motoneurons that survived but that also regenerated axons (double labeled with FB and Fluoro-Gold). RESULTS A mean number of 350 and 392 FB-labeled motoneurons were found after 8 weeks of nerve injury on the right and the left sides, respectively. This indicated no significant cell death due to initial nerve injury alone. A similar number (mean 390) of motoneurons were counted at final end point at 14 weeks, indicating no significant cell death after sequential and chronic nerve injury. However, only 50% (mean 180) of the surviving motoneurons were double labeled, indicating that only half of the population regenerated their axons. CONCLUSIONS The hypothesis that significant motoneuron cell death occurs after chronic and or sequential nerve injury was rejected. Despite cell survival, only 50% of motoneurons are capable of exhibiting a regenerative response, consistent with our previous findings of reduced regeneration after chronic axotomy.

A novel method for establishing daily in vivo concentration gradients of soluble nerve growth factor (NGF)

Despite the capacity for spontaneous axonal regeneration, recovery following injuries to the peripheral nervous system (PNS) following transection are often incomplete and limited to short distances. Nerve growth factor (NGF) has been previously shown to support neuron survival, and direct growth of both developing and regenerating nerve fibers along a concentration gradient, based largely on in vitro studies. Here, we present a novel in vivo model of administering daily concentration gradients of NGF by directly manipulating the placement of the catheter-nerve conduit junction. Our results show that a dose of 800 pg NGF can be reliably used to establish a chemotactic concentration gradient over both a transient time period, and chronically through repeated daily administrations of the drug. Results from these studies may lead to a better mechanistic understanding of how concentration gradients of soluble NGF influence in vivo peripheral nerve regeneration.