Surgical repair in humans after traumatic nerve injury provides limited functional neural regeneration in adults

Accepted Manuscript Surgical repair in humans after traumatic nerve injury provides limited functional neural regeneration in adults

Winnie A. Palispis, Ranjan Gupta PII: DOI: Reference:

S0014-4886(17)30018-3 doi: 10.1016/j.expneurol.2017.01.009 YEXNR 12464

To appear in:

Experimental Neurology

Received date: Revised date: Accepted date:

2 October 2016 18 January 2017 18 January 2017

Please cite this article as: Winnie A. Palispis, Ranjan Gupta , Surgical repair in humans after traumatic nerve injury provides limited functional neural regeneration in adults. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Yexnr(2017), doi: 10.1016/j.expneurol.2017.01.009

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ACCEPTED MANUSCRIPT Surgical Repair in Humans After Traumatic Nerve Injury Provides Limited Functional Neural Regeneration in Adults

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Winnie A. Palispisa,b, MD & Ranjan Guptaa,b,c, MD a Department of Orthopaedic Surgery, University of California, Irvine, Orange, California, USA b Peripheral Nerve Research Lab, Gillespie Neuroscience Research Facility, Irvine, California, USA c VA Long Beach Healthcare System, Long Beach, CA 90822, USA

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Research Highlights  Surgical intervention alone does not provide satisfactory nerve repair results  Injured nerves encounter barriers that limit their ability to regenerate  Denervation atrophy of target tissue is associated with peripheral nerve repair and regeneration

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Abstract

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Traumatic nerve injuries result in devastating loss of neurologic function with unpredictable functional recovery despite optimal medical management. After traumatic nerve injury and denervation, regenerating axons must traverse a complex environment in which they encounter numerous barriers on the way to reinnervation of their target muscle. Outcomes of surgical intervention alone have unfortunately reached a plateau, resulting in often unsatisfactory functional recovery. Over the past few decades, many improvements were developed to supplement and boost the results of surgical repair. Biological optimization of Schwann cells, macrophages, and degradation enzymes have been studied due to the key roles of these components in axonal development, maintenance and response to injury. Moreover, surgical techniques such as nerve grafting, conduits, and growth factor supplementation are also employed to enhance the microenvironment and nerve regeneration. Yet, most of the roadblocks to recovery after nerve injury remain unsolved. These roadblocks include, but are not limited to: slow regeneration rates and specificity of target innervation, the presence of a segmental nerve defect, and degeneration of the target end-organ after prolonged periods of denervation. A recognition of these limitations is necessary so as to develop new strategies to improve functional regeneration for these life changing injuries. Keywords Peripheral nerve injury, neural regeneration, target end-organ atrophy, neuromuscular junctions; neural agrin Abbreviations NMJ, neuromuscular junctions; AChRs, acetylcholine receptors; PNS, peripheral nervous system; SC, Schwann cells; ECM, extracellular matrix

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Introduction Traumatic nerve injuries are severe injuries that occur in up to 2.8% of all polytrauma patients (Noble, Munro, Prasad, & Midha, 1998). These injuries have life-altering impact on patients, causing them to suffer months, or even years, of uncertainty while waiting for an often unpredictable, marginal level of recovery (Grinsell and Keating, 2014; Kang et al., 2011). Patients may be left with devastating and disabling sensory and motor deficits such as numbness of a limb, cold intolerance, dysethesias, paralysis, and neuropathic pain. The management of traumatic nerve injuries is influenced by multiple factors including the location of the injury, the type of injury, the size of segmental nerve deficit, the timing of injury presentation, and accompanying soft tissue injury. Despite the permissive growth environment of the peripheral nervous system (PNS), major nerve injuries in humans have very limited potential for spontaneous recovery. On a molecular level, both physiological and histopathological changes to the nerve and its surrounding soft tissue occur, including demyelination, degeneration, remyelination and regeneration. Despite a significant amount of research addressing the molecular biology of nerve injury, and numerous surgical advances in peripheral nerve repair, these improvements have achieved only partial recovery of the affected limb, with plateauing of functional outcomes. In a recent meta-analysis of 2,997 digital nerve repairs, good outcomes were seen with respect to sensory recovery in just 42% of repairs, with excellent outcomes seen in only 25% of repairs (Paprottka et al., 2013). The regenerating nerve encounters numerous unsolved obstacles that need to be overcome to allow for efficient and effective neural regeneration. Many unexplored barriers to regrowth remain unaddressed by current practice; these include the rate of regeneration, specificity of regeneration, segmental nerve defects and degeneration of the target-end organ.

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Microanatomy Peripheral nerves are heterogeneous composite structures that are comprised of neurons, Schwann cells, fibroblasts and macrophages, and are heavily dependent on a complex blood supply. The neuron itself is a polarized cell that forms the foundation of the nerve. It consists of dendrites, the cell body, and a single axon. The axon originates from a unique region of the cell body called the axon hillock, which is also responsible for initial generation of the action potential. Axons project towards their sites of innervation, where they form synapses with target end-organs. Schwann cells produce myelin to encapsulate the axon and aid in action potential transmission. If the axonal diameter is greater than or equal to 1 µm, each Schwann cell will wrap its plasma membrane around a single region of an axon, thereby forming myelin. Myelin acts as an insulator, allowing fast and efficient conduction and propagation of an action potential down an axon. The blood supply to the nerve is a complex vascular plexus formed from anastomoses of epineural, perineural and endoneurial plexi (Yegiyants et al., 2010) as well as a segmental blood supply derived from a number of nutrient arteries. The blood supply to the nerve is quite fragile, and may be disrupted due to trauma or to tension during nerve repair. In addition, peripheral nerves have connective tissue layers that provide strength and protection to the nerve: namely, the epineurium, perineurium, and endoneurium. It is crucial to recognize that all surgical interventions are strictly directed at these connective tissue layers, leaving the axon and Schwann cells to respond to injury and regenerate via their inherent biology. Nerve response to injury In contrast to chronic nerve injuries which are Schwann cell driven, acute nerve injuries are axonally mediated. In the initial stages of injury, Wallerian degeneration begins with granular disintegration of the axonal cytoskeleton. Within 48 hours of injury, Schwann cells (SC) break down myelin and phagocytose axonal debris from the distal stump. Macrophages are then recruited to the area, which release growth factors that in turn encourage SC and fibroblast proliferation. SCs begin the reparative process by forming longitudinal bands of Bungner, which

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are essentially growth-promoting conduits for regenerating axons. Injection of pre-differentiated SCs near injured nerves has been shown to aid remyelination in regenerating axons and reduce the amount of myelin debris, thereby improving functional recovery in rodents (Khuong et al., 2014). At the tip of the regenerating axon is the growth cone, which is composed of cellular matrix from which fingerlike projections called filopodia extrude to explore the microenvironment. Proteases, under the influence of various factors, are released from the growth cone to clear a path towards a target organ. Simulataneously, SCs upregulate neurotrophic factors including nerve growth factor (NGF) and brain-derived growth factor (BDNF), as well as their corresponding receptors in the distal stumps (Flores et al., 2000; Stoll and Müller, 1999). This increase in expression of NGF and its low density receptors is believed to promote extensive proliferation and migration of SCs (Anton et al., 1994) and mainly affects properties of sensory neurons. BDNF levels are also increased and are postulated to act as an anterograde trophic messenger under the influence of NGF. Interestingly, the neurotrophic factor ciliary neurotrophic factor (CNTF) which is believed to affect survival and regeneration of motor neurons, is found to be reduced significantly in the SCs of the distal stump, with the reduction in CNTF levels extending to the neuromuscular junction (Hiruma et al., 1997). Furthermore, there is increased retrograde axonal transport of CNTF after nerve injury (Curtis et al., 1993). Neurite-promoting factors such as laminin and fibronectin, and matrix-forming precursors such as fibrinogen, are all synthesized in response to nerve injury (Yegiyants et al., 2010). In addition to this complex millieu of pro- and anti-neurotrophic factors, microtubulin is another molecule that plays a crucial role with respect to axonal integrity and regeneration. Following traumatic nerve injury, calcium-dependent activation of the histone deacetylases HDAC5 and HDAC6, in particular, leads to tubulin degeneration that likely serves to inhibit axonal regeneration (Cho and Cavalli, 2012; Rivieccio et al., 2009). Interaction between axons and SCs has also emerged as an important regulator of peripheral nervous system development and regeneration. Fleming et al. identified that the receptor tyrosine kinase Ret genetically interacts with Er81 to control Nrg1-Ig in promoting the formation of Pacinian corpuscles (Fleming et al., 2016). Taken together, these factors have the potential to promote regeneration and to provide signaling for cell survival, neuronal differentiation and proliferation, as well as to influence synaptic function (Rummler and Gupta, 2004). Neurons in the peripheral nervous system also upregulate a number of regenerationassociated genes (RAGs) that may have direct role in neurite outgrowth following peripheral nerve injury. For example, the overexpression of transcription factor ATF-3 has been shown to promote neurite outgrowth after peripheral nerve injury (Seijffers et al., 2006). In their animal study, Bomze et al. concluded that growth-associated protein 43 (GAP-43) and cytoskeletonassociated protein 23 (CAP 23) were expressed after nerve injury, and together, were able to induce a dramatic increase in the number of regenerated axons (Bomze et al., 2001). Pathways associated with optimization of regeneration after nerve injury have also been identified. The ERK pathway was shown to mediate axonal elongation, with the kinases ERK and Akt promoting regeneration after axonal injury (Chierzi et al., 2005). Additionally, the cytokine interleukin-6 has been shown to work through the JAK-STAT3 pathway to overcome some of the inhibitory molecules that inhibit axonal regeneration (Cao et al., 2006). In addition to pathways that are involved in axonal regeneration, there are also pathways that have been associated with inhibition of axonal regeneration. The small GTPase Rho signaling pathway, for instance, has a role in cytoskeletal reorganization and cell motility. Studies have shown that activation of Rho results to collapse of growth cones, and inhibiting Rho pathway allows for contractility and promotes neurite outgrowth (Jalink et al., 1994; Wahl et al., 2000). Surgical Repair

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The current mainstay of treatment for traumatic peripheral nerve injury involves surgical intervention. The primary goal of nerve repair is to correctly align and approximate severed nerve segments to allow reinnervation of the target organs with hopes of achieving functional recovery. Historically, it was thought best to wait 3 weeks for the completion of the Wallerian degeneration process before nerve repair. However, studies by Fu (Fu and Gordon, 1995) and Mackinnon (Mackinnon, 1989) suggested that immediate repair produced better outcomes. Major prerequisites to nerve repair include a clean wound, viable blood supply, no crush component to the injured nerve, adequate soft tissue coverage, skeletal stability, and minimal tension on the nerve repair. The single most important technical factor in successful peripheral nerve repair has long been regarded to be coaptation of nerve tissue without tension (McDonald and Bell, 2010; Millesi, 1985). Epineurial repair is the traditional method of repair for severed peripheral nerves. Successful repair entails proper rotational alignment without any tension. This can be achieved by using external markers (i.e vessels), or by matching mirror images of the fascicular pattern in the proximal and distal ends. Monofilament nylon suture is preferred for repair because of its ease of use and minimal foreign-body reactivity (Lee & Wolfe, 2000). The number of sutures needed depends on the thickness of the nerve; for instance, a digital nerve may only require 2-3 sutures, whereas a median nerve may require up to 10 sutures. While the epineurial suture technique is considered relatively atraumatic, it does not ensure appropriate matching of the deeper fascicular structures of the nerve. As a result, some believe that results are better with grouped fascicular repair, with sutures placed through the internal epineurium or perineurium due to more accurate axonal alignment. Using this method, each group of fascicle in the proximal nerve stump is coapted to its corresponding group in the distal nerve (Rowshan et al., 2004). In theory, a group fascicular repair offers a more accurate technique of repair as it ensures correct orientation of the regenerating axons. Different techniques have been reported to intraoperatively verify correct fascicular matching for both immediate and delayed primary nerve repairs. Electrical stimulation can be used to identify sensory fascicles by inducing sensation in an awake patient. Alternatively, nerve endings can be stained to help differentiate between motor and sensory axons. Motor neurons can be stained with cholinesterase, while sensory neurons can be stained with carbonic anhydrase (Sanger et al., 1991). While much has been written about these stains, neither technique has been widely adopted in clinical practice. Despite the theoretical advantage of fascicular matching, there is limited clinical data to support such claims as group fascicular repair may actually increase trauma to the nerve due to damage to the blood supply and increased scar formation as a result of additional fascicle manipulation and the presence of permanent sutures. In addition, repair of fascicular sutures alone do not adequately resist tension and thus can only be utilized as an adjunct (Lundborg, 1987). To date, there have been no randomized clinical trials to compare epineurial and group fascicular repair in humans; hence, no concrete evidence exists that group fascicular repair is superior to simple epineurial repair when it comes to functional outcomes (Lundborg, 2000). Although epineurial alignment is less precise, the less stringent repair may permit the neurotrophic effects described above to influence the direction of axonal sprouting (Rowshan et al., 2004). Tension at the site of nerve repair leads to poor outcomes as localized glial scar forms rather than permitting neural regeneration. With any injury that has a crush component, a segment of the nerve must be excised and bridged with autologous nerve grafts. Autografts remain the gold standard as they contain host SCs and neurotrophic growth factors, and lack immunogenicity (Ray and Mackinnon, 2010). The sural nerve is considered the workhorse autograft material as it is easy to harvest, provides length of up to 40 cm when taken from both legs, provides an appropriate diameter for grafting, and is relatively dispensable. Other autograft sources include the medial antebrachial nerve, superficial radial sensory nerve and the femoral lateral cutaneous nerve. Nerve autografts are classified into single, cable, trunk, interfascicular

ACCEPTED MANUSCRIPT or vascularized. A single graft joins nerve gaps with a segment of a donor nerve that is of similar diameter. Cable grafts are multiple small-caliber nerve grafts that span a gap between fascicular groups. True vascularized grafts are mainly utilized in irreversible brachial plexus injuries with the ulnar nerve as the donor nerve of the graft. While there was hope that there would be significant improvement in results when vascularized grafts are used, newer studies report that outcomes are no superior to well-performed non-vascularized grafts. Processed nerve allografts are also a newer option that have been utilized with some success, but result in donor site morbidity and immunogenicity risks.

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Results of Surgical Repair While early studies detailed poor functional results after surgical repair, advances in microsurgical technology and refinement of surgical techniques have led to more promising outcomes of nerve repair. Overall results; however, still remain unimpressive. In a study by Wood of 11 peroneal nerve reconstructions, 9 were treated with grafts and 2 with direct neurorrhaphy. In the nerve graft treated group, results were reported as fair in 3 patients and poor in 2 patients (Wood, 1991). Kallio et al. looked at a series of primary repairs and fascicular grafts in 132 patients with median nerve injuries. In their study, 48% of the patients that were grafted and 50% of those treated with neurorrhaphy reported good to excellent results. Overall, 53 (40%) of the 132 patients had poor results (Kallio and Vastamaki, 1993). Vastamaki et al. assessed 110 patients with injuries to the ulnar nerve after secondary nerve repair and fascicular grafting and found useful recovery in only 51.8% of the patients (Vastamaki et al., 1993). The criteria to determine meaningful recovery has been determined in literature and is defined as return of motor function to M3 or greater, and sensory recovery to S3 or greater. Unfortunately this method of assessing recovery is fraught with observer bias and significant variability. Processed nerve allografts (i.e., Avance, AxoGen, Alachua) consist of human nerve tissue that has been rendered acellular. While allografts may induce immunogenic host response, the processed nerve grafts also many contain beneficial characteristics of nerve autografts such as physical microarchitecture and protein components that are inherent to nerve tissue (Brooks et al., 2012; Cho et al., 2012). While processed nerve allografts are acellular, they are able to revascularize and repopulate with host cells thus providing an environment that is conducive for regeneration (Cho et al., 2012). Despite their popularity secondary to ease of use and lack of donor site morbidity, allograft use is typically limited to severe nerve injuries as they have not been shown to be superior to, and at best are equivalent to, autografts. In a multicenter study looking at reconstruction with processed nerve allografts in 132 individual nerve injuries, meaningful recovery was reported in 87% of the repairs, with a 5% revision rate (Brooks et al., 2012). Critical analysis of these data revealed that the best results were seen with digital nerves, i.e. pure sensory nerves with limited gapping. In a study by Kato el al involving low median or ulnar nerve repairs, satisfactory sensory improvement (S3+ or S4 function) was obtained in 29 nerves (78%), with M4 or M5 motor functions achieved in 29 nerves (78%) (Kato et al., 1998). Cho et al. analyzed the outcomes for upper extremity nerve repairs listed in the RANGER Study database and reported an overall recovery of 86%. In this study that consisted of 71 nerves repaired with processed nerve allografts, they found meaningful recovery in 89% of digital nerve repairs, 75% of median nerve repairs and 67% of ulnar nerve repairs. While those authors concluded that the use of processed nerve allografts could be effectively be used in nerve gaps of 5 to 50 cm in length (Cho et al., 2012), this claim has not been widely accepted in clinical practice. With defects of short distance, nerve conduits can be used to provide the framework for nerve regeneration, particularly for nerve defects of less than 3 cm (Agnew and Dumanian, 2010; Chiu and Strauch, 1990; Weber et al., 2000). Conduits have emerged as alternatives to autografts, but their use has mainly been tested in small diameter defects (Moore et al., 2009)

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and smaller caliber nerves. Lundborg initiated a clinical prospective randomized study comparing tubular nerve repair and conventional microsurgical repair in patients with median or ulnar nerve injuries. They reported significant initial axonal growth in the tubular group 3 months after surgery, but at 1- year follow-up, there were no noted differences in sensory and motor recovery between the tubular and conventional repair group (Lundborg, Rosen, Dahlin, Danielsen, & Holmberg, 1997). At the 5-year follow up study, they similarly reported no significant difference in sensory and motor recovery, except that cold intolerance was significantly less prominent in the tubular repair group. As such, they concluded that tubular repair of the median and ulnar nerves is at least as good as conventional nerve repair (Lundborg, Rosén, Dahlin, Holmberg, & Rosén, 2004). A randomized, controlled trial by Boeckstyns et al. comparing collagen conduits to conventional neurorrhaphy in 43 patients with ulnar or median nerve injury found that conduit use reduced operation time compared to neurorrhaphy without significant differences in infection rates, conduction velocities, or total Rosen scores 24 months after surgery (Boeckstyns et al., 2013). Some studies have noted limitations with conduit use, however. Wangensteen et al. studied 126 repairs using collagen conduits and reported 2-point discrimination improvement in only 24% of the repairs (Wangensteen and Kalliainen, 2010). The mixed success of functional recovery may stem from the major limitations of conduits for segmental nerve defects. In another study, 75% of collagen tube conduit repairs provided meaningful recovery; however, the patients with a gap of greater than 15 millimeters failed to recover sensation (Lohmeyer et al., 2009). Individual case studies have also highlighted the limited efficacy of nerve conduits for the repair of large-diameter nerves (Moore et al., 2009) and for large segmental defects (Berrocal, Almeida, & Levi (2013). These limitations are likely a result of deficient Schwann cell migration, which is important for the regeneration of long segmental defects in animal models (Berrocal, Almeida, Gupta, & Levi, 2013). Traction injuries frequently involve nerve root avulsion from the spinal cord, and due to the very proximal level of injury, the regenerating nerves must grow across considerable distances to reinnervate target tissue. In this situation, nerve transfers are often performed. This technique takes advantage of neuroplasticity, which allows a functionally redundant nerve to be used as a donor for another function that was lost as a result of the injury (Brown et al., 2009). In the Oberlin transfer, the ulnar nerve is used to reinnervate the biceps muscle. The free end of the donor nerve is connected near the terminal branches of the injured nerve, converting a proximal nerve lesion into a distal nerve lesion and reducing the distance and time required for reinnervation (Kang et al., 2011). A retrospective study evaluated elbow flexion and shoulder abduction in 74 patients who underwent nerve transfer surgery for adult brachial plexus injury. Shoulder abduction recovery to LSU grade 2 (Medical Research Council grade 3) after spinal accessory to suprascapular and/or thoracodorsal to axillary nerve transfer was 95% and 36%, respectively (Sulaiman et al., 2009). Limitations of Peripheral Nerve Regeneration As detailed by the above described studies, despite the promise of improved functional recovery, results are still limited with currently available techniques. Additionally, outcomes of nerve repair following traumatic injury are influenced by multiple factors that are outside of a surgeon’s control, such as the age of the patient, location of injury, and the timing of presentation and nerve repair following injury. As initially reported by Sunderland and subsequently by many others, nerve reconstruction outcomes are better with younger patients, early repairs, repairs of single function nerves, distal injuries, and short nerve grafts (Lee & Wolfe, 1999). Successful nerve repair faces numerous challenges, and optimization of outcomes has plateaued with surgical manipulations alone. As such, the current medical and surgical state of the art offers limited capacity to effect true functional neural regeneration. Neural repair and regeneration is a complex biological process that we are just beginning to

ACCEPTED MANUSCRIPT understand through clinical experiences as well as animal experimental studies. Rate of regeneration, specificity of regeneration, segmental nerve deficits, and degeneration of the target end-organ are challenges that need to be overcome in order to achieve meaningful functional recovery (Figure 1).

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Rate of Regeneration In adults, it is well accepted that neural regeneration occurs at a slow rate of about 1 millimeter per day (Sulaiman and Gordon, 2014). The rate of regeneration can be monitored with an advancing Tinel sign as it progresses from proximal to distal (Flores et al., 2000). When a nerve is injured, the nerve must grow over substantial distances and traverse over scar and areas of fibrosis in order to reach its target. For example, a brachial plexus injury requires regeneration of nerve from the spinal cord to the digits, which can involve a distance of up to one meter; thus it can take up to 3 years for regenerating axons to reach the hand muscles. Many factors can limit and influence the rate of nerve regeneration. The type of nerve injury can influence the probability of successful regeneration. For instance, a crush nerve lesion has a continuous basal lamina structure that provides guidance for regenerating nerve. . Conversely, after axotomy, the nerve sheath discontinuity impedes reinnervation and can lead to neuroma formation. Studies have implicated SCs and basal lamina as major players in inhibition of nerve regeneration (Fugleholm et al., 1994; Son and Thompson, 1995). This is because after a long period of denervation, SC atrophy and the basal lamina degenerate, thus physically removing guiding tracks for axonal growth (Vuorinen et al., 1995). Further evidence of the crucial role in SC in nerve regeneration is found in a study by Son et al., who reported that nerve regeneration occurs more rapidly in the presence of pre-established SC processes (Son and Thompson, 1995). The extent of scar tissue formation is another factor impeding nerve regeneration following repair. Nerve injury and repair are both associated with extensive deposition of fibrotic connective tissue that obstructs axonal regeneration. It is now well known that the site of nerve injury is exposed to an evolving molecular milieu including growth inhibitory chondroitin sulfate proteoglycans (CSPGs) and neural-glial antigen 2 (NG2) proteoglycans. CSPGs are well studied in the central nervous system (CNS), but are now recently been found in the PNS as well. CSPGs after nerve injury has been shown to contribute to poor quality of regeneration and functional recovery (Braunewell et al., 1995; Graham et al., 2007). The application of chondroitinase ABC (ChABC), an enzyme that digests scar tissue, may allow for robust nerve regeneration as it may help degrade already formed CSPGs or may help axons grow before fibrosis ensues. Studies have already shown ChABC to be effective at promoting functional recovery following CNS injury, and it follows that it may be useful following PNS injury as well. Animal studies have also shown that production of neurotrophic factors in the distal segments of a nerve gradually decreases to the point where sufficient levels are not present to support nerve growth. As such, as nerve regeneration progresses distally, the slower it proceeds. (Eggers, Tannemaat, Ehlert, & Verhaagen, 2010; Fu & Gordon, 1997; Höke et al., 2006; Höke, Gordon, Zochodne, & Sulaiman, 2002). Furthermore, this non-growth permissive state promotes axonal retraction and “wandering axons”, which are thought to prevent regeneration to the neuromuscular junction (Gordon et al., 2009; Luo and O’Leary, 2005). Many candidate therapeutic molecules and nerve stimulation methods have been proposed to accelerate the rate of regeneration (Grinsell and Keating, 2014). Neurotrophins are molecules that are naturally released from nerve endings following a nerve injury that influence growth and differentiation. Nerve growth factor, brain derived neurotrophic factor and glialderived neurotrophic factor are just a few examples of the molecules that are being integrated and applied to the proximal stump after injury to enhance axonal regeneration (Grinsell and Keating, 2014). Extracellular matrix (ECM) molecules are other critical players in neural regeneration because the interaction between the ECM and neurons and glial cells plays an

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instrumental role in neurite outgrowth, elongation and remyelination. ECM molecules such as fibronectin, collagen, and fibrin have been incorporated into guidance channels for peripheral nerve regeneration (Rummler and Gupta, 2004). Another proposed method is the use of SCs, as they are critical to regenerative processes such as Wallerian degeneration, and are able to produce and secrete factors that make up a growth-permissive microenvironment. The use of stem cells is also being investigated to improve nerve repair. Stem cells provide an abundant source of SCs without the complications of graft rejection and allograft immunosensitivity (Kang et al., 2011). Immune system modulators are also being investigated as possible method of increasing regeneration rate. Several studies suggest that FK506, a pharmacologic agent to prevent organ transplant rejection, promotes neurite outgrowth by decreasing the amount of immune response (Kang et al., 2011). There have been some reports of applying electrical gradient across a repaired nerve to speed neural regeneration. Application of a brief, one-hour low-frequency electrical stimulation directly after nerve repair in animal rat model promotes improvement of target muscle reinnervation (Gordon et al., 2009). Electrical stimulation was reported to enhance and accelerate sensory and motoneuron regeneration after peripheral nerve injury by increasing the expression of regeneration-associated genes (Al-Majed et al., 2000; Geremia et al., 2007). In their animal study, Al-Majed et al. reported a correlation between accelerated axonal regeneration and upregulation of BDNF and trkB receptors after electrical stimulation. The positive regenerative response of electrical stimulation can be explained by the upregulation of BDNF during membrane depolarization and the recruitment of trkB receptors to cell membranes. BDNF then acts in an autocrine fashion to provide trophic support for injured neurons (Al-Majed et al., 2000). Moreover, the application of brief electrical stimulation has also been shown to increase the expression of GAP-43, which correlated to enhanced predilection for axonal regeneration (Geremia et al., 2007). The utilization of electrical stimulation has been translated to the clinical setting, where a subset of patients with carpal tunnel syndrome received one hour of electrical stimulation. Compared to control subjects, the experimental group showed promising results of accelerated axonal and target reinnervation (Gordon et al., 2010). A randomized controlled trial by Wong et al. also tested low-frequency postsurgical electrical stimulation lasting 1 hour and found improvements to sensory modalities along with a trend towards functional recovery outcomes in the electrical stimulation group (Wong, Olson, Morhart, & Chan, 2015). Despite these innovative studies, there still exists no clinically proven molecular therapeutic to speed the inherent rate of regeneration (Grinsell and Keating, 2014).

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Specificity of Regeneration Axonal misdirection also plays a significant role in poor functional recovery after severe nerve injuries (Alant et al., 2013). Nerve injury induces rapid axonal sprouting so as to facilitate anterograde, target-directed axonal regeneration. Moreover, the degenerating distal nerve segment has growth-promoting potential and may enhance the specificity of regeneration (Brushart, Gerber, Kessens, Chen, & Royall, 1998). Quantitative retrograde labeling techniques have been utilized to define the number of regenerating motor neurons and the specificity of their peripheral connections (O’Daly et al., 2016). It has been shown that motor nerves tend to reinnervate motor pathways. Even in injured mixed sensory-motor nerves, it was observed that motor axons preferentially reinnervate motor pathways (Brushart, 1993). This involves recognition molecules of the L2/HNK-1 family that are detectable in ventral spinal roots and motor axons but not in dorsal root or sensory cutaneous nerves (Martini et al., 1994). As a nerve tries to regenerate, motor axons explore different pathways by sending out collateral branches. In the regenerating rat femoral nerve, motor axons send out collateral branches into both appropriate and inappropriate paths. Specificity of pathway regeneration is subsequently gained by “pruning off” those collaterals which have grown into inappropriate nerve branches (Brushart, 1993). After injury, muscle fibers previously belonging to a specific motor unit will likely be

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innervated by a different motor axon. This mismatch between central commands to motor neurons and the actual distribution of muscle fibers innervated by those motor neurons leads to unsuccessful functional recovery, as well as sensorimotor disturbances (Monti, Roy, & Edgerton, 2001). Furthermore, after injury, some axons compete for innervation as they grow in various directions, which will lead to loss of pre-lesional innervation selectivity (Valls-Sole et al., 2011). A study visualizing regenerating axons reported axons exposed to as many as 150 different potential distal pathways (Witzel et al., 2005) While preferential motor reinnervation has been detailed in animals, it remains unclear as to what level this occurs in the human condition. The phenomenon of regeneration is a fundamental biological process, and a process that is species-specific. Decades of research has continued to challenge our understanding as to what influences, controls and enables restoration of tissue, skin, muscle, organs and limbs in certain organisms. While whole appendage regeneration has been documented in arthropods and echinoderms, mammals have been found to at least be capable of cellular and physiological regeneration. The MRL mouse strain, also called the “healer mouse”, was found to have profound regenerative capacity for a mammal, being able to grow cartilage, skin, and myocardium without scarring (Heber-Katz et al., 2004). Fingertip regeneration after amputation past the distal interphalangeal joint distal to the nail bed in children has been documented as well (Carlson, 2007). Every species is capable of regeneration; however, large-scale replacement of tissues or body parts in mammals has not been considered possible despite examples of this regenerative processes in many species (Bryant et al., 2002; Carlson, 2007). As such, it is critical to focus efforts on the exploration of human neural regeneration and repair, in addition to the groundbreaking work being done in animal models.

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Segmental Nerve Defects Intuitively, regeneration over a segmental gap is quite challenging and has been demonstrated with animal studies. For example, epineural neurorrhaphy without a gap showed better nerve regeneration when compared to neurorrhaphy with a gap. Functional restoration after a nerve injury requires the growth of axons over the distance between the lesion and the end target, but following injury, fibrosis and edema cause nerve fibers to lose their elasticity and extensibility, as well as a certain amount of retraction. If a nerve tissue is destroyed, the distortion of the fascicular pattern of the proximal and distal stumps may differ in relation to the length of the defect (Millesi, 1986). Injury with a segmental gap precludes tension-free, end-to-end coaptation repair. There has been some disagreement regarding whether or not there is an ideal nerve defect length that will allow neural regeneration and achieve functional recovery. Nerve injury repairs of short defects between 0.5 and 5.0 millimeters have produced varying results (Brushart, 1990; Hasegawa, Shibata, & Takahashi, 1996; Heijke, Klopper, & Dutrieux, 1993; Scherman, Lundborg, Kanje, & Dahlin, 2000). Studies of segmental nerve defects have revealed the existence of a critical nerve gap length where the efficacy of conduits starts to decline. Conduits have been used to provide foundation for nerve regeneration to occur in nerve gaps that are less than 3 centimeters; however, animal models examining nerve regeneration in nerve gaps 3 centimeters or more have not shown promising results (Kim et al., 2010; Moore et al., 2009). Mackinnon and Dellon detailed that the primate peripheral nerve could regenerate across a 3 centimeter nerve gap when guided appropriately. They extrapolated their findings to humans with nerve gaps of 3 centimeters or less and demonstrated excellent recovery in 33 percent of the patients (Mackinnon and Dellon, 1990). Other experiments have suggested that axons may be able to align themselves in response to neurotrophic factors when allowed to grow across a conduit. This finding suggested that conduit repairs might lead to improved functional results compared to standard end-to-end repair (Brushart & Seiler, 1987; Seckel, Ryan, Gagne, Chiu, & Watkins, 1986; Weber et al.,

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2000).Towards that end, specialized entubulation chambers were developed. These are are hollow tubes that serve as nerve conduits. Entubulation chambers leave a small intentional gap between the nerve ends, which allows for fascicular rerouting (Yegiyants et al., 2010). Furthermore, they decrease surgical manipulation of nerve ends and thereby decrease scarring in theory. Unforunately, these experimental data have not successfully translated into clinical practice. Major engineering challenges remain unsolved, particularly with respect to guiding axons with target specificity in larger deficits. Vascularized nerve grafts can be considered if a long graft is needed in a poorly vascularized bed, but the results are mixed (Yegiyants et al., 2010). Different growth factors in conjunction with different conduit luminal scaffolds such as collagen and laminin have also been attempted. These modifications however, did not offer substantial benefit over using autografts (Pfister et al., 2011); thus continued investigation is being conducted to find the effective combination of scaffold, cells and signaling factors that will yield better neural regeneration outcomes.

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Degeneration of the Target End-Organ Denervation atrophy of target tissue perhaps the most significant issue that is associated with peripheral nerve repair and regeneration. Aside from the long distances that nerves need to traverse to reach their target organs and the fibrotic obstacles along their path, prolonged target deprivation is found to reduce the ability of motor neurons to regenerate and causes SCs to lose their growth-supportive phenotypes (Fu and Gordon, 1995; Furey et al., 2007). Once Wallerian degeneration occurs distal to the injury site, communication is lost between the nerves and the muscles that they innervate. It has been shown in animal models that following loss of innervation, the acetylcholine receptor end-organs (AChRs) located on the muscle membrane degenerate, as evidenced by decreased density and altered morphology. Furthermore, long term denervation leads to the disassembly of the motor endplates causing the AChRs to redistribute throughout the muscle fiber (Frank et al., 1975; Hartzell and Fambrough, 1972; Steinbach, 1981). The degradation of motor endplates render the target organ nonviable for the regenerating nerve, even when the regenerating nerve is able to reach its target. There are findings that suggest that after injury, regrowth into the muscle becomes successful. A recent study confirmed that regenerating fibers can reinnervate distal muscles and can reestablish structurally reformed NMJ even after several weeks of denervation (Sakuma et al., 2016). But even with successful regeneration, there is still often failure to reestablish pre-injury motor function, suggesting a possible critical role of the neuromuscular junction. The problem of end-organ atrophy is partially addressed with nerve transfer procedures as there is less time for degeneration with the shorter distance that regeneration must occur; and thus affords less time for the AChRs to degenerate. Yet, a successful nerve transfer only restores partial muscle function, and pre-injury strength is not fully recovered (Noaman et al., 2004). It is therefore of crucial importance to determine if stabilization of the NMJ could potentially reduce end-organ atrophy following nerve injury. Chao et al. provided the first evidence that preservation of the NMJ after traumatic nerve injury improves functional recovery after surgical repair (Chao et al., 2013). Agrin is a molecule secreted by terminal SCs that stabilizes muscle endplates; Matrix metalloproteinase 3 (MMP3) is the major enzyme responsible for its degradation in denervated muscles. Genetic deletion of MMP3 leads to sustained levels of agrin in denervated muscle endplates, which in turn is able to preserve motor endplate integrity for at least 2 months following nerve degeneration (Chao et al., 2013). Given these findings, it may be possible to extend the window of time allowed for regeneration beyond which destabilization of the motor endplate limits reinnervation. Pharmacological inhibition of MMP3 and local augmentation of neural agrin are a few innovations that could feasibly stabilize the NMJ and diminish end-organ atrophy after nerve injury. Moreover, the canonical Wnt/beta-catenin pathway has also been implicated as a potential source of motor endplate instability after long term denervation (Kurimoto et al., 2015), Therefore, targeting the

ACCEPTED MANUSCRIPT Wnt/beta-catenin pathway may also offer game-changing therapeutic opportunities for more effective reinnervation of endplates, and subsequent meaningful functional recovery (Figure 2).

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Conclusions The reasons for poor prognosis following traumatic nerve injury are multifactorial. Over the last decade there has been extensive research into new innovations and alternatives for surgical repair. Regardless of repair strategy, functional recovery is commonly absymal and failure is not uncommon. Functional neural recovery relies on motor axons matching their endplates and sensory axons reaching their sensory receptors. Functional neural regeneration has proven to be a challenge as optimization of outcomes has plateaued with surgical manipulation alone. Roadblocks that are encountered include, but are not limited to, slow rates of regeneration, poor specificity of regeneration, the presence of a segmental nerve defect and perhaps most crucially, degeneration of target-end-organ after prolonged periods of denervation. Advances in methods of axonal regeneration, preservation of NMJs, and methods that decrease inflammation and fibrosis and that promote a growth-permissive microenvironment hold promise for future treatment options for these devastating injuries. Despite the current advancements in peripheral nerve repair and regeneration, there is a need to focus experiments to develop effective therapeutic strategies that will promote accurate nerve regeneration to optimize human functional recovery in a meaningful way.

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ACCEPTED MANUSCRIPT Captions for Figures: Figure 1: Roadblocks to recovery after nerve injury include A) the presence of a segmental nerve defect, B) variable rates of regeneration, C) the need for specificity of regeneration, D) glial scar formation, and E) the degeneration of the target end –organ.

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Figure 2: Schematic representation of the neuromuscular junction with its three components (pre-synaptic, peri-synaptic, and post-synaptic) along with their associated signaling cascades

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Fig. 2