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Introduction: Peripheral nerve traumas are common injuries in young adult population. The myriad of techniques and medications have been defined to obtain better recovery but none of them was proved to have superior effect. This study aims to determine the anti-fibrotic effect of the decorin on sciatic nerve injury in order to enhance functional outcome., Materials and Methods: 24 12-week-old male Sprague-Dawley rats (350-400 gr) were divided into four groups. The sciatic nerve was dissected and exposed; a full-thickness laceration was created 1.5 cm proximal to the bifurcation point and 1.5 cm distal to where it originated from the lumbosacral plexus. Motor and sensory tests were conducted before and after the operations for evaluating the nerve healing., Results: There was a statistically significant difference between DCN bolus and PBS bolus group. (p<0.0001, p&lt;0.05) in neuromotor tests. Increase of the latency was significantly lower in DCN bolus and infusion group when compared with the PBS bolus group. (p&lt;0,001). All operated gastrocnemius muscles were atrophic compared with the contralateral side. The differences between the averages in the sciatic functional index, the improvement of the DCN infusion group was 8.6 units better than the PBS group and 4.4 units better than the DCN bolus group. When the amount of stimulation was 10 mV at the proximal segment in electromyography, there was no significant difference between the DCN bolus and sham groups. (p&gt; 0.05, p = 0.6623)., Conclusion: Decorin protein reduces the fibrosis and enhances the motor and sensory recovery both clinically and histologically. Despite the high cost, short half-life and production issues, this protein could be administered after the microsurgical repair but more studies are required to overcome the limitations.

Objectives: Whole-body vibration (WBV) is commonly used to improve motor function, balance and functional performance, but its effects on the body are not fully understood. The main objective was to evaluate the morphometric and functional effects of WBV in an experimental nerve regeneration model., Methods: Wistar rats were submitted to unilateral sciatic nerve crush and treated with WBV (4-5 weeks), started at 3 or 10 days after injury. Functional performances were weekly assessed by sciatic functional index, horizontal ladder rung walking and narrow beam tests. Nerve histomorphometry analysis was assessed at the end of the protocol., Results: Injured groups, sedentary and WBV started at 3 days, had similar functional deficits. WBV, regardless of the start time, did not alter the histomorphometry parameters in the regeneration process., Conclusions: The earlier therapy did not change the expected and natural recovery after the nerve lesion, but when the WBV starts later it seems to impair function parameter of recovery., Competing Interests: The authors have no conflict of interest.

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and a lot of comparative data has been published in the literature. This includes a multitude of assessment methods to study functional recovery following nerve injury and repair. Besides evaluation of nerve regeneration by means of histology, electrophysiology, and other in vivo and in vitro assessment techniques, functional recovery is the most important criterion to determine the degree of neural regeneration. Automated gait analysis allows recording of a vast quantity of gait-related parameters such as Paw Print Area and Paw Swing Speed as well as measures of inter-limb coordination. Additionally, the method provides digital data of the rats' paws after neuronal damage and during nerve regeneration, adding to our understanding of how peripheral and central nervous injuries affect their locomotor behavior. Besides the predominantly used sciatic nerve injury model, other models of peripheral nerve injury such as the femoral nerve can be studied by means of this method. In addition to injuries of the peripheral nervous systems, lesions of the central nervous system, e.g., spinal cord contusion can be evaluated. Valid and reproducible data assessment is strongly dependent on meticulous adjustment of the hard- and software settings prior to data acquisition. Additionally, proper training of the experimental animals is of crucial importance. This work aims to illustrate the use of computerized automated gait analysis to assess functional recovery in different animal models of peripheral nerve injury as well as spinal cord contusion injury. It also emphasizes the method's limitations, e.g., evaluation of nerve regeneration in rats with sciatic nerve neurotmesis due to limited functional recovery. Therefore, this protocol is thought to help researchers interested in peripheral and central nervous injuries to assess functional recovery in rodent models.

Despite the regenerative capacity of the peripheral nerve system (PNS), functional recovery after mechanical nerve trauma is often incomplete, resulting in motor, sensory, and autonomic deficits. The elucidation of key molecules involved in trauma-induced Wallerian degeneration and the ensuing regeneration processes is a prerequisite for the development of disease modifying drugs. The chemokine (C-X-C motif) receptor 3 (CXCR3) has been implicated in the recruitment of macrophages, the major immune cell population during the process of Wallerian degeneration. In this study, we examined whether deletion of CXCR3 affects macrophage recruitment, the expression of the proinflammatory cytokine tumor necrosis factor (TNF)- α and the CXCR3 agonist interferon gamma-induced protein 10 (CXCL10), and functional recovery in the sciatic nerve crush model. CXCR3 mice displayed significantly reduced macrophage counts preceded by diminished expression of CXCL10 and TNF- α. Furthermore, functional recovery of sciatic nerve motor function was significantly accelerated. In summary, these data indicate that the deletion of CXCR3 leads to a diminished inflammatory response and an accelerated functional recovery following sciatic nerve crush injury. Therefore, CXCR3 may be an interesting target for therapeutic interventions after traumatic nerve lesions.

Rodent models of sciatic nerve lesion are regularly used to assess functional deficits in nerves. Impaired locomotor functions induced by sciatic nerve lesion are currently evaluated with scoring systems despite their limitations. To overcome these shortcomings, which includes low sensitivity, little significance, and the representation of only marginal components of motion profiles, some additional metrics have been introduced. However, a quantitative determination of motion deficits is yet to be established. We used a three-dimensional motion analysis to investigate gait deficits after sciatic nerve lesion in rats. This enabled us to depict the distorted gait motion using both traditional parameters and novel readouts that are specific for the three-dimensional analysis. Our results suggest that three-dimensional motion analysis facilitates a comprehensive understanding of the gait impairment specifically, but not limited to, a sciatic lesion rat model. A broad application of these methods will improve understanding and standardized motor assessment.

Introduction: Improving axonal outgrowth and remyelination is crucial for peripheral nerve regeneration. Miconazole appears to enhance remyelination in the central nervous system. In this study we assess the effect of miconazole on axonal regeneration using a sciatic nerve crush injury model in rats., Methods: Fifty Sprague-Dawley rats were divided into control and miconazole groups. Nerve regeneration and myelination were determined using histological and electrophysiological assessment. Evaluation of sensory and motor recovery was performed using the pinprick assay and sciatic functional index. The Cell Counting Kit-8 assay and Western blotting were used to assess the proliferation and neurotrophic expression of RSC 96 Schwann cells., Results: Miconazole promoted axonal regrowth, increased myelinated nerve fibers, improved sensory recovery and walking behavior, enhanced stimulated amplitude and nerve conduction velocity, and elevated proliferation and neurotrophic expression of RSC 96 Schwann cells., Discussion: Miconazole was beneficial for nerve regeneration and functional recovery after peripheral nerve injury. Muscle Nerve 57: 821-828, 2018., (© 2017 Wiley Periodicals, Inc.)

Introduction: Erythropoietin (EPO) has been identified as a neuroregenerative agent. We hypothesize that it may accelerate recovery after crush injury and may vary with crush severity., Methods: Mice were randomized to mild, moderate, or severe crush of the sciatic nerve and were treated with EPO or vehicle control after injury. The sciatic function index (SFI) was monitored over the first week. Microstructural changes were analyzed by immunofluorescence for neurofilament (NF) and myelin (P 0 ), and electron microscopy was used to assess ultrastructural changes., Results: In moderate crush injuries, EPO significantly improved SFI at 7 days post-injury, an effect not observed with other severity levels. Increases in the ratio of P 0 to NF were observed after EPO treatment in moderate crush injuries. Electron microscopy demonstrated endothelial cell hypertrophy in the EPO group., Conclusions: EPO accelerates recovery in moderately crushed nerves, which may be through effects on myelination and vascularization. Injury severity may influence the efficacy of EPO. Muscle Nerve 56: 143-151, 2017., (© 2016 Wiley Periodicals, Inc.)

The prototypical neurotrophin, nerve growth factor (NGF), plays an important role in the development and maintenance of many neurons in both the central and peripheral nervous systems, and can promote functional recovery after peripheral nerve injury in adulthood. However, repair of peripheral nerve defects is hampered by the short half-life of NGF in vivo, and treatment with either NGF alone or NGF contained in synthetic nerve conduits is inferior to the use of nerve autografts, the current gold standard. We tested the reparative ability of a single local injection of a polyvalent coacervate containing polycation-poly(ethylene argininylaspartate diglyceride; PEAD), heparin, and NGF, in adult rats following sciatic nerve crush injury, using molecular, histological and behavioral approaches. In vitro assays demonstrated that NGF was loaded into the coacervate at nearly 100% efficiency, and was protected from proteolytic degradation. In vivo, the coacervate enhanced NGF bioavailability, leading to a notable improvement in motor function (track walking analysis) after 30days. The NGF coacervate treatment was also associated with better weight gain and reduction in atrophy of the gastrocnemius muscle. Furthermore, light and electron microscopy showed that the number of myelinated axons and axon-to-fiber ratio (G-ratio) were significantly higher in NGF coacervate-treated rats compared with control groups. Expression of markers of neural tissue regeneration (MAP-2, S-100β, MBP and GAP-43), as well as proliferating Schwann cells and myelin-axon relationships (GFAP and NF200), were also increased. These observations suggest that even a single administration of NGF coacervate could have therapeutic value for peripheral nerve regeneration and functional recovery., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Peripheral nerve injury (PNI) can result in neurodegenerative changes leading to motor, sensory and autonomic dysfunction. Injury to the rat sciatic nerve is used to model pathophysiologic processes following PNI and assess the efficacy of therapeutic interventions. Frequently, temporal changes in the sciatic functional index (SFI), a measure of sensorimotor integration are measured in rats to assess functional recovery following sciatic nerve injury. However, multiple rat strains and behavioral endpoints have been employed to investigate pathophysiology of PNI and impact of therapeutic intervention on recovery, raising the possibility that rat strain may influence the outcome of such studies., New Method: The temporal course of recovery from sham, sciatic nerve crush or transection injury was assessed using SFI determined by two methods (footprint and DigiGait), and proprioceptive hind limb placement (a measure of proprioceptive integrity) of the sciatic nerve innervation, in male Sprague Dawley, Lewis, Fischer, Wistar and Long Evans rats., Results: The SFI profile, as assessed by both inked footprint analysis and DigiGait, following sciatic nerve injury was remarkably conserved across strains. Dramatic strain-related differences were observed in the latency to place the crush- or transection-injured hind limb following proprioceptive hind limb stimulation., Comparison With Existing Method: The novelty of this study is the parallel comparison of multiple strains using existing and novel tests., Conclusion: These results suggest that some sensorimotor function tests may be sensitive to the choice of strain, as evidenced by the differences between SFI and proprioceptive function outcomes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Introduction: Comprehensive assessment of the time course of functional recovery following peripheral nerve repair is critical for surgical management of peripheral nerve injuries. This study describes the design and implementation of a novel implantable wireless nerve stimulator capable of repeatedly interfacing peripheral nerve tissue and providing serial evaluation of functional recovery postoperatively., Methods: Thin-film wireless implants were fabricated and subcutaneously implanted into Lewis rats. Wireless implants were used to serially stimulate rat sciatic nerve and assess functional recovery over 3 months following various nerve injuries., Results: Wireless stimulators demonstrated consistent performances over 3 months in vivo and successfully facilitated serial assessment of nerve and muscle function following nerve crush and nerve transection injuries., Conclusions: This study highlights the ability of implantable wireless nerve stimulators to provide a unique view into the time course of functional recovery in multiple motor targets. Muscle Nerve 54: 1114-1119, 2016., (© 2016 Wiley Periodicals, Inc.)

Effect of combination of undifferentiated bone marrow stromal cells (BMSCs) and pulsed electromagnetic fields (PEMF) on transected sciatic nerve regeneration was assessed in rats. A 10 mm nerve segment was excised and a vein graft was used to bridge the gap. Twenty microliter undifferentiated BMSCs (2× 107 cells /mL) were administered into the graft inBMSC group with no exposure to PEMF. In BMSC/PEMF group the whole body was exposed to PEMF (0.3 mT, 2Hz) for 4h/day within 1-5 days. In PEMF group the transected nerve was bridged and phosphate buffered saline was administered into the graft. In authograft group (AUTO), the transected nervesegments were reimplanted reversely and the whole body was exposed to PEMF. The regenerated nerve fibers were studied within 12 weeks after surgery. Behavioral, functional, electrophysiological, biomechanical, gastrocnemius muscle mass findings, morphometric indices and immuonohistochemical reactions confirmed faster recovery of regenerated axons in BMSC/PEMF group compared to those in the other groups (P<0.05). The use of undifferentiated BMSCs with whole body exposure to PEMF improved functional recovery. Combination of local transplantation of in vitro bone-marrow stromal cells and pulsed electromagnetic fields could be considered as an effective, safe and tolerable treatment for peripheral nerve repair in clinical practice.

Sciatic nerve injury has been used for over a century to investigate the process of nerve damage, to assess the absolute and relative capacity of the central and peripheral nervous systems to recover after axotomy, and to understand the development of chronic pain in many pathologies. Here we provide a historical review of the contributions of this experimental model to our current understanding of fundamental questions in the neurosciences, and an assessment of its continuing capacity to address these and future problems. We describe the different degrees of nerve injury - neurapraxia, axonotmesis, neurotmesis - together with the consequences of selective damage to the different functional and anatomic components of this nerve. The varied techniques used to model different degrees of nerve injury and their relationship to the development of neuropathic pain states are considered. We also provide a detailed anatomical description of the sciatic nerve from the spinal cord to the peripheral branches in the leg. A standardized protocol for carrying out sciatic nerve axotomy is proposed, with guides to assist in the accurate and reliable dissection of the peripheral and central branches of the nerve. Functional, histological, and biochemical criteria for the validation of the injury are described. Thus, this paper provides a review of the principal features of sciatic nerve injury, presents detailed neuroanatomical descriptions of the rat's inferior limb and spine, compares different modes of injury, offers material for training purposes, and summarizes the immediate and longterm consequences of damage to the sciatic nerve., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Transgenic mice have been previously used to assess nerve regeneration following peripheral nerve injury. However, mouse models are limited by their small caliber nerves, short nerve lengths, and their inability to fully participate during behavioral assessments. The transgenic Thy1 GFP rat is a novel transgenic rat model designed to assess regeneration following peripheral nerve injury. However, return of functional and behavioral recovery following nerve injury has not yet been evaluated in these rats. In this study, we ask whether differences in anatomy, recovery of locomotion, myological, and histomorphological measures exist between transgenic Thy1 GFP rats when compared to wild type (WT) Sprague Dawley rats following unilateral sciatic nerve injury. We found that both motor and sensory neuronal architecture, overground and skilled locomotion, muscle force, motor unit number estimation (MUNE) and wet muscle weights, and histomorphometric assessments are similar between both genetic phenotypes. Overall, these data support the use of the transgenic Thy1-GFP rat in experiments assessing functional and behavioral recovery following nerve injury and repair., (© 2013 Peripheral Nerve Society.)

Ciliary neurotrophic factor (CNTF) administration maintains, protects, and promotes the regeneration of both motor neurons (MNs) and skeletal muscle in a wide variety of models. Expression of CNTF receptor α (CNTFRα), an essential CNTF receptor component, is greatly increased in skeletal muscle following neuromuscular insult. Together the data suggest that muscle CNTFRα may contribute to neuromuscular maintenance, protection, and/or regeneration in vivo. To directly address the role of muscle CNTFRα, we selectively-depleted it in vivo by using a "floxed" CNTFRα mouse line and a gene construct (mlc1f-Cre) that drives the expression of Cre specifically in skeletal muscle. The resulting mice were challenged with sciatic nerve crush. Counting of nerve axons and retrograde tracing of MNs indicated that muscle CNTFRα contributes to MN axonal regeneration across the lesion site. Walking track analysis indicated that muscle CNTFRα is also required for normal recovery of motor function. However, the same muscle CNTFRα depletion unexpectedly had no detected effect on the maintenance or regeneration of the muscle itself, even though exogenous CNTF has been shown to affect these functions. Similarly, MN survival and lesion-induced terminal sprouting were unaffected. Therefore, muscle CNTFRα is an interesting new example of a muscle growth factor receptor that, in vivo under physiological conditions, contributes much more to neuronal regeneration than to the maintenance or regeneration of the muscle itself. This novel form of muscle-neuron interaction also has implications in the therapeutic targeting of the neuromuscular system in MN disorders and following nerve injury. J. Comp. Neurol. 521: 2947-2965, 2013. © 2013 Wiley Periodicals, Inc., (Copyright © 2013 Wiley Periodicals, Inc.)

Introduction: Functional muscle recovery after peripheral nerve injury is far from optimal, partly due to atrophy of the muscle arising from prolonged denervation. We hypothesized that injecting regenerative cells into denervated muscle would reduce this atrophy., Methods: A rat sciatic nerve lesion was performed, and Schwann cells or adipose-derived stem cells, untreated or induced to a "Schwann-cell-like" phenotype (dASC), were injected into the gastrocnemius muscle. Nerves were either repaired immediately or capped to prevent muscle reinnervation. One month later, functionality was measured using a walking track test, and muscle atrophy was assessed by examining muscle weight and histology., Results: Schwann cells and dASC groups showed significantly better scores on functional tests when compared with injections of growth medium alone. Muscle weight and histology were also significantly improved in these groups., Conclusion: Cell injections may reduce muscle atrophy and could benefit nerve injury patients., (Copyright © 2012 Wiley Periodicals, Inc.)

After peripheral nerve injury, axons regenerate and become remyelinated by resident Schwann cells. However, myelin repair never results in the original myelin thickness, suggesting insufficient stimulation by neuronal growth factors. Upon testing this hypothesis, we found that axonal neuregulin-1 (NRG1) type III and, unexpectedly, also NRG1 type I restored normal myelination when overexpressed in transgenic mice. This led to the observation that Wallerian degeneration induced de novo NRG1 type I expression in Schwann cells themselves. Mutant mice lacking a functional Nrg1 gene in Schwann cells are fully myelinated but exhibit impaired remyelination in adult life. We suggest a model in which loss of axonal contact triggers denervated Schwann cells to transiently express NRG1 as an autocrine/paracrine signal that promotes Schwann cell differentiation and remyelination.

De-focused low energy extracorporeal shock wave therapy (ESWT) has been widely used in various clinical and experimental models for the treatment of painful conditions such as epicondylitis and plantar fascitis and also bone and wound healing. There is evidence that ESWT improves the metabolic activity of various cell types, e.g. chondrocytes and endothelial cells but little is known about its effects on nervous tissue. The aim of this study was to investigate whether ESWT improves the regeneration of injured nerves in an experimental rat model. Sprague-Dawley rats received an 8mm long homotopic nerve autograft into the right sciatic nerve, fixed with epineurial sutures. Two experimental groups were set up: the group 1 animals received ESWT (300 impulses, 3 Hz) immediately after nerve grafting whereas the group 2 (control) animals received only nerve autografts. Serial CatWalk automated gait analysis, electrophysiological studies and morphological investigations were carried out. The survival time was either 3 weeks or 3 months. At 6 to 8 weeks of survival the ESWT group of animals exhibited a significantly improved functional recovery relative to the controls. Electrophysiological observations at 3 weeks after surgery revealed marked values of amplitude (3.9±0.8 mV, S.E.M.) and compound nerve action potential (CNAP, 5.9±1.4 mV·ms, S.E.M.) in the ESWT group, whereas there were no detectable amplitudes in the control group. This finding was accompanied by significantly greater numbers of myelinated nerve fibres in the middle of the graft (4644±170 [S.E.M., ESWT] vs 877±68 [S.E.M., control]) and in the distal stump (1586±157 [S.E.M., ESWT] vs 308±29 [S.E.M., control]) of ESWT animals relative to the controls 3 weeks after surgery. Three weeks after surgery the nerve grafts of control animals contained great numbers of phagocytes and unmyelinated nerve fibres, while the ESWT nerve grafts were filled with well-myelinated regenerating axons. There was no significant difference between the numbers of endoneural vessels in the ESWT and the control nerves. Three months after surgery, no significant differences were observed in the functional and electrophysiological data. Equally high numbers of myelinated axons distal to the graft could be found in both groups (7693±673 [S.E.M., ESWT] vs 6090±716 [S.E.M., control]). These results suggest that ESWT induces an improved rate of axonal regeneration, this phenomenon probably involving faster Wallerian degeneration, the improved removal of degenerated axons and a greater capacity of the injured axons to regenerate., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Transplantation of autologous Schwann cells (SCs) is a promising approach for treating various peripheral nerve disorders, including chronic denervation. However, given their drawbacks, such as invasive biopsy and lengthy culture in vitro, alternative cell sources would be needed. Adipose-derived stem cells (ASCs) are a candidate, and in this study rat ASCs transdifferentiated into a SC phenotype (dASC) cocultured with dorsal root ganglion neurons were shown to associate with neurites and to express myelin basic protein (MBP)-positive myelin protein. Furthermore, dASCs transplanted into a chronically denervated rat common peroneal nerve survived for at least for 10 weeks, maintaining their differentiated state. Immunohistochemical analysis revealed that transplanted dASCs associated with regenerating axons, forming MBP-/protein zero-positive myelin sheaths. The cell survival and myelin expression assessed by double labelling with S100 and glial fibrillary acidic protein were similar between the dASC- and SC-transplanted nerves. Importantly, transplantation of dASCs resulted in dramatically improved motor functional recovery and nerve regeneration, with a level comparable to, or even superior to, transplantation of SCs. In conclusion, dASCs are capable of myelinating axons in vivo and enhancing functional outcome after chronic denervation., (Copyright © 2011 Wiley Periodicals, Inc.)

Behavioral function lost in mammals (including humans) after peripheral nerve severance is slowly (weeks to years) and often poorly restored by 1-2-mm/day, nonspecifically directed outgrowths from proximal axonal stumps. To survive, proximal stumps must quickly repair (seal) plasmalemmal damage. We report that, after complete cut- or crush-severance of rat sciatic nerves, morphological continuity, action potential conduction, and behavioral functions can be consistently (>98% of trials), rapidly (minutes to days), dramatically (70-85% recovery), and chronically restored and some Wallerian degeneration prevented. We assess axoplasmic and axolemmal continuity by intra-axonal dye diffusion and action potential conduction across the lesion site and amount of behavioral recovery by Sciatic Functional Index and Foot Fault tests. We apply well-specified sequences of solutions containing FDA-approved chemicals. First, severed axonal ends are opened and resealing is prevented by hypotonic Ca²⁺-free saline containing antioxidants (especially methylene blue) that inhibit plasmalemmal sealing in sciatic nerves in vivo, ex vivo, and in rat B104 hippocampal cells in vitro. Second, a hypotonic solution of polyethylene glycol (PEG) is applied to open closely apposed (by microsutures, if cut) axonal ends to induce their membranes to flow rapidly into each other (PEG-fusion), consistent with data showing that PEG rapidly seals (PEG-seals) transected neurites of B104 cells, independently of any known endogenous sealing mechanism. Third, Ca²⁺-containing isotonic saline is applied to induce sealing of any remaining plasmalemmal holes by Ca²⁺-induced accumulation and fusion of vesicles. These and other data suggest that PEG-sealing is neuroprotective, and our PEG-fusion protocols that repair cut- and crush-severed rat nerves might rapidly translate to clinical procedures., (Copyright © 2012 Wiley Periodicals, Inc.)

Introduction: Although nerves can spontaneously regenerate in the peripheral nervous system without treatment, functional recovery is generally poor, and thus there is a need for strategies to improve nerve regeneration., Methods: The left sciatic nerve of adult rats was transected and immediately repaired by epineurial sutures. Rats were then assigned to one of two experimental groups treated with either growth hormone (GH) or saline for 8 weeks. Sciatic nerve regeneration was estimated by histological evaluation, nerve conduction tests, and rotarod and treadmill performance., Results: GH-treated rats showed increased cellularity at the lesion site together with more abundant immunoreactive axons and Schwann cells. Compound muscle action potential (CMAP) amplitude was also higher in these animals, and CMAP latency was significantly lower. Treadmill performance increased in rats receiving GH., Conclusion: GH enhanced the functional recovery of the damaged nerves, thus supporting the use of GH treatment, alone or combined with other therapeutic approaches, in promoting nerve repair., (Copyright © 2011 Wiley Periodicals, Inc.)

Peripheral nerve injuries are frequently seen in trauma patients and due to delayed nerve repair, lifelong disabilities often follow this type of injury. Innovative therapies are needed to facilitate and expedite peripheral nerve regeneration. The purpose of this study was to determine the effects of a 1-time topical application of a 26-amino-acid fragment (C3(156-181)), derived from the Clostridium botulinum C3-exoenzyme, on peripheral nerve regeneration in 2 models of nerve injury and repair in adult rats. After sciatic nerve crush, different dosages of C3(156-181) dissolved in buffer or reference solutions (nerve growth factor or C3(bot)-wild-type protein) or vehicle-only were injected through an epineurial opening into the lesion sites. After 10-mm nerve autotransplantation, either 8.0 nmol/kg C3(156-181) or vehicle were injected into the proximal and distal suture sites. For a period of 3 to 10 postoperative weeks, C3(156-181)-treated animals showed a faster motor recovery than control animals. After crush injury, axonal outgrowth and elongation were activated and consequently resulted in faster motor recovery. The nerve autotransplantation model further elucidated that C3(156-181) treatment accounts for better axonal elongation into motor targets and reduced axonal sprouting, which are followed by enhanced axonal maturation and better axonal functionality. The effects of C3(156-181) are likely caused by a nonenzymatic down-regulation of active RhoA. Our results indicate the potential of C3(156-181) as a therapeutic agent for the topical treatment of peripheral nerve repair sites.

Both central and peripheral axons contain pivotal microRNA (miRNA) proteins. While recent observations demonstrated that miRNA biosynthetic machinery responds to peripheral nerve lesion in an injury-regulated pattern, the physiological significance of this phenomenon remains to be elucidated. In the current paper we hypothesized that deletion of Dicer would disrupt production of Dicer-dependent miRNAs and would negatively impact regenerative axon growth. Taking advantage of tamoxifen-inducible CAG-CreERt:Dicer(fl/fl) knockout (Dicer KO), we investigated the results of Dicer deletion on sciatic nerve regeneration in vivo and regenerative axon growth in vitro. Here we show that the sciatic functional index, an indicator of functional recovery, was significantly lower in Dicer KO mice in comparison to wild-type animals. Restoration of mechanical sensitivity recorded in the von Frey test was also markedly impaired in Dicer mutants. Further, Dicer deletion impeded the recovery of nerve conduction velocity and amplitude of evoked compound action potentials in vitro. Histologically, both total number of regenerating nerve fibers and mean axonal area were notably smaller in the Dicer KO mice. In addition, Dicer-deficient neurons failed to regenerate axons in dissociated dorsal root ganglia (DRG) cultures. Taken together, our results demonstrate that knockout of Dicer clearly impedes regenerative axon growth as well as anatomical, physiological and functional recovery. Our data suggest that the intact Dicer-dependent miRNA pathway is critical for the successful peripheral nerve regeneration after injury., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Walking analysis in the rat is increasingly used to assess functional recovery after peripheral nerve injury. Here we assess the sensitivity and specificity of hindlimb joint kinematics measures during the rat gait early after sciatic nerve crush injury (DEN), after twelve weeks of recovery (REINN) and in sham-operated controls (Sham) using discriminant analysis. The analysis addressed gait spatiotemporal variables and hip, knee and ankle angle and angular velocity measures during the entire walking cycle. In DEN animals, changes affected all studied joints plus spatiotemporal parameters of gait. Both the spatiotemporal and ankle kinematics parameters recovered to normality within twelve weeks. At this time point, some hip and knee kinematics values were still abnormal when compared to sham controls. Discriminant models based on hip, knee and ankle kinematics displayed maximal sensitivity to identify DEN animals. However, the discriminant models based on spatiotemporal and ankle kinematics data showed a poor performance when assigning animals to the REINN and Sham groups. Models using hip and knee kinematics during walking showed the best sensitivity to recognize the reinnervated animals. The model construed on the basis of hip joint kinematics was the one combining highest sensitivity with robustness and high specificity. It is concluded that ankle joint kinematics fails in detecting minor functional deficits after long term recovery from sciatic nerve crush and extending the kinematic analysis during walking to the hip and knee joints improves the sensitivity of this functional test., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Here, we established a program of low-intensity aerobic exercise and compared the effects of exercise preoperative, postoperative, and a combination of both pre- and postoperative protocols on recovery from sciatic nerve crush injury in mice using behavioral, biochemical, and morphological assays. Sciatic nerve crush was performed in adult male mice. The animals were submitted to preoperative (for 2 weeks), postoperative (for 2 weeks), and a combination of preoperative-postoperative (for 4 weeks) training protocols. During the training period, functional recovery was monitored using the Sciatic Functional Index, the Sciatic Static Index, and mechanical and cold hypersensitivity analyses. Morphological and biochemical alterations were analyzed on the 14th day post-crushing. The functional recovery values of all of the exercised groups were significantly better than the nonexercised group. Biochemically, all of the exercise groups showed a reduction in the increase of interleukin-1β (IL-1β) in the sciatic nerve and in the IL-1β and interleukin-6 receptor (IL-6R) levels in the spinal cord. However, the levels of tumor necrosis factor alpha (TNF-α) decreased only in the postoperative group and in the combination exercise protocols. In the morphological analysis, the combination exercise subjects presented an increase in fiber and axon diameter, in the myelination degree and in the number of myelinated fibers. The present study showed that pre- and postoperative exercise achieved values for functional and morphological sciatic nerve regeneration that were significantly better than either the preoperative or postoperative protocols. This experimental study suggests that physical exercise can restore motor and nerve function to a substantial degree when performed using a prophylactic and therapeutic approach., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Objective: Topical cytidine-5'-diphosphocholine (CDP-choline) has been shown to improve the functional recovery and promote the nerve regeneration of injured sciatic nerves in rats. The aims of this study were to test whether CDP-choline was effective at promoting nerve healing when the surgery to repair an injury was delayed and to determine whether the cytidine and/or the choline moieties of CDP-choline contribute to its beneficial actions., Methods: One hundred and fifty Sprague-Dawley rats underwent a surgical procedure that involved damaging the right sciatic nerve and suturing the epineurium. The injured sciatic nerve was either repaired immediately (on the first day) or on the third day after surgery. Rats were assigned to one of five groups and received a topical application of either 0.4 ml of saline (control) or 0.4 ml of 100 μM CDP-choline, cytidine, choline, or cytidine+choline., Results: The sciatic function index (SFI) of the rats in both groups (those who had their nerve repair immediately versus those on day 3) improved gradually by 4, 8, and 12 weeks after surgery. The percentage recovery in SFI score was significantly higher in rats treated with CDP-choline or cytidine+choline at all time points. Axon count increased by ∼50% in rats treated either with CDP-choline or cytidine+choline. Treatment with CDP-choline or cytidine+choline reduced scar formation and decreased nerve adherence when the sciatic nerve was repaired immediately, and rats treated with CDP-choline or cytidine+choline had better axonal organization than control rats. Treatment with choline or cytidine alone led to a less marked improvement in SFI score and failed to increase axon count., Conclusion: Our results demonstrate that CDP-choline, as well as the combination of its metabolites, cytidine+choline, improves the functional recovery and promotes the regeneration of injured sciatic nerves treated with immediate or delayed surgical repair in rats.

Introduction: The advantage of minimally invasive electrodiagnostic methods for periodic evaluation of reinnervation at predefined time intervals does not seem to be widely recognized. In this study, using a rat model, we assessed the utility of periodic electrodiagnostic measurements for monitoring ongoing motor recovery after peripheral nerve injuries of differing severity., Methods: In a comparative study, either unilateral sciatic nerve crush injury (n = 10), end-to-end coaptation (n = 5), or 10-mm nerve autotransplantation (n = 10) were performed. During the 6-16-week period thereafter compound muscle action potentials (CMAPs) were recorded percutaneously every week in anesthetized animals. Motor nerve conduction velocity and percentage of axon loss were calculated and compared with footprint analyses (static sciatic index, SSI) and evaluations of sensory recovery., Results: Our results clearly demonstrate that, although SSI measurements reliably demonstrated progress of regeneration after nerve crush injury only, differences in electrodiagnostically determined values precisely remodeled differences in axonal regeneration, which was confirmed by histomorphometric analysis of axonal regeneration., Conclusions: Percutaneous electrodiagnostic measurements enable reliable estimation of axonal regeneration parameters such as myelination and nerve fiber density and display in close proximity the actual status of axonal regeneration., (Copyright © 2011 Wiley Periodicals, Inc.)

Introduction: Numerous therapeutic interventions have been tested to enhance functional recovery after peripheral nerve injuries., Methods: After sciatic nerve crush in rats we tested balance and coordination and motor control training in sensorimotor tests and analyzed nerve and muscle histology., Results: The balance and coordination training group and the sham group had better results than the sedentary and motor control groups in sensorimotor tests. The sham and balance and coordination groups had a significantly larger muscle area than the other groups, and the balance and coordination group showed significantly better values than the sedentary and motor control groups for average myelin sheath thickness and g-ratio of the distal portion of the nerve., Conclusions: The findings indicate that balance and coordination training improves sciatic nerve regeneration, suggesting that it is possible to revert and/or prevent soleus muscle atrophy and improve performance on sensorimotor tests., (Copyright © 2011 Wiley Periodicals, Inc.)

Sciatic nerve injury results in axon damage, muscle degeneration, and loss of function. We compared the potential of Schwann cell (SC), olfactory ensheathing cell (OEC), or mixed SC/OEC transplants for anatomical and functional restoration after adult rat sciatic nerve transection. The cells were seeded into a 20mm long macroporous poly(dl-lactide-co-glycolide) acid conduit and grafted between the sciatic nerve stumps. Some rats received a conduit without cells (controls) or an autologous nerve graft, the clinical standard of care. Compared with SC transplants, axon regeneration was 25% less with OEC transplants but 28% more with SC/OEC transplants. Gastrocnemius muscle restoration was similar with a SC or OEC transplant and 35% better with a SC/OEC transplant. With SC transplants, motor and sensory function recovery and electrophysiological outcomes were similar as with OEC transplants and 33% better with SC/OEC transplants. Compared with the mixed SC/OEC transplants, axon regeneration was 21% better and gastrocnemius muscle restoration was 18% better with autologous peripheral nerve transplants, but these improvements did not translate into increased function and electrophysiological outcomes. Our results revealed that OEC synergistically improve SC mediated sciatic nerve repair. The data emphasized the promise of SC/OEC transplants as artificial nerves for peripheral nerve repair. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The postsurgical period during which neurons remain without target connections (chronic axotomy) and distal nerve stumps and target muscles are denervated (chronic denervation) deleteriously affects functional recovery. An autologous nerve graft and cross-suture paradigm in Sprague Dawley rats was used to systematically and independently control time of motoneuron axotomy, denervation of distal nerve sheaths, and muscle denervation to determine relative contributions of each factor to recovery failure. Tibial (TIB) nerve was cross-sutured to common peroneal (CP) nerve via a contralateral 15 mm nerve autograft to reinnervate the tibialis anterior (TA) muscle immediately or after prolonging TIB axotomy, CP autograft denervation, or TA muscle denervation. Numbers of motoneurons that reinnervated TA muscle declined exponentially from 99 ± 15 to asymptotic mean (± SE) values of 35 ± 1, 41 ± 10, and 13 ± 5, respectively. Enlarged reinnervated motor units fully compensated for reduced motoneuron numbers after prolonged axotomy and autograft denervation, but the maximal threefold enlargement did not compensate for the severe loss of regenerating nerves through chronically denervated nerve stumps and for failure of reinnervated muscle fibers to recover from denervation atrophy. Muscle force, weight, and cross-sectional area declined. Our results demonstrate that chronic denervation of the distal stump plays a key role in reduced nerve regeneration, but the denervated muscle is also a contributing factor. That chronic Schwann cell denervation within the nerve autograft reduced regeneration less than after the denervation of both CP nerve stump and TA muscle, argues that chronic muscle denervation negatively impacts nerve regeneration.

Background and Objective: Post-traumatic nerve repair is still a challenge for rehabilitation. It is particularly important to develop clinical protocols to enhance nerve regeneration. The present study investigated the effects of 660 and 780 nm low-level laser therapy (LLLT) using different energy densities (10, 60, and 120 J/cm²) on neuromuscular and functional recovery as well as on matrix metalloproteinase (MMP) activity after crush injury in rat sciatic nerve., Materials and Methods: Rats received transcutaneous LLLT irradiation at the lesion site for 10 consecutive days post-injury and were sacrificed 28 days after injury. Both the sciatic nerve and tibialis anterior muscles were analyzed. Nerve analyses consisted of histology (light microscopy) and measurements of myelin, axon, and nerve fiber cross-sectional area (CSA). S-100 labeling was used to identify myelin sheath and Schwann cells. Muscle fiber CSA and zymography were carried out to assess the degree of muscle atrophy and MMP activity, respectively. Statistical significance was set at 5% (P≤0.05)., Results: Six hundred sixty nanometer LLLT either using 10 or 60 J/cm² restored muscle fiber, myelin and nerve fiber CSA compared to the normal group (N). Furthermore, it increased MMP-2 activity in nerve and decreased MMP-2 activity in muscle and MMP-9 activity in nerve. In contrast, 780 nm LLLT using 10 J/cm² decreased MMP-9 activity in nerve compared to the crush group (CR) and N; it also restored normal levels of myelin and nerve fiber CSA. Both 60 and 120 J/cm² decreased MMP-2 activity in muscle compared to CR and N. 780 nm did not prevent muscle fiber atrophy. Functional recovery in the irradiated groups did not differ from the non-irradiated CR., Conclusion: Data suggest that 660 nm LLLT with low (10 J/cm²) or moderate (60 J/cm²) energy densities is able to accelerate neuromuscular recovery after nerve crush injury in rats., (© 2010 Wiley-Liss, Inc.)

Background: The timing of electrical stimulation (ES) after peripheral nerve transection may enhance axonal regeneration and functional recovery., Objective: The authors examined whether percutaneous ES at 1 mA and 2 Hz affects regeneration between the proximal and distal nerve stumps., Methods: Four groups of adult rats were subjected to sciatic nerve section followed by repair using silicone rubber conduits across a 10-mm gap. All groups received ES for 15 minutes every other day for 2 weeks. Stimulation was initiated on day 1 following the nerve repair for group A, day 8 for group B, and day 15 for group C. The control group D received no ES., Results: At 6 weeks after surgery in groups B and C, histological evaluations showed a significantly higher number of regenerated myelinated fibers in the sciatic nerve, and the electrophysiological results showed higher levels of reinnervation with relatively larger mean values of amplitudes, durations, and areas of compound muscle action potentials compared with A and D., Conclusion: A short delay in the onset of ES may improve the recovery of a severe peripheral nerve injury, which should be considered as a way of augmenting rehabilitative approaches.

Background: Electrical stimulation (ES) can enhance the regenerative capacity of axotomized motor and sensory neurons. However, the impact of ES on axonal regeneration and functional recovery has not been investigated in an animal model of a lengthy peripheral nerve defect., Objective: To determine whether ES accelerates axonal regeneration and functional recovery of a 15-mm sciatic nerve defect in rats., Methods: A 15-mm excision of the sciatic nerve was bridged with a chitosan scaffold with longitudinally or randomly oriented pores or with autologous grafting of the segment. In half of the animals with chitosan grafts, the proximal nerve stump was electrically stimulated for 1 hour at 20 Hz immediately after the nerve repair with the scaffolds. Axonal regeneration was investigated by retrograde labeling and morphometric analysis. The rate of motor functional recovery was evaluated by electrical nerve stimulation, behavioral tests of stepping, and histological appearance of the target muscles., Results: Axonal regeneration and motor functional recovery were improved by ES in animals that received longitudinal pore grafts as compared with others. The maximal number of axons that regenerated across the longitudinal graft was achieved 2 to 4 weeks earlier in rats with ES. In addition, the latency of compound muscle action potentials (CMAPs), the peak amplitude of CMAPs, and nerve conduction velocity were improved by ES. Stepping indices were better, with less atrophy of target muscle in ES rats managed with longitudinal pores., Conclusion: Brief ES may accelerate axonal regeneration and motor recovery after focal peripheral nerve transection when repaired with optimally tissue-engineered grafts.

Glial cells are a key element to the process of axonal regeneration, either promoting or inhibiting axonal growth. The study of glial derived factors induced by injury is important to understand the processes that allow or preclude regeneration, and can explain why the PNS has a remarkable ability to regenerate, while the CNS does not. In this work we focus on Apolipoprotein D (ApoD), a Lipocalin expressed by glial cells in the PNS and CNS. ApoD expression is strongly induced upon PNS injury, but its role has not been elucidated. Here we show that ApoD is required for: (1) the maintenance of peripheral nerve function and tissue homeostasis with age, and (2) an adequate and timely response to injury. We study crushed sciatic nerves at two ages using ApoD knock-out and transgenic mice over-expressing human ApoD. The lack of ApoD decreases motor nerve conduction velocity and the thickness of myelin sheath in intact nerves. Following injury, we analyze the functional recovery, the cellular processes, and the protein and mRNA expression profiles of a group of injury-induced genes. ApoD helps to recover locomotor function after injury, promoting myelin clearance, and regulating the extent of angiogenesis and the number of macrophages recruited to the injury site. Axon regeneration and remyelination are delayed without ApoD and stimulated by excess ApoD. The mRNA and protein expression profiles reveal that ApoD is functionally connected in an age-dependent manner to specific molecular programs triggered by injury.

Improvement of nerve regeneration and functional recovery following nerve injury is a challenging problem in clinical research. We have already shown that following rat sciatic nerve transection, the local administration of triiodothyronine (T3) significantly increased the number and the myelination of regenerated axons. Functional recovery is a sum of the number of regenerated axons and reinnervation of denervated peripheral targets. In the present study, we investigated whether the increased number of regenerated axons by T3-treatment is linked to improved reinnervation of hind limb muscles. After transection of rat sciatic nerves, silicone or biodegradable nerve guides were implanted and filled with either T3 or phosphate buffer solution (PBS). Neuromuscular junctions (NMJs) were analyzed on gastrocnemius and plantar muscle sections stained with rhodamine alpha-bungarotoxin and neurofilament antibody. Four weeks after surgery, most end-plates (EPs) of operated limbs were still denervated and no effect of T3 on muscle reinnervation was detected at this stage of nerve repair. In contrast, after 14 weeks of nerve regeneration, T3 clearly enhanced the reinnervation of gastrocnemius and plantar EPs, demonstrated by significantly higher recovery of size and shape complexity of reinnervated EPs and also by increased acetylcholine receptor (AChRs) density on post synaptic membranes compared to PBS-treated EPs. The stimulating effect of T3 on EP reinnervation is confirmed by a higher index of compound muscle action potentials recorded in gastrocnemius muscles. In conclusion, our results provide for the first time strong evidence that T3 enhances the restoration of NMJ structure and improves synaptic transmission.

Genetic engineering of mice has become a major tool in understanding the roles of individual molecules in regeneration of nerves, and will play an increasing role in the future. Mice are in many ways well suited to assessment both of nerve regeneration after axotomy and of collateral sprouting of intact fibers into areas of denervation. However, mouse models present special challenges because of their small size, their inherent capacity for regeneration, and the potential strain effects. The most widely used model of regeneration, sciatic nerve injury, has its inherent limitations, and there is a need for other models of injury to long nerves. Measures of regeneration in the mouse can be divided into those that assess the latency to initiate growth, those sensitive to the rate of growth and the proportion of fibers growing at fast rates, those that assess the time to reinnervation of specific targets and the completeness of reinnervation, and those that assess specificity of reinnervation and functional recovery. The short length of nerve available in the mouse limits measures of the rates of outgrowth, and thus introduces a greater potential for "noise" of measurement than is seen in larger animals such as the rat. For both regeneration of interrupted fibers and collateral regeneration from intact fibers histological and physiological measures of "time to target" have the advantages of direct correlation with restoration of function, the ability to assess regeneration of different fiber types efficiently, and the fact that most of these measures are easier in the mouse than in the rat., (Copyright 2010. Published by Elsevier Inc.)

Neurotrophic factors have been shown to stimulate and support peripheral nerve repair. One of these factors is basic fibroblast growth factor (FGF-2), which is up-regulated after peripheral nerve injury and influences early sciatic nerve regeneration by regulating Schwann cell proliferation. Our previous study on FGF-2 deficient mice indicated that FGF-2 is important for axonal maturation and remyelination one week after sciatic nerve crush (Jungnickel, J., Claus, P., Gransalke, K., Timmer, M. and Grothe, C., 2004. Targeted disruption of the FGF-2 gene affects the response to peripheral nerve injury. Mol. Cell. Neurosci. 25, 444-452). However, the functional impact of these effects on sensory and motor fibers was not clear. After performing pinch test, walking track analysis and rotarod, we found faster recovery of mechanosensory but not of motor function in mutant mice. To elucidate the role of FGF-2 on structural recovery, we analyzed FGF-2 deficient mice and wild-type littermates 2 and 4 weeks after sciatic nerve crush. Two weeks after peripheral nerve injury, regenerating fibers of mutant mice showed both significantly increased axon and myelin size, but no difference in the number of myelinated and unmyelinated fibers. Molecular analysis indicated that the expression level of myelin protein zero was significantly enhanced in lesioned nerves in the absence of FGF-2. These results suggest that loss of FGF-2 could positively influence restoration of mechanosensory function by accelerating structural recovery transiently., (Copyright 2009 Elsevier Inc. All rights reserved.)

Neuromuscular recovery after peripheral nerve lesion depends on the regeneration of severed axons that re-establish their functional connection with the denervated muscle. The aim of this study was to determine the effects of electrical stimulation (ES) on the neuromuscular recovery after nerve crush injury in rats. Electrical stimulation was carried out on the tibialis anterior (TA) muscle after sciatic nerve crush injury in a rat model. Six ES sessions were administered every other day starting from day 3 postinjury until the end of the experiment (day 14). The sciatic functional index was calculated. Muscle excitability, neural cell adhesion molecule (N-CAM) expression, and muscle fiber cross-sectional area (CSA) were accessed from TA muscle. Regenerated sciatic nerves were analyzed by light and confocal microscopy. Both treated (crush+ES) and untreated (crush) groups had their muscle weight and CSA decreased compared with the normal group (P < 0.05). Electrical stimulation accentuated muscle fiber atrophy more in the crush+ES than in the crush group (P < 0.05). N-CAM expression increased in both crush and crush+ES groups compared with the normal group (P < 0.05). Regenerated nerves revealed no difference between the crush and crush+ES groups. Nevertheless, functional recovery at day 14 post-injury was significantly lower in crush+ES group compared with the crush group. In addition, the crush+ES group had chronaxie values significantly higher on days 7 and 13 compared with the crush group, which indicates a decrease in muscle excitability in the crush+ES animals. The results of this study do not support a benefit of the tested protocol of ES during the period of motor nerve recovery following injury.

Assessment of motor abilities after sciatic nerve injury in rodents, in particular mice, relies exclusively on walking track (footprint) analysis despite known limitations of this method. Using principles employed recently for video-based motion analyses after femoral nerve and spinal cord injuries, we have designed and report here a novel approach for functional assessments after sciatic nerve lesions in mice. Functional deficits are estimated by angle and distance measurements on single video frames recorded during beam-walking and inclined ladder climbing. Analyses of adult C57BL/6J mice after crush of the sciatic, tibial, or peroneal nerve allowed the identification of six numerical parameters, detecting impairments of the plantar flexion of the foot and the toe spread. Some of these parameters, as well as footprint functional indices, revealed severe impairment after crush injury of the sciatic or tibial, but not the peroneal nerve, and complete recovery within 3 weeks after lesion. Other novel estimates, however, showed that complete recovery is reached as late as 2-3 months after sciatic nerve crush. These measures detected both tibial and peroneal dysfunction. In contrast to the complete restoration of function in wild-type mice (100%), our new parameters, in contrast to the sciatic functional index, showed incomplete recovery (85%) 90 days after sciatic nerve crush in mice deficient in the neural cell adhesion molecule (NCAM). We conclude that the novel video-based approach is more precise, sensitive, and versatile than established tests, allowing objective numerical assessment of different motor functions in a sciatic nerve injury paradigm in mice.

Preventing the harm caused by nerve degeneration is a major challenge in neurodegenerative diseases and in various forms of trauma to the nervous system. The aim of the current work was to investigate the effects of systemic administration of 2,4-dinitrophenol (DNP), a compound with newly recognized neuroprotective properties, on sciatic-nerve degeneration following a crush injury. Sciatic-nerve injury was induced by unilateral application of an aneurysm clip. Four groups of mice were used: uninjured, injured treated with vehicle (PBS), injured treated with two intraperitoneal doses of DNP (0.06 mg DNP/kg every 24 h), and injured treated with four doses of DNP (every 12 h). Animals were sacrificed 48 h post injury and both injured and uninjured (contralateral) sciatic nerves were processed for light and electron microscopy. Morphometric, ultrastructural, and immunohistochemical analysis of injured nerves established that DNP prevented axonal degeneration, blocked cytoskeletal disintegration, and preserved the immunoreactivity of amyloid precursor protein (APP) and Neuregulin 1 (Nrg1), proteins implicated in neuronal survival and myelination. Functional tests revealed preservation of limb function following injury in DNP-treated animals. Results indicate that DNP prevents nerve degeneration and suggest that it may be a useful small-molecule adjuvant in the development of novel therapeutic approaches in nerve injury.

Background and Purpose: EPO has been shown to have beneficial effects in a variety of CNS injury models. The purpose of this study was to evaluate the effects of EPO on nerve regeneration and functional recovery in a rat model of peripheral nerve surgery., Materials and Methods: The sciatic nerve of the rat with a 10-mm defect was bridged with a silicone rubber tube. Forty adult male Sprague-Dawley rats were assigned to the control or experimental groups to receive an intraperitoneal injection of NGF (2000 U/kg daily for 2 weeks) or EPO (5000 U/kg daily for 2 weeks), respectively. Macroscopic, functional, electrophysiologic, ultraminiature, and histologic assessments of nerves were performed 4-8 weeks after surgery., Results: The results showed that in EPO-treated rats, there was a significant increase in the axon diameter, myelin thickness, and total number of nerve fibers as well as the degree of maturity of regenerated myelinated nerve fibers in comparison with those rats not treated with EPO. In addition, as measured by the SFI and MNCV, the motor function of the re-innervated hind limbs of rats with EPO treatment significantly improved at week 8, whereas there was no significant difference in the motor function between the 2 groups at 4 weeks., Conclusions: Our results demonstrated that EPO is able to enhance nerve regeneration and promote functional recovery after peripheral nerve injury in the rat, suggesting the potential clinical application of EPO for the treatment of peripheral nerve injury in humans.

Objective: To describe a refined technique for performing electrical impedance myography (EIM) in the rat and assess its reproducibility, long-term stability, and the effects of sciatic nerve injury., Methods: EIM at 50kHz was performed on the gastrocnemius-soleus complex of the rat hind limb in 12 rats, followed from 6 weeks of age for up to 6 months. Eight additional rats underwent sciatic nerve crush and 6 underwent a sham procedure., Results: The EIM variables of resistance, reactance and phase demonstrated substantial change with growth until approximately 14 weeks of age, at which point the measurements stabilized, giving mean values of 6.0+/-5.7Omega, 22.1+/-2.1Omega, and 16.5+/-1.1 degrees , respectively, at 16 weeks of age. Immediate reproducibility of technique was high with an intraclass correlation coefficient of 0.91 and higher for all three parameters. Sciatic crush produced marked reductions in the reactance and phase that reversed over a several week period., Conclusions: These results support that 50kHz EIM can be performed effectively in adult rat models of neuromuscular disease with a straightforward experimental technique and that it is sensitive to neurogenic injury., Significance: EIM can serve as a new approach to the study of neuromuscular disease in the rat.

The success of peripheral nerve regeneration depends on intrinsic properties of neurons and a favorable environment, although the mechanisms underlying the molecular events during degeneration and regeneration are still not elucidated. Schwann cells are considered one of the best candidates to be closely involved in the success of peripheral nerve regeneration. These cells and invading macrophages are responsible for clearing myelin and axon debris, creating an appropriate route for a successful regeneration. After injury, Schwann cells express galectin-3, and this has been correlated with phagocytosis; also, in the presence of galectin-3, there is inhibition of Schwann-cell proliferation in vitro. In the present study we explored, in vivo, the effects of the absence of galectin-3 on Wallerian degeneration and nerve-fiber regeneration. We crushed the sciatic nerves of galectin-3 knockout and wild-type mice, and followed the pattern of degeneration and regeneration from 24 h up to 3 weeks. We analyzed the number of myelinated fibers, axon area, fiber area, myelin area, G-ratio and immunofluorescence for beta-catenin, macrophages and Schwann cells in DAPI counterstained sections. Galectin-3 knockout mice showed earlier functional recovery and faster regeneration than the wild-type animals. We concluded that the absence of galectin-3 allowed faster regeneration, which may be associated with increased growth of Schwann cells and expression of beta-catenin. This would favor neuron survival, followed by faster myelination, culminating in a better morphological and functional outcome.

This study assessed the neuroprotective effects of angelica injection in the rat sciatic nerve crush injury (SCI). Forty eight male Sprague Dawley rats were randomly divided into 4 groups: one was the sham group (S), which received sham surgery and given saline injection and the others were received SCI surgery and given saline injection, high and low dose angelica injection for 4 weeks, respectively. The sciatic functional index (SFI) in walking-track analysis, conductive velocity (CV), the number of fluorogold labeled motoneurons, and the expression patterns of brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in the sciatic nerve and spine were examined. The results showed that SFI descended gradually on day 7, and dropped more quickly on day 28 in treatment groups (Low and High dose group). The CV in treatment groups was higher than control group (C). The numbers of motoneurons in treatment groups were larger than C group (P<0.05), but less than that in S group (P<0.01). The expressions of BDNF and NGF protein in the groups received SCI surgery were significantly lower than in S group, but the protein expressions in the groups received angelica injections were significantly higher than that in C group (P<0.01). These findings suggested that angelica injection can improve the sciatic nerve crush injury, and the mechanism might be through the increase of BDNF and NGF protein expression.

The Sciatic Functional Index (SFI) is widely used to evaluate functional recovery after sciatic nerve injury, primarily in the rat, and more recently shown useful in the mouse. This quantitative, non-invasive method allows tracking of regeneration capability, visible in the gait of the animal. Using a Martin micro needle holder, carrying a force measured to be 49.2 N, the left sciatic nerve was crushed for 60 s. We accumulated data from walking tracks collected preoperatively and 1, 7, 14, 21, and 28 days after injury. SFI values were first calculated in the traditional manner. Then using the preoperative values as the normal value in the postoperative calculations, SFI was again calculated; this isolated the calculations to either injured or contra lateral leg giving a "split" plot. The traditional SFI calculations resulted in typical shaped graphs for both rats and mice. However, the "split" SFI calculations showed how rats and mice differ in their recovery from sciatic nerve injury. The mouse graph shows the intact leg remaining stable and the injured leg having functional impairment, which then recovers. The rat graph showed functional impairment of the injured leg, however, the intact leg had an increase in SFI values as if to compensate until the injured leg showed recovery.

Following peripheral nerve injury repair, improved behavioural outcome may be the most important evidence of functionality of axon regeneration after any repair strategy. A range of behavioural testing paradigms have been developed for peripheral nerve injury research. Complete injury of the adult rat sciatic nerve is frequently used in combination with walking track analysis. Despite its wide-spread use, these walking track analyses are unsuitable for the simultaneous assessment of both dynamic and static gait parameters. Conversely, a novel automated gait analysis system, i.e. CatWalk can simultaneously measure dynamic as well as static gait parameters and, importantly, it's easy to control for the speed of locomotion which can strongly affect gait parameters. In a previous study, CatWalk was already successfully used to examine deficits in both dynamic and static gait parameters using the sciatic nerve lesion model with a 1cm gap characterized by absence of recovery [Deumens R, Jaken RJ, Marcus MA, Joosten EA. The CatWalk gait analysis in assessment of both dynamic and static gait changes after adult rat sciatic nerve resection. J Neurosci Methods 2007;164:120-30]. Using the sciatic nerve crush injury model (validated with the static sciatic index) and a follow-up period of 12 weeks, we now show that CatWalk can also measure behavioural recovery. In particular dynamic gait parameters, coordination measures, and the intensity of paw prints are of interest in detecting recovery as far as these parameters completely return to pre-operative values after crush injury. We conclude that CatWalk can be used as a complementary approach to other behavioural testing paradigms to assess clinically relevant behavioural benefits, with a main advantage that CatWalk demonstrates both static and dynamic gait parameters at the same time.

Toll-like receptors (TLRs) bind specific components conserved among microorganisms as well as endogenous ligands produced by necrotic cells, injured axons, and the extracellular matrix. Here, we investigated whether TLRs are involved in regulating the immune response, Wallerian degeneration (WD), and nerve regeneration after sciatic nerve lesion. Early expression of interleukin-1beta and monocyte chemoattractant protein-1 was compromised in the sciatic nerve distal stump of mice deficient in TLR signaling. In addition, significantly fewer macrophages were recruited and/or activated in the sciatic nerve distal stump of TLR2-, TLR4-, and MyD88-deficient mice compared with wild-type littermates, whereas WD, axonal regeneration, and recovery of locomotor function were impaired. In contrast, animals that received a single microinjection of TLR2 and TLR4 ligands at the site of sciatic nerve lesion had faster clearance of the degenerating myelin and recovered earlier than saline-injected control rats. Finally, rats that had altered innate immune response through dexamethasone treatment exhibited three times more myelin debris in their sciatic nerve distal stump and a significant delay in recovery of locomotor function. Our results provide strong evidence that TLR signaling plays a critical role in orchestrating the innate immune response leading to efficient and rapid clearance of inhibitory myelin debris and nerve regeneration.

The mature peripheral nervous system (PNS) generally shows better regeneration of injured axons as opposed to the central nervous system (CNS). However, complete functional recovery is rarely achieved even in the PNS although morphologically good axonal regeneration often occurs. This mainly results from aberrant reinnervation due to extensive branching of cut axons with consequent failure of synchronized movements of the muscles. Myelin-associated glycoprotein (MAG), a well-characterized molecule existing both in the CNS and PNS myelin, is considered to be a potent inhibitor of axonal regeneration especially in the CNS. In the present study, we investigated whether MAG has any effects not only on axonal elongation, but also on axonal branching. We show herein that MAG minimized branching of the peripheral axons both in vitro and in vivo via activation of RhoA. Furthermore, after sciatic nerve transection in rats, focal and temporary application of MAG to the lesion dramatically enhanced the functional recovery. Using double retrograde labeling and preoperative/postoperative labeling of spinal neurons, reduced hyperinnervation and improved accuracy of target reinnervation was confirmed, respectively. In conclusion, as MAG significantly improves the quality of axonal regeneration, it can be used as a new therapeutic approach for peripheral nerve repair with possible focal and temporary application.

We have recently described the sequence of functional and morphologic changes occurring after a standardized sciatic nerve crush injury. An 8-week post-injury time was used because this end point is the far most used. Unexpectedly, both functional and morphological data revealed that animals had still not recovered to normal pre-injury levels. Therefore, the present study was designed in order to prolong the observation up to 12 weeks. Functional recovery was evaluated using sciatic functional index (SFI), static sciatic index (SSI), extensor postural thrust (EPT), withdrawal reflex latency (WRL) and ankle kinematics. In addition, quantitative morphology was carried out on regenerated nerve fibers. A full functional recovery was predicted by SFI/SSI, EPT and WRL but not all ankle kinematics parameters. Moreover, only two morphological parameters (myelin thickness/axon diameter ratio and fiber/axon diameter ratio) returned to normal values. Data presented in this paper provide a baseline for selecting the adequate end-point and methods of recovery assessment for a rat sciatic nerve crush study and suggest that the combined use of functional and morphological analysis should be recommended in this experimental model.

Multiple molecular mechanisms influence nerve regeneration. Because serine proteases were shown to affect peripheral nerve regeneration, we performed nerve crush experiments to study synapse reinnervation in adult mice lacking the serpin protease nexin-1 (PN-1). PN-1 is a potent endogenous inhibitor of thrombin, trypsin, tissue plasminogen activators (tPAs), and urokinase plasminogen activators. Compared with the wild type, a significant delay in synapse reinnervation was detected in PN-1 knock-out (KO) animals, which was associated with both reduced proliferation and increased apoptosis of Schwann cells. Various factors known to affect Schwann cells were also altered. Fibrin deposits, tPA activity, mature BDNF, and the low-affinity p75 neurotrophin receptor were increased in injured sciatic nerves of mutant mice. To test whether the absence of PN-1 in Schwann cells or in the axon caused delay in reinnervation, PN-1 was overexpressed exclusively in the nerves of PN-1 KO mice. Neuronal PN-1 expression did not rescue the delayed reinnervation. The results suggest that Schwann cell-derived PN-1 is crucial for proper reinnervation through its contribution to the autocrine control of proliferation and survival. Thus, the precise balance between distinct proteases and serpins such as PN-1 can modulate the overall impact on the kinetics of recovery.

In injured adult neurons, the process of axonal regrowth and reestablishment of the neuronal function have to be activated. We assessed in this study whether RhoA, a key regulator of neurite elongation, is activated after injury to the peripheral nervous system. RhoA is activated in motoneurons but not in Schwann cells after mouse sciatic nerve injury. To examine whether the activation of RhoA and its effector, Rho-kinase, retards axon regeneration of injured motoneurons, we employed a Rho-kinase inhibitor, fasudil. Amplitudes of distally evoked compound muscle action potentials are increased significantly faster after axonal injury in mice treated with fasudil compared with controls. Histological analysis shows that fasudil treatment increases the number of regenerating axons with large diameter, suggesting that axon maturation is facilitated by Rho-kinase inhibition. In addition, fasudil does not suppress the myelination of regenerating axons. These findings suggest that RhoA/Rho-kinase may be a practical molecular target to enhance axonal regeneration in human peripheral neuropathies.