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This article highlights the use of rodents as preclinical models to evaluate the management of nerve injuries, describing the pitfalls and value from rodent nerve injury and regeneration outcomes, as well as treatments derived from these rodent models. The anatomic structure, size, and cellular and molecular differences and similarities between rodent and human nerves are summarized. Specific examples of success and failure when assessing outcome metrics are presented for context. Evidence for translation to clinical practice includes the topics of electrical stimulation, Tacrolimus (FK506), and acellular nerve allografts., Competing Interests: Disclosure M.D. Wood has been the recipient of sponsored research agreements from Checkpoint Surgical, Inc. and has consulted for AxoGen Inc, KLIS Bio Inc, Renerva, LLC, and Tissium, S.A. No consulting relationship affected the materials in this manuscript and no personal compensation was provided. None of the other authors has any conflict of interest to disclose. This work was supported in part by the National Institutes of Neurological Disorders and Stroke of the National Institutes of Health, United States (NIH) under award number R01 NS115960 (MDW) to Washington University., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Introduction/aims: Promoting regeneration after segmental nerve injury repair is a challenge, but improving angiogenesis could be beneficial. Macrophages facilitate regeneration after injury by promoting angiogenesis. Our aim in this study was to evaluate the feasibility and effects of transplanting exogenous macrophages to a segmental nerve injury., Methods: Bone marrow-derived cells were harvested from donor mice and differentiated to macrophages (BMDM), then suspended within fibrin hydrogels to facilitate BMDM transplantation. BMDM survival was characterized in vitro. The effect of this BMDM fibrin hydrogel construct at a nerve injury site was assessed using a mouse sciatic nerve gap injury. Mice were equally distributed to "fibrin+Mφ" (fibrin hydrogels containing culture medium and BMDM) or "fibrin" hydrogel control (fibrin hydrogels containing culture medium alone) groups. Flow cytometry (n = 3/group/endpoint) and immunohistochemical analysis (n = 5/group/endpoint) of the nerve gap region were performed at days 3, 5, and 7 after repair., Results: Incorporating macrophage colony-stimulating factor (M-CSF) improved BMDM survival and expansion. Transplanted BMDM survived for at least 7 days in a nerve gap (~40% retained at day 3 and ~15% retained at day 7). From transplantation, macrophage quantities within the nerve gap were elevated when comparing fibrin+Mφ with fibrin control (~25% vs. 3% at day 3 and ~14% vs. 6% at day 7). Endothelial cells increased by about fivefold within the nerve gap, and axonal extension into the nerve gap increased almost twofold for fibrin+Mφ compared with fibrin control., Discussion: BMDM suspended within fibrin hydrogels at a nerve gap do not impair regeneration., (© 2023 Wiley Periodicals LLC.)

The immune system has garnered attention for its role in peripheral nerve regeneration, particularly as it pertains to regeneration across segmental injuries. Previous work demonstrated that eosinophils are recruited to regenerating nerve and express interleukin-4, amongst potential cytokines. These results suggest a direct role for eosinophils in promoting nerve regeneration. Therefore, we further considered eosinophils roles in nerve regeneration using a segmental nerve injury and Gata1 knockout (KO) mice, which are severely eosinophil deficient, compared to wild-type BALB/c mice (WT). Mice receiving a sciatic nerve gap injury demonstrated distinct cytokine expression and leukocytes within regenerating nerve. Compared to controls, Gata1 KO regenerated nerves contained decreased expression of type 2 cytokines, including Il-5 and Il-13, and decreased recruitment of eosinophils and macrophages. At this early time point during ongoing regeneration, the macrophages within Gata1 KO nerves also demonstrated significantly less M2 polarization compared to controls. Subsequently, motor and sensory axon regeneration across the gap injury was decreased in Gata1 KO compared to WT during ongoing nerve regeneration. Over longer observation to allow for more complete nerve regeneration, behavioral recovery measured by grid-walk assessment was not different comparing groups but modestly delayed in Gata1 KO compared to WT. The extent of final axon regeneration was not different amongst groups. Our data provide additional evidence suggesting eosinophils contribute to nerve regeneration across a nerve gap injury, but are not essential to regeneration in this context. Our evidence also suggests eosinophils may regulate cytokines that promote distinct macrophage phenotypes and axon regeneration., Competing Interests: Declaration of Competing Interest M.D.W. has been the recipient of sponsored research agreements from Checkpoint Surgical, Inc. and has consulted for Renerva, LLC and Silk Biomaterials, SRL. No research agreements or consulting relationships affected the materials in this manuscript. No personal compensation was provided. None of the other authors have any conflicts of interest to disclosure., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Acellular nerve allografts have been used successfully and with increasing frequency to reconstruct nerve injuries. As their use has been expanded to treat longer gap, larger diameter nerve injuries, some failed cases have been reported. We present the histomorphometry of 5 such cases illustrating these limitations and review the current literature of acellular nerve allografts., Methods: Between 2014 and 2019, 5 patients with iatrogenic nerve injuries to the median or ulnar nerve reconstructed with an AxoGen AVANCE nerve allograft at an outside hospital were treated in our center with allograft excision and alternative reconstruction. These patients had no clinical or electrophysiological evidence of recovery, and allograft specimens at the time of surgery were sent for histomorphological examination., Results: Three patients with a median and 2 with ulnar nerve injury were included. Histology demonstrated myelinated axons present in all proximal native nerve specimens. In 2 cases, axons failed to regenerate into the allograft and in 3 cases, axonal regeneration diminished or terminated within the allograft., Conclusions: The reported cases demonstrate the importance of evaluating the length and the function of nerves undergoing acellular nerve allograft repair. In long length, large-diameter nerves, the use of acellular nerve allografts should be carefully considered.

Background: Clinical outcomes following nerve injury repair can be inadequate. Pulsed-current electrical stimulation (ES) is a therapeutic method that facilitates functional recovery by accelerating axon regeneration. However, current clinical ES protocols involve the application of ES for 60 minutes during surgery, which can increase operative complexity and time. Shorter ES protocols could be a strategy to facilitate broader clinical adoption. The purpose of the present study was to determine if a 10-minute ES protocol could improve outcomes., Methods: C57BL/6J mice were randomized to 3 groups: no ES, 10 minutes of ES, and 60 minutes of ES. In all groups, the sciatic nerve was transected and repaired, and, in the latter 2 groups, ES was applied after repair. Postoperatively, changes to gene expression from dorsal root ganglia were measured after 24 hours. The number of motoneurons regenerating axons was determined by retrograde labeling at 7 days. Histomorphological analyses of the nerve were performed at 14 days. Function was evaluated serially with use of behavioral tests up to 56 days postoperatively, and relative muscle weight was evaluated., Results: Compared with the no-ES group, both ES groups demonstrated increased regeneration-associated gene expression within dorsal root ganglia. The 10-minute and 60-minute ES groups demonstrated accelerated axon regeneration compared with the no-ES group based on increased numbers of labeled motoneurons regenerating axons (mean difference, 202.0 [95% confidence interval (CI), 17.5 to 386.5] and 219.4 [95% CI, 34.9 to 403.9], respectively) and myelinated axon counts (mean difference, 559.3 [95% CI, 241.1 to 877.5] and 339.4 [95% CI, 21.2 to 657.6], respectively). The 10-minute and 60-minute ES groups had improved behavioral recovery, including on grid-walking analysis, compared with the no-ES group (mean difference, 11.9% [95% CI, 3.8% to 20.0%] and 10.9% [95% CI, 2.9% to 19.0%], respectively). There was no difference between the ES groups in measured outcomes., Conclusions: A 10-minute ES protocol accelerated axon regeneration and facilitated functional recovery., Clinical Relevance: The brief (10-minute) ES protocol provided similar benefits to the 60-minute protocol in an acute sciatic nerve transection/repair mice model and merits further studies., Competing Interests: Disclosure: The Disclosure of Potential Conflicts of Interest forms are provided with the online version of the article (http://links.lww.com/JBJS/G615)., (Copyright © 2021 by The Journal of Bone and Joint Surgery, Incorporated.)

Following traumatic peripheral nerve injury, adequate restoration of function remains an elusive clinical goal. Recent research highlights the complex role that the immune system plays in both nerve injury and regeneration. Pro-regenerative processes in wounded soft tissues appear to be significantly mediated by cytokines of the type 2 immune response, notably interleukin (IL)-4. While IL-4 signaling has been firmly established as a critical element in general tissue regeneration during wound healing, it has also emerged as a critical process in nerve injury and regeneration. In this context of peripheral nerve injury, endogenous IL-4 signaling has recently been confirmed to influence more than leukocytes, but including also neurons, axons, and Schwann cells. Given the role IL-4 plays in nerve injury and regeneration, exogenous IL-4 and/or compounds targeting this signaling pathway have shown encouraging preliminary results to treat nerve injury or other neuropathy in rodent models. In particular, the exogenous stimulation of the IL-4 signaling pathway appears to promote postinjury neuron survival, axonal regeneration, remyelination, and thereby improved functional recovery. These preclinical data strongly suggest that targeting IL-4 signaling pathways is a promising translational therapy to augment treatment approaches of traumatic nerve injury. However, a better understanding of the type 2 immune response and associated signaling networks functioning within the nerve injury microenvironment is still needed to fully develop this promising therapeutic avenue.

Despite advances in surgery, the reconstruction of segmental nerve injuries continues to pose challenges. In this review, current neurobiology regarding regeneration across a nerve defect is discussed in detail. Recent findings include the complex roles of nonneuronal cells in nerve defect regeneration, such as the role of the innate immune system in angiogenesis and how Schwann cells migrate within the defect. Clinically, the repair of nerve defects is still best served by using nerve autografts with the exception of small, noncritical sensory nerve defects, which can be repaired using autograft alternatives, such as processed or acellular nerve allografts. Given current clinical limits for when alternatives can be used, advanced solutions to repair nerve defects demonstrated in animals are highlighted. These highlights include alternatives designed with novel topology and materials, delivery of drugs specifically known to accelerate axon growth, and greater attention to the role of the immune system., (© 2020 Wiley Periodicals, Inc.)

Surgical intervention followed by physical therapy remains the major way to repair damaged nerves and restore function. Imaging constitutes promising, yet underutilized, approaches to improve surgical and postoperative techniques. Dedicated methods for imaging nerve regeneration will potentially provide surgical guidance, enable recovery monitoring and postrepair intervention, elucidate failure mechanisms and optimize preclinical procedures. Herein, we present an outline of promising innovations in imaging-based tracking of in vivo peripheral nerve regeneration. We emphasize optical imaging because of its cost, versatility, relatively low toxicity and sensitivity. We discuss the use of targeted probes and contrast agents (small molecules and nanoparticles) to facilitate nerve regeneration imaging and the engineering of grafts that could be used to track nerve repair. We also discuss how new imaging methods might overcome the most significant challenges in nerve injury treatment.

Providing temporally regulated glial cell line-derived neurotrophic factor (GDNF) to injured nerve can promote robust axon regeneration. However, it is poorly understood why providing highly elevated levels of GDNF to nerve can lead to axon entrapment in the zone containing elevated GDNF. This limited understanding represents an obstacle to the translation of GDNF therapies to treat nerve injuries clinically. Here, we investigated how transgenic Schwann cells (SCs) overexpressing GDNF-IRES-DsRed impact nerve regeneration. Cultured primary SCs were transduced with lentiviruses (GDNF-overexpressing transgenic SCs), one of which provides the capability to express high levels of GDNF and regulate temporal GDNF expression. These SC groups were transplanted into acellular nerve allografts (ANAs) bridging a 14 mm rat sciatic nerve defect. GDNF-overexpressing transgenic SCs expressing GDNF for as little as 1 week decreased axon regeneration across ANAs and caused extensive extracellular matrix (ECM) remodeling. To determine whether additional gene expression changes beyond GDNF transgene expression occurred in GDNF-overexpressing transgenic SCs, microarray analysis of GDNF-overexpressing transgenic SCs compared to untreated SCs was performed. Microarray analysis revealed a set of common genes regulated in transgenic SC groups expressing high levels of GDNF compared to untreated SCs. A co-culture model of GDNF-overexpressing transgenic SCs with fibroblasts (FBs) revealed differential FB ECM-related gene expression compared to untreated SCs. These data suggest a component of axon entrapment is independent of GDNF's impact on axons. Biotechnol. Bioeng. 2017;114: 2121-2130. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Background: The goal of this study was to develop a partial, nonregenerative nerve injury model in a rat that results in permanently reduced motoneuron numbers and function. This model could serve as a platform for the study of therapeutics, such as a reverse end-to-side nerve transfer (i.e., supercharge). The authors hypothesized that transection of one or more of the L4 to L6 nerve roots supplying the sciatic nerve would cause a permanent reduction in muscle force., Methods: Rats were randomized into five groups that underwent variations of nerve root transections or sham injury. The L4 to L6 nerve roots were selectively transected and capped to prevent regeneration. Tibial and common peroneal nerves were harvested for quantitative histology and retrograde-labeled to assess the number of motoneurons projecting axons. Muscle force and relative muscle mass were assessed as metrics of postinjury motor function., Results: At 6 months, the number of motoneurons projecting axons and myelinated axon counts were reduced in both the tibial and common peroneal nerves after injury in all groups. Transecting both L4 and L5 or both L4 and L6 reduced motoneuron numbers sufficiently below sham numbers to reduce muscle force and mass in major muscles of the hindlimb innervated by both nerves. Transecting L4 reduced muscle force and mass in common peroneal-innervated muscles, whereas transecting L5 reduced muscle force and mass in tibial-innervated muscles. These findings were stable over time., Conclusion: Transection of nerve roots produces stable (time-independent) partial nerve injury models with a selective decrease in motor function.

Peripheral nerve injury evokes a complex cascade of chemical reactions including generation of molecular radicals. Conversely, the reactions within nerve induced by stress are difficult to directly detect or measure to establish causality. Monitoring these reactions in vivo would enable deeper understanding of the nature of the injury and healing processes. Here, we utilized near-infrared fluorescence molecular probes delivered via intra-neural injection technique to enable live, in vivo imaging of tissue response associated with nerve injury and stress. These initially quenched fluorescent probes featured specific sensitivity to hydroxyl radicals and become fluorescent upon encountering reactive oxygen species (ROS). Intraneurally delivered probes demonstrated rapid activation in injured rat sciatic nerve but minimal activation in normal, uninjured nerve. In addition, these probes reported activation within sciatic nerves of living rats after a stress caused by a pinprick stimulus to the abdomen. This imaging approach was more sensitive to detecting changes within nerves due to the induced stress than other techniques to evaluate cellular and molecular changes. Specifically, neither histological analysis of the sciatic nerves, nor the expression of pain and stress associated genes in dorsal root ganglia could provide statistically significant differences between the control and stressed groups. Overall, the results demonstrate a novel imaging approach to measure ROS in addition to the impact of ROS within nerve in live animals., Competing Interests: No benefit of any kind will be received either directly or indirectly by the author(s). The authors declare no competing financial interests., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The use of growth factors, such as glial cell line-derived neurotrophic factor (GDNF), for the treatment of peripheral nerve injury has been useful in promoting axon survival and regeneration. Unfortunately, finding a method that delivers the appropriate spatial and temporal release profile to promote functional recovery has proven difficult. Some release methods result in burst release profiles too short to remain effective over the regeneration period; however, prolonged exposure to GDNF can result in axonal entrapment at the site of release. Thus, GDNF was delivered in both a spatially and temporally controlled manner using a two-phase system comprised of an affinity-based release system and conditional lentiviral GDNF overexpression from Schwann cells (SCs). Briefly, SCs were transduced with a tetracycline-inducible (Tet-On) GDNF overexpressing lentivirus before transplantation. Three-centimeter acellular nerve allografts (ANAs) were modified by injection of a GDNF-releasing fibrin scaffold under the epineurium and then used to bridge a 3 cm sciatic nerve defect. To encourage growth past the ANA, GDNF-SCs were transplanted into the distal nerve and doxycycline was administered for 4, 6, or 8 weeks to determine the optimal duration of GDNF expression in the distal nerve. Live imaging and histomorphometric analysis determined that 6 weeks of doxycycline treatment resulted in enhanced regeneration compared to 4 or 8 weeks. This enhanced regeneration resulted in increased gastrocnemius and tibialis anterior muscle mass for animals receiving doxycycline for 6 weeks. The results of this study demonstrate that strategies providing spatial and temporal control of delivery can improve axonal regeneration and functional muscle reinnervation.

Accumulating evidence suggests that neuregulin, a potent Schwann cell mitogen, and its receptor, ErbB2, have an important role in regulating peripheral nerve regeneration. We hypothesized that Herceptin (Trastuzumab), a monoclonal antibody that binds ErbB2, would disrupt ErbB2 signaling, allowing us to evaluate ErbB2's importance in peripheral nerve regeneration. In this study, the extent of peripheral motor and sensory nerve regeneration and distal axonal outgrowth was analyzed two and four weeks after common peroneal (CP) nerve injury in rats. Outcomes analyzed included neuron counts after retrograde labeling, histomorphometry, and protein analysis. The data analysis revealed that there was no impact of Herceptin administration on either the numbers of motor or sensory neurons that regenerated their axons but histomorphometry revealed that Herceptin significantly increased the number of regenerated axons in the distal repaired nerve after 4 weeks. Protein analysis with Western blotting revealed no difference in either expression levels of ErbB2 or the amount of activated, phosphorylated ErbB2 in injured nerves. In conclusion, administration of the ErbB2 receptor inhibitor after nerve transection and surgical repair did not alter the number of regenerating neurons but markedly increased the number of regenerated axons per neuron in the distal nerve stump. Enhanced axon outgrowth in the presence of this ErbB2 inhibitor indicates that ErbB2 signaling may limit the numbers of axons that are emitted from each regenerating neuron., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Despite the inherent capability for axonal regeneration, recovery following severe peripheral nerve injury remains unpredictable and often very poor. Surgeons typically use autologous nerve grafts taken from the patient's own body to bridge long nerve gaps. However, the amount of suitable nerve available from a given patient is limited, and using autologous grafts leaves the patient with scars, numbness, and other forms of donor-site morbidity. Therefore, surgeons and engineers have sought off-the-shelf alternatives to the current practice of autologous nerve grafting. Decellularized nerve allografts have recently become available as an alternative to traditional nerve autografting. In this review, we provide a critical analysis comparing the advantages and limitations of the three major experimental models of decellularized nerve allografts: cold preserved, freeze-thawed, and chemical detergent based. Current tissue engineering-based techniques to optimize decellularized nerve allografts are discussed. We also evaluate studies that supplement decellularized nerve grafts with exogenous factors such as Schwann cells, stem cells, and growth factors to both support and enhance axonal regeneration through the decellularized allografts. In examining the advantages and disadvantages of the studies of decellularized allografts, we suggest that experimental methods, including the animal model, graft length, follow-up time, and outcome measures of regenerative progress and success be consolidated. Finally, all clinical studies in which decellularized nerve allografts have been used to bridge nerve gaps in patients are reviewed.

Introduction: Neuroenhancing therapies are desired because repair of nerve injuries can fail to achieve recovery. We compared two neuroenhancing therapies, electrical stimulation (ES) and systemic tacrolimus (FK506), for their capabilities to enhance regeneration in the context of a rat model.Methods: Rats were randomized to four groups: ES 0.5 mA, ES 2.0 mA, FK506, and repair alone. All groups underwent tibial nerve transection and repair, and outcomes were assessed by using twice per week walking track analysis, cold allodynia response, relative muscle mass, and nerve histology.Results: Electrical stimulation and FK506 groups demonstrated improved functional recovery and myelinated axon counts distal to the repair compared with repair alone. Electrical stimulation provided improvements in nerve regeneration that were not different from optimized FK506 systemic administration.Discussion: Providing ES after nerve repair improved regeneration and recovery in rats, with minimal differences in therapeutic efficacy to FK506, further demonstrating its clinical potential to improve management of nerve injuries. [ABSTRACT FROM AUTHOR]

Following peripheral nerve injury, the distal nerve is primed for regenerating axons by generating a permissive environment replete with glial cells, cytokines, and neurotrophic factors to encourage axonal growth. However, increasing evidence demonstrates that regenerating axons within peripheral nerves still encounter axonal-growth inhibitors, such as chondroitin sulfate proteoglycans. Given the generally poor clinical outcomes following peripheral nerve injury and reconstruction, the use of pharmacological therapies to augment axonal regeneration and overcome inhibitory signals has gained considerable interest. Joshi et al. (2014) have provided evidence for preferential or modality-specific (motor versus sensory) axonal growth and regeneration due to inhibitory signaling from Rho-associated kinase (ROCK) pathway regulation. By providing inhibition to the ROCK signaling pathway through Y-27632, they demonstrate that motor neurons regenerating their axons are impacted to a greater extent compared to sensory neurons. In light of this evidence, we briefly review the literature regarding modality-specific axonal regeneration to provide context to their findings. We also describe potential and novel barriers, such as senescent Schwann cells, which provide additional axonal-growth inhibitory factors for future consideration following peripheral nerve injury. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: Animal models of nerve compression, crush, and transection injuries of peripheral nerves have been subject to extensive study in order to understand the mechanisms of injury and axon regeneration and to investigate methods to promote axon regeneration and improve functional outcomes following nerve injury. Six outcome measures of regenerative success including axon and neuron counts, muscle and motor unit contractile forces, and behavior are reviewed in the context of nerve injury types, crush, transection and nerve repair by direct coaptation, or transection and repair via a nerve graft or conduit. The measures are evaluated for sciatic, tibial, common peroneal, femoral, single nerve branches such as the soleus nerve, and facial nerves. Their validity is discussed in the context of study objectives and the nerve branch. The case is made that outcome measures of axon counts and muscle contractile forces may be valid during the early phases of axon regeneration when regenerating sprouts emerge asynchronously from the proximal nerve stump and regenerate towards their denervated targets. However, care must be taken especially when experimental interventions differentially affect how many neurons regenerate axons and the number of axons per neuron that sprout from the proximal nerve stumps. Examples of erroneous conclusions are given to illustrate the need for researchers to ensure that the appropriate outcome measures are used in the evaluation of the success of peripheral nerve regeneration. [Copyright &y& Elsevier]