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Introduction: Peripheral nerves are frequently affected by lesions caused by traumatic or iatrogenic damages, resulting in loss of motor and sensory function, crucial in orthopedic outcomes and with a significant impact on patients' quality of life. Many strategies have been proposed over years to repair nerve injuries with substance loss, to achieve musculoskeletal reinnervation and functional recovery. Allograft have been tested as an alternative to the gold standard, the autograft technique, but nerves from donors frequently cause immunogenic response. For this reason, several studies are focusing to find the best way to decellularize nerves preserving either the extracellular matrix, either the basal lamina, as the key elements used by Schwann cells and axons during the regenerative process., Methods: This study focuses on a novel decellularization protocol for porcine nerves, aimed at reducing immunogenicity while preserving essential elements like the extracellular matrix and basal lamina, vital for nerve regeneration. To investigate the efficacy of the decellularization protocol to remove immunogenic cellular components of the nerve tissue and to preserve the basal lamina and extracellular matrix, morphological analysis was performed through Masson's Trichrome staining, immunofluorescence, high resolution light microscopy and transmission electron microscopy. Decellularized porcine nerve graft were then employed in vivo to repair a rat median nerve lesion. Morphological analysis was also used to study the ability of the porcine decellularized graft to support the nerve regeneration., Results and Discussion: The decellularization method was effective in preparing porcine superficial peroneal nerves for grafting as evidenced by the removal of immunogenic components and preservation of the ECM. Morphological analysis demonstrated that four weeks after injury, regenerating fibers colonized the graft suggesting a promising use to repair severe nerve lesions. The idea of using a porcine nerve graft arises from a translational perspective. This approach offers a promising direction in the orthopedic field for nerve repair, especially in severe cases where conventional methods are limited., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Muratori, Crosio, Ronchi, Molinaro, Tos, Lovati and Raimondo.)

Gut microbiota is responsible for essential functions in human health. Several communication axes between gut microbiota and other organs via neural, endocrine, and immune pathways have been described, and perturbation of gut microbiota composition has been implicated in the onset and progression of an emerging number of diseases. Here, we analyzed peripheral nerves, dorsal root ganglia (DRG), and skeletal muscles of neonatal and young adult mice with the following gut microbiota status: a) germ-free (GF), b) gnotobiotic, selectively colonized with 12 specific gut bacterial strains (Oligo-Mouse-Microbiota, OMM12), or c) natural complex gut microbiota (CGM). Stereological and morphometric analyses revealed that the absence of gut microbiota impairs the development of somatic median nerves, resulting in smaller diameter and hypermyelinated axons, as well as in smaller unmyelinated fibers. Accordingly, DRG and sciatic nerve transcriptomic analyses highlighted a panel of differentially expressed developmental and myelination genes. Interestingly, the type III isoform of Neuregulin1 (NRG1), known to be a neuronal signal essential for Schwann cell myelination, was overexpressed in young adult GF mice, with consequent overexpression of the transcription factor Early Growth Response 2 ( Egr2 ), a fundamental gene expressed by Schwann cells at the onset of myelination. Finally, GF status resulted in histologically atrophic skeletal muscles, impaired formation of neuromuscular junctions, and deregulated expression of related genes. In conclusion, we demonstrate for the first time a gut microbiota regulatory impact on proper development of the somatic peripheral nervous system and its functional connection to skeletal muscles, thus suggesting the existence of a novel 'Gut Microbiota-Peripheral Nervous System-axis.'

Genital herpes, most frequently caused by herpes simplex virus 2 (HSV-2) infection, is one of the most prevalent sexually transmitted infections. The current rationale for the treatment of HSV-2 infection involves nucleoside analogs (e.g. acyclovir) to suppress reactivation. Enzymatic oxysterols are endogenous 27-carbon atoms molecules produced by enzymatic cholesterol oxidation, and recently emerged as a broad-spectrum host targeting antivirals. In this study, we screened selected members of an in-house synthesized library of oxysterol analogs for their activity against HSV-2, identifying three compounds, named PFM064, PFM067, and PFM069, endowed with 50% effective concentrations (EC 50 ) in the micromolar range, without exerting any apparent cytotoxicity. Moreover, the results obtained showed the ability of the novel derivatives to inhibit both cell-to-cell fusion induced by HSV-2, and the production of an intracellular viral progeny. Further experiments performed with PFM067 (which was selected for more-in-depth studies as the most effective synthetic analog) showed that these molecules act in a late stage of HSV-2 replicative cycle, by sequestering viral glycoproteins in the Golgi compartment, and likely inhibiting the nuclear egress of neo-synthetized viral capsids. Taken together, these results point to PFM067 as a promising chemical scaffold for the development of novel herpetic antivirals., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Andrea Civra, David Lembo, Giuseppe Poli are the founders of Panoxyvir Ltd., a start-up developing the use of specific oxysterols as broad-spectrum antiviral agents. All other authors: none to declare., (Copyright © 2023 Elsevier B.V. All rights reserved.)

It has been widely demonstrated that the gut microbiota is responsible for essential functions in human health and that its perturbation is implicated in the development and progression of a growing list of diseases. The number of studies evaluating how the gut microbiota interacts with and influences other organs and systems in the body and vice versa is constantly increasing and several 'gut-organ axes' have already been defined. Recently, the view on the link between the gut microbiota (GM) and the peripheral nervous system (PNS) has become broader by exceeding the fact that the PNS can serve as a systemic carrier of GM-derived metabolites and products to other organs. The PNS as the communication network between the central nervous system and the periphery of the body and internal organs can rather be affected itself by GM perturbation. In this review, we summarize the current knowledge about the impact of gut microbiota on the PNS, with regard to its somatic and autonomic divisions, in physiological, regenerative and pathological conditions.

Regeneration of damaged peripheral nerves remains one of the main challenges of neurosurgery and regenerative medicine, a nerve functionality is rarely restored, especially after severe injuries. Researchers are constantly looking for innovative strategies for tackling this problem, with the development of advanced tissue-engineered nerve conduits and new pharmacological and physical interventions, with the aim of improving patients' life quality. Different evaluation methods can be used to study the effectiveness of a new treatment, including functional tests, morphological assessment of regenerated nerve fibers and biomolecular analyses of key factors necessary for good regeneration. The number and diversity of protocols and methods, as well as the availability of innovative technologies which are used to assess nerve regeneration after experimental interventions, often makes it difficult to compare results obtained in different labs. The purpose of the current review is to describe the main morphological approaches used to evaluate the degree of nerve fiber regeneration in terms of their usefulness and limitations.

Cell secretome has been explored as a cell-free technique with high scientific and medical interest for Regenerative Medicine. In this work, the secretome produced and collected from Olfactory Mucosa Mesenchymal Stem Cells and Olfactory Ensheating Cells was analyzed and therapeutically applied to promote peripheral nerve regeneration. The analysis of the conditioned medium revealed the production and secretion of several factors with immunomodulatory functions, capable of intervening beneficially in the phases of nerve regeneration. Subsequently, the conditioned medium was applied to sciatic nerves of rats after neurotmesis, using Reaxon ® as tube-guides. Over 20 weeks, the animals were subjected to periodic functional assessments, and after this period, the sciatic nerves and cranial tibial muscles were evaluated stereologically and histomorphometrically, respectively. The results obtained allowed to confirm the beneficial effects resulting from the application of this therapeutic combination. The administration of conditioned medium from Olfactory Mucosal Mesenchymal Stem Cells led to the best results in motor performance, sensory recovery, and gait patterns. Stereological and histomorphometric evaluation also revealed the ability of this therapeutic combination to promote nervous and muscular histologic reorganization during the regenerative process. The therapeutic combination discussed in this work shows promising results and should be further explored to clarify irregularities found in the outcomes and to allow establishing the use of cell secretome as a new therapeutic field applied in the treatment of peripheral nerves after injury.

Background: Somatic nerve injuries are a rising problem leading to disability associated with neuropathic pain commonly reported as mechanical allodynia (MA) and hyperalgesia. These symptoms are strongly dependent on specific processes in the dorsal root ganglia (DRG). Neurodynamic treatment (NDT), consisting of selective uniaxial nerve repeated tension protocols, effectively reduces pain and disability in neuropathic pain patients even though the biological mechanisms remain poorly characterized. We aimed to define, both in vivo and ex vivo, how NDT could promote nerve regeneration and modulate some processes in the DRG linked to MA and hyperalgesia., Methods: We examined in Wistar rats, after unilateral median and ulnar nerve crush, the therapeutic effects of NDT and the possible protective effects of NDT administered for 10 days before the injury. We adopted an ex vivo model of DRG organotypic explant subjected to NDT to explore the selective effects on DRG cells., Results: Behavioural tests, morphological and morphometrical analyses, and gene and protein expression analyses were performed, and these tests revealed that NDT promotes nerve regeneration processes, speeds up sensory motor recovery, and modulates mechanical pain by affecting, in the DRG, the expression of TACAN, a mechanosensitive receptor shared between humans and rats responsible for MA and hyperalgesia. The ex vivo experiments have shown that NDT increases neurite regrowth and confirmed the modulation of TACAN., Conclusions: The results obtained in this study on the biological and molecular mechanisms induced by NDT will allow the exploration, in future clinical trials, of its efficacy in different conditions of neuropathic pain.

Peripheral nerves are frequently affected by lesions caused by trauma (work accidents, car incidents, combat injuries) and following surgical procedures (for instance cancer resection), resulting in loss of motor and sensory function with lifelong impairments. Irrespective of the intrinsic capability of the peripheral nervous system for regeneration, spontaneous or surgically supported regeneration is often unsatisfactory with the limited functional success of nerve repair. For this reason, many efforts have been made to improve the regeneration process. Beyond innovative microsurgical methods that, in certain cases, are necessary to repair nerve injuries, different nonsurgical treatment approaches and adjunctive therapies have been investigated to enhance nerve regeneration. One possibility could be taking advantage of a healthy diet or lifestyle and their relation with proper body functions. Over the years, scientific evidence has been obtained on the benefits of the intake of polyphenols or polyphenol-rich foods in humans, highlighting the neuroprotective effects of these compounds in many neurodegenerative diseases. In order to improve the available knowledge about the potential beneficial role of polyphenols in the process of peripheral nerve regeneration, this review assessed the biological effects of polyphenol administration in supporting and promoting the regenerative process after peripheral nerve injury.

The repair of severe nerve injuries requires an autograft or conduit to bridge the gap and avoid axon dispersion. Several conduits are used routinely, but their effectiveness is comparable to that of an autograft only for short gaps. Understanding nerve regeneration within short conduits could help improve their efficacy for longer gaps. Since Schwann cells are known to migrate on endothelial cells to colonize the "nerve bridge", the new tissue spontaneously forming to connect the injured nerve stumps, here we aimed to investigate whether this migratory mechanism drives Schwann cells to also proceed within the nerve conduits used to repair large nerve gaps. Injured median nerves of adult female rats were repaired with 10 mm chitosan conduits and the regenerated nerves within conduits were analyzed at different time points using confocal imaging of sequential thick sections. Our data showed that the endothelial cells formed a dense capillary network used by Schwann cells to migrate from the two nerve stumps into the conduit. We concluded that angiogenesis played a key role in the nerve conduits, not only by supporting cell survival but also by providing a pathway for the migration of newly formed Schwann cells.

Ghrelin is a circulating peptide hormone released by enteroendocrine cells of the gastrointestinal tract as two forms, acylated and unacylated. Acylated ghrelin (AG) binds to the growth hormone secretagogue receptor 1a (GHSR1a), thus stimulating food intake, growth hormone release, and gastrointestinal motility. Conversely, unacylated GHR (UnAG), through binding to a yet unidentified receptor, protects the skeletal muscle from atrophy, stimulates muscle regeneration, and protects cardiomyocytes from ischemic damage. Recently, interest about ghrelin has raised also among neuroscientists because of its effect on the nervous system, especially the stimulation of neurogenesis in spinal cord, brain stem, and hippocampus. However, few information is still available about its effectiveness on peripheral nerve regeneration. To partially fill this gap, the aim of this study was to assess the effect of UnAG on peripheral nerve regeneration after median nerve crush injury and after nerve transection immediately repaired by means of an end-to-end suture. To this end, we exploited FVB1 Myh6/Ghrl transgenic mice in which overexpression of the ghrelin gene (Ghrl) results in selective up-regulation of circulating UnAG levels, but not of AG. Regeneration was assessed by both functional evaluation (grasping test) and morphometrical analysis of regenerated myelinated axons. Results obtained lead to conclude that UnAG could have a role in development of peripheral nerves and during more severe lesions.

Peripheral nerves are highly susceptible to injuries induced from everyday activities such as falling or work and sport accidents as well as more severe incidents such as car and motorcycle accidents. Many efforts have been made to improve nerve regeneration, but a satisfactory outcome is still unachieved, highlighting the need for easy to apply supportive strategies for stimulating nerve growth and functional recovery. Recent focus has been made on the effect of the consumed diet and its relation to healthy and well-functioning body systems. Normally, a balanced, healthy daily diet should provide our body with all the needed nutritional elements for maintaining correct function. The health of the central and peripheral nervous system is largely dependent on balanced nutrients supply. While already addressed in many reviews with different focus, we comprehensively review here the possible role of different nutrients in maintaining a healthy peripheral nervous system and their possible role in supporting the process of peripheral nerve regeneration. In fact, many dietary supplements have already demonstrated an important role in peripheral nerve development and regeneration; thus, a tailored dietary plan supplied to a patient following nerve injury could play a non-negotiable role in accelerating and promoting the process of nerve regeneration.

Nerve guidance conduits (NGCs) are used as an alternative to the "gold standard" nerve autografting, preventing the need for surgical intervention required to harvest autologous nerves. However, the regeneration outcomes achieved with the current NGCs are only comparable with autografting when the gap is short (less than 10 mm). In the present study, we have developed NGCs made from a blend of polyhydroxyalkanoates, a family of natural resorbable polymers. Hollow NGCs made from a 75:25 poly(3-hydroxyoctanoate)/poly(3-hydroxybutyrate) blend (PHA-NGCs) were manufactured using dip-molding. These PHA-NGCs showed appropriate flexibility for peripheral nerve regeneration. In vitro cell studies performed using RT4-D6P2T rat Schwann cell line confirmed that the material is capable of sustaining cell proliferation and adhesion. PHA-NGCs were then implanted in vivo to repair 10 mm gaps of the median nerve of female Wistar rats for 12 weeks. Functional evaluation of the regenerated nerve using the grasping test showed that PHA-NGCs displayed similar motor recovery as the autograft, starting from week 7. Additionally, nerve cross-sectional area, density and number of myelinated cells, as well as axon diameter, fiber diameter, myelin thickness and g-ratio obtained using the PHA-NGCs were found comparable to an autograft. This preclinical data confirmed that the PHA-NGCs are indeed highly promising candidates for peripheral nerve regeneration., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Bioengineering & Translational Medicine published by Wiley Periodicals LLC on behalf of The American Institute of Chemical Engineers.)

As a consequence of trauma or surgical interventions on peripheral nerves, scar tissue can form, interfering with the capacity of the nerve to regenerate properly. Scar tissue may also lead to traction neuropathies, with functional dysfunction and pain for the patient. The search for effective antiadhesion products to prevent scar tissue formation has, therefore, become an important clinical challenge. In this review, we perform extensive research on the PubMed database, retrieving experimental papers on the prevention of peripheral nerve scarring. Different parameters have been considered and discussed, including the animal and nerve models used and the experimental methods employed to simulate and evaluate scar formation. An overview of the different types of antiadhesion devices and strategies investigated in experimental models is also provided. To successfully evaluate the efficacy of new antiscarring agents, it is necessary to have reliable animal models mimicking the complications of peripheral nerve scarring and also standard and quantitative parameters to evaluate perineural scars. So far, there are no standardized methods used in experimental research, and it is, therefore, difficult to compare the results of the different antiadhesion devices.

Silk fibroin ( Bombyx mori ) was used to manufacture a nerve conduit (SilkBridge TM ) characterized by a novel 3D architecture. The wall of the conduit consists of two electrospun layers (inner and outer) and one textile layer (middle), perfectly integrated at the structural and functional level. The manufacturing technology conferred high compression strength on the device, thus meeting clinical requirements for physiological and pathological compressive stresses. As demonstrated in a previous work, the silk material has proven to be able to provide a valid substrate for cells to grow on, differentiate and start the fundamental cellular regenerative activities in vitro and, in vivo , at the short time point of 2 weeks, to allow the starting of regenerative processes in terms of good integration with the surrounding tissues and colonization of the wall layers and of the lumen with several cell types. In the present study, a 10 mm long gap in the median nerve was repaired with 12 mm SilkBridge TM conduit and evaluated at middle (4 weeks) and at longer time points (12 and 24 weeks). The SilkBridge TM conduit led to a very good functional and morphological recovery of the median nerve, similar to that observed with the reference autograft nerve reconstruction procedure. Taken together, all these results demonstrated that SilkBridge TM has an optimized balance of biomechanical and biological properties, which allowed proceeding with a first-in-human clinical study aimed at evaluating safety and effectiveness of using the device for the reconstruction of digital nerve defects in humans., (Copyright © 2020 Fregnan, Muratori, Bassani, Crosio, Biagiotti, Vincoli, Carta, Pierimarchi, Geuna, Alessandrino, Freddi and Ronchi.)

Conduits for the repair of peripheral nerve gaps are a good alternative to autografts as they provide a protected environment and a physical guide for axonal re-growth. Conduits require colonization by cells involved in nerve regeneration (Schwann cells, fibroblasts, endothelial cells, macrophages) while in the autograft many cells are resident and just need to be activated. Since it is known that soluble Neuregulin1 (sNRG1) is released after injury and plays an important role activating Schwann cell dedifferentiation, its expression level was investigated in early regeneration steps (7, 14, 28 days) inside a 10 mm chitosan conduit used to repair median nerve gaps in Wistar rats. In vivo data show that sNRG1, mainly the isoform α, is highly expressed in the conduit, together with a fibroblast marker, while Schwann cell markers, including NRG1 receptors, were not. Primary culture analysis shows that nerve fibroblasts, unlike Schwann cells, express high NRG1α levels, while both express NRG1β. These data suggest that sNRG1 might be mainly expressed by fibroblasts colonizing nerve conduit before Schwann cells. Immunohistochemistry analysis confirmed NRG1 and fibroblast marker co-localization. These results suggest that fibroblasts, releasing sNRG1, might promote Schwann cell dedifferentiation to a "repair" phenotype, contributing to peripheral nerve regeneration.

The rabbit has been proposed to represent an animal model that allows studying peripheral nerve regeneration across extended gap lengths. We describe here our experiences with the rabbit median nerve model and the obstacles it comes along with. This short communication is meant to inform the community and to prevent other researcher from investing time and animal lives in a model with low translational power., (© 2020 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons Ltd.)

In this work, dextran-based nerve tube-guides were prepared, characterized and used in a standardized animal model of neurotmesis injury. Non-porous and porous transparent tube-guides were obtained by photocrosslinking of two co-macromonomers based on dextran and poly(ε-caprolactone) (PCL). Swelling capacity of the tube-guides ranged from 40-60% with no visible constriction of their inner diameter. In vitro hydrolytic degradation tests showed that the tube-guides maintained their structural integrity up to 6 months. The in vivo performance of the tube-guides was evaluated by entubulation of the rat sciatic nerve after a neurotmesis injury, with a 10 mm-gap between the nerve stumps. The results showed that the tube-guides were able to promote the regeneration of the nerve in a similar manner to what was observed with conventional techniques (nerve graft and end-to-end suture). Stereological analysis proved that nerve regeneration occurred, and both tube-guides presented fibre diameter and g-ratio closer to healthy sciatic nerves. The histomorphometric analysis of Tibialis anterior (TA) skeletal muscle showed decreased neurogenic atrophy in the porous tube-guides treated group, presenting measurements that are similar to the uninjured control.

Objectives: To evaluate: (i) the neuro-regenerative potential of chitosan membrane (CS-Me) on acutely axotomised autonomic neurones in vitro; (ii) to exclude the possibility that a pro-regenerative biomaterial could interfere with the proliferation activity of prostate cancer cell lines; (iii) to provide an in vivo proof of the biocompatibility and regeneration promoting effect of CS-Me in a standardised rat model of peripheral nerve injury and repair; (iv) finally, to evaluate the tissue reaction induced by the degrading material; as previous studies have shown promising effects of CS-Me for protection of the neurovascular bundles for potency recovery in patients that undergo nerve-sparing radical prostatectomy (RP)., Materials and Methods: Addressing aim (i), the neuro-regenerative potential, organotypic cultures derived from primary sympathetic ganglia were cultured on CS-Me over 3 days and neurite extension and axonal sprouting were evaluated. Addressing aim (ii), effects of CS on cancer cells, different human prostate cancer cell lines (PC3, DU-145, LN-Cap) were seeded on CS-coated plates or cultured in the presence of CS-Me dissolution products. Addressing aims (iii) and (iv), functional recovery of peripheral nerve fibres and tissue reaction with the biomaterial, CS-Me and CS nerve guides were used to repair a median nerve injury in the rat. Functional recovery was evaluated during the post-recovery time by the behavioural grasping test., Results: CS-Me significantly stimulated axon elongation from autonomic ganglia in comparison to control conditions in organotypic three-dimensional cultures. CS coating, as well as the dissolution products of CS-Me, led to a significantly lower proliferation rate of prostate cancer cell lines in vitro. Tissue reaction towards CS-Me and standard CS nerve guides was similar in the rat median nerve model, as was the outcome of nerve fibre regeneration and functional recovery., Conclusion: The results of this study provide the first experimental evidence in support of the clinical safety of CS-Me and of their postulated effectiveness for improving functional recovery after RP. The presented results are coherent in demonstrating that acutely axotomised autonomic neurones show increased neurite outgrowth on CS-Me substrate, whilst the same substrate reduces prostate cancer cell line proliferation in vitro. Furthermore, CS-Me do not demonstrate any disadvantage for peripheral nerve repair in a standard animal model., (© 2019 The Authors BJU International © 2019 BJU International Published by John Wiley & Sons Ltd.)

The successful introduction of innovative treatment strategies into clinical practise strongly depends on the availability of effective experimental models and their reliable pre-clinical assessment. Considering pre-clinical research for peripheral nerve repair and reconstruction, the far most used nerve regeneration model in the last decades is the sciatic nerve injury and repair model. More recently, the use of the median nerve injury and repair model has gained increasing attention due to some significant advantages it provides compared to sciatic nerve injury. Outstanding advantages are the availability of reliable behavioural tests for assessing posttraumatic voluntary motor recovery and a much lower impact on the animal wellbeing. In this article, the potential application of the median nerve injury and repair model in pre-clinical research is reviewed. In addition, we provide a synthetic overview of a variety of methods that can be applied in this model for nerve regeneration assessment. This article is aimed at helping researchers in adequately adopting this in vivo model for pre-clinical evaluation of peripheral nerve reconstruction as well as for interpreting the results in a translational perspective.

Introduction: The aim of this research was to test, in an animal model, the nerve regeneration technique with a hypoallergenic acellular dermal matrix used to wrap the microsurgical neural suture., Materials and Methods: Two groups of rats received the cut of limb right median nerves. The regeneration technique considers for both groups an end-to-end nerve suture. In the experimental group (A) was used also a wrapping protocol by a conduit of collagen matrix currently used in oral surgery. The animals underwent functional grasping tests (at 1, 3, 5, and 7 months) and a histological and quantitative analysis of distal nerve was performed at the end of experimental time., Result: After seven months, the grasping test reveals functional recovery in each tested animal; this improvement is more evident in Group A. The fibers appear well organized with restored myelin sheaths in both groups. Group A showed a great quantity of connective tissue surrounding the nerve. The quantitative morphology analysis in both groups shows a similar fibers density, fiber diameter, and myelin thickness. The differences between the groups in axon mean diameter are significant. In Group A M/d, D/d, and g-ratio is significantly higher compared to control group., Conclusions: Histological and functional assessments show a functional recovery of the injured nerve in the test groups, stressed by the results of the grasping tests and the meaningful increasing in fiber diameter and higher g-ratio. Moreover, a connective tissue cuff distinguishes the distal portion of the injured nerve. Considering the easy availability and handling of the material used in this study we can conclude that this experimental technique can be considered as a valid alternative to protect nerves in nerve wrap surgery.

Nerve regeneration after delayed nerve repair is often unsuccessful. Indeed, the expression of genes associated with regeneration, including neurotrophic and gliotrophic factors, is drastically reduced in the distal stump of chronically transected nerves; moreover, Schwann cells undergo atrophy, losing their ability to sustain regeneration. In the present study, to provide a three-dimensional environment and trophic factors supporting Schwann cell activity and axon re-growth, we combined the use of an effective conduit (a chitosan tube) with a promising intraluminal structure (fresh longitudinal skeletal muscle fibers). This enriched conduit was used to repair a 10-mm rat median nerve gap after 3-month delay and functional and morphometrical analyses were performed 4 months after nerve reconstruction. Our data show that the enriched chitosan conduit is as effective as the hollow chitosan conduit in promoting nerve regeneration, and its efficacy is not statistically different from the autograft, considered the "gold standard" technique for nerve reconstruction. Since hollow tubes not always lead to good results after long defects (> 20 mm), we believe that the conduit enriched with fresh muscle fibers could be a promising strategy to repair longer gaps, as muscle fibers create a favorable three-dimensional environment and release trophic factors. All procedures were approved by the Bioethical Committee of the University of Torino and by the Italian Ministry of Health (approval number: 864/2016/PR) on September 14, 2016., Competing Interests: None

Spinocerebellar ataxia 28 is an autosomal dominant neurodegenerative disorder caused by missense mutations affecting the proteolytic domain of AFG3L2, a major component of the mitochondrial m-AAA protease. However, little is known of the underlying pathogenetic mechanisms or how to treat patients with SCA28. Currently available Afg3l2 mutant mice harbour deletions that lead to severe, early-onset neurological phenotypes that do not faithfully reproduce the late-onset and slowly progressing SCA28 phenotype. Here we describe production and detailed analysis of a new knock-in murine model harbouring an Afg3l2 allele carrying the p.Met665Arg patient-derived mutation. Heterozygous mutant mice developed normally but adult mice showed signs of cerebellar ataxia detectable by beam test. Although cerebellar pathology was negative, electrophysiological analysis showed a trend towards increased spontaneous firing in Purkinje cells from heterozygous mutants with respect to wild-type controls. As homozygous mutants died perinatally with evidence of cardiac atrophy, for each genotype we generated mouse embryonic fibroblasts (MEFs) to investigate mitochondrial function. MEFs from mutant mice showed altered mitochondrial bioenergetics, with decreased basal oxygen consumption rate, ATP synthesis and mitochondrial membrane potential. Mitochondrial network formation and morphology was altered, with greatly reduced expression of fusogenic Opa1 isoforms. Mitochondrial alterations were also detected in cerebella of 18-month-old heterozygous mutants and may be a hallmark of disease. Pharmacological inhibition of de novo mitochondrial protein translation with chloramphenicol caused reversal of mitochondrial morphology in homozygous mutant MEFs, supporting the relevance of mitochondrial proteotoxicity for SCA28 pathogenesis and therapy development., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: In this study, we evaluate the role of RGMa (Repulsive Guidance Molecule a) during peripheral nerve regeneration using the mouse median nerve model., Methods: By real-time PCR and Western Blot analysis, we examined expression changes of RGMa mRNA and RGMa protein in neural tissue after transection and microsurgical repair of the mouse median nerve distal to the transection site. We evaluated histomorphometrical changes in neural tissue distal to the injury site and functional recovery of the grasping force after median nerve transection and repair in wild-type mice and RGMa+/- heterozygous mice., Results: RT-PCR revealed a 1,8 fold increase of RGMa mRNA two weeks and a 4,4 fold increase of RGMa mRNA 3 weeks after nerve transection and repair in the nerve segment distal to the injury site. In Western blot analysis, we could show a high increase of RGMa in the nerve segment distal to the injury site at day 14. Histomorphometrical analysis showed significant differences between wild-type animals and heterozygous animals. The absolute number of myelinated fibres was significantly higher in operated heterozygous RGMa+/- animals compared to operated wildtye animals. Using the functional grasping test, we could demonstrate that peripheral nerve regeneration is significantly diminished in heterozygous RGMa+/- mice., Conclusions: Employing the mouse median nerve model in transgenic animals, we demonstrate that RGMa plays an important role during peripheral nerve regeneration.

Muscle-in-vein conduit is successfully employed for repairing nerve injuries: the vein prevents muscle fiber dispersion, while the muscle prevents the vein collapse and creates a favorable environment for Schwann cell migration and axon regrowth. However, it requires microsurgical skills. In this study we show a simple strategy to improve the performance of a chitosan hollow tube by the introduction of fresh skeletal muscle fibers. The hypothesis is to overcome the technical issue of the muscle-in-vein preparation and to take advantage of fiber muscle properties to create an easy and effective conduit for nerve regeneration. Rat median nerve gaps were repaired with chitosan tubes filled with skeletal muscle fibers (muscle-in-tube graft), hollow chitosan tubes, or autologous nerve grafts. Our results demonstrate that the fresh skeletal muscle inside the conduit is an endogenous source of soluble Neuregulin 1, a key factor for Schwann cell survival and dedifferentiation, absent in the hollow tube during the early phase of regeneration. However, nerve regeneration assessed at late time point was similar to that obtained with the hollow tube. To conclude, the muscle-in-tube graft is surgically easy to perform and we suggest that it might be a promising strategy to repair longer nerve gap or for secondary nerve repair, situations in which Schwann cell atrophy is a limiting factor for recovery.

Peripheral nerves are characterised by the ability to regenerate after injury. Schwann cell activity is fundamental for all steps of peripheral nerve regeneration: immediately after injury they de-differentiate, remove myelin debris, proliferate and repopulate the injured nerve. Soluble Neuregulin1 (NRG1) is a growth factor that is strongly up-regulated and released by Schwann cells immediately after nerve injury. To identify the genes regulated in Schwann cells by soluble NRG1, we performed deep RNA sequencing to generate a transcriptome database and identify all the genes regulated following 6 h stimulation of primary adult rat Schwann cells with soluble recombinant NRG1. Interestingly, the gene ontology analysis of the transcriptome reveals that NRG1 regulates genes belonging to categories that are regulated in the peripheral nerve immediately after an injury. In particular, NRG1 strongly inhibits the expression of genes involved in myelination and in glial cell differentiation, suggesting that NRG1 might be involved in the de-differentiation (or "trans-differentiation") process of Schwann cells from a myelinating to a repair phenotype. Moreover, NRG1 inhibits genes involved in the apoptotic process, and up-regulates genes positively regulating the ribosomal RNA processing, thus suggesting that NRG1 might promote cell survival and stimulate new protein expression. This in vitro transcriptome analysis demonstrates that in Schwann cells NRG1 drives the expression of several genes which partially overlap with genes regulated in vivo after peripheral nerve injury, underlying the pivotal role of NRG1 in the first steps of the nerve regeneration process.

Neuregulin 1 (NRG1) is a growth factor produced by both peripheral nerves and skeletal muscle. In muscle, it regulates neuromuscular junction gene expression, acetylcholine receptor number, muscle homeostasis and satellite cell survival. NRG1 signalling is mediated by the tyrosine kinase receptors ErbB3 and ErbB4 and their co-receptors ErbB1 and ErbB2. The NRG1/ErbB system is well studied in nerve tissue after injury, but little is known about this system in skeletal muscle after denervation/reinnervation processes. Here, we performed a detailed time-course expression analysis of several NRG1 isoforms and ErbB receptors in the rat superficial digitorum flexor muscle after three types of median nerve injuries of different severities. We found that ErbB receptor expression was correlated with the innervated state of the muscle, with upregulation of ErbB2 clearly associated with the denervation state. Interestingly, the NRG1 isoforms were differently regulated depending on the nerve injury type, leading to the hypothesis that both the NRG1α and NRG1β isoforms play a key role in the muscle reaction to injury. Indeed, in vitro experiments with C2C12 atrophic myotubes revealed that both NRG1α and NRG1β treatment influences the best-known atrophic pathways, suggesting that NRG1 might play an anti-atrophic role.

Neuregulin1 (NRG1) is a growth factor playing a pivotal role in peripheral nerve development through the activation of the transmembrane co-receptors ErbB2-ErbB3. Soluble NRG1 isoforms, mainly secreted by Schwann cells, are strongly and transiently up-regulated after acute peripheral nerve injury, thus suggesting that they play a crucial role also in the response to nerve damage. Here we show that in the rat experimental model of the peripheral demyelinating neuropathy Charcot-Marie-Tooth 1A (CMT1A) the expression of the different NRG1 isoforms (soluble, type α and β, type a and b) is strongly up-regulated, as well as the expression of NRG1 co-receptors ErbB2-ErbB3, thus showing that CMT1A nerves have a gene expression pattern highly reminiscent of injured nerves. Because it has been shown that high concentrations of soluble NRG1 negatively affect myelination, we suggest that soluble NRG1 over-expression might play a negative role in the pathogenesis of CMT1A disease, and that a therapeutic approach, aimed to interfere with NRG1 activity, might be beneficial for CMT1A patients. Further studies will be necessary to test this hypothesis in animal models and to evaluate NRG1 expression in human patients. Impact statement Charcot-Marie-Tooth1A (CMT1A) is one of the most frequent inherited neurological diseases, characterized by chronic demyelination of peripheral nerves, for which effective therapies are not yet available. It has been recently proposed that the treatment with soluble Neuregulin1 (NRG1), a growth factor released by Schwann cells immediately after acute nerve injury, might be effective in CMT1A treatment. However, the expression of the different isoforms of endogenous NRG1 in CMT1A nerves has not been yet investigated. In this preliminary study, we demonstrate that different isoforms of soluble NRG1 are strongly over-expressed in CMT1A nerves, thus suggesting that a therapeutic approach based on NRG1 treatment should be carefully reconsidered. If soluble NRG1 is over-expressed also in human CMT1A nerves, a therapeutic approach aimed to inhibit (instead of stimulate) the signal transduction pathways driven by NRG1 might be fruitfully developed. Further studies will be necessary to test these hypotheses.

OBJECTIVE Multiple factors may affect functional recovery after peripheral nerve injury, among them the lesion site and the interval between the injury and the surgical repair. When the nerve segment distal to the lesion site undergoes chronic degeneration, the ensuing regeneration (when allowed) is often poor. The aims of the current study were as follows: 1) to examine the expression changes of the neuregulin 1/ErbB system during long-term nerve degeneration; and 2) to investigate whether a chronically denervated distal nerve stump can sustain nerve regeneration of freshly axotomized axons. METHODS This study used a rat surgical model of delayed nerve repair consisting of a cross suture between the chronically degenerated median nerve distal stump and the freshly axotomized ulnar proximal stump. Before the suture, a segment of long-term degenerated median nerve stump was harvested for analysis. Functional, morphological, morphometric, and biomolecular analyses were performed. RESULTS The results showed that neuregulin 1 is highly downregulated after chronic degeneration, as well as some Schwann cell markers, demonstrating that these cells undergo atrophy, which was also confirmed by ultrastructural analysis. After delayed nerve repair, it was observed that chronic degeneration of the distal nerve stump compromises nerve regeneration in terms of functional recovery, as well as the number and size of regenerated myelinated fibers. Moreover, neuregulin 1 is still downregulated after delayed regeneration. CONCLUSIONS The poor outcome after delayed nerve regeneration might be explained by Schwann cell impairment and the consequent ineffective support for nerve regeneration. Understanding the molecular and biological changes occurring both in the chronically degenerating nerve and in the delayed nerve repair may be useful to the development of new strategies to promote nerve regeneration. The results suggest that neuregulin 1 has an important role in Schwann cell activity after denervation, indicating that its manipulation might be a good strategy for improving outcome after delayed nerve repair.

Background: Delayed reconstruction of transection or laceration injuries of peripheral nerves is inflicted by a reduced regeneration capacity. Diabetic conditions, more frequently encountered in clinical practice, are known to further impair regeneration in peripheral nerves. Chitosan nerve guides (CNGs) have recently been introduced as a new generation of medical devices for immediate peripheral nerve reconstruction. Here, CNGs were used for 45 days delayed reconstruction of critical length 15 mm rat sciatic nerve defects in either healthy Wistar rats or diabetic Goto-Kakizaki rats; the latter resembling type 2 diabetes. In short and long-term investigations, we comprehensively analyzed the performance of one-chambered hollow CNGs (hCNGs) and two-chambered CNGs (CFeCNGs) in which a chitosan film has been longitudinally introduced. Additionally, we investigated in vitro the immunomodulatory effect provided by the chitosan film., Results: Both types of nerve guides, i.e. hCNGs and CFeCNGs, enabled moderate morphological and functional nerve regeneration after reconstruction that was delayed for 45 days. These positive findings were detectable in generally healthy as well as in diabetic Goto-Kakizaki rats (for the latter only in short-term studies). The regenerative outcome did not reach the degree as recently demonstrated after immediate reconstruction using hCNGs and CFeCNGs. CFeCNG-treatment, however, enabled tissue regrowth in all animals (hCNGs: only in 80% of animals). CFeCNGs did further support with an increased vascularization of the regenerated tissue and an enhanced regrowth of motor axons. One mechanism by which the CFeCNGs potentially support successful regeneration is an immunomodulatory effect induced by the chitosan film itself. Our in vitro results suggest that the pro-regenerative effect of chitosan is related to the differentiation of chitosan-adherent monocytes into pro-healing M2 macrophages., Conclusions: No considerable differences appear for the delayed nerve regeneration process related to healthy and diabetic conditions. Currently available chitosan nerve grafts do not support delayed nerve regeneration to the same extent as they do after immediate nerve reconstruction. The immunomodulatory characteristics of the biomaterial may, however, be crucial for their regeneration supportive effects.

Nerve fibers are attracted by sutureless end-to-side nerve coaptation into the recipient nerve. Opening a window in the epineurium enhances axon attraction and myelination. The authors analyze the features of nerve repair by end-to-side coaptation. They highlight the known mechanisms of axon sprouting and different hypotheses of start up signals (presence or absence of an epineurial window, role of Schwann cells, signaling from the distal trunk). The clinical literature is also presented and differences between experimental and clinical applications are pointed out. The authors propose their point of view and perspectives deriving from recent experimental and clinical experiences., Competing Interests: Conflicts of interest: None declared.

Schwann cells (SCs) generate the myelin wrapping of peripheral nerve axons and are promising candidates for cell therapy. However, to date a renewable source of SCs is lacking. In this study, we show the conversion of skin fibroblasts into induced Schwann cells (iSCs) by driving the expression of two transcription factors, Sox10 and Egr2. iSCs resembled primary SCs in global gene expression profiling and PNS identity. In vitro, iSCs wrapped axons generating compact myelin sheaths with regular nodal structures. Conversely, iSCs from Twitcher mice showed a severe loss in their myelinogenic potential, demonstrating that iSCs can be an attractive system for in vitro modelling of PNS diseases. The same two factors were sufficient to convert human fibroblasts into iSCs as defined by distinctive molecular and functional traits. Generating iSCs through direct conversion of somatic cells offers opportunities for in vitro disease modelling and regenerative therapies.

Object: This study evaluated a chitosan tube for regeneration of the injured peripheral nerve in a rodent transected sciatic nerve model in comparison to autologous nerve graft repair., Methods: Chitosan hollow tube was used to bridge a 10-mm gap between the proximal and distal ends in 11 rats. In the control group, an end-to-end coaptation of 10-mm long autologous nerve graft was performed in 10 rats for nerve reconstruction., Results: SFI showed an insignificant advantage to the autologous group both at 30 days (P = 0.177) and at 90 days post procedure (P = 0.486). Somato-sensory evoked potentials (SSEP) and compound muscle action potentials (CMAP) tests showed similar results between chitosan tube (group 1) and autologous (group 2) groups with no statistically significant differences. Both groups presented the same pattern of recovery with 45% in group 1 and 44% in group 2 (P = 0.96) showing SSEP activity at 30 days. At 90 days most rats showed SSEP activity (91% vs.80% respectively, P = 0.46). The CMAP also demonstrated no statistically significant differences in latency (1.39 ms in group 1 vs. 1.63 ms in group 2; P = 0.48) and amplitude (6.28 mv vs. 6.43 mv respectively; P = 0.8). Ultrasonography demonstrated tissue growth inside the chitosan tube. Gastrocnemius muscle weight showed no statistically significant difference. Histomorphometry of the distal sciatic nerve, 90 days post reconstructive procedure, showed similar number of myelinated fibers and size parameters in both groups (P ≥ 0.05)., Conclusions: Chitosan hollow tube used for peripheral nerve reconstruction of rat sciatic nerve showed similar results in comparison to autologous nerve grafting. © 2015 Wiley Periodicals, Inc. Microsurgery 36:664-671, 2016., (© 2015 Wiley Periodicals, Inc.)

End-to-side nerve coaptation brings regenerating axons from the donor to the recipient nerve. Several techniques have been used to perform coaptation: microsurgical sutures with and without opening a window into the epi(peri)neurial connective tissue; among these, window techniques have been proven more effective in inducing axonal regeneration. The authors developed a sutureless model of end-to-side coaptation in the rat upper limb. In 19 adult Wistar rats, the median and the ulnar nerves of the left arm were approached from the axillary region, the median nerve transected and the proximal stump sutured to the pectoral muscle to prevent regeneration. Animals were then randomly divided in two experimental groups (7 animals each, 5 animals acting as control): Group 1: the distal stump of the transected median nerve was fixed to the ulnar nerve by applying cyanoacrylate solution; Group 2: a small epineurial window was opened into the epineurium of the ulnar nerve, caring to avoid damage to the nerve fibres; the distal stump of the transected median nerve was then fixed to the ulnar nerve by applying cyanoacrylate solution. The grasping test for functional evaluation was repeated every 10-11 weeks starting from week-15, up to the sacrifice (week 36). At week 36, the animals were sacrificed and the regenerated nerves harvested and processed for morphological investigations (high-resolution light microscopy as well as stereological and morphometrical analysis). This study shows that a) cyanoacrylate in end-to-side coaptation produces scarless axon regeneration without toxic effects; b) axonal regeneration and myelination occur even without opening an epineurial window, but c) the window is related to a larger number of regenerating fibres, especially myelinated and mature, and better functional outcomes.

The peripheral nervous system has an intrinsic capability to regenerate, crucially related to the ability of Schwann cells (SC) to create a permissive environment, for example, through production of regeneration-promoting neurotrophic factors. Survival, proliferation, migration and differentiation of SC into a myelinating phenotype during development and after injury is regulated by different Neuregulin1 (NRG1) isoforms. This study investigates the expression of different NRG1 isoforms and of their ErbB receptors in distal rat median nerve samples under regenerating conditions after a mild (crush) and more severe (end-to-end repair) injury and under degenerating condition. The expression of the NRG1/ErbB system was evaluated at mRNA and protein level, and demonstrated to be specific for distinct and consecutive phases following nerve injury and regeneration or the progress in degeneration. For the first time a detailed analysis of expression profiles not only of soluble and transmembrane NRG1 isoforms, but also of alpha and beta as well as type a, b and c isoforms is presented. The results of mRNA and protein expression pattern analyses were related to nerve ultrastructure changes evaluated by electron microscopy. In particular, transmembrane NRG1 isoforms are differentially regulated and proteolytically processed under regeneration and degeneration conditions. Soluble NRG1 isoforms alpha and beta, as well as type a and b, are strongly upregulated during axonal regrowth, while type c NRG1 isoform is downregulated. This is accompanied by an upregulation of ErbB receptors. This accurate regulation suggests that each molecule plays a specific role that could be clinically exploited to improve nerve regeneration., (© 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Biosynthetic nerve grafts are developed in order to complement or replace autologous nerve grafts for peripheral nerve reconstruction. Artificial nerve guides currently approved for clinical use are not widely applied in reconstructive surgery as they still have limitations especially when it comes to critical distance repair. Here we report a comprehensive analysis of fine-tuned chitosan nerve guides (CNGs) enhanced by introduction of a longitudinal chitosan film to reconstruct critical length 15 mm sciatic nerve defects in adult healthy Wistar or diabetic Goto-Kakizaki rats. Short and long term investigations demonstrated that the CNGs enhanced by the guiding structure of the introduced chitosan film significantly improved functional and morphological results of nerve regeneration in comparison to simple hollow CNGs. Importantly, this was detectable both in healthy and in diabetic rats (short term) and the regeneration outcome almost reached the outcome after autologous nerve grafting (long term). Hollow CNGs provide properties likely leading to a wider clinical acceptance than other artificial nerve guides and their performance can be increased by simple introduction of a chitosan film with the same advantageous properties. Therefore, the chitosan film enhanced CNGs represent a new generation medical device for peripheral nerve reconstruction., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Sprouty (Spry) proteins are negative feedback inhibitors of receptor tyrosine kinase signaling. Downregulation of Spry2 has been demonstrated to promote elongative axon growth of cultured peripheral and central neurons. Here, we analyzed Spry2 global knockout mice with respect to axon outgrowth in vitro and peripheral axon regeneration in vivo. Neurons dissociated from adult Spry2 deficient sensory ganglia revealed stronger extracellular signal-regulated kinase activation and enhanced axon outgrowth. Prominent axon elongation was observed in heterozygous Spry2(+/-) neuron cultures, whereas homozygous Spry2(-/-) neurons predominantly exhibited a branching phenotype. Following sciatic nerve crush, Spry2(+/-) mice recovered faster in motor but not sensory testing paradigms (Spry2(-/-) mice did not tolerate anesthesia required for nerve surgery). We attribute the improvement in the rotarod test to higher numbers of myelinated fibers in the regenerating sciatic nerve, higher densities of motor endplates in hind limb muscles and increased levels of GAP-43 mRNA, a downstream target of extracellular regulated kinase signaling. Conversely, homozygous Spry2(-/-) mice revealed enhanced mechanosensory function (von Frey's test) that was accompanied by an increased innervation of the epidermis, elevated numbers of nonmyelinated axons and more IB4-positive neurons in dorsal root ganglia. The present results corroborate the functional significance of receptor tyrosine kinase signaling inhibitors for axon outgrowth during development and nerve regeneration and propose Spry2 as a novel potential target for pharmacological inhibition to accelerate long-distance axon regeneration in injured peripheral nerves., (© 2014 The Authors Developmental Neurobiology Published by Wiley Periodicals, Inc.)

The evidence of neurons generated ex novo in sensory ganglia of adult animals is still debated. In the present study, we investigated, using high resolution light microscopy and stereological analysis, the changes in the number of neurons in dorsal root ganglia after 30 days from a crush lesion of the rat brachial plexus terminal branches. Results showed, as expected, a relevant hypertrophy of dorsal root ganglion neurons. In addition, we reported, for the first time in the literature, that neuronal hypertrophy was accompanied by massive neuronal hyperplasia leading to a 42% increase of the number of primary sensory neurons. Moreover, ultrastructural analyses on sensory neurons showed that there was not a relevant neuronal loss as a consequence of the nerve injury. The evidence of BrdU-immunopositive neurons and neural progenitors labeled with Ki67, nanog, nestin, and sox-2 confirmed the stereological evidence of posttraumatic neurogenesis in dorsal root ganglia. Analysis of morphological changes following axonal damage in addition to immunofluorescence characterization of cell phenotype suggested that the neuronal precursors which give rise to the newly generated neurons could be represented by satellite glial cells that actively proliferate after the lesion and are able to differentiate toward the neuronal lineage.

Quantitative estimation of myelinated nerve fiber number, together with fiber size parameters, is one of the most important tools for nerve regeneration research. In this study we used a design-based stereological method to evaluate the regenerative process in two experimental paradigms: crush injury and autograft repair. Samples were embedded in resin and morphometric counting and measurements were performed using both light and electron microscopes. Results show a significant difference in myelinated fiber number estimation between light and electron microscopes, especially after autograft repair; light microscope significantly underestimates the number of fibers because of the large number of very small axons that can be detected only in electron microscope. The analysis of the size parameters also shows a higher number of small fibers in electron microscopic analysis, especially in regenerated nerves. This comparative study shows that the integration of data obtained in light microscope with those obtained in electron microscope is necessary in revealing very small myelinated fibers that cannot be detected otherwise. Moreover, the difference in the estimation of total number of myelinated fibers between light and electron microscopes must be considered in data analysis to ensure accurate interpretation of the results., (© 2014 Peripheral Nerve Society.)

Injury to the peripheral nerve induces dramatic changes in terms of cellular composition that are reflected on RNA quality and quantity, making messenger RNA expression analysis very complex. Several commonly used housekeeping genes are regulated following peripheral nerve injury and are thus not suitable for quantitative real-time PCR normalization; moreover, the presence of pseudogenes in some of them impairs their use. To deal with this problem, we have developed a new method to identify new stable housekeeping genes based on publicly available microarray data on normal and injured nerves. Four new candidate stable genes were identified and validated by quantitative real-time PCR analysis on nerves during the different phases after nerve injury: nerve degeneration, regeneration and remyelination. The stability measure of these genes was calculated with both NormFinder and geNorm algorithms and compared with six commonly used housekeeping genes. This procedure allowed us to identify two new and highly stable genes that can be employed for normalizing injured peripheral nerve data: ANKRD27 and RICTOR. Besides providing a tool for peripheral nerve research, our study also describes a simple and cheap procedure that can be used to identify suitable housekeeping genes in other tissues and organs.

Sciatic nerve crush injury in rat animal model is one of the most common experimental models used in regenerative research. However, the availability of transgenic mouse for nerve regeneration studies is constantly increasing and, therefore, the shift from rat model to mouse model is, in some cases, necessary. Moreover, since most of the human nerve lesions occur in the upper limb, it is also advantageous to shift from sciatic nerve to median nerve. In this study we described an experimental model which involves lesions of the median nerve in the mouse. Data showed that the finger flexor muscle contraction strength, assessed to evaluate the motor function recovery, and reached values not different from the control already 20 days after injury. The degree of nerve regeneration evaluated with stereological methods in light microscopy showed that, 25 days after injury, the number of regenerated myelinated fibers was comparable to the control, but they were smaller with a thinner myelin thickness. Stereological analysis made in electron microscopy confirmed these results, although the total number of fibers quantified was significantly higher compared to light microscopy analysis, due to the very small size of some fibers that can be detected only in electron microscopy.

Background: There is abundant evidence suggesting that growth factors may play a key role in the healing process, especially in the early stages of inflammation. Despite the reported clinical successes with the use of growth factors there is still a lack of knowledge on the biological mechanism underlying the activity of platelet-rich plasma during the process of muscle healing. The aim of this study was to analyse the early effects of platelet- rich plasma in an easily reproducible animal model., Materials and Methods: Wistar male adult rats (n=102) were used in this study. The muscle lesion was created with a scalpel in the flexor sublimis muscles. Platelet-rich plasma was applied immediately after surgery. Treated, untreated and contralateral muscles were analysed by morphological evaluation and western blot assay., Results: Leucocyte infiltration was significantly greater in muscles treated with platelet-rich plasma than in both untreated and contralateral muscles. The latter showed greater leucocyte infiltration when compared to the untreated muscles. Platelet-rich plasma treatment also modified the cellular composition of the leucocyte infiltration leading to increased expression of CD3, CD8, CD19 and CD68 and to decreased CD4 antigen expression in both platelet-rich plasma treated and contralateral muscles. Blood vessel density and blood vessel diameters were not statistically significantly different between the three groups analysed., Discussion: The results of this study showed that treatment with platelet-rich plasma magnified the physiological early inflammatory response following a muscle injury, modifying the pattern of cellular recruitment. Local platelet-rich plasma treatment may exert a direct or, more plausibly, indirect systemic effect on healing processes, at least in the earliest inflammatory phase.

In spite of great recent advancements, the definition of the optimal strategy for bridging a nerve defect, especially across long gaps, still remains an open issue since the amount of autologous nerve graft material is limited while the outcome after alternative tubulization techniques is often unsatisfactory. The aim of this study was to investigate a new tubulization technique based on the employment of vein conduits filled with whole subcutaneous adipose tissue obtained by lipoaspiration. In adult rats, a 1cm-long defect of the left median nerve was repaired by adipose tissue-vein-combined conduits and compared with fresh skeletal muscle tissue-vein-combined conduits and autologous nerve grafts made by the excised nerve segment rotated by 180°. Throughout the postoperative period, functional recovery was assessed using the grasping test. Regenerated nerve samples were withdrawn at postoperative month-6 and processed for light and electron microscopy and stereology of regenerated nerve fibers. Results showed that functional recovery was significantly slower in the adipose tissue-enriched group in comparison to both control groups. Light and electron microscopy showed that a large amount of adipose tissue was still present inside the vein conduits at postoperative month-6. Stereology showed that all quantitative morphological predictors analyzed performed significantly worse in the adipose tissue-enriched group in comparison to the two control groups. On the basis of this experimental study in the rat, the use of whole adipose tissue for tissue engineering of peripheral nerves should be discouraged. Pre-treatment of adipose tissue aimed at isolating stromal vascular fraction and/or adipose derived stem/precursor cells should be considered a fundamental requisite for nerve repair., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Introduction: In this study, we evaluated the role of the Netrin-1 receptor UNC5b (Uncoordinated), a neuronal guidance molecule, during peripheral nerve regeneration using the mouse median nerve model., Materials and Methods: Using Western blot analysis, we examined the expression changes of UNC5b after transection and microsurgical repair of the mouse median nerve distal to the transection site. We evaluated the histomorphometrical changes and functional recovery of the grasping force after median nerve transection and repair in wild-type (WT) mice and UNC5b(+/-) heterozygous mice., Results: In Western blot analysis, we could show a high increase of UNC5b in the nerve segment distal to the injury site at day 14. Histomorphometrical analysis did not show any significant differences between WT animals and heterozygous animals. Using the functional grasping test, we could demonstrate that peripheral nerve regeneration is significantly diminished in heterozygous UNC5b(+/-) mice., Conclusion: By using the mouse median nerve model in transgenic animals, we demonstrate that the Netrin-1 receptor UNC5b plays an important role during peripheral nerve regeneration., (Copyright © 2012 Wiley Periodicals, Inc.)

Cachexia is a wasting syndrome associated with cancer, AIDS, multiple sclerosis, and several other disease states. It is characterized by weight loss, fatigue, loss of appetite, and skeletal muscle atrophy and is associated with poor patient prognosis, making it an important treatment target. Ghrelin is a peptide hormone that stimulates growth hormone (GH) release and positive energy balance through binding to the receptor GHSR-1a. Only acylated ghrelin (AG), but not the unacylated form (UnAG), can bind GHSR-1a; however, UnAG and AG share several GHSR-1a-independent biological activities. Here we investigated whether UnAG and AG could protect against skeletal muscle atrophy in a GHSR-1a-independent manner. We found that both AG and UnAG inhibited dexamethasone-induced skeletal muscle atrophy and atrogene expression through PI3Kβ-, mTORC2-, and p38-mediated pathways in myotubes. Upregulation of circulating UnAG in mice impaired skeletal muscle atrophy induced by either fasting or denervation without stimulating muscle hypertrophy and GHSR-1a-mediated activation of the GH/IGF-1 axis. In Ghsr-deficient mice, both AG and UnAG induced phosphorylation of Akt in skeletal muscle and impaired fasting-induced atrophy. These results demonstrate that AG and UnAG act on a common, unidentified receptor to block skeletal muscle atrophy in a GH-independent manner.

After peripheral nerve injuries, the process of nerve regeneration and target reinnervation is very complex and depends on many different events occurring not only at the lesion site but also proximally and distally to it. In spite of the recent scientific and technological advancements, the need to find out new strategies to improve clinical nerve repair and regeneration remains. To reach this goal, the therapeutic strategy should thus exert its effects at different levels in order to simultaneously potentiate axonal regeneration, increase neuronal survival, modulate central reorganization, and inhibit or reduce target organ atrophy. It is expected that this multilevel approach might lead to significant improvement in the functional outcome and thus the quality of life of the patients suffering from peripheral nerve injury., (© 2013 Elsevier Inc. All rights reserved.)

Promoting neuromuscular recovery after neural injury is a major clinical issue. While techniques for nerve reconstruction are continuously improving and most peripheral nerve lesions can be repaired today, recovery of the lost function is usually unsatisfactory. This evidence claims for innovative nonsurgical therapeutic strategies that can implement the outcome after neural repair. Although no pharmacological approach for improving posttraumatic neuromuscular recovery has still entered clinical practice, various molecules are explored in experimental models of neural repair. One of such molecules is the circulating peptide hormone ghrelin. This hormone has proved to have a positive effect on neural repair after central nervous system lesion, and very recently its effectiveness has also been demonstrated in preventing posttraumatic skeletal muscle atrophy. By contrast, no information is still available about its effectiveness on peripheral nerve regeneration although preliminary data from our laboratory suggest that this molecule can have an effect also in promoting axonal regeneration after nerve injury and repair. Should this be confirmed, ghrelin might represent an ideal candidate as a therapeutic agent for improving posttraumatic neuromuscular recovery because of its putative effects at all the various structural levels involved in this regeneration process, namely, the central nervous system, the peripheral nerve, and the target skeletal muscle., (© 2013 Elsevier Inc. All rights reserved.)