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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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., Introduction Skeletal muscle atrophy involves massive loss of muscle structural proteins, which leads to muscle weight decrease and progressive loss of muscle function. Skeletal muscle atrophy is induced by muscle [...]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The article focuses on the development of a new decellularization protocol for pig nerves that preserves the nerve extracellular matrix (ECM) and its subsequent use in repairing rat median nerves, demonstrating its potential for treating severe nerve injuries.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The interest in perforator vessels is continuously increasing due to the increasing employment of perforator flaps in reconstructive surgery. While the gross anatomy of perforator pedicles has been extensively described, little is known about their histological organization. To fill this gap, we carried out a light and transmission electron microscopy analysis of superior and inferior epigastric perforator pedicles in the rat and of thoraco-dorsal, intercostal and gluteal perforators in the pig. Results showed that, in both animal models, perforating pedicles have a rather complex histological structure, based on a refined organization of the connective tissue surrounding the vessels. The number of veins accompanying each perforating artery varied among specimens, without a clear relation to the anatomical site. Yet, veins are sometimes very small and difficult to be recognized. Finally, the comparison between rat and pig did not reveal major inter-species histological differences. These results point to the perforator pedicle as quite complex and organized anatomical entity that, also considering the emerging relevance of perforator-based flap surgery, should be referred to as an independent organ rather than a appendix of muscles and skin only. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Less is known about the role of Netrin-1 in the peripheral nervous system. In this study, we evaluated the role of Netrin-1 using the mouse median nerve model for assessment of peripheral nerve regeneration. Methods: Using real-time PCR and Western Blot analysis, we examined expression changes of Netrin-1 mRNA and Netrin-1 protein after transection and repair of the mouse median nerve in Wild-type animals. We further evaluated histomorphometrical changes as well as the functional recovery of the grasping force after median nerve transection and repair in WT mice and Netrin-1+/- heterozygous mice. Results: RT-PCR revealed a 1, 9 fold increase of Netrin-1 mRNA two 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 Netrin-1 in the nerve segment distal to the injury site at day 14. Histomorphometrical analysis showed significantly higher cross sectional area and a lower fibre density in heterozygous Netrin-1+/- mice. Using the functional grasping test, we could show that peripheral nerve regeneration is significantly diminished in heterozygous Netrin-1+/- mice. Conclusions: Employing the mouse median nerve model in transgenic animals, we demonstrate that Netrin-1 plays an important role during peripheral nerve regeneration. [ABSTRACT FROM AUTHOR]

In a transgenic mice (BALB-neuT) over-expressing ErbB2 receptor, we investigated the adult mouse median nerve in physiological and pathological conditions. Results showed that, in physiological conditions, the grip function controlled by the median nerve in BALB-neuT mice was similar to wild-type (BALB/c). Stereological assessment of ErbB2-overexpressing intact nerves revealed no difference in number and size of myelinated fibers compared to wild-type mice. By contrast, after a nerve crush injury, the motor recovery was significantly faster in BALB-neuT compared to BALB/c mice. Moreover, stereological assessment revealed a significant higher number of regenerated myelinated fibers with a thinner axon and fiber diameter and myelin thickness in BALB-neuT mice. At day-2 post-injury, the level of the mRNAs coding for all the ErbB receptors and for the transmembrane (type III) Neuregulin 1 (NRG1) isoforms significantly decreased in both BALB/c and BALB-neuT mice, as shown by quantitative real time PCR. On the other hand, the level of the mRNAs coding for soluble NRG1 isoforms (type I/II, alpha and beta) increased at the same post-traumatic time point though, intriguingly, this response was significantly higher in BALB-neuT mice with respect to BALB/c mice. Altogether, these results suggest that constitutive ErbB2 receptor over-expression does not influence the physiological development of peripheral nerves, while it improves nerve regeneration following traumatic injury, possibly through the up-regulation of soluble NRG1 isoforms. [ABSTRACT FROM AUTHOR]

This paper critically overviews the main procedures used for carrying out morphological analysis of peripheral nerve fibers in light, confocal, and electron microscopy. In particular, this paper emphasizes the importance of osmium tetroxide post‐fixation as a useful procedure to be adopted independently from the embedding medium. In order to facilitate the use of any described techniques, all protocols are presented in full details. The pros and cons for each method are critically addressed and practical indications on the different imaging approaches are reported. Moreover, the basic rules of morpho‐quantitative stereological analysis of nerve fibers are described addressing the important concepts of design‐based sampling and the disector. Finally, a comparison of stereological analysis on myelinated nerve fibers between paraffin‐ and resin‐embedded rat radial nerves is reported showing that different embedding procedures might influence the distribution of size parameters. [Copyright &y& Elsevier]

This paper addresses several basic issues that are important for the experimental model design to investigate peripheral nerve regeneration. First, the importance of carrying out adequate preliminary in vitro investigation is emphasized in light of the ethical issues and with particular emphasis on the concept of the Three Rs (Replacement, Reduction, and Refinement) for limiting in vivo animal studies. Second, the various options for the selection of the animal species for nerve regeneration research are reviewed. Third, the two main experimental paradigms of nerve lesion (axonotmesis vs. neurotmesis followed by microsurgical reconstruction) are critically outlined and compared. Fourth, the various nerve models that have most commonly been employed are overviewed focusing in particular on forearm mixed nerves and on behavioural tests for assessing their function: the ulnar test and the grasping test which is useful for assessing both median and radial nerves in the rat. Finally, the importance of considering the influence of various factors and diseases which could interfere with the nerve regeneration process is emphasized in the perspective of a wider adoption of experimental models which more closely mimic the environmental and clinical conditions found in patients. [Copyright &y& Elsevier]

Peripheral nerves are complex organs that can be found throughout the body reaching almost all tissues and organs to provide motor and/or sensory innervation. A parenchyma (the noble component made by the nerve fibers, i.e., axons and Schwann cells) and a stroma (the scaffold made of various connective elements) can be recognized. Although morphological analysis is the most common approach for studying peripheral nerve regeneration, researchers are not always aware of several histological peculiarities of these organs. Therefore, the aim of this review is to describe, at a structural and ultrastructural level, the main features of the parenchyma and the stroma of the normal undamaged nerve as well as the most important morphological changes that occur after nerve damage and during posttraumatic nerve regeneration. The paper is aimed at providing the reader with the basic framework information on nerve morphology. This would enable the correct interpretation of morphological data obtained by many experimental studies on peripheral nerve repair and regeneration such as those outlined in several other papers included in this special issue of the International Review of Neurobiology. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]