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Peripheral nerve injuries are commonly encountered in clinical practice. Peripheral nerve injuries most commonly result in serious problems affecting quality of life. The present study is designed to research the possible neuroprotective effects of Ankaferd blood stopper (ABS), platelet-rich plasma (PRP), and Momordica charantia (MC) on regeneration using unbiased stereological techniques. In total, 30 female 8-10 week Sprague Dawley rats were randomly divided into five equal groups; control (CG), the group exposed to sciatic nerve resection (Gap) (CGG), additionally following the surgical process ABS, MC, and PRP were injected into collagen tube (ABSG, MCG, PRPG). Nucleator and fractionator methods were used to estimate the number of myelinated and unmyelinated axons, axon area, and myelin sheath thickness. Also, an electron microscopic evaluation was performed. Regarding the number of myelinated axons, significant increases were found in the ABSG, MCG, and PRPG compared with CG (p < 0.05). The protective effects of ABS, PRP, and MC on peripheral nerve regeneration in experimental models in rats were shown using electrophysiological and stereological assessment parameters., Competing Interests: Declarations Conflict of interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The gut microbiome plays a fundamental role in metabolism, as well as the immune and nervous systems. Microbial imbalance (dysbiosis) can contribute to subsequent physical and mental pathologies. As such, interest has been growing in the microbiota-gut-brain brain axis and the bioelectrical communication that could exist between bacterial and nervous cells. The aim of this study was to investigate the bioelectrical profile (electrome) of two bacterial species characteristic of the gut microbiome: a Proteobacteria Gram-negative bacillus Escherichia coli ( E. coli ), and a Firmicutes Gram-positive coccus Enterococcus faecalis ( E. faecalis ). We analyzed both bacterial strains to (i) validate the fluorescent probe bis-(1,3-dibutylbarbituric acid) trimethine oxonol, DiBAC4(3), as a reliable reporter of the changes in membrane potential (Vmem) for both bacteria; (ii) assess the evolution of the bioelectric profile throughout the growth of both strains; (iii) investigate the effects of two neural-type stimuli on Vmem changes: the excitatory neurotransmitter glutamate (Glu) and the inhibitory neurotransmitter γ-aminobutyric acid (GABA); (iv) examine the impact of the bioelectrical changes induced by neurotransmitters on bacterial growth, viability, and cultivability using absorbance, live/dead fluorescent probes, and viable counts, respectively. Our findings reveal distinct bioelectrical profiles characteristic of each bacterial species and growth phase. Importantly, neural-type stimuli induce Vmem changes without affecting bacterial growth, viability, or cultivability, suggesting a specific bioelectrical response in bacterial cells to neurotransmitter cues. These results contribute to understanding the bacterial response to external stimuli, with potential implications for modulating bacterial bioelectricity as a novel therapeutic target.

Optimal recovery of muscle function after proximal nerve injuries remains a complex and challenging problem. After a nerve injury, alterations in the affected muscles lead to atrophy, and later degeneration and replacement by fat-fibrous tissues. At present, several different strategies for the preservation of skeletal muscle have been reported, including various sets of physical exercises, muscle massage, physical methods (e.g. electrical stimulation, magnetic field and laser stimulation, low-intensity pulsed ultrasound), medicines (e.g. nutrients, natural and chemical agents, anti-inflammatory and antioxidants, hormones, enzymes and enzyme inhibitors), regenerative medicine (e.g. growth factors, stem cells and microbiota) and surgical procedures (e.g. supercharge end-to-side neurotization). The present review will focus on methods that aimed to minimize the damage to muscles after denervation based on our present knowledge., Competing Interests: Declaration of conflicting interestsThe authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Aim: Peripheral nerve injury regeneration studies using animal models are crucial to different pre-clinical therapeutic approaches efficacy evaluation whatever the surgical technique explored. Materials & methods: A 944 articles systematic review on 'peripheral nerve injury in animal models' over the last 9 years was carried out. Results: It was found that 91% used rodents, and only 9% employed large animals. Different nerves are studied, with generated gaps (10,78 mm) and methods applied for regeneration evaluation uniformed. Sciatic nerve was the most used (88%), followed by median and facial nerves (2.6%), significantly different. Conclusion: There has not been a significant scale-up of the in vivo testing to large animal models (anatomically/physiologically closer to humans), allowing an improvement in translational medicine for clinical cases.

Inter-cellular communication is mediated by a sum of biochemical, biophysical, and bioelectrical signals. This might occur not only between cells belonging to the same tissue and/or animal species but also between cells that are, from an evolutionary point of view, far away. The possibility that bioelectrical communication takes place between bacteria and nerve cells has opened exciting perspectives in the study of the gut microbiota-brain axis. The aim of this paper is (i) to establish a reliable method for the assessment of the bioelectrical state of two bacterial strains: Bacillus subtilis ( B. subtilis ) and Limosilactobacillus reuteri ( L. reuteri ); (ii) to monitor the bacterial bioelectrical profile throughout its growth dynamics; and (iii) to evaluate the effects of two neurotransmitters (glutamate and γ-aminobutyric acid-GABA) on the bioelectrical signature of bacteria. Our results show that membrane potential ( Vmem ) and the proliferative capacity of the population are functionally linked in B. subtilis in each phase of the cell cycle. Remarkably, we demonstrate that bacteria respond to neural signals by changing Vmem properties. Finally, we show that Vmem changes in response to neural stimuli are present also in a microbiota-related strain L. reuteri . Our proof-of-principle data reveal a new methodological approach for the better understanding of the relation between bacteria and the brain, with a special focus on gut microbiota. Likewise, this approach will open exciting perspectives in the study of the inter-cellular mechanisms which regulate the bi-directional communication between bacteria and neurons and, ultimately, for designing gut microbiota-brain axis-targeted treatments for neuropsychiatric diseases.

Silicon (Si) is an essential trace element in the human body and it exists in connective tissue as aqueous orthosilicic acid. Porous chitosan-3-glycidoxypropyltrimethoxysilane (GPTMS) hybrids can regenerate nerve tissue and recover sensor and motor functions. However, the structures and roles of the degradation products with Si extracted from the hybrids in nerve regeneration are not clear. In this study, we prepared porous chitosan-GPTMS hybrids with different amounts of GPTMS to amino groups of chitosan (chitosan:GPTMS = 1:0.5 and 1:1 molar ratios). The structures of the degradation products with Si from the hybrids were examined using time-of-flight mass spectrometry, and biological assessments were conducted in order to evaluate their potential use in the preparation of devices for nerve repair. Glial and motor cell lines and ex vivo explants of dorsal root ganglia were used in this study for evaluating their behavior in the presence of the different degradation products with Si. The structure of the degradation products with Si depended on the starting composition. The results showed that glial cell proliferation was lower in the medium with the higher-molecular-weight degradation products with Si. Moreover, motor cell line differentiation and the neurite outgrowth of dorsal root ganglion explants were improved with the lower-molecular-weight degradation products with Si. The results obtained could be useful for designing a new nerve regeneration scaffold including silicon components.

Exosomes have shown promising potential as a therapeutic approach for wound healing. Nevertheless, the translation from experimental studies to commercially available treatments is still lacking. To assess the current state of research in this field, a systematic review was performed involving studies conducted and published over the past five years. A PubMed search was performed for English-language, full-text available papers published from 2018 to June 2023, focusing on exosomes derived from mammalian sources and their application in wound healing, particularly those involving in vivo assays. Out of 531 results, 148 papers were selected for analysis. The findings revealed that exosome-based treatments improve wound healing by increasing angiogenesis, reepithelization, collagen deposition, and decreasing scar formation. Furthermore, there was significant variability in terms of cell sources and types, biomaterials, and administration routes under investigation, indicating the need for further research in this field. Additionally, a comparative examination encompassing diverse cellular origins, types, administration pathways, or biomaterials is imperative. Furthermore, the predominance of rodent-based animal models raises concerns, as there have been limited advancements towards more complex in vivo models and scale-up assays. These constraints underscore the substantial efforts that remain necessary before attaining commercially viable and extensively applicable therapeutic approaches using exosomes.

Hollow conduits of natural or synthetic origins have shown acceptable regeneration results in short nerve gap repair; however, results are still not comparable with the current gold standard technique "autografts". Hollow conduits do not provide a successful regeneration outcome when it comes to critical nerve gap repair. Enriching the lumen of conduits with different extracellular materials and cells could provide a better biomimicry of the natural nerve regenerating environment and is expected to ameliorate the conduit performance. In this study, we evaluated nerve regeneration in vivo using hollow chitosan conduits or conduits enriched with fibrin-collagen hydrogels alone or with the further addition of adipose-derived mesenchymal stem cells in a 15 mm rat sciatic nerve transection model. Unexpected changes in the hydrogel consistency and structural stability in vivo led to a failure of nerve regeneration after 15 weeks. Nevertheless, the molecular assessment in the early regeneration phase (7, 14, and 28 days) has shown an upregulation of useful regenerative genes in hydrogel enriched conduits compared with the hollow ones. Hydrogels composed of fibrin-collagen were able to upregulate the expression of soluble NRG1, a growth factor that plays an important role in Schwann cell transdifferentiation. The further enrichment with adipose-derived mesenchymal stem cells has led to the upregulation of other important genes such as ErbB2, VEGF-A, BDNF, c-Jun, and ATF3., Competing Interests: None

As a reliable alternative to autografts, decellularized peripheral nerve allografts (DPNAs) should mimic the complex microstructure of native nerves and be immunogenically compatible. Nevertheless, there is a current lack of decellularization methods able to remove peripheral nerve cells without significantly altering the nerve extracellular matrix (ECM). The aims of this study are firstly to characterize ex vivo , in a histological, biochemical, biomechanical and ultrastructural way, three novel chemical-enzymatic decellularization protocols (P1, P2 and P3) in rat sciatic nerves and compared with the Sondell classic decellularization method and then, to select the most promising DPNAs to be tested in vivo . All the DPNAs generated present an efficient removal of the cellular material and myelin, while preserving the laminin and collagen network of the ECM (except P3) and were free from any significant alterations in the biomechanical parameters and biocompatibility properties. Then, P1 and P2 were selected to evaluate their regenerative effectivity and were compared with Sondell and autograft techniques in an in vivo model of sciatic defect with a 10-mm gap, after 15 weeks of follow-up. All study groups showed a partial motor and sensory recovery that were in correlation with the histological, histomorphometrical and ultrastructural analyses of nerve regeneration, being P2 the protocol showing the most similar results to the autograft control group., 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., (Copyright © 2023 García-García, El Soury, Campos, Sánchez-Porras, Geuna, Alaminos, Gambarotta, Chato-Astrain, Raimondo and Carriel.)

Background: Lumbrical muscles originate in the palm from the 4 tendons of the flexor digitorum profundus and course distally along the radial side of the corresponding metacarpophalangeal joints, in front of the deep transverse metacarpal ligament. The first and second lumbrical muscles are typically innervated by the median nerve, and third and fourth by the ulnar nerve. A plethora of lumbrical muscle variants has been described, ranging from muscles' absence to reduction in their number or presence of accessory slips. The current cadaveric study highlights typical and variable neural supply of lumbrical muscles., Materials: Eight (3 right and 5 left) fresh frozen cadaveric hands of 3 males and 5 females of unknown age were dissected. From the palmar wrist crease, the median and ulnar nerve followed distally to their terminal branches. The ulnar nerve deep branch was dissected and lumbrical muscle innervation patterns were noted., Results: The frequency of typical innervations of lumbrical muscles is confirmed. The second lumbrical nerve had a double composition from both the median and ulnar nerves, in 12.5% of the hands. The thickest branch (1.38 mm) originated from the ulnar nerve and supplied the third lumbrical muscle, and the thinnest one (0.67 mm) from the ulnar nerve and supplied the fourth lumbrical muscle. In 54.5%, lumbrical nerve bifurcation was identified., Conclusion: The complex innervation pattern and the peculiar anatomy of branching to different thirds of the muscle bellies are pointed out. These findings are important in dealing with complex and deep injuries in the palmar region, including transmetacarpal amputations.

Competing Interests: None

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.

Aim of this study is to investigate effects of stem cells derived from the peripheral nerve and adipose tissues following the nerve crush injury in control and obese rats. For this aim, 41 Wistar Albino female rats were separated into eight equal groups; non-obese control (NOC) obese control (OC), non-obese injury (NOH), obese injury (OH), non-obese adipose (NOY), obese adipose (OY), non-obese nerve (NOPS), obese nerve (OPS). At the end of 8 weeks, all experimental animals without control groups were subjected to nerve crush procedure and sciatic nerve or fat stem cell homogenates were injected on the treatment group rats, and then, recovery process has been observed and histopathological, stereological, electrophysiological analyses and bioinformatic evaluation were made on removed sciatic nerves. Stereological results showed that adipose homogenate gave more successful results than peripheral nerve homogenates in the NOY group in comparison to the NOPS group in terms of myelinated axon number. Peripheral nerve homogenate has shown more successful results in the OPS group in comparison to the OY group. The number of unmyelinated axons was increased following treatment with adipose tissue homogenate in NOY and OY groups. In terms of myelin sheath thickness; we detected that treatments by peripheral nerve and especially adipose tissue homogenates lead to increase in the thickness of the axons of the peripheral nerves belong to the control and obese injury groups. All results showed that mesenchymal stem cell treatment by fresh tissue homogenates is successful in peripheral nerve regeneration and fat tissue is a considerable source of the stem cells for clinical applications.

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.

In Italy, recent legislation (Law No. 10/2020) has tuned regulations concerning the donation of one's postmortem body and tissues for study, training, and scientific research purposes. This study discusses several specific issues to optimise the applicability and effectiveness of such an important, novel regulatory setting. Critical issues arise concerning the learners, the type of training and teaching activities that can be planned, the position of academic anatomy institutes, the role of family members in the donation process, the time frame of the donation process, the eligibility of partial donation, or the simultaneous donation of organs and tissues to patients awaiting transplantation. In particular, a universal time limit for donations (i.e., one year) makes it impossible to plan the long-term use of specific body parts, which could be effectively preserved for the advanced teaching and training of medical students and surgeons. The abovementioned conditions lead to the limited use of corpses, thus resulting in the inefficiency of the whole system of body donation. Overall, the donors' scope for the donation of their body could be best honoured by a more flexible and tuneable approach that can be used on a case-by-case basis. Furthermore, it is deemed necessary to closely monitor the events scheduled for corpses in public nonacademic institutions or private enterprises. This paper presents useful insights from Italian anatomists with the hope of providing inspiration for drafting the regulations. In conclusion, this paper focuses on the critical issues derived from the recently introduced Italian law on the donation and use of the body after death and provides suggestions to lawmakers for future implementations., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Axonal count is the base for efficient nerve transfer; despite its capital importance, few studies have been published on human material, most research approaches being performed on experimental animal models of nerve injury. Thus, standard analysis methods are still lacking. Quantitative data obtained have to be reproducible and comparable with published data by other research groups. To share results with the scientific community, the standardization of quantitative analysis is a fundamental step. For this purpose, the experiences of the Italian, Austrian, German, Greek, and Iberian-Latin American groups have been compared with each other and with the existing literature to reach a consensus in the fiber count and draw up a protocol that can make future studies from different centers comparable. The search for a standardization of the methodology was aimed to reduce all the factors that are associated with an increase in the variability of the results. All the preferential methods to be used have been suggested. On the other hand, alternative methods and different methods have been identified to achieve the same goal, which in our experience are completely comparable; therefore, they can be used indifferently by the different centers according to their experience and availability., Competing Interests: Declaration of Competing Interest The authors report no conflicts of interest to disclose., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: The vagus nerve (VN), also called the pneumogastric nerve, connects the brainstem to organs contained in the chest and abdomen. Physiologically, VN stimulation can rapidly affect cardiac activity and heart rate (HR). VN neuropathy can increase the risk of arrhythmias and sudden death. Therefore, a selective test of VN function may be very useful. Since peripheral neurodynamic tests (NDT) are reliable for the assessment of neuropathies in somatic nerves, we aimed to validate a novel NDT to assess VN activity, namely, the VN-NTD., Methods: In this cross-sectional double-blind, sex-balanced study, 30 participants (15 females) completed a checklist of autonomic dysfunction symptoms. During the VN-NDT administration, HR and symptoms (i.e., mechanical allodynia) were monitored in parallel to a real-time ultrasonography imaging (USI) and motion capture analysis of the neck. The VN-NDT impact on HR and its accuracy for autonomic symptoms reported in the last 7 days were tested., Results: The VN-NDT induced a significant HR reduction of about 12 and 8 bpm in males and females [ t (1, 119) = 2.425; p < 0.017; η p 2 = 0.047, 95% confidence interval (CI): 0.93-9.18], respectively. No adverse events were observed during VN-NDT. A substantial interexaminer agreement between the evaluators in symptoms induction by VN-NDT was detected [ F (1, 119) = 0.540; p = 0.464; η p 2 = 0.005, low effect]. Notably, mechanical allodynia accuracy for gastrointestinal dysfunctions was excellent ( p < 0.05; 95% CI: 0.52-0.73; p < 0.001; 95% CI: 0.81-0.96)., Conclusions: The novel VN-NDT is a valid and accurate test capable of detecting VN activation with high sensitivity. Data provided are suitable for both sexes as a hallmark of HR variation due to VN normal response. The proposed VN-NDT may be reliable as daily routine neurological examination tests for the evaluation of neuropathic signs related to neuroinflammation of the VN., Clinical Trial Registration: www.ClinicalTrials.gov, identifier NCT04192877., 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., (Copyright © 2021 Carta, Seregni, Casamassima, Galli, Geuna, Pagliaro and Zago.)

The accumulating evidence linking bacteria in the gut and neurons in the brain (the microbiota-gut-brain axis) has led to a paradigm shift in the neurosciences. Understanding the neurobiological mechanisms supporting the relevance of actions mediated by the gut microbiota for brain physiology and neuronal functioning is a key research area. In this review, we discuss the literature showing how the microbiota is emerging as a key regulator of the brain's function and behavior, as increasing amounts of evidence on the importance of the bidirectional communication between the intestinal bacteria and the brain have accumulated. Based on recent discoveries, we suggest that the interaction between diet and the gut microbiota, which might ultimately affect the brain, represents an unprecedented stimulus for conducting new research that links food and mood. We also review the limited work in the clinical arena to date, and we propose novel approaches for deciphering the gut microbiota-brain axis and, eventually, for manipulating this relationship to boost mental wellness.

Nerves are subjected to tensile forces in various paradigms such as injury and regeneration, joint movement, and rehabilitation treatments, as in the case of neurodynamic treatment (NDT). The NDT induces selective uniaxial repeated tension on the nerve and was described to be an effective treatment to reduce pain in patients. Nevertheless, the biological mechanisms activated by the NDT promoting the healing processes of the nerve are yet still unknown. Moreover, a dose-response analysis to define a standard protocol of treatment is unavailable. In this study, we aimed to define in vitro whether NDT protocols could induce selective biological effects on sensory and motor neurons, also investigating the possible involved molecular mechanisms taking a role behind this change. The obtained results demonstrate that NDT induced significant dose-dependent changes promoting cell differentiation, neurite outgrowth, and neuron survival, especially in nociceptive neurons. Notably, NDT significantly upregulated PIEZO1 gene expression. A gene that is coding for an ion channel that is expressed both in murine and human sensory neurons and is related to mechanical stimuli transduction and pain suppression. Other genes involved in mechanical allodynia related to neuroinflammation were not modified by NDT. The results of the present study contribute to increase the knowledge behind the biological mechanisms activated in response to NDT and to understand its efficacy in improving nerve regenerational physiological processes and pain reduction.

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.)

Thousands of people worldwide suffer from peripheral nerve injuries and must deal daily with the resulting physiological and functional deficits. Recent advances in this field are still insufficient to guarantee adequate outcomes, and the development of new and compelling therapeutic options require the use of valid preclinical models that effectively replicate the characteristics and challenges associated with these injuries in humans. In this study, we established a sheep model for common peroneal nerve injuries that can be applied in preclinical research with the advantages associated with the use of large animal models. The anatomy of the common peroneal nerve and topographically related nerves, the functional consequences of its injury and a neurological examination directed at this nerve have been described. Furthermore, the surgical protocol for accessing the common peroneal nerve, the induction of different types of nerve damage and the application of possible therapeutic options were described. Finally, a preliminary morphological and stereological study was carried out to establish control values for the healthy common peroneal nerves regarding this animal model and to identify preliminary differences between therapeutic methods. This study allowed to define the described lateral incision as the best to access the common peroneal nerve, besides establishing 12 and 24 weeks as the minimum periods to study lesions of axonotmesis and neurotmesis, respectively, in this specie. The post-mortem evaluation of the harvested nerves allowed to register stereological values for healthy common peroneal nerves to be used as controls in future studies, and to establish preliminary values associated with the therapeutic performance of the different applied options, although limited by a small sample size, thus requiring further validation studies. Finally, this study demonstrated that the sheep is a valid model of peripheral nerve injury to be used in pre-clinical and translational works and to evaluate the efficacy and safety of nerve injury therapeutic options before its clinical application in humans and veterinary patients.

Peripheral nerve injury remains a clinical challenge with severe physiological and functional consequences. Despite the existence of multiple possible therapeutic approaches, until now, there is no consensus regarding the advantages of each option or the best methodology in promoting nerve regeneration. Regenerative medicine is a promise to overcome this medical limitation, and in this work, chitosan nerve guide conduits and olfactory mucosa mesenchymal stem/stromal cells were applied in different therapeutic combinations to promote regeneration in sciatic nerves after neurotmesis injury. Over 20 weeks, the intervened animals were subjected to a regular functional assessment (determination of motor performance, nociception, and sciatic indexes), and after this period, they were evaluated kinematically and the sciatic nerves and cranial tibial muscles were evaluated stereologically and histomorphometrically, respectively. The results obtained allowed confirming the beneficial effects of using these therapeutic approaches. The use of chitosan NGCs and cells resulted in better motor performance, better sciatic indexes, and lower gait dysfunction after 20 weeks. The use of only NGGs demonstrated better nociceptive recoveries. The stereological evaluation of the sciatic nerve revealed identical values in the different parameters for all therapeutic groups. In the muscle histomorphometric evaluation, the groups treated with NGCs and cells showed results close to those of the group that received traditional sutures, the one with the best final values. The therapeutic combinations studied show promising outcomes and should be the target of new future works to overcome some irregularities found in the results and establish the combination of nerve guidance conduits and olfactory mucosa mesenchymal stem/stromal cells as viable options in the treatment of peripheral nerves after injury., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this article., (Copyright © 2021 Rui D. Alvites et al.)

Neurotrophins play a major role in the regulation of neuronal growth such as neurite sprouting or regeneration in response to nerve injuries. The role of nerve growth factor, neurotrophin-3, and brain-derived neurotrophic factor in maintaining the survival of peripheral neurons remains poorly understood. In regenerative medicine, different modalities have been investigated for the delivery of growth factors to the injured neurons, in search of a suitable system for clinical applications. This study was to investigate the influence of nerve growth factor, neurotrophin-3 and brain-derived neurotrophic factor on the growth of neurites using two in vitro models of dorsal root ganglia explants and dorsal root ganglia-derived primary cell dissociated cultures. Quantitative data showed that the total neurite length and tortuosity were differently influenced by trophic factors. Nerve growth factor and, indirectly, brain-derived neurotrophic factor stimulate the tortuous growth of sensory fibers and the formation of cell clusters. Neurotrophin-3, however, enhances neurite growth in terms of length and linearity allowing for a more organized and directed axonal elongation towards a peripheral target compared to the other growth factors. These findings could be of considerable importance for any clinical application of neurotrophic factors in peripheral nerve regeneration. Ethical approval was obtained from the Regione Piemonte Animal Ethics Committee ASLTO1 (file # 864/2016-PR) on September 14, 2016., Competing Interests: None

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.

Stem/stromal cell-based therapies are a branch of regenerative medicine and stand as an attractive option to promote the repair of damaged or dysfunctional tissues and organs. Olfactory mucosa mesenchymal stem/stromal cells have been regarded as a promising tool in regenerative therapies because of their several favorable properties such as multipotency, high proliferation rate, helpful location, and few associated ethical issues. These cells are easily accessible in the nasal cavity of most mammals, including the rat, can be easily applied in autologous treatments, and do not cope with most of the obstacles associated with the use of other stem cells. Despite this, its application in preclinical trials and in both human and animal patients is still limited because of the small number of studies performed so far and to the nonexistence of a standard and unambiguous protocol for collection, isolation, and therapeutic application. In the present work a validation of a protocol for isolation, culture, expansion, freezing, and thawing of olfactory mucosa mesenchymal stem/stromal cells was performed, applied to the rat model, as well as a biological characterization of these cells. To investigate the therapeutic potential of OM-MSCs and their eventual safe application in preclinical trials, the main characteristics of OMSC stemness were addressed., Competing Interests: The author(s) declare(s) that there are no conflicts of interest regarding the publication of this article., (Copyright © 2020 Rui D. Alvites et al.)

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.)

Background: The aim of the current study is to investigate the first and second lumbrical nerves as potential fibers donors to the deep motor branch of the ulnar nerve to avoid intrinsic atrophy in high ulnar nerve injuries., Methods: Sixteen fresh frozen cadaveric hands were dissected, the radial lumbrical nerves accessed, and a coaptation, either in reverse end-to-side or in double end-to-side through a bridge nerve graft, was created to the deep motor branch of ulnar nerve. Semithin sections were taken from samples of donor and recipient nerves for qualitative (nerve architecture) and quantitative studies (fiber count and donor/recipient ratio)., Results: The first lumbrical showed a robust trunk and a superior axon density (9,126.50 ± 2,923.41 axons/mm 2 ) to the ulnar motor branch (7,506.50 ± 1,137.50 axons/mm 2 distal to the opponens tunnel and 7,947.75 ± 1,741.24 axons/mm 2 before its terminal branching); the ulnar motor branch showed a higher axon number (2,633.51 ± 410.00 distal to the opponens tunnel and 2,345.75 ± 2,101.56 before its terminal branching) than the first lumbrical (1,410.56 ± 823.89); section areas occupied by axons were higher in proximal (0.20 ± 0.16) and distal (0.26 ± 0.20) ulnar samples than the first lumbrical (0.17 ± 0.16). Donor/recipient ratio first lumbrical/deep motor branch of the ulnar nerve were 1:1.86 (distal to the opponens tunnel) and 1:1.67 (at its terminal branching); data about the second lumbrical were ruled out because of bias., Conclusions: A transfer from the first lumbrical nerve to the deep motor branch of the ulnar nerve in palm is suitable to avoid intrinsic atrophy., (© 2018 Wiley Periodicals, Inc.)

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.)

The recovery of walking function following spinal cord injury (SCI) is of major importance to patients and clinicians. In experimental SCI studies, a rat model is widely used to assess walking function, following thoracic spinal cord lesion. In an effort to provide a resource which investigators can refer to when seeking the most appropriate functional assay, the authors have compiled and categorized the behavioral assessments used to measure the deficits and recovery of the gait in thoracic SCI rats. These categories include kinematic and kinetic measurements. Within this categorization, we discuss the advantages and disadvantages of each type of measurement. The present review includes the type of outcome data that they produce, the technical difficulty and the time required to potentially train the animals to perform them, and the need for expensive or highly specialized equipment. The use of multiple kinematic and kinetic parameters is recommended to identify subtle deficits and processes involved in the compensatory mechanisms of walking function after experimental thoracic SCI in rats., (Copyright © 2019 Elsevier Ltd. 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.

Peripheral nerves are complex organs that spread throughout the entire human body. They are frequently affected by lesions not only as a result of trauma but also following radical tumor resection. In fact, despite the advancement in surgical techniques, such as nerve-sparing robot assisted radical prostatectomy, some degree of nerve injury may occur resulting in erectile dysfunction with significant impairment of the quality of life. The aim of this review was to provide an overview on the mechanisms of the regeneration of injured peripheral nerves and to describe the potential strategies to improve the regeneration process and the functional recovery. Yet, the recent advances in bio-engineering strategies to promote nerve regeneration in the urological field are outlined with a view on the possible future regenerative therapies which might ameliorate the functional outcome after radical prostatectomy.

Peripheral nerve regeneration after severe traumatic nerve injury is a relevant clinical problem. Several different strategies have been investigated to solve the problem of bridging the nerve gap. Among these, the use of decellularized nerve grafts has been proposed as an alternative to auto/isografts, which represent the current gold standard in the treatment of severe nerve injury. This study reports the results of a systematic review of the literature published between January 2007 and October 2017. The aim was to quantitatively analyze the effectiveness of decellularized nerve grafts in rat experimental models. The review included 33 studies in which eight different decellularization protocols were described. The decellularized nerve grafts were reported to be immunologically safe and able to support both functional and morphological regeneration after nerve injury. Chemical protocols were found to be superior to physical protocols. However, further research is needed to optimize preparation protocols, including recellularization, improve their effectiveness, and substitute the current gold standard, especially in the repair of long nerve defects.

Functional recovery following general nerve reconstruction is often associated with poor results. Comparing to rat and mice experimental studies, there are much fewer investigations on nerve regeneration and repair in the sheep, and there are no studies on this subject using gait analysis in the sheep model as an assessment tool. Additionally, this is the first study evaluating obstacle negotiation and the compensatory strategies that take place at each joint in response to the obstacle during locomotion in the sheep model. This study aims to get kinematic data to serve as a template for an objective assessment of the ankle joint motion in future studies of common peroneal nerve (CP) injury and repair in the ovine model. Our results show that a moderately high obstacle set to 10% of the sheep's hindlimb length was associated to several spatial and temporal strategies in order to increase hoof height during obstacle negotiating. Sheep efficiently cleared an obstacle by increasing knee, ankle and metatarsophalangeal flexion during swing, whereas the hip joint is not affected. This study establishes the bounds of normal motion in the neurologically intact hindlimb when approached and cleared an obstacle and provides baseline data for further studies of peripheral nerve research in the ovine model.

Injuries to the peripheral nerves result in loss of motor, sensory and autonomic functions in the denervated segments of the body, thus having strong impact in the quality of life of affected patients. Neurons are able to regenerate their injured axons in the peripheral nerves; however, the endogenous repair mechanisms usually do not allow for a satisfactory functional recovery, especially after severe nerve injuries. The interest on regeneration after peripheral nerve injuries has increased in the recent years due to the numerous advances derived from studies of neurobiology, cell therapy, and tissue engineering. This Thematic Papers Issue brings together a number of papers, authored by researchers in the field, which cover a wide spectrum of topics related to regeneration and repair of peripheral nerve injuries. The Issue proposal originated from the recent 4th International Symposium on Peripheral Nerve Regeneration (ISPNR2017) which was hosted by Xavier Navarro and the European Society for the Study of Peripheral Nerve Repair and Regeneration in Barcelona, Spain. Anat Rec, 301:1614-1617, 2018. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Nerve guidance channels facilitate nerve regeneration and represent an attractive alternative to nerve graft. Actually, nano- and microstructured biomaterials for nerve reconstruction have gained much attention, thanks to recent discoveries about topography effects on cell behavior and morphology. Electrospun fibres have been proposed as filler or structural component for nerve guidance channels, principally due to their similarity with extracellular matrices which facilitate nerve regeneration. Among several tested biomaterials, gelatin has been used to prepare fibres able to support Schwann cell migration and neurite outgrowth. In this work, the effects of gelatin fibre size on axon elongation and Schwann cell migration have been tested using dorsal root ganglia cultures. Moreover, we analyzed how fibres might affect the expression of specific neuronal subtype markers in sensory neuron cultures and how the combined effect of substrate and biological cues affects neurite growth and gene expression. Data show that fibre topography differentially affects both neurite outgrowth and gene expression and suggest that fibre size and topography associated to specific growth factor exposure might be used to select neuron subpopulations and favor the axonal growth of specific neurons. Anat Rec, 301:1668-1677, 2018. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Vascular endothelial growth factor (VEGF) represents one of the main factors involved not only in angiogenesis and vasculogenesis but also in neuritogenesis. VEGF plays its function acting via different receptors: VEGF receptor1 (VEGFR-1), VEGF receptor2 (VEGFR-2), VEGF receptor3 (VEGFR-3), and co-receptors Neuropilin-1 (NRP1) and Neuropilin-2 (NRP2). This study reports on the first in vivo analysis of the expression of VEGF and VEGF family molecules in peripheral nerve degeneration and regeneration: for this purpose, different models of nerve lesion in rat were adopted, the median nerve crush injury and the median nerve transaction followed or not by end-to end microsurgical repair. Results obtained by real time polymerase chain reaction showed that VEGF and VEGF family molecules are differentially expressed under regenerating and degenerating condition, furthermore, in order to study the modulation and involvement of these factors in two different regenerative models, crush injury and end-to-end repair, protein expression analysis was evaluated. In addition, immunohistochemical analysis allowed to state a glial localization of VEGF and VEGFR-2 after peripheral nerve crush injury. Finally in vitro assay on primary Schwann cells culture show that VEGF165 stimulation increases Schwann cells migration, a major process in the promotion of neurite outgrowth. Anat Rec, 301:1646-1656, 2018. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

The amnion muscle combined graft (AMCG) conduits showed good clinical results in peripheral nerves gap repair. It combines the human amniotic membrane with autologous skeletal muscle fibres. These results seem attributable to the biological characteristics of human amniotic membrane: Pluripotency, anti-inflammatory and low immunogenicity.We here evaluate the final outcome of nerve regeneration morphologically and functionally, across the AMCG compared to nerve autograft. Fourteen Wistar rats were divided into two groups: In Group A, including 6 rats, the left forelimb was treated performing a 1.5 cm length gap on median nerve that was then reconstructed with a reverse autograft. In Group B, including 8 rats, the gap was reconstructed with AMCG. Functional results were evaluated at 30, 60 and 90 days performing grasping tests. Morphological and stereological analyses were performed at T90 using high-resolution light microscopy and design-based stereology. The AMCG conduits revealed nerve fibres regeneration and functional recovery. Functional recovery was observed in both groups with AMCG conduits group showing lower values and a regeneration of median nerves with more myelinated fibres with the same axon size, but thinner myelin than the autograft group. Though the autograft remains the gold standard to restore wide nerve gaps, the AMCG conduit has proved to be effective in enabling nerve regeneration through a critical rat's nerve gap of 15 mm. These findings empirically support the great clinical results obtained using AMCG conduit to restore traumatic nerve's gap from 3 to 6 cm of mixed forearm nerves.

Platelets affect myocardial damage from ischemia/reperfusion. Redox-dependent sphingosine-1-phosphate production and release are altered in diabetic platelets. Sphingosine-1-phosphate is a double-edged sword for ischemia/reperfusion injury. Therefore, we aimed to verify whether: (1) human healthy- or diabetic-platelets are cardioprotective, (2) sphingosine-1-phosphate receptors and downstream kinases play a role in platelet-induced cardioprotection, and (3) a correlation between platelet redox status and myocardial ischemia/reperfusion injury exists. Isolated rat hearts were subjected to 30-min ischemia and 1-h reperfusion. Infarct size was studied in hearts pretreated with healthy- or diabetic-platelets. Healthy-platelets were co-infused with sphingosine-1-phosphate receptor blocker, ERK-1/2 inhibitor, PI3K antagonist or PKC inhibitor to ascertain the cardioprotective mechanisms. In platelets we assessed (i) aggregation response to ADP, collagen, and arachidonic-acid, (ii) cyclooxygenase-1 levels, and (iii) AKT and ERK-phosphorylation. Platelet sphingosine-1-phosphate production and platelet levels of reactive oxygen species (ROS) were quantified and correlated to infarct size. Infarct size was reduced by about 22% in healthy-platelets pretreated hearts only. This cardioprotective effect was abrogated by either sphingosine-1-phosphate receptors or ERK/PI3K/PKC pathway blockade. Cyclooxygenase-1 levels and aggregation indices were higher in diabetic-platelets than healthy-platelets. Diabetic-platelets released less sphingosine-1-phosphate than healthy-platelets when mechanical or chemically stimulated in vitro . Yet, ROS levels were higher in diabetic-platelets and correlated with infarct size. Cardioprotective effects of healthy-platelet depend on the platelet's capacity to activate cardiac sphingosine-1-phosphate receptors and ERK/PI3K/PKC pathways. However, diabetic-platelets release less S1P and lose cardioprotective effects. Platelet ROS levels correlate with infarct size. Whether these redox alterations are responsible for sphingosine-1-phosphate dysfunction in diabetic-platelets remains to be ascertained.

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.