Peripheral nerve injuries significantly impair quality of life due to limited regenerative capacity, which is affected by factors such as neuroma formation, injury severity, scarring, and comorbidities. The Muscle-in-Vein (MIV) repair technique, consisting of a vein filled with skeletal muscle fibers, has emerged as a promising alternative to nerve autografts. This approach supports regeneration by providing growth factors, guiding axonal growth, enhancing Schwann cell migration, and limiting scar and neuroma formation. However, its clinical use is currently restricted mainly to short gaps in sensory digital nerves, and the biological mechanisms underlying its effectiveness remain incompletely understood. In this study, we investigated the role of muscle fibers in the early phases of nerve regeneration, with a particular focus on vascularization. An 8 mm gap in rat median nerves was repaired using the MIV technique and analyzed at 3, 7, 14, and 21 days post-injury. Immunofluorescence analysis demonstrated complete macrophage infiltration and well-organized vascularization throughout the entire graft as early as 7 days post-injury. Consistently, RNA sequencing at early time points revealed significant enrichment of pathways associated with vascular development and identified key angiogenesis-related genes. Notably, our findings indicate partial anastomosis between vessels originating from the nerve stumps and those within the muscle component of the graft. These results suggest that the success of muscle-in-vein nerve repair strategy may be due to an early vascularization process mediated by the synergistic contribution of both muscle and vein.

Peripheral nerve injury is a significant clinical challenge due to its limited regenerative capacity. While immediate repair is generally preferred, delayed repair is often necessary in clinical scenarios where primary intervention is not feasible. Understanding the molecular and genetic mechanisms underlying both conditions is essential for optimizing functional recovery. Thus, this study aimed to investigate key pathways involved in nerve regeneration by conducting the first transcriptomic analysis of a regenerating nerve within a conduit, while also comparing immediate and delayed nerve repair over time in a rat model. Immediately after injury or following a delay of 3 months, microsurgical intervention with a chitosan tube was performed to repair an 8-mm median nerve gap, and regenerated nerves inside the conduit were collected at 14 and 21 days for morphometric analysis and at 7, 14, and 21 days post-repair for RNA sequencing. Morphometric analysis based on absolute values showed a significant reduction in Schwann cell and axonal areas at 14 days, along with a decreased number of blood vessels and an overall smaller section area at 21 days in the delayed repair group. However, when normalized to the total section area to assess the relative proportions occupied by Schwann cells, axons, and vessels, no significant differences were observed between the immediate repair and delayed repair groups at 21 days. To correlate morphometric data with transcriptomic profiles, RNA sequencing was conducted. When comparing regenerating nerves at different time points with a healthy nerve, 25,596 genes were differentially expressed in the immediate group and 25,868 genes in the delayed repair group. These genes were mainly involved in pathways related to inflammatory response, phagocytosis, cell signaling, and response to lipoprotein particles. However, only 137 genes were differentially expressed when comparing the delayed repair group versus immediate repair groups. Gene ontology analysis revealed that the most enriched pathways were related to angiogenesis, particularly at 7 days, which aligned with the higher vessel density observed at 14 days in the delayed group. Overall, the comparison between the experimental groups indicated that immediate repair initiated a more rapid regenerative response, while delayed repair followed a slower, yet ultimately convergent trajectory, highlighting that regeneration is postponed and partially impaired. Notably, the nerve caliber was reduced in the delayed repair group compared to the immediate repair group. These findings emphasize the importance of early intervention and address a critical gap in the field by providing the first transcriptomic comparison of delayed versus immediate repair within conduits, thereby contributing to the development of novel molecular therapies to enhance recovery.

The extracellular matrix (ECM) is a non-cellular and gelatinous component of tissues, rich in proteins and proteoglycans, that provides information about the environment, forms a reservoir of trophic factors and regulates cell behavior by binding and activating cell surface receptors. This important network acts as a scaffold for tissues and organs throughout the body, playing an essential role in their structural and functional integrity. It is essential for cells to connect and communicate with each other and play an active role in intracellular signaling. Due to these properties, in recent decades the potential of the extracellular matrix in tissue engineering has begun to be explored with the aim of developing innovative biomaterials to be used in regenerative medicine. This review will first outline the components of the extracellular matrix in the peripheral nerve, followed by an exploration of its role in the regeneration process after injury, with a focus on the mechanisms underlying its interactions with nerve cells. Qualitative and quantitative methods used for extracellular matrix analysis will be described, and finally an overview will be given of recent advances in nerve repair strategies that exploit the potential of the extracellular matrix to enhance regeneration, highlighting the critical issues of extracellular matrix molecule use and proposing new directions for future research.

Before clinical approval of chitosan nerve conduits (CNCs) less than 10 years ago, substantial material degradation was not reported from pre-clinical research. The current study investigated the biodegradation of two different CNC variants in the median nerve model. In adult rats, 10 mm long CNCs were secured covering end-to-end repair sites. After 2, 6, 12, and 18 months, the implantation sites were inspected, and samples harvested. Histology was performed in order to analyze local immune response or foreign body tissue reaction around the devices or within nerve samples. Further, the number of myelinated nerve fibers and the condition of the chitosan material was evaluated. Data confirmed that different CNC variants did not induce tissue reaction or negatively impact the condition of the regenerated nerve. At late time points, some specific variants were demonstrated to have resulted in encapsulated material debris in the vicinity of the original implantation side. The reported degradation behavior resembles recent clinical reports and indicates that implantation sites for CNCs need to be chosen carefully. Nerve regeneration itself is undisturbed, but narrow implantation sites should be avoided for eliminating the risk of foreign body sensation with immunologically inert material degradation.

Introduction: Regulators of G Protein Signaling (RGS) form a gene family that modulates G protein-coupled receptor signaling by enhancing the GTPase activity of the Gα-GTP complex, effectively inhibiting G protein-dependent signal transduction cascades. While RGSs are expressed across many organs, including the central nervous system, few data are available for the peripheral nervous system (PNS).Methods and Results: To investigate potential links between RGS and PNS, open-access single-cell RNA sequencing datasets were analyzed, focusing on mice intact sciatic nerves and distal stumps at 3 and 9 days post-transection. Rgs16 emerged as the RGS member most highly expressed by Schwann cells after injury, suggesting its involvement in nerve degeneration. To further explore Rgs16 behavior in nerve injury, its expression was assessed at mRNA level at different time points in the median nerve of adult rats under regenerating conditions following mild (crush) or more severe (end-to-end repair) traumatic injury, and under degenerating conditions. Results revealed that Rgs16 expression increased 3 days after injury, declining under regenerating conditions, but remaining high in degenerating conditions. To examine the role of Rgs16 in chronic nerve degeneration, its expression was evaluated in a pathological model of Charcot-Marie-Tooth disease type-1A (CMT1A), a chronic demyelinating peripheral neuropathy. Analysis of publicly available RNA sequencing data from sciatic nerves of wild-type and CMT1A rats during development showed a significant upregulation of Rgs16 in transgenic rats at P18. Interestingly, this upregulation mirrored the expression pattern of Neuregulin1 (Nrg1), a gene critical for Schwann cell dedifferentiation and demyelination, strongly upregulated in traumatic and chronic nerve injuries. To explore a potential NRG1-RGS16 link, primary Schwann cell cultures were treated with recombinant NRG1β1, which induced an increase in Rgs16 expression.Discussion: These findings suggest a potential feedback mechanism where transient Rgs16 upregulation in response to injury and/or NRG1 might negatively regulate NRG1 release through RGS16-mediated inhibition of GPCR/ErbB transactivation. This study highlights the dynamic role of Rgs16 in traumatic and chronic nerve injuries, implicating its involvement in processes of nerve degeneration, regeneration, and possibly neuropathic pain. Further investigations are needed to clarify RGS16 function, which could pave the way for novel therapeutic strategies to enhance nerve regeneration and alleviate neuropathic pain.

MOTIVATION:Computational models are crucial for addressing critical questions about systems evolution and deciphering system connections. The pivotal feature of making this concept recognizable from the biological and clinical community is the possibility of quickly inspecting the whole system, bearing in mind the different granularity levels of its components. This holistic view of system behaviour expands the evolution study by identifying the heterogeneous behaviours applicable, e.g. to the cancer evolution study. RESULTS:To address this aspect, we propose a new modelling paradigm, UnifiedGreatMod, which allows modellers to integrate fine-grained and coarse-grained biological information into a unique model. It enables functional studies by combining the analysis of the system's multi-level stable states with its fluctuating conditions. This approach helps to investigate the functional relationships and dependencies among biological entities. This is achieved, thanks to the hybridization of two analysis approaches that capture a system's different granularity levels. The proposed paradigm was then implemented into the open-source, general modelling framework GreatMod, in which a graphical meta-formalism is exploited to simplify the model creation phase and R languages to define user-defined analysis workflows. The proposal's effectiveness was demonstrated by mechanistically simulating the metabolic output of Escherichia coli under environmental nutrient perturbations and integrating a gene expression dataset. Additionally, the UnifiedGreatMod was used to examine the responses of luminal epithelial cells to Clostridium difficile infection. AVAILABILITY AND IMPLEMENTATION:GreatMod https://qbioturin.github.io/epimod/, epimod_FBAfunctions https://github.com/qBioTurin/epimod_FBAfunctions, first case study E. coli  https://github.com/qBioTurin/Ec_coli_modelling, second case study C. difficile  https://github.com/qBioTurin/EpiCell_CDifficile.

>The gut microbiota: The human body is colonized by a diverse and complex microbial community – including bacteria, viruses, archaea, and unicellular eukaryotes – that plays a central role in human wellbeing. Indeed, microbiota is crucial for several functions, including host metabolism, physiology, maintenance of the intestinal epithelial integrity, nutrition, and immune function, earning it the designation of a “vital organ”(Guinane and Cotter, 2013).

IntroductionRecent evidence supports the hypothesis of an association between gut microbiota and the pathogenesis of retinal and eye diseases, suggesting the existence of a gut-eye axis. However, no data are available on the possible effect of the gut microbiota on the optic nerve fiber maturation and myelin development.MethodsWe investigated the impact of gut microbiota on the optic nerves collected from neonatal and young adult germ-free (GF), gnotobiotic (stably colonized with 12 bacteria strains, OMM12) and control (colonized with a complex gut microbiota, CGM) mice, by performing stereological and morphoquantitative analyses with transmission electron microscopy and gene expression analysis by quantitative real-time PCR.ResultsYoung adult GF and OMM12 optic nerve axons are smaller and hypermyelinated compared to CGM ones, while no such differences were detected in neonatal optic nerves. The transcription factors Olig1, Olig2, and Sox10 (oligodendrocyte myelination positive regulators) are downregulated in CGM and OMM12 young adult mice compared to the respective neonates. Such developmental downregulation was not observed in GF optic nerves, suggesting that the absence of the gut microbiota prolongs the stimulation of optic nerve fiber myelination, possibly through mechanisms that are yet to be identified.DiscussionAltogether, these data underscore the gut microbiota pivotal role in driving optic nerve myelination, contributing to our knowledge about both the gut-eye axis and the gut-brain axis, and opening new horizons for further investigations that will explore the role of the microbiota also in pathologies, injuries and regeneration associated with the optic nerve.

IntroductionPeripheral 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.MethodsThis 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 DiscussionThe 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.

The core component of the class III phosphatidylinositol 3-kinase complex, Beclin 1, takes part in different protein networks, thus switching its role from inducing autophagy to regulating autophagosomal maturation and endosomal trafficking. While assessed in neurons, astrocytes, and microglia, its role is far less investigated in myelinating glia, including Schwann cells (SCs), responsible for peripheral nerve myelination. Remarkably, the dysregulation in endosomal trafficking is emerging as a pathophysiological mechanism underlying peripheral neuropathies, such as demyelinating Charcot-Marie-Tooth (CMT) diseases. By knocking out Beclin 1 in SCs here a novel mouse model (Becn1 cKO) is generated, developing a severe and progressive neuropathy, accompanied by involuntary tremors, body weight loss, and premature death. Ultrastructural analysis revealed abated myelination and SCs displaying enlarged cytoplasm with progressive accumulation of intracellular vesicles. Transcriptomic and histological analysis from sciatic nerves of 10-day and 2-month-old mice revealed pro-mitotic gene deregulation and increased SCs proliferation at both stages with axonal loss and increased immune infiltration in adults, well reflecting the progressive motor and sensory functional impairment that characterizes Becn1 cKO mice, compared to controls. The study establishes a further step in understanding key mechanisms in SC development and points to Beclin 1 and its regulated pathways as targets for demyelinating CMT forms.

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

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.

Genital herpes, most frequently caused by herpes simplex virus 2 (HSV-2) infection, is one of the most prevalent sexually transmitted infections. The current rationale for the treatment of HSV-2 infection involves nucleoside analogs (e.g. acyclovir) to suppress reactivation. Enzymatic oxysterols are endogenous 27-carbon atoms mole-cules produced by enzymatic cholesterol oxidation, and recently emerged as a broad-spectrum host targeting antivirals. In this study, we screened selected members of an in-house synthesized library of oxysterol analogs for their activity against HSV-2, identifying three compounds, named PFM064, PFM067, and PFM069, endowed with 50% effective concentrations (EC50) in the micromolar range, without exerting any apparent cytotoxicity. Moreover, the results obtained showed the ability of the novel derivatives to inhibit both cell-to-cell fusion induced by HSV-2, and the production of an intracellular viral progeny. Further experiments performed with PFM067 (which was selected for more-in-depth studies as the most effective synthetic analog) showed that these molecules act in a late stage of HSV-2 replicative cycle, by sequestering viral glycoproteins in the Golgi compartment, and likely inhibiting the nuclear egress of neo-synthetized viral capsids.Taken together, these results point to PFM067 as a promising chemical scaffold for the development of novel herpetic antivirals.

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 The core component of the class III phosphatidylinositol 3-kinase complex, Beclin 1, takes part in different protein networks, thus switching its role from inducing autophagy to regulating autophagosomal maturation and endosomal trafficking. While assessed in neurons, astrocytes and microglia, its role was far less investigated in myelinating glia, including Schwann cells (SCs), responsible for peripheral nerve myelination. Remarkably, the dysregulation in endosomal trafficking is emerging as a pathophysiological mechanism underlying peripheral neuropathies, such as demyelinating Charcot-Marie-Tooth diseases. By knocking out Beclin 1 in SCs we here generated a novel mouse model (Becn1 cKO), developing a severe and progressive neuropathy, accompanied by involuntary tremors, body weight loss and premature death. Ultrastructural analysis revealed abated myelination and SCs displaying enlarged cytoplasm with progressive accumulation of intracellular vesicles. Transcriptomic and histological analysis from sciatic nerves of 10-day and 2-month-old mice revealed pro-mitotic gene deregulation and increased SCs proliferation at both stages with axonal loss and increased immune infiltration in adults, well reflecting the progressive motor and sensory functional impairment that characterizes Becn1 cKO mice, compared to controls. Our study establishes a further step in understanding key mechanisms in SC development and points to Beclin 1 and its regulated pathways as targets for demyelinating CMT forms.

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.