The Role of Dietary Nutrients in Peripheral Nerve Regeneration.

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

Histological, Biomechanical, and Biological Properties of Genipin-Crosslinked Decellularized Peripheral Nerves.

Acellular nerve allografts (ANGs) represent a promising alternative in nerve repair. Our aim is to improve the structural and biomechanical properties of biocompatible Sondell (SD) and Roosens (RS) based ANGs using genipin (GP) as a crosslinker agent ex vivo. The impact of two concentrations of GP (0.10% and 0.25%) on Wistar rat sciatic nerve-derived ANGs was assessed at the histological, biomechanical, and biocompatibility levels. Histology confirmed the differences between SD and RS procedures, but not remarkable changes were induced by GP, which helped to preserve the nerve histological pattern. Tensile test revealed that GP enhanced the biomechanical properties of SD and RS ANGs, being the crosslinked RS ANGs more comparable to the native nerves used as control. The evaluation of the ANGs biocompatibility conducted with adipose-derived mesenchymal stem cells cultured within the ANGs confirmed a high degree of biocompatibility in all ANGs, especially in RS and RS-GP 0.10% ANGs. Finally, this study demonstrates that the use of GP could be an efficient alternative to improve the biomechanical properties of ANGs with a slight impact on the biocompatibility and histological pattern. For these reasons, we hypothesize that our novel crosslinked ANGs could be a suitable alternative for future in vivo preclinical studies.

Comparison of Decellularization Protocols to Generate Peripheral Nerve Grafts: A Study on Rat Sciatic Nerves.

In critical nerve gap repair, decellularized nerve allografts are considered a promising tissue engineering strategy that can provide superior regeneration results compared to nerve conduits. Decellularized nerves offer a well-conserved extracellular matrix component that has proven to play an important role in supporting axonal guiding and peripheral nerve regeneration. Up to now, the known decellularized techniques are time and effort consuming. The present study, performed on rat sciatic nerves, aims at investigating a novel nerve decellularization protocol able to combine an effective decellularization in short time with a good preservation of the extracellular matrix component. To do this, a decellularization protocol proven to be efficient for tendons (DN-P1) was compared with a decellularization protocol specifically developed for nerves (DN-P2). The outcomes of both the decellularization protocols were assessed by a series of in vitro evaluations, including qualitative and quantitative histological and immunohistochemical analyses, DNA quantification, SEM and TEM ultrastructural analyses, mechanical testing, and viability assay. The overall results showed that DN-P1 could provide promising results if tested in vivo, as the in vitro characterization demonstrated that DN-P1 conserved a better ultrastructure and ECM components compared to DN-P2. Most importantly, DN-P1 was shown to be highly biocompatible, supporting a greater number of viable metabolically active cells.

Neuregulin1 alpha activates migration of neuronal progenitors expressing ErbB4.

Deficits in neuronal migration during development in the central nervous system may contribute to psychiatric diseases. The ligand neuregulin1 (NRG1) and its receptor ErbB4 are genes conferring susceptibility to schizophrenia, playing a key role in the control of neuronal migration both during development and adulthood. Several NRG1 and ErbB4 isoforms were identified, which deeply differ in their characteristics. Here we focused on the four ErbB4 isoforms and the two NRG1 isoforms differing in their EGF-like domain, namely α and ß. We hypothesized that these isoforms, which are differently regulated in schizophrenic patients, could play different roles in neuronal migration. Our hypothesis was strengthened by the observation that both NRG1α and NRG1ß and the four ErbB4 isoforms are expressed in the medial and lateral ganglionic eminences and in the cortex during development in rat. We analysed in vitro the signal transduction pathways activated by the different ErbB4 isoforms following the treatment with soluble recombinant NRG1α or NRG1ß and the ability to stimulate migration. Our data show that two ErbB4 isoforms, namely JMa-cyt2 and JMb-cyt1, following NRG1α and NRG1ß treatment, strongly activate AKT phosphorylation, conferring high migratory activity to neuronal progenitors, thus demonstrating that both NRG1α and NRG1ß can play a role in neuronal migration.

Bone-nerve crosstalk: a new state for neuralizing bone tissue engineering-A mini review.

Neuro bone tissue engineering is a multidisciplinary field that combines both principles of neurobiology and bone tissue engineering to develop innovative strategies for repairing and regenerating injured bone tissues. Despite the fact that regeneration and development are considered two distinct biological processes, yet regeneration can be considered the reactivation of development in later life stages to restore missing tissues. It is noteworthy that the regeneration capabilities are distinct and vary from one organism to another (teleost fishes, hydra, humans), or even in the same organism can vary dependent on the injured tissue itself (Human central nervous system vs. peripheral nervous system). The skeletal tissue is highly innervated, peripheral nervous system plays a role in conveying the signals and connecting the central nervous system with the peripheral organs, moreover it has been shown that they play an important role in tissue regeneration. Their regeneration role is conveyed by the different cells' resident in it and in its endoneurium (fibroblasts, microphages, vasculature associated cells, and Schwann cells) these cells secrete various growth factors (NGF, BDNF, GDNF, NT-3, and bFGF) that contribute to the regenerative phenotype. The peripheral nervous system and central nervous system synchronize together in regulating bone homeostasis and regeneration through neurogenic factors and neural circuits. Receptors of important central nervous system peptides such as Serotonin, Leptin, Semaphorins, and BDNF are expressed in bone tissue playing a role in bone homeostasis, metabolism and regeneration. This review will highlight the crosstalk between peripheral nerves and bone in the developmental stages as well as in regeneration and different neuro-bone tissue engineering strategies for repairing severe bone injuries.

Chitosan conduits enriched with fibrin-collagen hydrogel with or without adipose-derived mesenchymal stem cells for the repair of 15-mm-long sciatic nerve defect.

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.

Comprehensive ex vivo and in vivo preclinical evaluation of novel chemo enzymatic decellularized peripheral nerve allografts.

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.

The neurodynamic treatment induces biological changes in sensory and motor neurons in vitro.

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.

Fibroblasts Colonizing Nerve Conduits Express High Levels of Soluble Neuregulin1, a Factor Promoting Schwann Cell Dedifferentiation.

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 NRG1ß. 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.

Peripheral Nerve Regeneration Is Independent From Schwann Cell p75NTR Expression.

Schwann cell reprogramming and differentiation are crucial prerequisites for neuronal regeneration and re-myelination to occur following injury to peripheral nerves. The neurotrophin receptor p75NTR has been identified as a positive modulator for Schwann cell myelination during development and implicated in promoting nerve regeneration after injury. However, most studies base this conclusion on results obtained from complete p75NTR knockout mouse models and cannot dissect the specific role of p75NTR expressed by Schwann cells. In this present study, a conditional knockout model selectively deleting p75NTR expression in Schwann cells was generated, where p75NTR expression is replaced with that of an mCherry reporter. Silencing of Schwann cell p75NTR expression was confirmed in the sciatic nerve in vivo and in vitro, without altering axonal expression of p75NTR. No difference in sciatic nerve myelination during development or following sciatic nerve crush injury was observed, as determined by quantification of both myelinated and unmyelinated nerve fiber densities, myelinated axonal diameter and myelin thickness. However, the absence of Schwann cell p75NTR reduced motor nerve conduction velocity after crush injury. Our data indicate that the absence of Schwann cell p75NTR expression in vivo is not critical for axonal regrowth or remyelination following sciatic nerve crush injury, but does play a key role in functional recovery. Overall, this represents the first step in redefining the role of p75NTR in the peripheral nervous system, suggesting that the Schwann cell-axon unit functions as a syncytium, with the previous published involvement of p75NTR in remyelination most likely depending on axonal/neuronal p75NTR and/or mutual glial-axonal interactions.

Soluble Neuregulin1 Down-Regulates Myelination Genes in Schwann Cells.

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.