Benedetta Elena Fornasari

The Effect of Electrospun Gelatin Fibers Alignment on Schwann Cell and Axon Behavior and Organization in the Perspective of Artificial Nerve Design

Electrospun fibrous substrates mimicking extracellular matrices can be prepared by electrospinning, yielding aligned fibrous matrices as internal fillers to manufacture artificial nerves. Gelatin aligned nano-fibers were prepared by electrospinning after tuning the collector rotation speed. The effect of alignment on cell adhesion and proliferation was tested in vitro using primary cultures, the Schwann cell line, RT4-D6P2T, and the sensory neuron-like cell line, 50B11. Cell adhesion and proliferation were assessed by quantifying at several time-points. Aligned nano-fibers reduced adhesion and proliferation rate compared with random fibers. Schwann cell morphology and organization were investigated by immunostaining of the cytoskeleton. Cells were elongated with their longitudinal body parallel to the aligned fibers. B5011 neuron-like cells were aligned and had parallel axon growth when cultured on the aligned gelatin fibers. The data show that the alignment of electrospun gelatin fibers can modulate Schwann cells and axon organization in vitro, suggesting that this substrate shows promise as an internal filler for the design of artificial nerves for peripheral nerve reconstruction.

The Neuregulin1/ErbB system is selectively regulated during peripheral nerve degeneration and regeneration

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

The influence of electrospun fibre size on Schwann cell behaviour and axonal outgrowth

Fibrous substrates functioning as temporary extracellular matrices can be prepared easily by electrospinning, yielding fibrous matrices suitable as internal fillers for nerve guidance channels. In this study, gelatin micro- or nano-fibres were prepared by electrospinning by tuning the gelatin concentration and solution flow rate. The effect of gelatin fibre diameter on cell adhesion and proliferation was tested in vitro using explant cultures of Schwann cells (SC) and dorsal root ganglia (DRG). Cell adhesion was assessed by quantifying the cell spreading area, actin cytoskeleton organization and focal adhesion complex formation. Nano-fibres promoted cell spreading and actin cytoskeleton organization, increasing cellular adhesion and the proliferation rate. However, both migration rate and motility, quantified by transwell and time lapse assays respectively, were greater in cells cultured on micro-fibres. Finally, there was more DRG axon outgrowth on micro-fibres. These data suggest that the topography of electrospun gelatin fibres can be adjusted to modulate SC and axon organization and that both nano- and micro-fibres are promising fillers for the design of devices for peripheral nerve repair.

Irreversible changes occurring in long-term denervated Schwann cells affect delayed nerve repair.

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

In vitro evaluation of gelatin and chitosan electrospun fibers as artificial guide in peripheral nerve repair: a comparative study.

Random and aligned gelatin (GL) and chitosan (CS) nano‐fibres have been prepared by electrospinning tuning the collector rotation speed. The effect of fibre alignment on cell adhesion and proliferation was assessed in vitro by using different Schwann cell (SC) and neuronal models. Moreover, actin cytoskeleton organization, lamellipodia and filipodia formation, and axon outgrowth were evaluated. GL and CS fibres induced similar adhesion and proliferation rates. GL and CS random fibres promoted higher adhesion and proliferation rates induction in comparison to the aligned ones, although GL and CS fibres alignment resulted in SC and axon‐oriented growth. Filipodia formation was higher on aligned fibres, suggesting that these substrates can promote higher cell migration in comparison to random ones. 50B11 (neuronal cell line) differentiation was higher on GL fibres, whereas no differences were observed in dorsal root ganglia explants model. These data suggest that both GL and CS fibres can be promising substrates to be used in peripheral nerve reconstruction. Copyright

Modulation of the Neuregulin 1/ErbB system after skeletal muscle denervation and reinnervation

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.

Soluble Neuregulin1 is strongly up-regulated in the rat model of Charcot-Marie-Tooth 1A disease:

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

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 β. 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 NRG1β 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 NRG1β and the ability to stimulate migration. Our data show that two ErbB4 isoforms, namely JMa-cyt2 and JMb-cyt1, following NRG1α and NRG1β treatment, strongly activate AKT phosphorylation, conferring high migratory activity to neuronal progenitors, thus demonstrating that both NRG1α and NRG1β can play a role in neuronal migration.

Combined Influence of Gelatin Fibre Topography and Growth Factors on Cultured Dorsal Root Ganglia Neurons: EFFECTS OF SUBSTRATE AND DRUGS ON DRG NEURONS

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.

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.