Axonal GABAA stabilizes excitability in unmyelinated sensory axons secondary to NKCC1 activity.

KEY POINTS: GABA depolarized sural nerve axons and increased the electrical excitability of C-fibres via GABAA receptor. Axonal excitability responses to GABA increased monotonically with the rate of action potential firing. Action potential activity in unmyelinated C-fibres is coupled to Na-K-Cl cotransporter type 1 (NKCC1) loading of axonal chloride. Activation of axonal GABAA receptor stabilized C-fibre excitability during prolonged low frequency (2.5 Hz) firing. NKCC1 maintains intra-axonal chloride to provide feed-forward stabilization of C-fibre excitability and thus support sustained firing. ABSTRACT: GABAA receptor (GABAA R)-mediated depolarization of dorsal root ganglia (DRG) axonal projections in the spinal dorsal horn is implicated in pre-synaptic inhibition. Inhibition, in this case, is predicated on an elevated intra-axonal chloride concentration and a depolarizing GABA response. In the present study, we report that the peripheral axons of DRG neurons are also depolarized by GABA and this results in an increase in the electrical excitability of unmyelinated C-fibre axons. GABAA R agonists increased axonal excitability, whereas GABA excitability responses were blocked by GABAA R antagonists and were absent in mice lacking the GABAA R ß3 subunit selectively in DRG neurons (AdvillinCre or snsCre ). Under control conditions, excitability responses to GABA became larger at higher rates of electrical stimulation (0.5-2.5 Hz). However, during Na-K-Cl cotransporter type 1 (NKCC1) blockade, the electrical stimulation rate did not affect GABA response size, suggesting that NKCC1 regulation of axonal chloride is coupled to action potential firing. To examine this, activity-dependent conduction velocity slowing (activity-dependent slowing; ADS) was used to quantify C-fibre excitability loss during a 2.5 Hz challenge. ADS was reduced by GABAA R agonists and exacerbated by either GABAA R antagonists, ß3 deletion or NKCC1 blockade. This illustrates that activation of GABAA R stabilizes C-fibre excitability during sustained firing. We posit that NKCC1 acts in a feed-forward manner to maintain an elevated intra-axonal chloride in C-fibres during ongoing firing. The resulting chloride gradient can be utilized by GABAA R to stabilize axonal excitability. The data imply that therapeutic strategies targeting axonal chloride regulation at peripheral loci of pain and itch may curtail aberrant firing in C-fibres.

Peripheral nerve regeneration following injury is altered in mice lacking P2X7 receptor.

Peripheral nerve injuries are debilitating, and current clinical management is limited to surgical intervention, which often leads to poor functional outcomes. Development of pharmacological interventions aimed at enhancing regeneration may improve this. One potential pharmacological target is the P2X purinergic receptor 7 (P2X7R) expressed in Schwann cells, which is known to play a role during the development of the peripheral nerves. Herein, we analysed differences in regeneration between genetically engineered P2X7 knockout mice and wild-type controls, using in vivo and ex vivo models of peripheral nerve regeneration. We have found that the speed of axonal regeneration is unaltered in P2X7 knockout mice, nevertheless regenerated P2X7 knockout nerves are morphologically different to wild-type nerves following transection and immediate repair. Indeed, the detailed morphometric analysis at 4 and 8 weeks after injury showed evidence of delayed remyelination in P2X7 knockout mice, compared to the wild-type controls. Furthermore, the Wallerian degeneration phase was unaltered between the two experimental groups. We also analysed gene expression changes in the dorsal root ganglia neurones as a result of the peripheral nerve injury, and found changes in pathways related to pain, inflammation and cell death. We conclude that P2X7 receptors in Schwann cells may be a putative pharmacological target to control cell fate following injury, thus enhancing nerve re-myelination.

Functional Characterization of Muscarinic Receptors in Human Schwann Cells.

Functional characterization of muscarinic cholinergic receptors in myelinating glial cells has been well described both in central and peripheral nervous system. Rat Schwann cells (SCs) express different muscarinic receptor subtypes with the prevalence of the M2 subtype. The selective stimulation of this receptor subtype inhibits SC proliferation, improving their differentiation towards myelinating phenotype. In this work, we describe for the first time that human SCs are cholinoceptive as they express several muscarinic receptor subtypes and, as for rat SCs, M2 receptor is one of the most abundant. Human SCs, isolated from adult nerves, were cultured in vitro and stimulated with M2 muscarinic agonist arecaidine propargyl ester (APE). Similarly to that observed in rat, M2 receptor activation causes a decreased cell proliferation and promotes SC differentiation as suggested by increased Egr2 expression with an improved spindle-like shape cell morphology. Conversely, the non-selective stimulation of muscarinic receptors appears to promote cell proliferation with a reduction of SC average cell diameter. The data obtained demonstrate that human SCs are cholinoceptive and that human cultured SCs may represent an interesting tool to understand their physiology and increase the knowledge on how the cholinergic stimulation may contribute to address human SC development in normal and pathological conditions.

Human Schwann-like cells derived from adipose-derived mesenchymal stem cells rapidly de-differentiate in the absence of stimulating medium.

Finding a viable cell-based therapy to address peripheral nerve injury holds promise for enhancing the currently suboptimal microsurgical approaches to peripheral nerve repair. Autologous nerve grafting is the current gold standard for surgical repair of nerve gaps; however, this causes donor nerve morbidity in the patient, and the results remain unsatisfactory. Transplanting autologous Schwann cells (SCs) results in similar morbidity, as well as limited cell numbers and restricted potential for expansion in vitro. Adipose-derived stem cells (ASCs), 'differentiated' towards an SC-like phenotype in vitro (dASCs), have been presented as an alternative to SC therapies. The differentiation protocol stimulates ASCs to mimic the SC phenotype; however, the efficacy of dASCs in nerve repair is not yet convincing, and the practicality of the SC-like phenotype is unproven. Here, we examined the stability of dASCs by withdrawing differentiation medium for 72 h after the full 18-day differentiation protocol, and measuring changes in morphology, gene expression, and protein levels. Withdrawal of differentiation medium from dASCs resulted in a rapid reversion to stem cell-like characteristics. Quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay analyses demonstrated a significant reduction in gene and protein expression of growth factors that were expressed at high levels following 'differentiation'. Therefore, we question the relevance of differentiation to an SC-like phenotype, as withdrawal of differentiation medium, a model of transplantation into an injured nerve, results in rapid reversion of the dASC phenotype to stem cell-like characteristics. Further investigation into the differentiation process and the response of dASCs to an injured environment must be undertaken prior to the use of dASCs in peripheral nerve repair therapies.

Deletion of GABA-B receptor in Schwann cells regulates remak bundles and small nociceptive C-fibers.

The mechanisms regulating the differentiation into non-myelinating Schwann cells is not completely understood. Recent evidence indicates that GABA-B receptors may regulate myelination and nociception in the peripheral nervous system. GABA-B receptor total knock-out mice exhibit morphological and molecular changes in peripheral myelin. The number of small myelinated fibers is higher and associated with altered pain sensitivity. Herein, we analyzed whether these changes may be produced by a specific deletion of GABA-B receptors in Schwann cells. The conditional mice (P0-GABA-B1(fl/fl)) show a morphological phenotype characterized by a peculiar increase in the number of small unmyelinated fibers and Remak bundles, including nociceptive C-fibers. The P0-GABA-B1(fl/fl) mice are hyperalgesic and allodynic. In these mice, the morphological and behavioral changes are associated with a downregulation of neuregulin 1 expression in nerves. Our findings suggest that the altered pain sensitivity derives from a Schwann cell-specific loss of GABA-B receptor functions, pointing to a role for GABA-B receptors in the regulation of Schwann cell maturation towards the non-myelinating phenotype.

Schwann-like adult stem cells derived from bone marrow and adipose tissue express γ-aminobutyric acid type B receptors.

γ-Aminobutyric acid type B receptors (GABA-B) are expressed in glial cells of the central and peripheral nervous systems, and recent evidence has shown their importance in modulating physiological parameters of Schwann cell (SC). SC play essential roles in peripheral nerve regeneration, but several drawbacks prevent their use for nerve repair. Adult stem cells from adipose tissue (ASC) or bone marrow (BM-MSC) can be differentiated into an SC-like phenotype and used as SC replacements. The aim of this study was to investigate GABA-B receptor functional expression in differentiated stem cells by assessing the similarity to SC. By means of RT-PCR and Western blot methodologies, BM-MSC and ASC were found to express both GABA-B1 and GABA-B2 receptor subunits. The expression levels of GABA-B1b and GABA-B2 receptors were influenced by SC-like differentiation, as shown by Western blot studies. GABA-B receptor stimulation with baclofen reduced the proliferation rate of SC and differentiated ASC (dASC) but not that of dBM-MSC. In conclusion, both of the subunits that assemble into a functional GABA-B receptor are present in differentiated stem cells. Furthermore, GABA-B receptors in dASC are functionally active, regulating a key process such as proliferation. The presence of functional GABA-B receptors on differentiated stem cells opens new opportunities for a possible pharmacological modulation of their physiology and phenotype.

Graphene Oxide Substrate Promotes Neurotrophic Factor Secretion and Survival of Human Schwann-Like Adipose Mesenchymal Stromal Cells.

Mesenchymal stromal cells from adipose tissue (AD-MSCs) exhibit favorable clinical traits for autologous transplantation and can develop 'Schwann-like' phenotypes (sAD-MSCs) to improve peripheral nerve regeneration, where severe injuries yield insufficient recovery. However, sAD-MSCs regress without biochemical stimulation and detach from conduits under unfavorable transplant conditions, negating their paracrine effects. Graphene-derived materials support AD-MSC attachment, regulating cell adhesion and function through physiochemistry and topography. Graphene oxide (GO) is a suitable substrate for human sAD-MSCs incubation toward severe peripheral nerve injuries by evaluating transcriptome changes, neurotrophic factor expression over a 7-days period, and cell viability in apoptotic conditions is reported. Transcriptome changes from GO incubation across four patients are minor compared to biological variance. Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and glial-derived neurotrophic factor (GDNF) gene expression is unchanged from sAD-MSCs on GO substrates, but NGF and GDNF protein secretion increase at day 3 and 7. Secretome changes do not improve dorsal root ganglia neuron axon regeneration in conditioned media culture models. Fewer sAD-MSCs detach from GO substrates compared to glass following phosphate buffer saline exposure, which simulates apoptotic conditions. Overall, GO substrates are compatible with sAD-MSC primed for peripheral nerve regeneration strategies and protect the cell population in harsh environments.

The angiogenic potential of CD271+ human adipose tissue-derived mesenchymal stem cells.

BACKGROUND: Autologous fat grafting is often a crucial aspect of reconstructive and aesthetic surgeries, yet poor graft retention is a major issue with this technique. Enriching fat grafts with adipose tissue-derived mesenchymal stem cells (AD-MSCs) improves graft survival-however, AD-MSCs represent a heterogeneous population. Selection of subpopulations of AD-MSCs would allow the targeting of specific AD-MSCs that may benefit fat graft survival more than the general AD-MSC population. METHODS: Human AD-MSCs were selected for the surface marker CD271 using magnetic-activated cell sorting and compared to the CD271 negative phenotype.  These subpopulations were analysed for gene expression using Real-Time qPCR and RNA sequencing; surface marker characteristics using immunostaining; ability to form tubules when cultured with endothelial cells; and gene and protein expression of key angiogenic mediators when cultured with ex-vivo adipose tissue. RESULTS: Human AD-MSCs with the surface marker CD271 express angiogenic genes at higher levels, and inflammatory genes at lower levels, than the CD271- AD-MSC population. A greater proportion of CD271+ AD-MSCs also possess the typical complement of stem cell surface markers and are more likely to promote effective neoangiogenesis, compared to CD271- AD-MSCs. CONCLUSION: Enriching grafts with the CD271+ AD-MSC subpopulation holds potential for the improvement of reconstructive and aesthetic surgeries involving adipose tissue.

Transcriptomic Profile Reveals Deregulation of Hearing-Loss Related Genes in Vestibular Schwannoma Cells Following Electromagnetic Field Exposure.

Hearing loss (HL) is the most common sensory disorder in the world population. One common cause of HL is the presence of vestibular schwannoma (VS), a benign tumor of the VIII cranial nerve, arising from Schwann cell (SC) transformation. In the last decade, the increasing incidence of VS has been correlated to electromagnetic field (EMF) exposure, which might be considered a pathogenic cause of VS development and HL. Here, we explore the molecular mechanisms underlying the biologic changes of human SCs and/or their oncogenic transformation following EMF exposure. Through NGS technology and RNA-Seq transcriptomic analysis, we investigated the genomic profile and the differential display of HL-related genes after chronic EMF. We found that chronic EMF exposure modified the cell proliferation, in parallel with intracellular signaling and metabolic pathways changes, mostly related to translation and mitochondrial activities. Importantly, the expression of HL-related genes such as NEFL, TPRN, OTOGL, GJB2, and REST appeared to be deregulated in chronic EMF exposure. In conclusion, we suggest that, at a preclinical stage, EMF exposure might promote the transformation of VS cells and contribute to HL.

Cross-talk between motor neurons and myotubes via endogenously secreted neural and muscular growth factors.

Neuromuscular junction (NMJ) research is vital to advance the understanding of neuromuscular patho-physiology and development of novel therapies for diseases associated with NM dysfunction. In vivo, the micro-environment surrounding the NMJ has a significant impact on NMJ formation and maintenance via neurotrophic and differentiation factors that are secreted as a result of cross-talk between muscle fibers and motor neurons. Recently we showed the formation of functional NMJs in vitro in a co-culture of immortalized human myoblasts and motor neurons from rat-embryo spinal-cord explants, using a culture medium free from serum and neurotrophic or growth factors. The aim of this study was to assess how functional NMJs were established in this co-culture devoid of exogenous neural growth factors. To investigate this, an ELISA-based microarray was used to compare the composition of soluble endogenously secreted growth factors in this co-culture with an a-neural muscle culture. The levels of seven neurotrophic factors brain-derived neurotrophic factor (BDNF), glial-cell-line-derived neurotrophic factor (GDNF), insulin-like growth factor-binding protein-3 (IGFBP-3), insulin-like growth factor-1 (IGF-1), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and vascular endothelial growth factor (VEGF) were higher (p < 0.05) in the supernatant of NMJ culture compared to those in the supernatant of the a-neural muscle culture. This indicates that the cross-talk between muscle and motor neurons promotes the secretion of soluble growth factors contributing to the local microenvironment thereby providing a favourable regenerative niche for NMJs formation and maturation.

Hyaluronic Acid (HA) Receptors and the Motility of Schwann Cell(-Like) Phenotypes.

The cluster of differentiation 44 (CD44) and the hyaluronan-mediated motility receptor (RHAMM), also known as CD168, are perhaps the most studied receptors for hyaluronic acid (HA); among their various functions, both are known to play a role in the motility of a number of cell types. In peripheral nerve regeneration, the stimulation of glial cell motility has potential to lead to better therapeutic outcomes, thus this study aimed to ascertain the presence of these receptors in Schwann cells (rat adult aSCs and neonatal nSCs) and to confirm their influence on motility. We included also a Schwann-like phenotype (dAD-MSCs) derived from adipose-derived mesenchymal stem cells (uAD-MSCs), as a possible basis for an autologous cell therapy. CD44 was expressed similarly in all cell types. Interestingly, uAD-MSCs were RHAMM(low), whereas both Schwann cells and dASCs turned out to be similarly RHAMM(high), and indeed antibody blockage of RHAMM effectively immobilized (in vitro scratch wound assay) all the RHAMM(high) Schwann(-like) types, but not the RHAMM(low) uAD-MSCs. Blocking CD44, on the other hand, affected considerably more uAD-MSCs than the Schwann(-like) cells, while the combined blockage of the two receptors immobilized all cells. The results therefore indicate that Schwann-like cells have a specifically RHAMM-sensitive motility, where the motility of precursor cells such as uAD-MSCs is CD44- but not RHAMM-sensitive; our data also suggest that CD44 and RHAMM may be using complementary motility-controlling circuits.

Effects mediated by the α7 nicotinic acetylcholine receptor on cell proliferation and migration in rat adipose-derived stem cells.

Adipose-derived stem cells (ASCs) are an attractive source for regenerative medicine as they can be easily isolated, rapidly expandable in culture and show excellent in vitro differentiation potential. Acetylcholine (ACh), one of the main neurotransmitters in central and peripheral nervous systems, plays key roles in the control of several physiological processes also in non-neural tissues. As demonstrated in our previous studies, ACh can contribute to the rat ASCs physiology, negatively modulating ASCs proliferation and migration via M2 muscarinic receptor (mAChR) activation. In the present work we show that rat ASCs also express α7 nicotinic receptors (nAChRs). In particular, we have investigated the effects mediated by the selective activation of α7 nAChRs, which causes a reduction of ASC proliferation without affecting cell survival and morphology, and significantly promotes cell migration via upregulation of the CXCR4 expression. Interestingly, the activation of the α7 nAChR also upregulates the expression of M2 mAChR protein, indicating a cooperation between muscarinic and nicotinic receptors in the inhibition of ASC proliferation.

Development and Characterisation of an in vitro Model of Wallerian Degeneration.

Following peripheral nerve injury, a sequence of events termed Wallerian degeneration (WD) takes place at the distal stump in order to allow the regenerating axons to grow back toward the target organs. Schwann cells (SCs) play a lead role in this by initiating the inflammatory response attracting macrophages and immune cells, as well as producing neurotrophic signals that are essential for nerve regeneration. The majority of existing research has focused on tools to improve regeneration, overlooking the critical degeneration phase. This is also due to the lack of in vitro models recapitulating the features of in vivo WD. In particular, to understand the initial SC response following injury, and to investigate potential interventions, a model that isolates the nerve from other systemic influences is required. Stem cell intervention has been extensively studied as a potential therapeutic intervention to augment regeneration; however, data regarding their role in WD is lacking. Thus, in this study we describe an in vitro model using rat sciatic nerve explants degenerating up to 14 days. Characterisation of this model was performed by gene and protein expression for key markers of WD, in addition to immunohistochemical analysis and electron microscopy. We found changes in keeping with WD in vivo: upregulation of repair program protein CJUN, downregulation of myelin protein genes and subsequent disorganisation and breakdown of myelin structure. As a means of testing the effects of stem cell intervention on WD we established indirect co-cultures of human adipose-derived mesenchymal stem cells (AD-MSC) with the degenerating nerve explants. The stem cell intervention potentiated neurotrophic factors and Cjun expression. We conclude that our in vitro model shares the main features of in vivo WD, and we provide proof of principle on its effectiveness to study experimental approaches for nerve regeneration focused on the events happening during WD.

Schwann Cell Autocrine and Paracrine Regulatory Mechanisms, Mediated by Allopregnanolone and BDNF, Modulate PKCε in Peripheral Sensory Neurons.

Protein kinase type C-ε (PKCε) plays important roles in the sensitization of primary afferent nociceptors, such as ion channel phosphorylation, that in turn promotes mechanical hyperalgesia and pain chronification. In these neurons, PKCε is modulated through the local release of mediators by the surrounding Schwann cells (SCs). The progesterone metabolite allopregnanolone (ALLO) is endogenously synthesized by SCs, whereas it has proven to be a crucial mediator of neuron-glia interaction in peripheral nerve fibers. Biomolecular and pharmacological studies on rat primary SCs and dorsal root ganglia (DRG) neuronal cultures were aimed at investigating the hypothesis that ALLO modulates neuronal PKCε, playing a role in peripheral nociception. We found that SCs tonically release ALLO, which, in turn, autocrinally upregulated the synthesis of the growth factor brain-derived neurotrophic factor (BDNF). Subsequently, glial BDNF paracrinally activates PKCε via trkB in DRG sensory neurons. Herein, we report a novel mechanism of SCs-neuron cross-talk in the peripheral nervous system, highlighting a key role of ALLO and BDNF in nociceptor sensitization. These findings emphasize promising targets for inhibiting the development and chronification of neuropathic pain.

A Novel Bioengineered Functional Motor Unit Platform to Study Neuromuscular Interaction.

BACKGROUND: In many neurodegenerative and muscular disorders, and loss of innervation in sarcopenia, improper reinnervation of muscle and dysfunction of the motor unit (MU) are key pathogenic features. In vivo studies of MUs are constrained due to difficulties isolating and extracting functional MUs, so there is a need for a simplified and reproducible system of engineered in vitro MUs. OBJECTIVE: to develop and characterise a functional MU model in vitro, permitting the analysis of MU development and function. METHODS: an immortalised human myoblast cell line was co-cultured with rat embryo spinal cord explants in a serum-free/growth fact media. MUs developed and the morphology of their components (neuromuscular junction (NMJ), myotubes and motor neurons) were characterised using immunocytochemistry, phase contrast and confocal microscopy. The function of the MU was evaluated through live observations and videography of spontaneous myotube contractions after challenge with cholinergic antagonists and glutamatergic agonists. RESULTS: blocking acetylcholine receptors with α-bungarotoxin resulted in complete, cessation of myotube contractions, which was reversible with tubocurarine. Furthermore, myotube activity was significantly higher with the application of L-glutamic acid. All these observations indicate the formed MU are functional. CONCLUSION: a functional nerve-muscle co-culture model was established that has potential for drug screening and pathophysiological studies of neuromuscular interactions.

Self-Assembling Peptide Hydrogel Matrices Improve the Neurotrophic Potential of Human Adipose-Derived Stem Cells.

Despite advances in microsurgical techniques, treatment options to restore prior function following peripheral nerve injury remain unavailable, and autologous nerve grafting remains the therapy of choice. Recent experimental work has focused on the development of artificial constructs incorporating smart biomaterials and stem cells, aspiring to match/improve the outcomes of nerve autografting. Chemically stimulated human adipose-derived stem cells (dhASC) can improve nerve regeneration outcomes; however, these properties are lost when chemical stimulation is withdrawn, and survival rate upon transplantation is low. It is hypothesized that interactions with synthetic hydrogel matrices could maintain and improve neurotrophic characteristics of dhASC. dhASC are cultured on PeptiGel-Alpha 1 and PeptiGel-Alpha 2 self-assembling peptide hydrogels, showing comparable viability to collagen I control gels. Culturing dhASC on Alpha 1 and Alpha 2 substrates allow the maintenance of neurotrophic features, such as the expression of growth factors and neuroglial markers. Both Alpha 1 and Alpha 2 substrates are suitable for the culture of peripheral sensory neurons, permitting sprouting of neuronal extensions without the need of biological extracellular matrices, and preserving neuronal function. PeptiGel substrates loaded with hdASC are proposed as promising candidates for the development of tissue engineering therapies for the repair of peripheral nerve injuries.

Protocol for a phase I trial of a novel synthetic polymer nerve conduit 'Polynerve' in participants with sensory digital nerve injury (UMANC).

Background: Peripheral nerve injuries are common, with approximately 9,000 cases in the UK annually. Young working individuals are predominantly affected, leading to significant health and social implications. Functional recovery is often poor with impaired hand sensation, reduced motor function and pain and cold intolerance. Where a nerve gap exists, nerve grafting remains the gold-standard treatment but creates a second surgical site, sensory deficit at the donor site, possible neuroma formation and has limited availability. Current commercially available synthetic and resorbable nerve conduit alternatives are reported to be rigid and inflexible. This study will set out to examine the first-in-man use of a new nerve conduit device 'Polynerve' to repair small nerve gaps in digital sensory nerves of the hand. Polynerve is a degradable co-polymer of poly-ε-caprolactone and poly-l-lactic acid, which is shaped as a cylinder that has greater tensile strength, flexibility and less acidic degradation compared with current commercially available synthetic nerve conduits. In addition, it has a novel micro-grooved internal lumen that aids Schwann cell ingress and alignment to improve nerve regeneration. Methods: In total, 17 eligible participants will be recruited to undergo repair of a transected sensory nerve of the hand using the Polynerve device. All participants that receive the nerve conduit device will be followed for a period of 12 months post-surgery. The primary endpoint is safety of the device and the secondary endpoint is degree of sensory nerve regeneration through the conduit assessed using standard sensory testing (2-PD, WEST monofilament testing and locognosia). Discussion: The 'UMANC' trial is a single-centre UK-based, prospective, unblinded, phase I clinical trial of a novel nerve conduit device. We aim to demonstrate the safety of Polynerve as a synthetic, biodegradable nerve conduit and improve the treatment options available to patients with significant nerve injuries. Registration: Clinicaltrials.gov: NCT02970864; EudraCT: 2016-001667-37.

M2 receptors activation modulates cell growth, migration and differentiation of rat Schwann-like adipose-derived stem cells.

Schwann cells (SCs) play a central role in peripheral nervous system physiology and in the response to axon injury. The ability of SCs to proliferate, secrete growth factors, modulate immune response, migrate and re-myelinate regenerating axons has been largely documented. However, there are several restrictions hindering their clinical application, such as the difficulty in collection and a slow in vitro expansion. Adipose-derived stem cells (ASCs) present good properties for peripheral nerve regenerative medicine. When exposed to specific growth factors in vitro, they can acquire a SC-like phenotype (dASCs) expressing key SCs markers and assuming spindle-shaped morphology. Nevertheless, the differentiated phenotype is unstable and several strategies, including pharmacological stimulation, are being studied to improve differentiation outcomes. Cholinergic receptors are potential pharmacological targets expressed in glial cells. Our previous work demonstrated that muscarinic cholinergic receptors, in particular M2 subtype, are present in SCs and are able to modulate several physiological processes. In the present work, muscarinic receptors expression was characterised and the effects mediated by M2 muscarinic receptor were evaluated in rat dASCs. M2 receptor activation, by the preferred agonist arecaidine propargyl ester (APE), caused a reversible arrest of dASCs cell growth, supported by the downregulation of proteins involved in the maintenance of cell proliferation and upregulation of proteins involved in the differentiation (i.e., c-Jun and Egr-2), without affecting cell survival. Moreover, M2 receptor activation in dASCs enhances a pronounced spindle-shaped morphology, supported by Egr2 upregulation, and inhibits cell migration. Our data clearly demonstrate that rat dASCs express functional muscarinic receptors, in particular M2 subtype, which is able to modulate their physiological and morphological processes, as well as SCs differentiation. These novel findings could open new opportunities for the development of combined cell and pharmacological therapies for peripheral nerve regeneration, harnessing the potential of dASCs and M2 receptors.

Gene expression changes in dorsal root ganglia following peripheral nerve injury: roles in inflammation, cell death and nociception.

Subsequent to a peripheral nerve injury, there are changes in gene expression within the dorsal root ganglia in response to the damage. This review selects factors which are well-known to be vital for inflammation, cell death and nociception, and highlights how alterations in their gene expression within the dorsal root ganglia can affect functional recovery. The majority of studies used polymerase chain reaction within animal models to analyse the dynamic changes following peripheral nerve injuries. This review aims to highlight the factors at the gene expression level that impede functional recovery and are hence are potential targets for therapeutic approaches. Where possible the experimental model, specific time-points and cellular location of expression levels are reported.

Simplified in vitro engineering of neuromuscular junctions between rat embryonic motoneurons and immortalized human skeletal muscle cells.

BACKGROUND: Neuromuscular junctions (NMJs) consist of the presynaptic cholinergic motoneuron terminals and the corresponding postsynaptic motor endplates on skeletal muscle fibers. At the NMJ the action potential of the neuron leads, via release of acetylcholine, to muscle membrane depolarization that in turn is translated into muscle contraction and physical movement. Despite the fact that substantial NMJ research has been performed, the potential of in vivo NMJ investigations is inadequate and difficult to employ. A simple and reproducible in vitro NMJ model may provide a robust means to study the impact of neurotrophic factors, growth factors, and hormones on NMJ formation, structure, and function. METHODS: This report characterizes a novel in vitro NMJ model utilizing immortalized human skeletal muscle stem cells seeded on 35 mm glass-bottom dishes, cocultured and innervated with spinal cord explants from rat embryos at ED 13.5. The cocultures were fixed and stained on day 14 for analysis and assessment of NMJ formation and development. RESULTS: This unique serum- and trophic factor-free system permits the growth of cholinergic motoneurons, the formation of mature NMJs, and the development of highly differentiated contractile myotubes, which exhibit appropriate configuration of transversal triads, representative of in vivo conditions. CONCLUSION: This coculture system provides a tool to study vital features of NMJ formation, regulation, maintenance, and repair, as well as a model platform to explore neuromuscular diseases and disorders affecting NMJs.