Gut microbiota depletion delays somatic peripheral nerve development and impairs neuromuscular junction maturation.

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.'

Extracellular vesicles and their role in peripheral nerve regeneration.

Peripheral nerve injuries often result in sensory and motor dysfunction in respective parts of the body. Regeneration after peripheral nerve injuries is a complex process including the differentiation of Schwann cells, recruiting of macrophages, blood vessel growth and axonal regrowth. Extracellular vesicles (EVs) are considered to play a pivotal role in intercellular communication and transfer of biological information. Specifically, their bioactivity and ability to deliver cargos of various types of nucleic acids and proteins have made them a potential vehicle for neurotherapeutics. However, production, characterization, dosage and targeted delivery of EVs still pose challenges for the clinical translation of EV therapeutics. This review summarizes the current knowledge of EVs in the context of the healthy and injured peripheral nerve and addresses novel concepts for modification of EVs as therapeutic agents for peripheral nerve regeneration.

Hedging against Neuropathic Pain: Role of Hedgehog Signaling in Pathological Nerve Healing.

The peripheral nervous system has important regenerative capacities that regulate and restore peripheral nerve homeostasis. Following peripheral nerve injury, the nerve undergoes a highly regulated degeneration and regeneration process called Wallerian degeneration, where numerous cell populations interact to allow proper nerve healing. Recent studies have evidenced the prominent role of morphogenetic Hedgehog signaling pathway and its main effectors, Sonic Hedgehog (SHH) and Desert Hedgehog (DHH) in the regenerative drive following nerve injury. Furthermore, dysfunctional regeneration and/or dysfunctional Hedgehog signaling participate in the development of chronic neuropathic pain that sometimes accompanies nerve healing in the clinical context. Understanding the implications of this key signaling pathway could provide exciting new perspectives for future research on peripheral nerve healing.

Satellite glial cells promote regenerative growth in sensory neurons.

Peripheral sensory neurons regenerate their axon after nerve injury to enable functional recovery. Intrinsic mechanisms operating in sensory neurons are known to regulate nerve repair, but whether satellite glial cells (SGC), which completely envelop the neuronal soma, contribute to nerve regeneration remains unexplored. Using a single cell RNAseq approach, we reveal that SGC are distinct from Schwann cells and share similarities with astrocytes. Nerve injury elicits changes in the expression of genes related to fatty acid synthesis and peroxisome proliferator-activated receptor (PPARα) signaling. Conditional deletion of fatty acid synthase (Fasn) in SGC impairs axon regeneration. The PPARα agonist fenofibrate rescues the impaired axon regeneration in mice lacking Fasn in SGC. These results indicate that PPARα activity downstream of FASN in SGC contributes to promote axon regeneration in adult peripheral nerves and highlight that the sensory neuron and its surrounding glial coat form a functional unit that orchestrates nerve repair.

Acute pharmacological inhibition of matrix metalloproteinase-9 activity during development restores perineuronal net formation and normalizes auditory processing in Fmr1 KO mice.

Individuals with Fragile X Syndrome (FXS) and autism spectrum disorder (ASD) exhibit cognitive impairments, social deficits, increased anxiety, and sensory hyperexcitability. Previously, we showed that elevated levels of matrix metalloproteinase-9 (MMP-9) may contribute to abnormal development of parvalbumin (PV) interneurons and perineuronal nets (PNNs) in the developing auditory cortex (AC) of Fmr1 knock-out (KO) mice, which likely underlie auditory hypersensitivity. Thus, MMP-9 may serve as a potential target for treatment of auditory hypersensitivity in FXS. Here, we used the MMP-2/9 inhibitor, SB-3CT, to pharmacologically inhibit MMP-9 activity during a specific developmental period and to test whether inhibition of MMP-9 activity reverses neural oscillation deficits and behavioral impairments by enhancing PNN formation around PV cells in Fmr1 KO mice. Electroencephalography (EEG) was used to measure resting state and sound-evoked electrocortical activity in auditory and frontal cortices of postnatal day (P)22-23 male mice before and one-day after treatment with SB-3CT (25 mg/kg) or vehicle. At P27-28, animal behaviors were tested to measure the effects of the treatment on anxiety and hyperactivity. Results show that acute inhibition of MMP-9 activity improved evoked synchronization to auditory stimuli and ameliorated mouse behavioral deficits. MMP-9 inhibition enhanced PNN formation, increased PV levels and TrkB phosphorylation yet reduced Akt phosphorylation in the AC of Fmr1 KO mice. Our results show that MMP-9 inhibition during early postnatal development is beneficial in reducing some auditory processing deficits in the FXS mouse model and may serve as a candidate therapeutic for reversing sensory hypersensitivity in FXS and possibly other ASDs.

Simultaneous fluorescence imaging of distinct nerve and blood vessel patterns in dual Thy1-YFP and Flt1-DsRed transgenic mice.

Blood vessels and nerve tissues are critical to the development and functionality of many vital organs. However, little is currently known about their interdependency during development and after injury. In this study, dual fluorescence transgenic reporter mice were utilized to observe blood vessels and nervous tissues in organs postnatally. Thy1-YFP and Flt1-DsRed (TYFD) mice were interbred to achieve dual fluorescence in the offspring, with Thy1-YFP yellow fluorescence expressed primarily in nerves, and Flt1-DsRed fluorescence expressed selectively in blood vessels. Using this dual fluorescent mouse strain, we were able to visualize the networks of nervous and vascular tissue simultaneously in various organ systems both in the physiological state and after injury. Using ex vivo high-resolution imaging in this dual fluorescent strain, we characterized the organizational patterns of both nervous and vascular systems in a diverse set of organs and tissues. In the cornea, we also observed the dynamic patterns of nerve and blood vessel networks following epithelial debridement injury. These findings highlight the versatility of this dual fluorescent strain for characterizing the relationship between nerve and blood vessel growth and organization.

Nanofibrous Nerve Conduits with Nerve Growth Factors and Bone Marrow Stromal Cells Pre-Cultured in Bioreactors for Peripheral Nerve Regeneration.

Peripheral nerve injury (PNI) was the leading cause of permanent dysfunction in movement and sensation. Synthesized nerve guide conduits (NGCs) with Schwann Cells (SCs) can help peripheral nerve regeneration. However, poor accessibility of SCs and lack of full coverage of seeded cells on NGCs can lead to failure of nerve regeneration across long gaps and full functional recovery. To overcome these limitations, bone marrow stromal cells (BMSCs) and a novel culture method were proposed in the current study. BMSCs were harvested and seeded on a never growth factor (NGF)-loaded PCL nanofibrous NGCs and cultured with a rotary cell culture system (RCCS) before implantation. The NGCs were tested in vitro with PC-12 cells to validate the bioactivity of released NGF and to access its ability to promote neurite extension. Also, the NGCs were tested in vivo with rat sciatic nerve model to exam its potential in bridging the long gap (15 mm segmental defect). The efficacy of the NGCs was investigated based on the results of the functional test, electrophysiology test, muscle atrophy, and histological analysis. The results of in vitro PC-12 cell study confirmed the bioactivity of released NGF and showed a significant increase in the neurite extension with the help of PEG-diamine and BSA. These results showed that the novel loading method could preserve the bioactivity of growth factors and achieve a sustained release in vitro. Besides, the results of the in vivo study exhibited a significant increase with the combination of all additives. These results showed that with the help of NGF and RCCS, the NGCs with the seeded BMSCs could enhance peripheral nerve regeneration across long nerve injury gaps.

Elmo1 function, linked to Rac1 activity, regulates peripheral neuronal numbers and myelination in zebrafish.

Peripheral nervous system development involves a tight coordination of neuronal birth and death and a substantial remodelling of the myelinating glia cytoskeleton to achieve myelin wrapping of its projecting axons. However, how these processes are coordinated through time is still not understood. We have identified engulfment and cell motility 1, Elmo1, as a novel component that regulates (i) neuronal numbers within the Posterior Lateral Line ganglion and (ii) radial sorting of axons by Schwann cells (SC) and myelination in the PLL system in zebrafish. Our results show that neuronal and myelination defects observed in elmo1 mutant are rescued through small GTPase Rac1 activation. Inhibiting macrophage development leads to a decrease in neuronal numbers, while peripheral myelination is intact. However, elmo1 mutants do not show defective macrophage activity, suggesting a role for Elmo1 in PLLg neuronal development and SC myelination independent of macrophages. Forcing early Elmo1 and Rac1 expression specifically within SCs rescues elmo1-/- myelination defects, highlighting an autonomous role for Elmo1 and Rac1 in radial sorting of axons by SCs and myelination. This uncovers a previously unknown function of Elmo1 that regulates fundamental aspects of PNS development.

The mouse olfactory peduncle 4: Development of synapses, perineuronal nets, and capillaries.

Olfaction is critical for survival in neonatal mammals. However, little is known about the neural substrate for this ability as few studies of synaptic development in several olfactory processing regions have been reported. Odor information detected in the nasal cavity is first processed by the olfactory bulb and then sent via the lateral olfactory tract to a series of olfactory cortical areas. The first of these, the anterior olfactory nucleus pars principalis (AONpP), is a simple, two layered cortex with an outer plexiform and inner cell zone (Layers 1 and 2, respectively). Five sets of studies examined age-related changes in the AONpP. First, immunocytochemistry for glutamatergic (VGlut1 and VGlut2) and GABAergic (VGAT) synapses demonstrated that overall synaptic patterns remained uniform with age. The second set quantified synaptic development with electron microscopy and found different developmental patterns between Layers 1 and 2. As many of the interhemispheric connections in the olfactory system arise from AONpP, the third set examined the development of crossed projections using anterograde tracers and electron microscopy to explore the maturation of this pathway. A fourth study examined ontogenetic changes in immunostaining for the proteoglycans aggrecan and brevican, markers of mesh-like extracellular structures known as perineuronal nets whose maturation is associated with the end of early critical periods of synaptogenesis. A final study found no age-related changes in the density of vasculature in the peduncle from P5 to P30. This work is among the first to examine early postnatal changes in this initial cortical region of the olfactory system.

Engineering a 3D functional human peripheral nerve in vitro using the Nerve-on-a-Chip platform.

Development of "organ-on-a-chip" systems for neuroscience applications are lagging due in part to the structural complexity of the nervous system and limited access of human neuronal & glial cells. In addition, rates for animal models in translating to human success are significantly lower for neurodegenerative diseases. Thus, a preclinical in vitro human cell-based model capable of providing critical clinical metrics such as nerve conduction velocity and histomorphometry are necessary to improve prediction and translation of in vitro data to successful clinical trials. To answer this challenge, we present an in vitro biomimetic model of all-human peripheral nerve tissue capable of showing robust neurite outgrowth (~5 mm), myelination of hNs by primary human Schwann cells (~5%), and evaluation of nerve conduction velocity (0.13-0.28 m/sec), previously unrealized for any human cell-based in vitro system. To the best of our knowledge, this Human Nerve-on-a-chip (HNoaC) system is the first biomimetic microphysiological system of myelinated human peripheral nerve which can be used for evaluating electrophysiological and histological metrics, the gold-standard assessment techniques previously only possible with in vivo studies.

Iodine-Enhanced Micro-CT Imaging of Soft Tissue on the Example of Peripheral Nerve Regeneration.

Microcomputed tomography (µCT) is widely used for the study of mineralized tissues, but a similar use for soft tissues is hindered by their low X-ray attenuation. This limitation can be overcome by the recent development of different staining techniques. Staining with Lugol's solution, a mixture of one part iodine and two parts potassium iodide in water, stands out among these techniques for its low complexity and cost. Currently, Lugol staining is mostly used for anatomical examination of tissues. In the present study, we seek to optimize the quality and reproducibility of the staining for ex vivo visualization of soft tissues in the context of a peripheral nerve regeneration model in the rat. We show that the staining result not only depends on the concentration of the staining solution but also on the amount of stain in relation to the tissue volume and composition, necessitating careful adaptation of the staining protocol to the respective specimen tissue. This optimization can be simplified by a stepwise staining which we show to yield a similar result compared to staining in a single step. Lugol staining solution results in concentration-dependent tissue shrinkage which can be minimized but not eliminated. We compared the shrinkage of tendon, nerve, skeletal muscle, heart, brain, and kidney with six iterations of Lugol staining. 60 ml of 0.3% Lugol's solution per cm3 of tissue for 24 h yielded good results on the example of a peripheral nerve regeneration model, and we were able to show that the regenerating nerve inside a silk fibroin tube can be visualized in 3D using this staining technique. This information helps in deciding the region of interest for histological imaging and provides a 3D context to histological findings. Correlating both imaging modalities has the potential to improve the understanding of the regenerative process.

Calcium/calmodulin-dependent protein kinase II regulates mammalian axon growth by affecting F-actin length in growth cone.

While axon regeneration is a key determinant of functional recovery of the nervous system after injury, it is often poor in the mature nervous system. Influx of extracellular calcium (Ca2+ ) is one of the first phenomena that occur following axonal injury, and calcium/calmodulin-dependent protein kinase II (CaMKII), a target substrate for calcium ions, regulates the status of cytoskeletal proteins such as F-actin. Herein, we found that peripheral axotomy activates CaMKII in dorsal root ganglion (DRG) sensory neurons, and inhibition of CaMKII impairs axon outgrowth in both the peripheral and central nervous systems (PNS and CNS, respectively). Most importantly, we also found that the activation of CaMKII promotes PNS and CNS axon growth, and regulatory effects of CaMKII on axon growth occur via affecting the length of the F-actin. Thus, we believe our findings provide clear evidence that CaMKII is a critical modulator of mammalian axon regeneration.

Matrices, scaffolds, and carriers for protein and molecule delivery in peripheral nerve regeneration.

Local application of exogenous agents with neurotrophic properties enhances the regenerative capacity of injured neurons, especially following reconstructions of long nerve gaps and delayed nerve repairs. Recent advances in biomaterials and biomedical engineering have provided options for the sustained and controlled release of macromolecules to the peripheral nerve. Here, we review five methods for delivering macromolecules to the peripheral nerve including mini-osmotic pumps, hydrogel-based delivery systems, nerve guidance conduits, electrospun fibers, and nerve wraps. In addition to controlling the release of bioactive macromolecules, the ease of clinical use and versatility in implantation at a variety of "real-world" anatomical locations are key factors in designing an ideal delivery system. The incorporation of both mechanical and biological cues into such devices also helps optimize these systems.

Dental pulp-derived stem cell conditioned medium to regenerate peripheral nerves in a novel animal model of dysphagia.

In nerve regeneration studies, various animal models are used to assess nerve regeneration. However, because of the difficulties in functional nerve assessment, a visceral nerve injury model is yet to be established. The superior laryngeal nerve (SLN) plays an essential role in swallowing. Although a treatment for SLN injury following trauma and surgery is desirable, no such treatment is reported in the literature. We recently reported that stem cells derived from human exfoliated deciduous teeth (SHED) have a therapeutic effect on various tissues via macrophage polarization. Here, we established a novel animal model of SLN injury. Our model was characterized as having weight loss and drinking behavior changes. In addition, the SLN lesion caused a delay in the onset of the swallowing reflex and gain of laryngeal residue in the pharynx. Systemic administration of SHED-conditioned media (SHED-CM) promoted functional recovery of the SLN and significantly promoted axonal regeneration by converting of macrophages to the anti-inflammatory M2 phenotype. In addition, SHED-CM enhanced new blood vessel formation at the injury site. Our data suggest that the administration of SHED-CM may provide therapeutic benefits for SLN injury.

BIG1/Arfgef1 and Arf1 regulate the initiation of myelination by Schwann cells in mice.

During development of the peripheral nervous system in mammals, Schwann cells wrap their plasma membranes around neuronal axons, forming multiple myelin sheaths. A mature myelin sheath insulates axons and increases nerve conduction velocity while protecting nerve fibers from various stresses such as physical ones. Despite this functional importance, the molecular units that underlie dynamic morphological changes in formation of myelin sheaths are not sufficiently understood. Arf1 is a small guanosine triphosphate-binding protein that plays multiple roles in intracellular trafficking and related signaling, both of which are processes involved in cell morphogenesis. We demonstrate that the Arf1 guanine nucleotide exchange factor, brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1)/Arfgef1, and the effector Arf1 regulate the initiation of myelination of axons by Schwann cells. Schwann cell-specific BIG1 conditional knockout mice, which have been generated here, exhibit reduced myelin thickness and decreased localization of myelin protein zero in the myelin membrane, compared with their littermate controls. BIG1 knockout mouse nerves specifically decrease the amounts of Arf1 in the AP1 clathrin adaptor protein subunits but not the Arf1 binding to GGA1 (Golgi-localized, gamma-adaptin ear-containing, Arf-binding protein 1) transporting proteins. The amounts of Arf1 in the COPI coatomer protein subunits were comparable in the knockout mice and controls. Similar results in myelin thickness are observed in Arf1 conditional knockout mice, which have also been generated here. Thus, the BIG1 and Arf1 unit plays a key role in Schwann cell myelination, newly adding it to the list of molecular units controlling myelination.

Use of a capillary alginate gel (Capgel™) to study the three-dimensional development of sensory nerves reveals the formation of a rudimentary perineurium.

BACKGROUND: Peripheral neuropathies affect approximately 20 million people in the United States and often stem from other chronic conditions, such as diabetes. In vitro methodologies to facilitate the understanding and treatment of these disorders often lack the cellular and functional complexity required to accurately model peripheral neuropathies. In particular, they are often 2D and fail to faithfully reproduce the 3D in vivo microenvironment. NEW METHOD: Embryonic dorsal root ganglion (DRG) explants were inserted into laminin derivatized capillary alginate gel (Capgel™), a bioabsorbable, self-assembling biomaterial, possessing parallel microchannel architecture, and cultured to mimic normal nerve development, including Schwann cell myelination. RESULTS: Laminin derivatization of the microchannels improved nerve growth through the gel. Axon bundles containing myelinating Schwann cells migrated through the gel and were ensheathed by rudimentary perineurium up to 1 mm from the DRG explant site. COMPARISON WITH EXISTING METHODS: Other nerve models are two-dimensional in nature and/or fail to conserve the complicated architecture and cellular milieu observed in vivo. Our nerve model shows the simple culture technique of cells grown in 3D, which allows for a more advanced structural organization that more accurately mimics the in vivo nerve fascicle. CONCLUSIONS: When embryonic DRG explants are cultured in this system, they show a striking resemblance to in vivo peripheral nerve fascicles, including myelinated axons and the formation of a rudimentary perineurium, suggesting that both neuronal and non-neuronal cells within the DRG explant are capable of recreating the 3D structure of a developing sensory fascicle within the microchannel architecture.

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion.

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve microenvironment. This type of invasion is exhibited by a variety of cancer types, and very frequently is found in pancreatic cancer. The microscopic size of nerve fibers within mouse pancreas renders the study of PNI difficult in orthotopic murine models. Here, we describe a heterotopic in vivo model of PNI, where we inject syngeneic pancreatic cancer cell line Panc02-H7 into the murine sciatic nerve. In this model, sciatic nerves of anesthetized mice are exposed and injected with cancer cells. The cancer cells invade in the nerves proximally toward the spinal cord from the point of injection. The invaded sciatic nerves are then extracted and processed with OCT for frozen sectioning. H&E and immunofluorescence staining of these sections allow quantification of both the degree of invasion and changes in protein expression. This model can be applied to a variety of studies on PNI given its versatility. Using mice with different genetic modifications and/or different types of cancer cells allows for investigation of the cellular and molecular mechanisms of PNI and for different cancer types. Furthermore, the effects of therapeutic agents on nerve invasion can be studied by applying treatment to these mice.

A ketogenic diet reduces metabolic syndrome-induced allodynia and promotes peripheral nerve growth in mice.

Current experiments investigated whether a ketogenic diet impacts neuropathy associated with obesity and prediabetes. Mice challenged with a ketogenic diet were compared to mice fed a high-fat diet or a high-fat diet plus exercise. Additionally, an intervention switching to a ketogenic diet following 8 weeks of high-fat diet was performed to compare how a control diet, exercise, or a ketogenic diet affects metabolic syndrome-induced neural complications. When challenged with a ketogenic diet, mice had reduced bodyweight and fat mass compared to high-fat-fed mice, and were similar to exercised, high-fat-fed mice. High-fat-fed, exercised and ketogenic-fed mice had mildly elevated blood glucose; conversely, ketogenic diet-fed mice were unique in having reduced serum insulin levels. Ketogenic diet-fed mice never developed mechanical allodynia contrary to mice fed a high-fat diet. Ketogenic diet fed mice also had increased epidermal axon density compared all other groups. When a ketogenic diet was used as an intervention, a ketogenic diet was unable to reverse high-fat fed-induced metabolic changes but was able to significantly reverse a high-fat diet-induced mechanical allodynia. As an intervention, a ketogenic diet also increased epidermal axon density. In vitro studies revealed increased neurite outgrowth in sensory neurons from mice fed a ketogenic diet and in neurons from normal diet-fed mice given ketone bodies in the culture medium. These results suggest a ketogenic diet can prevent certain complications of prediabetes and provides significant benefits to peripheral axons and sensory dysfunction.

Peripheral nervous system maturation in preterm infants: longitudinal motor and sensory nerve conduction studies.

OBJECTIVE: To study the evolution of sensory-motor nerves in the upper and lower limbs in neurologically healthy preterm infants and to use sensory-motor studies to compare the rate of maturation in preterm infants at term age and full-term healthy neonates. METHODS: The study comprised 26 neurologically normal preterm infants born at 23-33 weeks of gestational age, who underwent sensory nerve conduction and motor nerve conduction studies from plantar medial and median nerves and from tibial and ulnar nerves, respectively. We repeated the same neurophysiological studies in 19 of the preterm infants every 2 weeks until postnatal term age. The data from the preterm infants at term was matched with a group of ten full-term babies a few days after birth. RESULTS: The motor nerve conduction velocity of the tibial and ulnar nerves showed progressive increases in values in relation to gestational age, but there was a decrease of values in distal latencies and F wave latencies. Similarly, there was a gradual increase of sensory nerve conduction velocity values of the medial plantar and median nerves and decreases in latencies in relation to gestational age. At term age, the preterm infants showed significantly lower values of conduction velocities and distal latencies than the full-term neonates. These results were probably because the preterm infants had significantly lower weights, total length and, in particular, distal segments of the limbs at term age. CONCLUSION: The sensory-motor conduction parameters were clearly related to gestational age, but extrauterine life did not affect the maturation of the peripheral nervous system in the very preterm babies who were neurologically healthy.