Dorsal Root Ganglion Explants Research Articles

Small-molecule GSK-3 inhibitor rescued apoptosis and neurodegeneration in anesthetics-injured dorsal root ganglion neurons.

Application of general anesthetics may induce neurotoxicity in dorsal root ganglia (DRG) neurons. In this study, we examined the possible protective mechanism and associated signaling pathways of small-molecule glycogen synthase kinase-3 (GSK-3) inhibitor, SB216763, in bupivacaine-injured mouse DRG neurons in vitro. In vitro DRG explant of 6-week old mice was treated with 5mM bupivacaine to induce neurotoxicity. The explants were also pre-treated with SB216763 for 72h. Neural protection of SB216763 on bupivacaine-injured DRG neurons was investigated by TUNEL assay, neurite outgrowth assay and western blot assay, respectively. Possible downstream gene of GSK-3 signaling pathway, protein kinase C (PKC) was knocked down by siRNA in DRG explant. Its function in regulating GSK-3 inhibition induced DRG neural protection was also examined by TUNEL, neurite outgrowth and western blot assays. Pre-treatment of SB216763 significantly ameliorated bupivacaine induced apoptosis and neurite loss in DRG neurons. Western blot showed that, in addition to the decrease of phosphorylated-GSK-3 α/β protein, SB216763 increased PKC and decreased caspase-3 (Casp-3) in bupivacaine-injured DRG neurons. SiRNA-mediated PKC knockdown was able to reverse the neural protection of SB216763 in bupivacaine-injured DRG neurons. Western blot showed that PKC knockdown increased phosphorylated-GSK-3 α/β and Casp-3 protein in DRG neurons, confirming that PKC was directly involved in GSK-3-inhibition induced neural protection in DRG. GSK-3 inhibitor SB216763, through PKC, is effective in protecting anesthetics-induced neurotoxicity in DRG.

Development of a new miniaturized bioreactor for axon stretch growth.

Peripheral nerve injury requires a physical bridge across the lesion, which is limited by the insufficient supply of donor nerves. Here, we developed a new miniaturized bioreactor system for axon stretch growth. Dorsal root ganglia explants were first placed on two adjoining substrates and formed new synaptic connections. The axon bundles across the border between the top and bottom membranes were then stretched in a stepwise fashion by a microstepper motor system. After several days of stretch, the axon tracts could reach lengths that could develop into living nervous tissue constructs. In order to achieve appropriate neuronal culture to stimulate physiological conditions during axon stretch, we tested a variety of coating methods. Based on these results, the elongator substrates were coated with both poly-D-lysine and rat-tail collagen to maximize the number of axon bundles. Additionally, we found that increasing the axon stretch by 1[Formula: see text][Formula: see text]m at each step resulted in the highest stability. The bridging axons adapted to the stretch by increasing their length from 500[Formula: see text][Formula: see text]m to 5.94[Formula: see text]mm over 7 days of stretch growth. Immunocytochemical analysis confirmed that beta-III-tubulin, a major cytoskeletal constituent and neuronal marker, was present along axons. The findings demonstrate that bioreactor has the potential to generate transplant materials to address neural repair.

An approach to comprehensive genome and proteome expression analyses in Schwann cells and neurons during peripheral nerve myelin formation

Peripheral nerve myelination is a complex event resulting from spatially and temporally regulated reciprocal interactions between the neuron and myelin-forming Schwann cells. The dynamic process and the protein functional modules and networks that operate throughout the myelination process are poorly understood because of a lack of methodologies suitable for observing specific changes in the Schwann cell/neuron-unit. The identification of the precise roles for the proteins participating in the functional modules and networks that participate in the myelination process is hindered by the cellular and molecular complexity of the nervous tissue itself. We have developed an approach based on a myelinating dorsal root ganglion explant model that allows distinguishing clear, reproducible and predictable differences between the biochemical properties and the genomic and proteomic expression profiles of both cellular components of the Schwann cell/neuron unit at different stages of the myelination process. This model, derived from E13.5 C57BL/6J mouse embryos, is sufficiently robust for use in identifying the protein functional networks and modules related to peripheral nerve myelin formation. The genomic expression profiles of the selected neuronal, Schwann cell and myelin-specific proteins in the cultures reflect invivo profiles reported in the literature, and the structural and ultrastructural properties of the myelin, as well as the myelination schedule of the cultures, closely resemble those observed in peripheral nerves insitu. The RNA expression data set is available through NCBI gene expression omnibus accession GSE60345. We have developed a reproducible and robust cell culture-based approach, accompanied by a genome-wide expression data set, which allows studying myelination in the peripheral nervous system at the proteomic and transcriptomic levels in Schwann cells and neurons. Myelinating dorsal root explant cultures, prepared from C57BL/6J mouse embryos, present distinct developmental stages comparable to those observed in a peripheral nerve insitu. This model can be used for identifying the protein functional networks and modules related to peripheral nerve myelin formation.

Effects of Chemically Doped Bioactive Borate Glass on Neuron Regrowth and Regeneration.

Peripheral nerve injuries present challenges to regeneration. Currently, the gold standard for nerve repair is an autograft that results in another region of the body suffering nerve damage. Previously, bioactive borate glass (BBG) has been studied in clinical trials to treat patients with non-healing wounds, and we have reported that BBG is conducive for soft tissue repair. BBG provides structural support, degrades in a non-cytotoxic manner, and can be chemically doped. Here, we tested a wide range of chemical compounds that are reported to have neuroprotective characteristics to promote regeneration of peripheral neurons after traumatic injury. We hypothesized that chemical dopants added in trace amounts to BBG would improve neuronal survival and neurite outgrowth from dorsal root ganglion (DRG) explants. We measured neurite outgrowth from whole DRG explants, and survival rates of dissociated neurons and support cells that comprise the DRG. Results show that chemically doped BBGs have differentially variable effects on neuronal survival and outgrowth, with iron, gallium, and zinc improving outgrowth of neurons, and iodine causing the most detriment to neurons. Because chemically doped BBGs support increased nerve regrowth and survival, they show promise for use in peripheral nerve regeneration.

Urokinase and urokinase receptor participate in regulation of neuronal migration, axon growth and branching

PurposeRecent findings indicate the significant contribution of urokinase and urokinase receptor (uPA and uPAR) in the processes of nerve regeneration, however, their role in axonal growth and branching is unclear. Using a 3D model of mouse Dorsal Root Ganglia (DRG) explants, differentiated into neurons Neuro 2a cells and transgenic mice lacking the urokinase gene, we studied the involvement of the uPA/uPAR system in the neural cell migration, neurite outgrowth, elongation and branching. ResultsuPA and uPAR are expressed in the growth cones of axons. Using an ex vivo model of DRG explants in Matrigel we have found that uPA inhibition attenuates neural cell migration and axonal growth, pointing to an important role of urokinase in these processes. Apparently, uPA mediates its effects through its specific receptor uPAR: anti-uPAR antibody, which blocks the uPA binding to uPAR, stimulates axon branching and attenuates neural cell migration from DRG explants. Simultaneous inhibition of uPA and uPAR almost completely prevents the axonal outgrowth from explants into the Matrigels. Experiments in vitro using Neuro 2a cells differentiated into neurons demonstrate that administration of exogenous uPA increases the neurite growth rate (elongation), most likely via the interaction of uPA with uPAR. Blocking of uPAR stimulates neurite formation and enhances branching of preexisting neurites. The results obtained on DRG explants from transgenic mice lacking uPA gene support the assumption that uPA stimulates neurite growth via uPA/uPAR interaction and uPAR role in axons branching and neural cell migration. ConclusionsThe uPA/uPAR system plays an essential role in neural cell migration, axonal growth and branching.

Inhibition of long non-coding RNA IGF2AS has profound effect on inducing neuronal growth and protecting local-anesthetic induced neurotoxicity in dorsal root ganglion neurons.

Long non-coding RNA IGF2AS was initially identified as a cancer regulator in wilm's tumors. In this study, IGF2AS was investigated of its functions in inducing neural development and protecting local-anesthetic induced neurotoxicity in dorsal root ganglion (DRG) in spinal cord. Explant of mouse spinal cord DRG was transfected with IGF2AS specific siRNA. The effect of IGF2AS inhibition on neural development was assessed by neurite growth assay, qRT-PCR and western blot assay, respectively. IGF2AS-downregulated DRG explant was then exposed to local anesthetic agent, lidocaine in vitro. The possible protective effects of IGF2AS inhibition on lidocaine-induced DRG neuron apoptosis and neurite loss were further assessed by TUNEL assay, neurite growth assay, qRT-PCR and western blot assays. SiRNA-mediated IGF2AS inhibition promoted neuronal growth, and induced IGF2, BDNF and NT3 upregulations at both gene and protein expressions. In lidocaine-exposed DRG neurons, endogenous IGF2AS inhibition was effective to protect local-anesthetic induced neuronal apoptosis and neurite loss. Further molecular characterization demonstrated that the neuronal protection of IGF2AS inhibition was also associated with upregulations of IGF2, BDNF and NT3 in DRG neurons. Inhibiting endogenous IGF2AS may promote neuronal growth and protect local-anesthetic induced neurotoxicity in DRG neurons, possibly through complimentary IGF2 upregulation and autocrine activation neurotrophin genes.

Adrenomedullin mediates tumor necrosis factor-α-induced responses in dorsal root ganglia in rats

Adrenomedullin (AM), a member of the calcitonin gene-related peptide (CGRP) family, has been demonstrated to be a pain peptide. This study investigated the possible involvement of AM in tumor necrosis factor-alpha (TNF-α)-induced responses contributing to neuronal plasticity in the dorsal root ganglia (DRG). Exposure of the DRG explant cultures to TNF-α (5nM) for 48h upregulated the expression of AM mRNA. The treatment with TNF-α also increased the level of CGRP, CCL-2 and MMP-9 mRNA in the cultured DRG. This increase was attenuated by the co-treatment with the selective AM receptor antagonist AM22-52 (2μM). The blockade of AM receptors inhibited TNF-α-induced increase of the glial fibrillary acidic protein (GFAP), interleukin-1β (IL-1β), phosphorylated cAMP response element binding protein (pCREB) and nuclear factor kappa B (pNF-κB) proteins. On the other hand, the treatment with the AM receptor agonist AM1-50 (10nM) for 96h induced an increase in the level of GFAP, IL-1β, pCREB and pNF-κB proteins. The inhibition of AM activity did not change TNF-α-induced phosphorylation of extracellular signal-related kinase (pERK) while the treatment with AM1-50 still increased the level of pERK in the cultured DRG. Immunofluorescence assay showed the colocalization of AM-like immunoreactivity (IR) with TNF-α-IR in DRG neurons. The present study suggests that the increased AM receptor signaling mediated the many, but not all, TNF-α-induced activities, contributing to peripheral sensitization in neuropathic pain.

Three-dimensional model of biomatrix as a method of studying blood vessels and nerve growth in tissue engineering structures

The method involves isolating and culturing ex vivo cultures of mouse dorsal root ganglia and abdominal aorta in a three-dimensional gel (Matrigel). Unlike other explant cultures, this technique allows to study the physiological and biochemical processes in the tissue explants of dorsal root ganglia and abdominal aorta in three-dimensional space but in contrast to a two-dimensional adhesive culture. Administration of the tested substances to the Matrigel allows analyzing their effects on the growth of blood vessels and neurites from the explants for 21 days. The developed method can be applied in modern cardiological and neurobiological research and explores how new therapeutic agents can accelerate the regeneration of blood vessels and nerves.

Amitriptyline Activates TrkA to Aid Neuronal Growth and Attenuate Anesthesia-Induced Neurodegeneration in Rat Dorsal Root Ganglion Neurons.

Tricyclic antidepressant amitriptyline (AM) has been shown to exert neurotrophic activity on neurons. We thus explored whether AM may aid the neuronal development and protect anesthesia-induced neuro-injury in young spinal cord dorsal root ganglion (DRG) neurons.The DRG explants were prepared from 1-day-old rats. The effect of AM on aiding DRG neural development was examined by immunohistochemistry at dose-dependent manner. AM-induced changes in gene and protein expressions, and also phosphorylation states of tyrosine kinases receptor A (TrkA) and B (TrkB) in DRG, were examined by quantitative real-time polymerase chain reaction and western blot. The effect of AM on attenuating lidocaine-induced DRG neurodegeneration was examined by immunohistochemistry, and small interfering RNA (siRNA)-mediated TrkA/B down-regulation.Amitriptyline stimulated DRG neuronal development in dose-dependent manner, but exerted toxic effect at concentrations higher than 10 M. AM activated TrkA in DRG through phosphorylation, whereas it had little effect on TrkB-signaling pathway. AM reduced lidocaine-induced DRG neurodegeneration by regenerating neurites and growth cones. Moreover, the neuroprotection of AM on lidocaine-injured neurodegeneration was blocked by siRNA-mediated TrkA down-regulation, but not by TrkB down-regulation.Amitriptyline facilitated neuronal development and had protective effect on lidocaine-induced neurodegeneration, very likely through the activation of TrkA-signaling pathway in DRG.

Reversal of neurochemical alterations in the spinal dorsal horn and dorsal root ganglia by Mas-related gene (Mrg) receptors in a rat model of spinal nerve injury

The rodent Mas-related gene (Mrg) receptor subtype C has been demonstrated to inhibit pathological pain. This study investigated the mechanisms underlying the reversal of pain hypersensitivity by the selective MrgC receptor agonist bovine adrenal medulla 8–22 (BAM8–22) in a rat model of L5 spinal nerve ligation (SNL). Intrathecal (i.t.) administration of BAM8–22 (0.1–10nmol) attenuated mechanical allodynia in a dose-dependent manner on day 10 after SNL. The antiallodynia effect of BAM8–22 was abolished by MrgC receptor antibody, but not by naloxone. I.t. BAM8–22 (10nmol) inhibited SNL-induced upregulation of neuronal nitric oxide synthesis (nNOS) and phosphorylation of cyclic AMP response element-binding protein (p-CREB) in the spinal dorsal horn. The BAM8–22 treatment reversed the SNL-induced astrocyte activation, increase of interleukin-1β (IL-1β) expression and phosphorylation of extracellular signal-regulated kinase (p-ERK) in the spinal cord. BAM8–22 also reversed the upregulation of fractalkine and IL-1β in small- and medium-sized dorsal root ganglion (DRG) neurons. Furthermore, the BAM8–22 exposure suppressed the lipopolysaccharide (LPS)-induced increase of nNOS and IL-1β in the DRG explant cultures and the BAM8–22-induced suppression disappeared in the presence of MrgC receptor antibody. The present study provides evidence that activation of MrgC receptors inhibits nerve injury-induced increase of pronociceptive molecules in DRG neurons, suppressing astrocyte activation, the upregulation of excitatory mediators and phosphorylation of transcription factors in the spinal dorsal horn. As MrgC receptors are unequally expressed in the dorsal root and trigeminal ganglia, this study suggests that targeting MrgC receptors could be a new therapy for neuropathic pain with limited unwanted effects.

Production of Highly Aligned Collagen Scaffolds by Freeze-drying of Self-assembled, Fibrillar Collagen Gels.

Matrix and cellular alignment are critical factors in the native function of many tissues, including muscle, nerve, and ligaments. Collagen is frequently a component of these aligned tissues, and collagen biomaterials are widely used in tissue engineering applications. However, the generation of aligned collagen scaffolds that maintain the native architecture of collagen fibrils has not been straightforward, with many methods requiring specialized equipment or technical procedures, extensive incubation times, or denaturing of the collagen. Herein, we present a simple, rapid method for fabrication of highly aligned collagen scaffolds. Collagen was assembled to form a fibrillar hydrogel in a cylindrical conduit with high aspect ratio and then frozen and lyophilized. The resulting collagen scaffolds demonstrated highly aligned topographical features along the scaffold surface. This presence of an initial fibrillar network and the high-aspect ratio vessel were both required to generate alignment. The diameter of fabricated scaffolds was found to vary significantly with both the collagen concentration of the hydrogel suspension and the diameter of conduits used for fabrication. Additionally, the size of individual aligned topographical features was significantly dependent on the conduit diameter and the freezing temperature. When cultured on aligned collagen scaffolds, both rat dermal fibroblasts and axons emerging from chick dorsal root ganglia explants demonstrated elongated, aligned morphology and growth on the aligned topographical features. Overall, this method presents a simple means for generating aligned collagen scaffolds that can be applied to a wide variety of tissue types, particularly those where such alignment is critical to native function.

Neurogenic potential of engineered mesenchymal stem cells overexpressing VEGF.

Numerous signaling molecules are altered following nerve injury, serving as a blueprint for drug delivery approaches that promote nerve repair. However, challenges with achieving the appropriate temporal duration of recombinant protein delivery have limited the therapeutic success of this approach. Genetic engineering of mesenchymal stem cells (MSCs) to enhance the secretion of proangiogenic molecules such as vascular endothelial growth factor (VEGF) may provide an alternative. We hypothesized that the administration of VEGF-expressing human MSCs would stimulate neurite outgrowth and proliferation of cell-types involved in neural repair. When cultured with dorsal root ganglion (DRG) explants in vitro, control and VEGF-expressing MSCs (VEGF-MSCs) increased neurite extension and proliferation of Schwann cells (SCs) and endothelial cells, while VEGF-MSCs stimulated significantly greater proliferation of endothelial cells. When embedded within a 3D fibrin matrix, VEGF-MSCs maintained overexpression and expressed detectable levels over 21 days. After transplantation into a murine sciatic nerve injury model, VEGF-MSCs maintained high VEGF levels for 2 weeks. This study provides new insight into the role of VEGF on peripheral nerve injury and the viability of transplanted genetically engineered MSCs. The study aims to provide a framework for future studies with the ultimate goal of developing an improved therapy for nerve repair.

17β-Estradiol Promotes Schwann Cell Proliferation and Differentiation, Accelerating Early Remyelination in a Mouse Peripheral Nerve Injury Model.

Estrogen induces oligodendrocyte remyelination in response to demyelination in the central nervous system. Our objective was to determine the effects of 17β-estradiol (E2) on Schwann cell function and peripheral nerve remyelination after injury. Adult male C57BL/6J mice were used to prepare the sciatic nerve transection injury model and were randomly categorized into control and E2 groups. To study myelination in vitro, dorsal root ganglion (DRG) explant culture was prepared using 13.5-day-old mouse embryos. Primary Schwann cells were isolated from the sciatic nerves of 1- to 3-day-old Sprague–Dawley rats. Immunostaining for myelin basic protein (MBP) expression and toluidine blue staining for myelin sheaths demonstrated that E2 treatment accelerates early remyelination in the “nerve bridge” region between the proximal and distal stumps of the transection injury site in the mouse sciatic nerve. The 5-bromo-2′-deoxyuridine incorporation assay revealed that E2 promotes Schwann cell proliferation in the bridge region and in the primary culture, which is blocked using AKT inhibitor MK2206. The in vitro myelination in the DRG explant culture determined showed that the MBP expression in the E2-treated group is higher than that in the control group. These results show that E2 promotes Schwann cell proliferation and myelination depending on AKT activation.

Phospholipase C gamma mediates endogenous brain-derived neurotrophic factor-regulated calcitonin gene-related peptide expression in colitis-induced visceral pain

BackgroundVisceral hypersensitivity is a complex pathophysiological paradigm with unclear mechanisms. Primary afferent neuronal plasticity marked by alterations in neuroactive compounds such as calcitonin gene-related peptide is suggested to underlie the heightened sensory responses. Signal transduction that leads to calcitonin gene-related peptide expression thereby sensory neuroplasticity during colitis remains to be elucidated.ResultsIn a rat model with colitis induced by 2,4,6-trinitrobenzene sulfonic acid, we found that endogenously elevated brain-derived neurotrophic factor elicited an up-regulation of calcitonin gene-related peptide in the lumbar L1 dorsal root ganglia. At seven days of colitis, neutralization of brain-derived neurotrophic factor with a specific brain-derived neurotrophic factor antibody reversed calcitonin gene-related peptide up-regulation in the dorsal root ganglia. Colitis-induced calcitonin gene-related peptide transcription was also inhibited by brain-derived neurotrophic factor antibody treatment. Signal transduction studies with dorsal root ganglia explants showed that brain-derived neurotrophic factor-induced calcitonin gene-related peptide expression was mediated by the phospholipase C gamma, but not the phosphatidylinositol 3-kinase/Akt or the mitogen-activated protein kinase/extracellular signal-regulated protein kinase pathway. Application of PLC inhibitor U73122 in vivo confirmed that colitis-induced and brain-derived neurotrophic factor-mediated calcitonin gene-related peptide up-regulation in the dorsal root ganglia was regulated by the phospholipase C gamma pathway. In contrast, suppression of the phosphatidylinositol 3-kinase activity in vivo had no effect on colitis-induced calcitonin gene-related peptide expression. During colitis, calcitonin gene-related peptide also co-expressed with phospholipase C gamma but not with p-Akt. Calcitonin gene-related peptide up-regulation during colitis correlated to the activation of cAMP-responsive element binding protein in the same neurons. Consistently, colitis-induced cAMP-responsive element binding protein activation in the dorsal root ganglia was attenuated by brain-derived neurotrophic factor antibody treatment.ConclusionThese results suggest that colitis-induced and brain-derived neurotrophic factor-mediated calcitonin gene-related peptide expression in sensory activation is regulated by a unique pathway involving brain-derived neurotrophic factor-phospholipase C gamma-cAMP-responsive element binding protein axis.

Cytosine Arabinoside 전처리가 척수후근절 외식편 배양에 미치는 영향

Explant culture condition of dorsal root ganglion have been used to investigate the pathophysiology of peripheral nerve injury, while applying for the various clinical symptom such as trauma, pressure, and stretch. However, explant culture is usually contaminated by mitotic cells, which may observed as a newly divided cells including fibroblast or glia. The mitotic cells could be able to interrupt and change the cell signaling that make it difficult to avoid detrimental effects during the experiments. To eliminate mitotic cells, anti-mitotic reagents like mixture of uridine and 5-fluorodeoxyuridine or cytosine arabinoside were added to the cultures on the following day, but there is no research that investigate viability of anti-mitotic reagent in dorsal root ganglion explant culture. In this study, we investigate inhibition effect of cytosine arabinoside to mitotic cells in dorsal root ganglion explant culture. Also we visualized and analyzed anti-mitotic effect and toxicity of cytosine arabinoside in various concentration condition. This dorsal root ganglion explant culture condition can be applied to research that effect and mechanism of various stimulation and chemical application which affect peripheral nerve regeneration.

Downregulation of miR-210 protected bupivacaine-induced neurotoxicity in dorsal root ganglion.

Local anesthetic may cause neurotoxicity in developing neurons. In this study, we examined the molecular mechanisms of microRNA-210 (miR-210) in regulating bupivacaine-induced dorsal root ganglia (DRG) neurotoxicity in vitro. Young mouse (P30) DRG explants were cultured in vitro and treated with 5 mM bupivacaine to induce neurotoxicity. QRT-PCR was used to evaluate the expression profiles of miRNAs within 24 h after bupivacaine treatment. MiR-210 was downregulated in DRG, and its effects on bupivacaine-induced neurotoxicity were evaluated by apoptosis and neurite growth assays, respectively. Putative downstream target of miR-210 in DRG, BDNF, was evaluated by dual-luciferase assay, qRT-PCR, and western blot, respectively. BDNF was then knocked down by siRNA to assess its associated effects in regulating DRG neurotoxicity. Within the initial 24 h after bupivacaine treatment, various patterns of miRNA expression were observed, whereas miR-210 was constantly upregulated. Application of miR-210 inhibitor efficiently downregulated endogenous miR-210, protected apoptosis and neurite retraction in bupivacaine damaged DRG neurons. Using dual-luciferase assay, qRT-PCR, and western blot, BDNF was confirmed to the downstream target of miR-210 in DRG. SiRNA-mediated BDNF downregulation reversed the effect of miR-210 downregulation in DRG neurotoxicity. MiR-210, through the regulation of BDNF, plays important role in anesthetics-induced DRG neurotoxicity.

Stimulation Frequency Alters the Dorsal Root Ganglion Neurite Growth and Directionality In Vitro.

To improve peripheral nerve repair, new techniques to increase the speed of regeneration are required. Studies have shown that the electrical stimulation can enhance nerve regeneration; however, stimulation parameters that regulate the growth increases are unknown. The objective of this study was to examine dorsal root ganglion (DRG) neurite extension, directionality, and density after using methods to specifically control ac electrical field intensity and frequency exposure. Chick DRG explants were exposed to 20-Hz,200-Hz, 1-MHz, and 20-MHz sinusoidal electric field of 17.86V/m, and tissue parameters were measured. Results show that neurite extension and directionality were influenced by frequency; however, the ratio of support cell emigration with respect to neurite extension from the DRG body was not. These results were further verified through finite-element modeling of intracellular calcium, which show that higher frequencies have minimal effect on intracellular calcium. In conclusion, these results demonstrate that 1) directional growth of neurites within EFs can be achieved, 2) high-frequency stimulation in megahertz does not enhance or impair the neurite growth, and 3) low-frequency stimulation affects the growth and directionality. The significance of this study is the direct comparison of neurite extension after high stimulation frequencies (megahertz) with typical low-frequency fields (20 and 200Hz), and modeling the results with finite-element modeling.

Substratum preferences of motor and sensory neurons in postnatal and adult rats.

After peripheral nerve injuries, damaged axons can regenerate but functional recovery is limited by the specific reinnervation of targets. In this study we evaluated if motor and sensory neurites have a substrate preference for laminin and fibronectin in postnatal and adult stages. In postnatal dorsal root ganglia (DRG) explants, sensory neurons extended longer neurites on collagen matrices enriched with laminin (~50%) or fibronectin (~35%), whereas motoneurons extended longer neurites (~100%) in organotypic spinal cord slices embedded in fibronectin-enriched matrix. An increased percentage of parvalbumin-positive neurites (presumptive proprioceptive) vs. neurofilament-positive neurites was also found in DRG in fibronectin-enriched matrix. To test if the different preference of neurons for extracellular matrix components was maintained in vivo, these matrices were used to fill a chitosan guide to repair a 6-mm gap in the sciatic nerve of adult rats. However, the number of regenerating motor and sensory neurons after 1 month was similar between groups. Moreover, none of the retrotraced sensory neurons in DRG was positive for parvalbumin, suggesting that presumptive proprioceptive neurons had poor regenerative capabilities compared with other peripheral neurons. Using real-time PCR we evaluated the expression of α5β1 (receptor for fibronectin) and α7β1 integrin (receptor for laminin) in spinal cord and DRG 2 days after injury. Postnatal animals showed a higher increase of α5β1 integrin, whereas both integrins were similarly expressed in adult neurons. Therefore, we conclude that motor and sensory axons have a different substrate preference at early postnatal stages but this difference is lost in the adult.

Laser fabricated discontinuous anisotropic microconical substrates as a new model scaffold to control the directionality of neuronal network outgrowth

Patterning of neuronal outgrowth in vitro is important in tissue engineering as well as for the development of neuronal interfaces with desirable characteristics. To date, this has been achieved with the aid of micro- and nanofabrication techniques giving rise to various anisotropic topographies, either in the form of continuous or discontinuous structures. In this study we propose a currently unexplored geometry of a 3D culture substrate for neuronal cell growth comprising discontinuous subcellular microstructures with anisotropic geometrical cross-section. Specifically, using laser precision 3D micro/nano fabrication techniques, silicon substrates comprising arrays of parallel oriented elliptical microcones (MCs) were fabricated to investigate whether a discontinuous geometry comprising anisotropic features at the subcellular level could influence the alignment of peripheral nervous system cell populations. It was shown that both Schwann cells and axons of sympathetic neurons were parallel oriented onto the MCs of elliptical shape, while they exhibited a random orientation onto the MCs of arbitrary shape. Notably, this topography-induced guidance effect was also observed in more complex cell culture systems, such as the organotypic culture whole dorsal root ganglia (DRG) explants. Our results suggest that a discontinuous topographical pattern could promote Schwann cell and axonal alignment, provided that it hosts anisotropic geometrical features, even though the sizes of those range at the subcellular lengthscale. The laser-patterned arrays of MCs presented here could potentially be a useful platform for patterning neurons into artificial networks, allowing the study of neuronal cells interactions under 3D ex-vivo conditions.

Bone Marrow Stromal Cells Combined With a Honeycomb Collagen Sponge Facilitate Neurite Elongation In Vitro and Neural Restoration in the Hemisected Rat Spinal Cord.

In the last decade, researchers and clinicians have reported that transplantation of bone marrow stromal cells (BMSCs) promotes functional recovery after brain or spinal cord injury (SCI). However, an appropriate scaffold designed for the injured spinal cord is needed to enhance the survival of transplanted BMSCs and to promote nerve regeneration. We previously tested a honeycomb collagen sponge (HC), which when applied to the transected spinal cord allowed bridging of the gap with nerve fibers. In this study, we examined whether the HC implant combined with rat BMSCs increases nerve regeneration in vitro and enhances functional recovery in vivo. We first evaluated the neurite outgrowth of rat dorsal root ganglion (DRG) explants cultured on HC with or without BMSCs in vitro. Regeneration of neurites from the DRGs was increased by BMSCs combined with HC scaffolds. In the in vivo study, 3-mm-long HC scaffolds with or without BMSCs were implanted into the hemisected rat thoracic spinal cord. Four weeks after the procedure, rats implanted with HC scaffolds containing BMSCs displayed better motor and sensory recovery than those implanted with HC scaffolds only. Histologically, more CGRP-positive sensory fibers at the implanted site and 5-HT-positive serotonergic fibers contralateral to the implanted site were observed in spinal cords receiving BMSCs. Furthermore, more rubrospinal neurons projected distally to the HC implant containing BMSCs. Our study indicates that the application of BMSCs in a HC scaffold in the injured spinal cord directly promoted sensory nerve and rubrospinal tract regeneration, thus resulting in functional recovery.