Primary Dorsal Root Ganglion Neurons Research Articles

Transcriptional profiling of neuronal ion channels in dorsal root ganglion-derived immortal cell line (F-11) under different culture conditions.

Pathological pain is a prevalent condition that affects majority of adults with a variety of underlying disease conditions. Current available pharmacological pain treatments have several negative and potentially life-threatening side effects associated with their long-term use. Due to the heterogeneity of pain perception and the diversity of neuronal mechanisms that contribute to pain, high-throughput screening of small molecules that may have underlying analgesic properties is essential for identifying new analgesic treatments that are both effective and safe. The F-11 hybrid immortalized cell line is one of the currently available dorsal root ganglion (DRG) cell lines used for drug screening. While F-11 cells are commonly used as analogs to primary DRG sensory neurons, they differ significantly in physiological properties. The present study investigated the impact of differentiation protocols on the expression of mature neuron ion channels and receptors in F-11 cells. Using a customized gene array of more than eighty neuronal ion channels and receptors including voltage-gated ion channels, transient receptor potential channels, and cannabinoid receptors, we assessed the following groups: control F-11 cells; F-11 cells cultured under different culture conditions, and murine DRG tissue. The expression profiles of majority of the investigated ion channels and receptors in F-11 cells were found to be lower compared to primary mouse DRG neurons. F-11 cells cultured under low serum (LSM) conditions had increased expression of several investigated targets including voltage-gated ion channels and cannabinoid receptors when compared to control F-11 cells. The study showed that the culture conditions significantly modulated the transcriptional expression of studied ion channels and receptors, and that long-term culture (21 days) may adversely affect the expression of many of the studied targets.

Selective inhibitors of SARM1 targeting an allosteric cysteine in the autoregulatory ARM domain

The nicotinamide adenine dinucleotide hydrolase (NADase) sterile alpha toll/interleukin receptor motif containing-1 (SARM1) acts as a central executioner of programmed axon death and is a possible therapeutic target for neurodegenerative disorders. While orthosteric inhibitors of SARM1 have been described, this multidomain enzyme is also subject to intricate forms of autoregulation, suggesting the potential for allosteric modes of inhibition. Previous studies have identified multiple cysteine residues that support SARM1 activation and catalysis, but which of these cysteines, if any, might be selectively targetable by electrophilic small molecules remains unknown. Here, we describe the chemical proteomic discovery of a series of tryptoline acrylamides that site-specifically and stereoselectively modify cysteine-311 (C311) in the noncatalytic, autoregulatory armadillo repeat (ARM) domain of SARM1. These covalent compounds inhibit the NADase activity of WT-SARM1, but not C311A or C311S SARM1 mutants, show a high degree of proteome-wide selectivity for SARM1_C311 and stereoselectively block vincristine- and vacor-induced neurite degeneration in primary rodent dorsal root ganglion neurons. Our findings describe selective, covalent inhibitors of SARM1 targeting an allosteric cysteine, pointing to a potentially attractive therapeutic strategy for axon degeneration-dependent forms of neurological disease.

Cultured dissociated primary dorsal root ganglion neurons from adult horses enable study of axonal transport.

Neurological disorders are prevalent in horses, but their study is challenging due to anatomic constraints and the large body size; very few host‐specific in vitro models have been established to study these types of diseases, particularly from adult donor tissue. Here we report the generation of primary neuronal dorsal root ganglia (DRG) cultures from adult horses: the mixed, dissociated cultures, containing neurons and glial cells, remained viable for at least 90 days. Similar to DRG neurons in vivo, cultured neurons varied in size, and they developed long neurites. The mitochondrial movement was detected in cultured cells and was significantly slower in glial cells compared to DRG‐derived neurons. In addition, mitochondria were more elongated in glial cells than those in neurons. Our culture model will be a useful tool to study the contribution of axonal transport defects to specific neurodegenerative diseases in horses as well as comparative studies aimed at evaluating species‐specific differences in axonal transport and survival.

Cisplatin Neurotoxicity Targets Specific Subpopulations and K+ Channels in Tyrosine-Hydroxylase Positive Dorsal Root Ganglia Neurons.

Among the features of cisplatin chemotherapy-induced peripheral neuropathy are chronic pain and innocuous mechanical hypersensitivity. The complete etiology of the latter remains unknown. Here, we show that cisplatin targets a heterogeneous population of tyrosine hydroxylase-positive (TH+) primary afferent dorsal root ganglion neurons (DRGNs) in mice, determined using single-cell transcriptome and electrophysiological analyses. TH+ DRGNs regulate innocuous mechanical sensation through C-low threshold mechanoreceptors. A differential assessment of wild-type and vitamin E deficient TH+ DRGNs revealed heterogeneity and specific functional phenotypes. The TH+ DRGNs comprise; fast-adapting eliciting one action potential (AP; 1-AP), moderately-adapting (≥2-APs), in responses to square-pulse current injection, and spontaneously active (SA). Cisplatin increased the input resistance and AP frequency but reduced the temporal coding feature of 1-AP and ≥2-APs neurons. By contrast, cisplatin has no measurable effect on the SA neurons. Vitamin E reduced the cisplatin-mediated increased excitability but did not improve the TH+ neuron temporal coding properties. Cisplatin mediates its effect by targeting outward K+ current, likely carried through K2P18.1 (Kcnk18), discovered through the differential transcriptome studies and heterologous expression. Studies show a potential new cellular target for chemotherapy-induced peripheral neuropathy and implicate the possible neuroprotective effects of vitamin E in cisplatin chemotherapy.

Enhancing Membrane Repair in Neuronal Cell Types as a Potential Therapeutic for Neurodegenerative Diseases

Cell membrane repair is an essential cellular process to repair damage to the membrane and maintain the integrity of the cell membrane. Impaired membrane resealing kinetics can potentially lead to neurodegenerative diseases. Enhancing membrane repair via therapeutic intervention could minimize neurodegeneration in neuronal disease states. Recombinant human MG53 (rhMG53) and poloxamers have been an effective therapeutic to enhance membrane repair in muscle and non‐muscle cell types. Here, we aimed to assess if the membrane repair response is active and the efficacy of enhancing membrane resealing in neuronal cells. To assess if neuronal cells have an active membrane repair response, mouse neuroblastoma N2a cells were transfected with GFP or GFP‐MG53. MG53 localization was tracked following multiphoton infrared laser injury. N2a cells were transfected with GFP or GFP‐MG53, and subjected to multiphoton infrared laser injury to investigate if MG53 expression enhances membrane resealing following damage. An infrared laser was used to damage the cell membrane of N2a cells and primary mouse dorsal root ganglion neurons (DRG) in the presence of FM4‐64 dye. To test the efficacy of therapeutics, N2as and DRGs were treated with 1μM rhMG53 or 100μM Poloxamer 188 (P188) and challenged with the multiphoton laser injury. Lastly, the therapeutic potential of rhMG53 was tested in vivo with a sciatic nerve crush injury model. C57Bl/6 mice sciatic nerves were mechanically crushed for 10 seconds and then injected with 1μL rhMG53 (1mg/mL) or saline. The sciatic nerves were harvested and immunostained with an axonal regeneration marker, SCG10. SCG10 staining intensity was measured to calculate the regeneration index. Overall, we observed MG53 trafficking to the injury site which indicates there is an activate membrane repair mechanism within neuronal cells. Therapeutic levels of rhMG53 and P188 increased membrane resealing in both N2as and DRGs. Lastly, rhMG53‐treated sciatic nerves displayed a two‐fold increase in regenerative capacity following sciatic nerve crush injury compared to the saline vehicle control. These results illustrate an active membrane repair process in neuronal cells and potential for therapeutic intervention in the context of neuronal diseases, specifically neurodegenerative diseases.

Inhibition of sodium conductance by cannabigerol contributes to a reduction of dorsal root ganglion neuron excitability.

Cannabigerol (CBG), a non-psychotropic phytocannabinoid and a precursor of ∆9 -tetrahydrocannabinol and cannabidiol, has been suggested to act as an analgesic. A previous study reported that CBG (10μM) blocks voltage-gated sodium (Nav ) currents in CNS neurons, although the underlying mechanism is not well understood. Genetic and functional studies have validated Nav 1.7 channels as an opportune target for analgesic drug development. The effects of CBG on Nav 1.7 channels, which may contribute to its analgesic properties, have not been previously investigated. To determine the effects of CBG on Nav channels, we used stably transfected HEK cells and primary dorsal root ganglion (DRG) neurons to characterize compound effects using experimental and computational techniques. These included patch-clamp, multielectrode array, and action potential modelling. CBG is a ~10-fold state-dependent Nav channel inhibitor (KI -KR : ~2-20 μM) with an average Hill-slope of ~2. We determined that, at lower concentrations, CBG predominantly blocks sodium Gmax and slows recovery from inactivation. However, as the concentration is increased, CBG also induces a hyperpolarizing shift in the half-voltage of inactivation. Our modelling and multielectrode array recordings suggest that CBG attenuates DRG excitability. Inhibition of Nav 1.7 channels in DRG neurons may underlie CBG-induced neuronal hypoexcitability. As most Nav 1.7 channels are inactivated at the resting membrane potential of DRG neurons, they are more likely to be inhibited by lower CBG concentrations, suggesting functional selectivity against Nav 1.7 channels, compared with other Nav channels (via Gmax block).

Nuclear Magnetic Resonance Treatment Accelerates the Regeneration of Dorsal Root Ganglion Neurons in vitro.

Functional recovery from peripheral nerve injuries depends on a multitude of factors. Schwann cells (SCs) are key players in the regenerative process as they develop repair-specific functions to promote axon regrowth. However, chronically denervated SCs lose their repair phenotype, which is considered as a main reason for regeneration failure. Previous studies reported a modulatory effect of low nuclear magnetic resonance therapy (NMRT) on cell proliferation and gene expression. To provide first insight into a possible effect of NMRT on cells involved in peripheral nerve regeneration, this study investigated whether NMRT is able to influence the cellular behavior of primary SC and dorsal root ganglion (DRG) neuron cultures in vitro. The effect of NMRT on rat SCs was evaluated by comparing the morphology, purity, proliferation rate, and expression levels of (repair) SC associated genes between NMRT treated and untreated SC cultures. In addition, the influence of (1) NMRT and (2) medium obtained from NMRT treated SC cultures on rat DRG neuron regeneration was examined by analyzing neurite outgrowth and the neuronal differentiation status. Our results showed that NMRT stimulated the proliferation of SCs without changing their morphology, purity, or expression of (repair) SC associated markers. Furthermore, NMRT promoted DRG neuron regeneration shown by an increased cell survival, enhanced neurite network formation, and progressed neuronal differentiation status. Furthermore, the medium of NMRT treated SC cultures was sufficient to support DRG neuron survival and neurite outgrowth. These findings demonstrate a beneficial impact of NMRT on DRG neuron survival and neurite formation, which is primarily mediated via SC stimulation. Our data suggest that NMRT could be suitable as a non-invasive auxiliary treatment option for peripheral nerve injuries and encourage future studies that investigate the effect of NMRT in a physiological context.

Development and Biological Characterization of a Novel Selective TrkA Agonist with Neuroprotective Properties against Amyloid Toxicity.

Neurotrophins are growth factors that exert important neuroprotective effects by preventing neuronal death and synaptic loss. Nerve Growth Factor (NGF) acts through the activation of its high-affinity, pro-survival TrkA and low-affinity, pro-apoptotic p75NTR receptors. NGF has been shown to slow or prevent neurodegenerative signals in Alzheimer’s Disease (AD) progression. However, its low bioavailability and its blood–brain-barrier impermeability limit the use of NGF as a potential therapeutic agent against AD. Based on our previous findings on synthetic dehydroepiandrosterone derivatives, we identified a novel NGF mimetic, named ENT-A013, which selectively activates TrkA and exerts neuroprotective, anti-amyloid-β actions. We now report the chemical synthesis, in silico modelling, metabolic stability, CYP-mediated reaction phenotyping and biological characterization of ENT-A013 under physiological and neurodegenerative conditions. We show that ENT-A013 selectively activates the TrkA receptor and its downstream kinases Akt and Erk1/2 in PC12 cells, protecting these cells from serum deprivation-induced cell death. Moreover, ENT-A013 promotes survival of primary Dorsal Root Ganglion (DRG) neurons upon NGF withdrawal and protects hippocampal neurons against Amyloid β-induced apoptosis and synaptic loss. Furthermore, this neurotrophin mimetic partially restores LTP impairment. In conclusion, ENT-A013 represents a promising new lead molecule for developing therapeutics against neurodegenerative disorders, such as Alzheimer’s Disease, selectively targeting TrkA-mediated pro-survival signals.

Development of Lipidoid Nanoparticles for siRNA Delivery to Neural Cells.

Lipidoid nanoparticles (LNPs) are the delivery platform in Onpattro, the first FDA-approved siRNA drug. LNPs are also the carriers in the Pfizer-BioNTech and Moderna COVID-19 mRNA vaccines. While these applications have demonstrated that LNPs effectively deliver nucleic acids to hepatic and muscle cells, it is unclear if LNPs could be used for delivery of siRNA to neural cells, which are notoriously challenging delivery targets. Therefore, the purpose of this study was to determine if LNPs could efficiently deliver siRNA to neurons. Because of their potential delivery utility in either applications for the central nervous system and the peripheral nervous system, we used both cortical neurons and sensory neurons. We prepared siRNA-LNPs using C12-200, a benchmark ionizable cationic lipidoid along with helper lipids. We demonstrated using dynamic light scattering that the inclusion of both siRNA and PEG-lipid provided a stabilizing effect to the LNP particle diameters and polydispersity indices by minimizing aggregation. We found that siRNA-LNPs were safely tolerated by primary dorsal root ganglion neurons. Flow cytometry analysis revealed that Cy5 siRNA delivered via LNPs into rat primary cortical neurons showed uptake levels similar to Lipofectamine RNAiMAX—the gold standard commercial transfection agent. However, LNPs demonstrated a superior safety profile, whereas the Lipofectamine-mediated uptake was concomitant with significant toxicity. Fluorescence microscopy demonstrated a time-dependent increase in the uptake of LNP-delivered Cy5 siRNA in a human cortical neuron cell line. Overall, our results suggest that LNPs are a viable platform that can be optimized for delivery of therapeutic siRNAs to neural cells.Graphical Supplementary InformationThe online version contains supplementary material available at 10.1208/s12248-021-00653-2.

Development of an Axon-Guiding Aligned Nanofiber-Integrated Compartmentalized Microfluidic Neuron Culture System

Microfluidic-based neuron cell culture systems have recently gained a lot of attention due to their efficiency in supporting the spatial and temporal control of cellular microenvironments. However, the lack of axon guidance is the key limitation in current culture systems. To combat this, we have developed electrospun aligned nanofiber-integrated compartmentalized microfluidic neuron culture systems (NIMSs), where the nanofibers have enabled axonal guidance and stability. The resulting platform significantly improved axon alignment, length, and stability for both rat primary embryonic motor neurons (MNs) and dorsal root ganglia (DRG) neurons compared to the conventional glass-based microfluidic systems (GMSs). The results showed that axonal growth covered more than two times the area on the axonal chamber of NIMSs compared to the area covered for GMSs. Overall, this platform can be used as a valuable tool for fundamental neuroscience research, drug screening, and biomaterial testing.

Comparing axon regeneration in male and female mice after peripheral nerve injury.

Axons in the adult mammalian central nervous system fail to regenerate after injury. By contrast, spontaneous axon regeneration occurs in the peripheral nervous system (PNS) due to a supportive PNS environment and an increase in the intrinsic growth potential induced by injury via cooperative activation of multifaceted biological pathways. This study compared axon regeneration and injury responses in C57BL/6 male and female mice after sciatic nerve crush (SNC) injury. The extent of axon regeneration in vivo was indistinguishable in male and female mice when observed at 3days after SNC injury, and primary dorsal root ganglion (DRG) neurons from injured, male and female mice extended axons to a similar length. Moreover, the induction of selected regeneration-associated genes (RAGs), such as Atf3, Sprr1a, Gap43, Sox11, Jun, Gadd45a, and Smad1 were comparable in male and female DRGs when assessed by quantitative real-time reverse transcription polymerase chain reaction. Furthermore, the RNA-seq analysis of male and female DRGs revealed that differentially expressed genes (DEGs) in SNC groups compared to sham-operated groups included many common genes associated with neurite outgrowth. However, we also found that a large number of genes in the DEGs were sex dependent, implicating the involvement of distinct gene regulatory network in the two sexes following peripheral nerve injury. In conclusion, we found that male and female mice mounted a comparable axon regeneration response and many RAGs were commonly induced in response to SNC. However, given that many DEGs were sex-dependently expressed, future studies are needed to investigate whether they contribute to peripheral axon regeneration, and if so, to what extent.

Ketogenic diet prevents paclitaxel-induced neuropathic nociception through activation of PPARγ signalling pathway and inhibition of neuroinflammation in rat dorsal root ganglion.

Chemotherapy-induced peripheral neuropathy (CIPN) is a common side effect during the course of cancer treatment, which is mainly manifested as a series of sensory abnormalities. At present, there are no recommended prevention or treatment strategies, and the underlying mechanisms are unclear. The ketogenic diet (KD), a special diet that is high in fat and low in carbohydrate intake, shows good therapeutic potential in children with epilepsy. In this study, it was found that KD significantly prevented paclitaxel-induced neuropathic nociception. Using the GSE113941 database, 281 differentially expressed genes (DEGs) were found in an animal model of CIPN and controls. The DEGs were mainly enriched in peroxisome proliferator activated receptor (PPAR) and oxidative phosphorylation signalling pathways. As a main regulatory pathway of lipid metabolism, the PPARγ signalling pathway was significantly upregulated in the KD model. In addition, KD also inhibited the expression of pro-inflammatory cytokines and the TLR4/NF-κB signalling pathway in the dorsal root ganglion (DRG) in paclitaxel-treated rats. In vitro, rat primary DRG neurons were used to investigate the role of PPARγ in paclitaxel-induced neurotoxicity. It was found that PPARγ agonist rosiglitazone significantly protected DRG neurons against cell apoptosis and reactive oxygen species generation induced by paclitaxel administration. Therefore, KD is a prospective treatment option when applied as a dietary intervention in the prevention and treatment of paclitaxel-induced neuropathic nociception, possibly through the activation of PPARγ and its neuroprotective functions.

Development of EQ-6, a Novel Analogue of Ethoxyquin to Prevent Chemotherapy-Induced Peripheral Neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a common and often dose-limiting side effect of many cancer drugs. Because the onset of neuronal injury is known, it is an ideal clinical target to develop neuroprotective strategies. Several years ago, we had identified ethoxyquin as a potent neuroprotective drug against CIPN through a phenotypic drug screening and demonstrated a novel mechanism of action, inhibition of chaperone domain of heat shock protein 90. To improve its drug-like properties we synthesized a novel analogue of ethoxyquin and named it EQ-6 (6-(5-amino)-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline hydrochloride). Here we show that EQ-6 prevents axon degeneration in primary dorsal root ganglion neurons in vitro, and this axon protection is associated with preserved levels of nicotinamide adenine dinucleotide, a key metabolite in programmed axon degeneration pathway. We also found that EQ-6 prevents loss of epidermal nerve fibers in a mouse model of CIPN induced by paclitaxel and that doses of EQ-6 that provide neuroprotection are associated with reduced tissue levels of SF3B2, a potential biomarker of target engagement. Furthermore, we show that EQ-6 is safe in vitro and in mice with daily administration for a month. We found that oral bioavailability is about 10%, partly due to rapid metabolism in liver, but EQ-6 appears to be concentrated in neural tissues. Given these findings, we propose EQ-6 as a first-in-class drug to prevent CIPN.

Development of lipid nanoparticles for delivery of siRNA to neural cells

Lipid nanoparticles (LNPs) are a clinically approved platform to deliver siRNA to the liver. We are optimizing this platform to deliver siRNA for the treatment of pain. siRNA against TRPV1 (siTRPV1) was chosen as a proof of concept. siTRPV1-LNPs were formulated using C12-200, an ionizable cationic lipid and helper lipids. Their particle size and surface charge was characterized using dynamic light scattering and siRNA encapsulation efficiency was determined using a Ribogreen assay. A panel of breast cancer cell lines (MCF-7, MDA-MB-231 and BT-549) that express TRPV1 were selected for in vitro screening of LNPs to determine their cytocompatibility and gene knockdown effects using an ATP assay, qPCR and western blotting. The effects of mixing speeds during LNP formulation, slow vs. fast, were compared for determining their effects on %siRNA encapsulation. The values increased from 74.4% at slow mixing to 83.8% at faster mixing speeds. siRNA-LNPs showed a particle diameter of ca. 251 nm compared to 434 nm for empty LNPs. A lower polydispersity index of 0.15 was observed for siRNA-LNPs compared to 0.41 for empty LNPs, suggesting narrower particle size distribution upon siRNA loading. Zeta potential of siRNA-LNPs (+0.15 mV) was slightly lower compared to empty LNPs (+0.41 mV). siRNA-LNPs had a negligible impact on cell viability, which was >95% in all three cell lines and primary rat dorsal root ganglion neurons four hours post-exposure. We are currently optimizing transfection parameters to determine TRPV1 knockdown in primary DRG cultures. LNPs were well-tolerated by TRPV1-expressing cell lines and primary DRG cultures, allowing their further exploration for TRPV1 silencing.

Bortezomib activation of mTORC1 pathway mediated by NOX2-drived reactive oxygen species results in apoptosis in primary dorsal root ganglion neurons

Bortezomib (Bort), a chemotherapeutic agent, is widely used for the clinical treatment of cancers. However, Bort-induced peripheral neurotoxicity (BIPN) significantly restricts its clinical application, which is difficult to deal with since the underlying mechanisms of BIPN are unclear. Here, we showed that Bort activates mTORC1 pathway leading to dorsal root ganglion (DRG) neuronal apoptosis. Inhibition of mTORC1 with rapamycin or knockdown of raptor, regulatory-associated protein of mTORC1, with shRNA dramatically rescued the cells from Bort-caused apoptosis. In addition, we found that Bort-activated mTORC1 pathway was attributed to Bort elevation of reactive oxygen species (ROS). This is supported by the evidence that using ROS scavenger N-acetyl cysteine (NAC) significantly alleviated Bort-activated mTORC1 pathway. Furthermore, we revealed that upregulation of NOX2 contributed to Bort-elicited ROS overproduction, leading to mTORC1 pathway-dependent apoptosis in DRG neurons. Inhibition of NOX2 with apocynin remarkably diminished Bort-induced overgeneration of ROS, activation of mTORC1 pathway and apoptosis in the cells. Taken together, these results indicate that Bort activation of mTORC1 pathway mediated by NOX2-drived ROS leads to apoptotic death in DRG neurons. Our findings highlight that manipulation of intracellular ROS level or NOX2 or mTORC1 activity may be exploited for prevention of BIPN.

The neuroprotective effect of oxytocin on vincristine-induced neurotoxicity in mice

Vincristine (VCR) is commonly used to treat a variety of hematological malignancies and solid tumors in pediatric and adult patients. However, peripheral neuropathy is a dose-limiting side effect that leaves some patients with functional disability and long-term pain. Oxytocin (OT) has demonstrated analgesic and anti-inflammatory properties, but there is no evidence regarding its effects on VCR-induced neurotoxicity. Therefore, we evaluated the potential protective effects of OT on VCR-induced neurotoxicity. In vitro, VCR (0.005 ∼ 0.1 μmol/l) and OT (10−8 ∼ 10−5 mol/l) were added into cultured primary dorsal root ganglion (DRG) neurons of mice. The length of neurites was counted by using immunofluorescence. In vivo, neurotoxicity was induced in mice by administration of VCR (0.1 mg/kg, intraperitoneal injection for 14 days) with or without pretreatment of OT (0.1 mg/kg or 1 mg/kg). Atosiban, an OT receptor (OTR) antagonist and OTR knockout (KO) mice were used for evaluating effects of OTR. Mechanical hyperalgesia was measured by using von Frey filaments. Histology of plantar skin, sciatic nerve and DRG was observed by using transmission electron microscopy (TEM) and hematoxylin-eosin (HE) staining. Results indicated that OT alleviated VCR-induced neurite damage in cultured primary DRG neurons in vitro. In vivo, OT ameliorated VCR-induced hyperalgesia. Histologically, OT attenuated the VCR-induced damages of nerve endings, myelin sheaths and Schwann cells in sciatic nerve and DRG. These effects were antagonized by atosiban. In addition, OTR knockout mice exhibited more severe hyperalgesia than wild-type mice. Globally, these results indicated that OT may have neuroprotective effects on vincristine-induced neurotoxicity in mice.

Human Embryonic Stem Cell–derived Neural Crest Cells Promote Sprouting and Motor Recovery Following Spinal Cord Injury in Adult Rats

Spinal cord injury results in irreversible tissue damage and permanent sensorimotor impairment. The development of novel therapeutic strategies that improve the life quality of affected individuals is therefore of paramount importance. Cell transplantation is a promising approach for spinal cord injury treatment and the present study assesses the efficacy of human embryonic stem cell–derived neural crest cells as preclinical cell-based therapy candidates. The differentiated neural crest cells exhibited characteristic molecular signatures and produced a range of biologically active trophic factors that stimulated in vitro neurite outgrowth of rat primary dorsal root ganglia neurons. Transplantation of the neural crest cells into both acute and chronic rat cervical spinal cord injury models promoted remodeling of descending raphespinal projections and contributed to the partial recovery of forelimb motor function. The results achieved in this proof-of-concept study demonstrates that human embryonic stem cell–derived neural crest cells warrant further investigation as cell-based therapy candidates for the treatment of spinal cord injury.

Defining the regenerative effects of native spider silk fibers on primary Schwann cells, sensory neurons, and nerve-associated fibroblasts.

The search for a suitable material to promote regeneration after long‐distance peripheral nerve defects turned the spotlight on spider silk. Nerve conduits enriched with native spider silk fibers as internal guiding structures previously demonstrated a regenerative outcome similar to autologous nerve grafts in animal studies. Nevertheless, spider silk is a natural material with associated limitations for clinical use. A promising alternative is the production of recombinant silk fibers that should mimic the outstanding properties of their native counterpart. However, in vitro data on the regenerative features that native silk fibers provide for cells involved in nerve regeneration are scarce. Thus, there is a lack of reference parameters to evaluate whether recombinant silk fiber candidates will be eligible for nerve repair in vivo. To gain insight into the regenerative effect of native spider silk, our study aims to define the behavioral response of primary Schwann cells (SCs), nerve‐associated fibroblasts (FBs), and dorsal root ganglion (DRG) neurons cultured on native dragline silk from the genus Nephila and on laminin coated dishes. The established multi‐color immunostaining panels together with confocal microscopy and live cell imaging enabled the analysis of cell identity, morphology, proliferation, and migration on both substrates in detail. Our findings demonstrated that native spider silk rivals laminin coating as it allowed attachment and proliferation and supported the characteristic behavior of all tested cell types. Axonal out‐growth of DRG neurons occurred along longitudinally aligned SCs that formed sustained bundled structures resembling Bungner bands present in regenerating nerves. The migration of SCs along the silk fibers achieved the reported distance of regenerating axons of about 1 mm per day, but lacked directionality. Furthermore, rFBs significantly reduced the velocity of rSCs in co‐cultures on silk fibers. In summary, this study (a) reveals features recombinant silk must possess and what modifications or combinations could be useful for enhanced nerve repair and (b) provides assays to evaluate the regenerative performance of silk fibers in vitro before being applied as internal guiding structure in nerve conduits in vivo.

3D micro-organisation printing of mammalian cells to generate biological tissues

Significant strides have been made in the development of in vitro systems for disease modelling. However, the requirement of microenvironment control has placed limitations on the generation of relevant models. Herein, we present a biological tissue printing approach that employs open-volume microfluidics to position individual cells in complex 2D and 3D patterns, as well as in single cell arrays. The variety of bioprinted cell types employed, including skin epithelial (HaCaT), skin cancer (A431), liver cancer (Hep G2), and fibroblast (3T3-J2) cells, all of which exhibited excellent viability and survivability, allowing printed structures to rapidly develop into confluent tissues. To demonstrate a simple 2D oncology model, A431 and HaCaT cells were printed and grown into tissues. Furthermore, a basic skin model was established to probe drug response. 3D tissue formation was demonstrated by co-printing Hep G2 and 3T3-J2 cells onto an established fibroblast layer, the functionality of which was probed by measuring albumin production, and was found to be higher in comparison to both 2D and monoculture approaches. Bioprinting of primary cells was tested using acutely isolated primary rat dorsal root ganglia neurons, which survived and established processes. The presented technique offers a novel open-volume microfluidics approach to bioprint cells for the generation of biological tissues.

125-OR: Polyunsaturated Omega-6 Fatty Acids Reduce Axonal Mitochondrial Trafficking in Dorsal Root Ganglion Sensory Neurons

Peripheral neuropathy is a prevalent complication of diabetes and prediabetes characterized by distal-to-proximal peripheral nerve damage. Dyslipidemia is a contributory factor that underlies neuropathy development in type 2 diabetes and prediabetes. However, the molecular pathways that underlie sensory neuron dysfunction in peripheral neuropathy associated with diabetes and prediabetes are unknown. Our recent lipidomic and transcriptomic analyses identified altered metabolism of polyunsaturated omega-6 fatty acid linoleic acid in the peripheral nerves of prediabetic mice with neuropathy. However, the molecular effect of linoleic acid on sensory neurons remains unknown. Since sensory dorsal root ganglion (DRG) neurons are dependent on trafficking mechanisms to transport healthy mitochondria throughout the length of the axon, we evaluated the effect of linoleic acid on axonal mitochondrial trafficking and function in DRG neurons. Primary DRG neurons from adult C57BL/6J mice were treated with physiological concentrations of linoleic acid ranging from 31.25-250 µM for 24 hours. Following treatment, we evaluated the effect of linoleic acid treatments on mitochondrial trafficking, mitochondrial membrane potential, ATP level, and apoptosis in DRG neurons. Interestingly, all concentrations of linoleic acid ranging from 31.25-250 µM preserved mitochondrial membrane potential and ATP level in DRG neurons. In line with these results, linoleic acid treatments did not stimulate neuronal apoptosis. However, physiological diabetic concentrations of 250 µM linoleic acid significantly impaired mitochondrial trafficking in the axon of DRG neurons. Overall, these results suggest that elevated levels of linoleic acid impair axonal mitochondrial trafficking without effecting mitochondrial function, and that defects in mitochondrial trafficking of sensory neurons may contribute to neuropathy pathogenesis in diabetes and prediabetes. Disclosure A. Rumora: None. G. LoGrasso: None. J.D. McGrath: None. S.I. Lentz: None. E.L. Feldman: Consultant; Self; Novartis Pharmaceuticals Corporation. Funding American Diabetes Association (7-12-BS-045 to E.L.F.); National Institutes of Health (1R24082841, 1DP3DK094292); National Institute of Diabetes and Digestive and Kidney Diseases (T32DK101357, F32DK112642, K99DK119366, P30DK020572); NeuroNetwork for Emerging Therapies; A. Alfred Taubman Medical Research Institute