Primary Dorsal Root Ganglion Cells Research Articles

Extracellular membrane vesicles from Limosilactobacillus reuteri strengthen the intestinal epithelial integrity, modulate cytokine responses and antagonize activation of TRPV1.

Bacterial extracellular membrane vesicles (MV) are potent mediators of microbe-host signals, and they are not only important in host-pathogen interactions but also for the interactions between mutualistic bacteria and their hosts. Studies of MV derived from probiotics could enhance the understanding of these universal signal entities, and here we have studied MV derived from Limosilactobacillus reuteri DSM 17938 and BG-R46. The production of MV increased with cultivation time and after oxygen stress. Mass spectrometry-based proteomics analyses revealed that the MV carried a large number of bacterial cell surface proteins, several predicted to be involved in host-bacteria interactions. A 5'-nucleotidase, which catalyze the conversion of AMP into the signal molecule adenosine, was one of these and analysis of enzymatic activity showed that L. reuteri BG-R46 derived MV exhibited the highest activity. We also detected the TLR2 activator lipoteichoic acid on the MV. In models for host interactions, we first observed that L. reuteri MV were internalized by Caco-2/HT29-MTX epithelial cells, and in a dose-dependent manner decreased the leakage caused by enterotoxigenic Escherichia coli by up to 65%. Furthermore, the MV upregulated IL-1β and IL-6 from peripheral blood mononuclear cells (PBMC), but also dampened IFN-γ and TNF-α responses in PBMC challenged with Staphylococcus aureus. Finally, we showed that MV from the L. reuteri strains have an antagonistic effect on the pain receptor transient receptor potential vanilloid 1 in a model with primary dorsal root ganglion cells from rats. In summary, we have shown that these mobile nanometer scale MV reproduce several biological effects of L. reuteri cells and that the production parameters and selection of strain have an impact on the activity of the MV. This could potentially provide key information for development of innovative and more efficient probiotic products.

ラット初代神経後根節細胞を用いた細胞外電位計測によるオキサリプラチン急性毒性の評価

ラット初代神経後根節細胞を用いた細胞外電位計測によるオキサリプラチン急性毒性の評価

Intrathecal Injection of Dual Zipper Kinase shRNA Alleviating the Neuropathic Pain in a Chronic Constrictive Nerve Injury Model.

Dual leucine zipper kinase (DLK) is a member of mitogen-activated protein kinase kinase kinase (MAP3K) family mainly involved in neuronal degeneration. However, the role of DLK signaling in the neuropathic pain has not yet been fully determined. Chronic constrictive injury (CCI) was conducted by four 3-0 chromic gut ligatures loosely ligated around the sciatic nerve. Escalated DLK expression over the dorsal root ganglion was observed from one to four rings of CCI. Remarkable expression of DLK was observed in primary dorsal root ganglion cells culture subjected to electrical stimulation and attenuated by DLK short hairpin RNA (shRNA) treatment. Intrathecal injection of DLK shRNA attenuates the expression of DLK over the dorsal root ganglion and hippocampus neurons and increased the threshold of mechanical allodynia and decreased thermal hyperalgesia. In CatWalk gait analysis, significant decreases of print area, maximum contact maximum intensity, stand phase, single stance, and regular index by CCI were alleviated by the DLK shRNA administration. In conclusion, the expression of DLK was up-regulated in chronic constrictive injury and attenuated by the administration of DLK shRNA, which paralleled the improvement of neurobehavior of neuropathic pain. The modulation of DLK expression is a potential clinic treatment option for neuropathic pain.

Late administration of high-frequency electrical stimulation increases nerve regeneration without aggravating neuropathic pain in a nerve crush injury

BackgroundHigh-frequency transcutaneous neuromuscular electrical nerve stimulation (TENS) is currently used for the administration of electrical current in denervated muscle to alleviate muscle atrophy and enhance motor function; however, the time window (i.e. either immediate or delayed) for achieving benefit is still undetermined. In this study, we conducted an intervention of sciatic nerve crush injury using high-frequency TENS at different time points to assess the effect of motor and sensory functional recovery.ResultsAnimals with left sciatic nerve crush injury received TENS treatment starting immediately after injury or 1 week later at a high frequency(100 Hz) or at a low frequency (2 Hz) as a control. In SFI gait analysis, either immediate or late admission of high-frequency electrical stimulation exerted significant improvement compared to either immediate or late administration of low-frequency electrical stimulation. In an assessment of allodynia, immediate high frequency electrical stimulation caused a significantly decreased pain threshold compared to late high-frequency or low-frequency stimulation at immediate or late time points. Immunohistochemistry staining and western blot analysis of S-100 and NF-200 demonstrated that both immediate and late high frequency electrical stimulation showed a similar effect; however the effect was superior to that achieved with low frequency stimulation. Immediate high frequency electrical stimulation resulted in significant expression of TNF-α and synaptophysin in the dorsal root ganglion, somatosensory cortex, and hippocampus compared to late electrical stimulation, and this trend paralleled the observed effect on somatosensory evoked potential. The CatWalk gait analysis also showed that immediate electrical stimulation led to a significantly high regularity index. In primary dorsal root ganglion cells culture, high-frequency electrical stimulation also exerted a significant increase in expression of TNF-α, synaptophysin, and NGF in accordance with the in vivo results.ConclusionImmediate or late transcutaneous high-frequency electrical stimulation exhibited the potential to stimulate the motor nerve regeneration. However, immediate electrical stimulation had a predilection to develop neuropathic pain. A delay in TENS initiation appears to be a reasonable approach for nerve repair and provides the appropriate time profile for its clinical application.

Effect of ninjin'yoeito and ginseng extracts on oxaliplatin-induced neuropathies in mice.

Oxaliplatin (L-OHP) is a platinum-based anticancer agent used to treat various types of cancer. It frequently causes acute and chronic peripheral neuropathies, such as cold allodynia and mechanical hyperalgesia. Ninjin'yoeito (NYT) is a formula used in traditional Japanese Kampo medicine to improve recovery from diseases and other medical disorders. We previously reported that treatment with a boiling-water extract of NYT prevented L-OHP-induced damage to neurite-like outgrowths from differentiated PC12 cells. The objectives of the present study were to evaluate the in vivo effects of NYT on L-OHP-induced neuropathic pain in mice and identify the active ingredients in NYT. Treatment with NYT extract significantly ameliorated both cold allodynia and mechanical hyperalgesia induced by L-OHP. While L-OHP treatment suppressed neurite outgrowths from primary dorsal root ganglion cells in vitro, NYT extract blocked this suppression in a concentration-dependent manner. Among the herbal components of NYT, the extract of ginseng (Panax ginseng roots) showed a protective effect against neurite damage induced by L-OHP, and one of its active ingredients was identified as ginsenoside Rg3. Ginseng extract partially relieved L-OHP-induced neuropathic pain in mice. Our results suggest that NYT could be an attractive agent for treating L-OHP-induced neuropathic pain, and that the active ingredient of NYT may be ginseng.

Tempol Ameliorates and Prevents Mechanical Hyperalgesia in a Rat Model of Chemotherapy-Induced Neuropathic Pain.

Chemotherapy-induced neuropathic pain is difficult to treat and prevent. Tempol decreases cellular superoxide radical levels and oxidative stress. The aims of our study were to investigate the analgesic and preventive effects of tempol on paclitaxel-induced neuropathic pain in rats and to identify the associated mechanisms of action. Neuropathic pain was induced with intraperitoneally injected paclitaxel on four alternate days in male Sprague–Dawley rats. Tempol was administered systemically as a single injection and a continuous infusion before or after the injection of paclitaxel. The mechanical threshold for allodynia, protein levels, and free radical levels were measured using von Frey filaments, Western blotting, and live cell imaging, respectively. After the rats developed neuropathic pain behavior, a single intraperitoneal injection and continuous infusion of tempol ameliorated paclitaxel-induced mechanical allodynia. Systemic infusion of tempol in the early phase of the development of pain behavior prevented the development of paclitaxel-induced pain behavior. Paclitaxel increased the levels of phosphorylated protein kinase C, phosphorylated nuclear factor κB, phosphodiesterase 4D (PDE4D), IL-1β, and monocyte chemoattractant protein-1 in the lumbar dorsal root ganglia; however, tempol decreased these levels. Paclitaxel also increased superoxide levels in a culture of primary dorsal root ganglion cells and tempol decreased these levels. In conclusion, tempol alleviates and prevents chemotherapy-induced neuropathic pain in rats by reducing the levels of inflammatory cytokines and free radicals in dorsal root ganglia.

JunB regulates angiogenesis and neurovascular parallel alignment in mouse embryonic skin.

Blood vessels and nerve fibers are often closely arranged in parallel throughout the body. Therefore, neurovascular interactions have been suggested to be important for the development of vascular networks. However, the molecular mechanisms and genes regulating this process remain unclear. In the present study, we investigated the genes that are activated in endothelial cells (ECs) following interactions with neurons during vascular development. Microarray analyses of human primary microvascular ECs co-cultured with mouse primary dorsal root ganglion cells showed that JunB is strongly upregulated in ECs by neurovascular interactions. Furthermore, the forced expression of JunB in ECs stimulated a tip-like cell formation and angiogenesis in vitro and induced vascular endothelial growth factor A (VEGFA) and the pro-angiogenic integrin subunit ITGB3 expression. Moreover, in vivo knockdown of JunB in ECs from developing mouse limb skin considerably decreased the parallel alignments of blood vessels and nerve fibers. Taken together, the present data demonstrates for the first time that JunB plays an important role in the formation of embryonic vascular networks. These results contribute to the molecular understanding of neurovascular interactions during embryonic vascular development.

Regulable Transgene Expression in Dorsal Root Ganglia of a Replication-Defective Herpes Simplex Virus Type 1 Vector by Means of Sciatic Nerve Injection.

Targeted and controllable gene delivery to neurons is essential to efforts to facilitate peripheral nerve regeneration. The authors investigated both the in vitro and in vivo expression profiles of a tetracycline-controlled, replication-defective, herpes simplex virus type 1-based vector. Mouse primary dorsal root ganglia cells were infected with QR9TO-LacZ in the absence or presence of tetracycline. LacZ gene expression was examined. It was also injected into sciatic nerves in CD-1 mice fed with and without tetracycline. LacZ expression in the upstream dorsal root ganglia was examined. Following inoculation with QR9TO-LacZ, approximately 40 percent of the cultured primary dorsal root ganglia cells exhibited strong LacZ activity in the presence of tetracycline at 48 and 72 hours, whereas little was detected in those in the absence of tetracycline. Quantitative analysis revealed that the β-galactosidase activity within cells exposed to tetracycline increased 181-fold at 48 hours (p < 0.001) and 47-fold at 72 hours after infection (p < 0.05) compared with those without tetracycline. However, this LacZ transgene activity in the presence of tetracycline tapered off to less than sevenfold over baseline 168 hours after infection (p < 0.05). Furthermore, successful uptake of this replication-defective viral vector was evident in upstream dorsal root ganglia after sciatic nerve injection in mice. In addition, its expression profile was similar to that in vitro, as strong β-galactosidase activity was evident only in mice fed with a doxycycline-containing diet, and it tapered off by 168 hours. The replication-defective herpes simplex virus type 1-based vector, which provides tightly regulated transgene expression in dorsal root ganglia by means of peripheral nerve injection, represents an appealing approach to improve peripheral nerve regeneration.

Neuronal merlin influences ERBB2 receptor expression on Schwann cells through neuregulin 1 type III signalling

Axonal surface proteins encompass a group of heterogeneous molecules, which exert a variety of different functions in the highly interdependent relationship between axons and Schwann cells. We recently revealed that the tumour suppressor protein merlin, mutated in the hereditary tumour syndrome neurofibromatosis type 2, impacts significantly on axon structure maintenance in the peripheral nervous system. We now report on a role of neuronal merlin in the regulation of the axonal surface protein neuregulin 1 important for modulating Schwann cell differentiation and myelination. Specifically, neuregulin 1 type III expression is reduced in sciatic nerve tissue of neuron-specific knockout animals as well as in biopsies from seven patients with neurofibromatosis type 2. In vitro experiments performed on both the P19 neuronal cell line and primary dorsal root ganglion cells demonstrate the influence of merlin on neuregulin 1 type III expression. Moreover, expression of ERBB2, a Schwann cell receptor for neuregulin 1 ligands is increased in nerve tissue of both neuron-specific merlin knockout animals and patients with neurofibromatosis type 2, demonstrating for the first time that axonal merlin indirectly regulates Schwann cell behaviour. Collectively, we have identified that neuronally expressed merlin can influence Schwann cell activity in a cell-extrinsic manner.

Generation of oligodendroglial cells by direct lineage conversion

Transplantation of oligodendrocyte precursor cells (OPCs) is a promising potential therapeutic strategy for diseases affecting myelin. However, the derivation of engraftable OPCs from human pluripotent stem cells has proven difficult and primary OPCs are not readily available. Here we report the generation of induced OPCs (iOPCs) by direct lineage conversion. Forced expression of the three transcription factors Sox10, Olig2 and Zfp536 was sufficient to reprogram mouse and rat fibroblasts into iOPCs with morphologies and gene expression signatures resembling primary OPCs. More importantly, iOPCs gave rise to mature oligodendrocytes that could ensheath multiple host axons when co-cultured with primary dorsal root ganglion cells and formed myelin after transplantion into shiverer mice. We propose direct lineage reprogramming as a viable alternative approach for the generation of OPCs for use in disease modeling and regenerative medicine.

The Transcription Factor Runx3 Represses the Neurotrophin Receptor TrkB during Lineage Commitment of Dorsal Root Ganglion Neurons

Runx3, a Runt domain transcription factor, determines neurotrophin receptor phenotype in dorsal root ganglion (DRG) neurons. Molecular mechanisms by which Runx3 controls distinct neurotrophin receptors are largely unknown. Here, we show that RUNX3 abolished mRNA induction of TRKB expression, and concomitantly altered the neurotrophin response in a differentiating neuroblastoma cell line. In contrast, RUNX3 did not play a significant role in TRKC regulation even under the relevant BMP signaling pathway. We identified putative regulatory elements of Ntrk2/NTRK2 (a gene that codes for TrkB) using an unbiased computational approach. One of these elements was a highly conserved intronic sequence that contains a cluster of Runx binding sites. In a primary culture of DRG neurons, endogenous Runx3 bound to the consensus cluster, which had repressor activity against the Ntrk2 promoter under the control of NT-3 signaling. Consistent with these findings, Runx3-deficient embryos showed an increased number of trkB+ DRG neurons and failed to maintain trkC expression. Taken together, Runx3 determines TrkC positive sensory neuron identities through the transcriptional repression of TrkB when Trk-BTrkC double positive neurons differentiate into TrkC single positive neurons.

Neuronal plasticity after spinal cord injury: identification of a gene cluster driving neurite outgrowth

Functional recovery after spinal cord injury (SCI) may result in part from axon outgrowth and related plasticity through coordinated changes at the molecular level. We employed microarray analysis to identify a subset of genes the expression patterns of which were temporally coregulated and correlated to functional recovery after SCI. Steady-state mRNA levels of this synchronously regulated gene cluster were depressed in both ventral and dorsal horn neurons within 24 h after injury, followed by strong re-induction during the following 2 wk, which paralleled functional recovery. The identified cluster includes neuritin, attractin, microtubule-associated protein 1a, and myelin oligodendrocyte protein genes. Transcriptional and protein regulation of this novel gene cluster was also evaluated in spinal cord tissue and in single neurons and was shown to play a role in axonal plasticity. Finally, in vitro transfection experiments in primary dorsal root ganglion cells showed that cluster members act synergistically to drive neurite outgrowth.