Primary Cultured Dorsal Root Ganglion Neurons Research Articles

Propionate alleviates itch in murine models of atopic dermatitis by modulating sensory TRP channels of dorsal root ganglion.

Itch is the most common symptom of atopic dermatitis (AD) and significantly decreases the quality of life. Skin microbiome is involved in AD pathogenesis, whereas its role in the regulation of itch remains elusive. In this study, we aimed to investigate the effects of skin microbial metabolite propionate on acute and chronic pruritus and to explore the mechanism. Using various mouse models of itch, the roles of propionate were explored by behavioral tests and histopathology/immunofluorescent analysis. Primary-cultured dorsal root ganglion neurons and HEK293 cells expressing recombinant human TRP channels were utilized for invitro calcium imaging/in vivo miniature two-photon imaging in combination with electrophysiology and molecular docking approaches for investigation of the mechanism. Propionate significantly alleviated itch and alloknesis in various mouse models of pruritus and AD and decreased the density of intraepidermal nerve fibers. Propionate reduced the responsiveness of dorsal root ganglion neurons to pruritogens invitro, attenuated the hyper-excitability in sensory neurons in MC903-induced AD model, and inhibited capsaicin-evoked hTRPV1 currents (IC50 = 20.08 ± 1.11 μM) via interacting with the vanilloid binding site. Propionate also decreased the secretion of calcitonin gene-related peptide by nerves in MC903-induced AD mouse model, which further attenuated itch and skin inflammation. Our study revealed a protective effect of propionate against persistent itch through direct modulation of sensory TRP channels and neuropeptide production in neurons. Regulation of itch via the skin microbiome might be a novel strategy for the treatment of AD.

Increased extracellular release of microRNAs from dorsal root ganglion cells in a rat model of neuropathic pain caused by peripheral nerve injury.

microRNAs (miRNAs) are extracellularly released by cells for intercellular communication, while intracellularly, they inhibit the expression of specific genes. An increasing number of studies suggest that extracellular miRNAs have great potential as both therapeutic targets and disease-specific biomarkers in a variety of diseases, including pain disorders. However, little is known about miRNA release from dorsal root ganglion (DRG) neurons in neuropathic pain caused by peripheral nerve injury. In this study, we investigated the changes in the extracellular release of miRNAs from DRG neurons in a rat model of neuropathic pain induced by chronic constriction injury of the sciatic nerve. We found increased release of six miRNAs (let-7d, miR-21, miR-142-3p, miR-146b, miR-203-3p and miR-221) from primary cultured DRG neurons prepared from rats 7 days after nerve injury. Among these, miR-221 was also increased in serum from days 7 to 28 after nerve injury. In contrast, serum miR-221 levels and its release from DRG neurons were unchanged in an inflammatory pain model produced by intraplantar injection of complete Freund's adjuvant. These results suggest that the increased release of specific miRNAs by DRG neurons may be involved in the pathophysiology of neuropathic pain through extracellular as well as intracellular mechanisms. Furthermore, serum miR-221 may be useful as a biomarker of neuropathic pain caused by peripheral nerve injury.

Novel neuron-Schwann cell co-culture models to study peripheral nerve degeneration and regeneration.

Novel neuron-Schwann cell co-culture models to study peripheral nerve degeneration and regeneration.

Intramuscular injection of a plasmid DNA vector expressing hepatocyte growth factor (HGF) ameliorated pain symptoms by controlling the expression of pro-inflammatory cytokines in the dorsal root ganglion

Hepatocyte growth factor (HGF) is a secretory protein that is involved in various biological activities such as angiogenesis, neuroprotection, and anti-inflammatory effects. Intramuscular injection of an HGF-encoding plasmid DNA (pCK-HGF-X7) has been shown to produce pain-relieving effects in a rodent model and patients with neuropathic pain.To further investigate the underlying mechanism, we investigated the anti-inflammatory effects of HGF in the context of neuropathic pain. Consistent with previous data, intramuscular injection of pCK-HGF-X7 showed pain relieving effects up to 8 weeks and pharmacological blockade of the c-Met receptor hindered this effect, which suggest that the analgesic effect was c-Met receptor-dependent. At the histological level, macrophage infiltration in the dorsal root ganglion (DRG) was significantly decreased in the pCK-HGF-X7 injected group. Moreover, HGF treatment significantly downregulated the LPS-mediated induction of pro-inflammatory cytokines in primary cultured DRG neurons. Taken together, these data suggest that HGF-encoding plasmid DNA attenuates neuropathic pain via controlling the expression of pro-inflammatory cytokines.

Formalin Itch Test: Low-Dose Formalin Induces Histamine-Independent, TRPA1-Mediated Itch in Mice.

Chronic itch is a common distressing symptom of many diseases, which reduced patient's quality of life. The mechanistic study on itch and screening for new anti-itch drugs require the development of new pre-clinical itch animal models. Herein, we established an acute itch model by intradermal (i.d.) injection of low-dose formalin into the neck or cheek in mice. In mice, i.d. injection of formalin (0.1–5%) in the nape of the neck evoked robust scratching behavior in a dose-dependent manner and the dose–response curves showed an inverted “U” shape. I.d. injection of formalin (0.3–0.6%) into the cheek evoked scratching in mice but wiping in rats, while formalin (1.25–5%) induced mixed wiping and scratching behavior in both mice and rats. Further, we found that 0.3% formalin-induced scratching was histamine-independent and significantly attenuated by transient receptor potential ion channel A1 (TRPA1) inhibitor (HC030031) or in TRPA1 knockout (KO) mice, but not affected by transient receptor potential ion channel V1 (TRPV1) inhibitor (capsazepine) or in TRPV1 KO mice. Additionally, 0.3% formalin-induced up-regulation of phosphorylation of extracellular regulated protein kinases (p-ERK) in the dorsal root ganglion (DRG) and scratching were suppressed by intrathecal injection of MEK inhibitor U0126 in mice. Incubation of 0.03% formalin induced the accumulation of intracellular reactive oxygen species (ROS) in the cultured DRG-derived cell line ND7-23, and formalin-induced itch was suppressed by antioxidants in mice. Finally, perfusion of 0.03% formalin induced elevation of intracellular calcium in a subset of primary cultured DRG neurons of mice. Thus, these results indicate that low-dose formalin induced non-histaminergic itch by activation of TRPA1 in mice, which may be employed as a useful acute itch model for screening potential anti-itch drugs.

Clearance of Senescent Cells Reverses Cisplatin-induced Peripheral Neuropathy in Mice

Radiation and chemotherapy-induced neuropathy represent some of the most common causes of dose reduction and discontinuation of cancer treatment, often leaving patients with major permanent impairment of quality of life. Moreover, chemotherapy-induced neuropathy acts as a risk factor for development of radiation-induced neuropathy in patients receiving concomitant or adjuvant radiation therapy. The lack of effective treatments for or prevention of this debilitating neuronal toxicity not only compromises optimal treatment, but also leaves cancer survivors with significant disability. Here, we sought to investigate the biological role of senescence in cisplatin neurotoxicity and to evaluate the efficacy of pharmacological clearance of senescent cells. A murine model was established to measure the development of cisplatin-induced peripheral neuropathy (CIPN) in wild-type mice. The hyperalgesia seen in neuropathy was measured in terms of mechanical and thermal thresholds. SA-β-gal staining, p16 expression, and Mmp-9 mRNA expression were used to monitor senescence in primary cultured dorsal root ganglion (DRG) neurons. The effect of senescent cell clearance by the senolytic agent ABT-263 was examined using wild-type mice in the neuropathy model. Finally, using transgenic p16-3MR mice that allow for tracking and elimination of senescent cells by ganciclovir (GCV), we verified the efficacy of senescent cell clearance by ABT-263. CIPN was induced and measured in mice, showing consistent reduction in thermal and mechanical tolerance. Cisplatin provoked a significant increase in senescence in primary cultured DRG neurons as demonstrated by increased SA-β-gal positive cells, p16 expression, and Mmp-9 mRNA expression. Clearance of senescent cells using ABT-263 abolished CIPN in wild-type mice as evidenced by reversal of decreased mechanical and thermal thresholds to pre-cisplatin levels. Lastly, ABT-263 inhibition of senescence showed equally effective treatment of CIPN compared to GCV-mediated clearance of senescent cells in p16-3MR mice. ABT-263 treatment reverses cisplatin-induced peripheral neuropathy through clearance of senescent cells. Identification of this novel function warrants future investigation of senolytic-mediated reversal of neurotoxicity following platinum-based cancer treatment.

Dexmedetomidine modulates transient receptor potential vanilloid subtype 1

Dexmedetomidine, a highly selective alpha-2 adrenergic receptor agonist and novel sedative drug with minimal respiratory suppression, have shown anti-nociceptive activity in various pain models by poorly understood mechanisms. Because alpha-2 adrenergic receptor is co-localized with TRPV1 polymodal nociceptive receptor in dorsal root ganglion neurons and up-regulated in neuropathic pain animal models, the analgesic activity might be mediated through inhibition of TRPV1 in the peripheral nervous system. In an effort to elucidate whether modulatory effect of dexmedetomidine on TRPV1 activity could be the potential peripheral mechanism underlying the antinociceptive effect of dexmedetomidine, intracellular calcium concentration after capsaicin application was investigated in mice dorsal root ganglion (DRG) neurons, with and without pretreatment of dexmedetomidine. Dexmedetomidine (10 μM) reduced capsaicin-induced calcium responses by 29.7 ± 7.39% (n = 34, p < 0.0001), in dose-dependent manner. Higher level of inhibition was observed with increased dose of dexmedetomidine (50 μM, 45.1 ± 8.58%, n = 15, p = 0.0002), and lower inhibition by decreased dose (1 μM, 18.8 ± 1.48%, n = 148, p = 0.004). RT-PCR analysis revealed expression of TRPV1 and alpha-2A, alpha-2B and alpha-2C subtypes of adrenergic receptor in mice DRG neurons, and immunocytochemical analysis revealed co-expression of TRPV1 and alpha-2A receptors in primary cultured DRG neurons. In summary, these results suggested the inhibition of TRPV1 expressed in the primary sensory neurons as a potential mechanism that contributes to the anti-nociceptive action of dexmedetomidine.

Low-affinity neurotrophin receptor p75 of brain-derived neurotrophic factor contributes to cancer-induced bone pain by upregulating mTOR signaling.

Crucial to the development and maintenance of pain sensations is neurotrophin receptor p75 (p75NTR), the low affinity receptor of brain-derived neurotrophic factor (BDNF). This receptor is widespread among dorsal root ganglion (DRG) neurons and the spinal cord. Few reports have demonstrated the specific role of p75NTR in the development of cancer-induced bone pain (CIBP). Therefore the present study examined whether p75NTR contributed to CIBP by upregulating mammalian target of rapamycin (mTOR) signaling. A CIBP rat model was induced and reverse transcription-quantitative polymerase chain reaction was employed to determine p75NTR and mTOR mRNA expression. Immunofluorescence analysis was performed to determine the coexpression of p75NTR and mTOR in DRG neurons, as well as the spinal cord. Von Frey filaments were used to measure the 50% likelihood of paw withdrawal thresholds (PWTs). Spontaneous pain was assessed by ambulatory score. The results demonstrated that compared with the control group, mTOR activation in primary cultured DRG neurons was significantly increased. In addition, mTOR and p75NTR expression was significantly enhanced in the BDNF-treated primary DRG in the BDNF group. In vivo experiments determined that mTOR and p75NTR levels were increased in the CIBP rats compared with the sham group. PWT, in response to mechanical stimulation, was significantly lower compared with that in sham rats and the ambulatory score was significantly higher than that in sham rats. Finally, intrathecal injection of a p75NTR-targeting small interfering RNA significantly decreased mTOR and p75NTR expression levels in DRG neurons and the spinal cord of CIBP rats, as well as partially reversing the decline in PWTs and the increase in ambulatory score. In conclusion, the present study determined that the activation of BDNF/p75NTR/mTOR signaling may participate in nociceptive transmission in CIBP, suggesting a novel mechanism and potential therapeutic target for CIBP treatment and management.

A TLR\u2013CXCL1 pathway in DRG neurons induces neutrophil accumulation in the DRG and mechanical allodynia in EAE mice

Multiple sclerosis (MS) is a potentially disabling disease of the central nervous system. Approximately half of the patients with MS experience severe pain; however, currently available therapeutics provide only insufficient relief. The mechanisms underlying the generation of neuropathic pain in patients with MS are not fully understood. Recently, we found that neutrophil elastase from accumulated neutrophils in the dorsal root ganglion (DRG) sensitizes DRG neurons and induces mechanical allodynia in a mouse model of experimental autoimmune encephalomyelitis (EAE). However, the mechanism underlying neutrophil accumulation in the DRG after myelin oligodendrocyte glycoprotein (MOG35–55, immunogenic peptide) immunization remains unclear. Here, we found that C-X-C motif ligand 1 (CXCL1) was upregulated in DRG neurons after MOG35–55 immunization. Increased expression of CXCL1 protein was also observed in primary cultured DRG neurons treated with MOG35–55, which was mediated through toll-like receptor 4 (TLR4). Gene silencing of TLR4 or CXCL1 in DRG neurons significantly attenuated neutrophil accumulation in the DRG and mechanical allodynia during the preclinical phase of EAE (around day 5 after immunization). Our results thus suggest that a TLR4–CXCL1 pathway in DRG neurons triggers neutrophil recruitment in the DRG and subsequent mechanical allodynia in response to MOG35–55.

Upregulation of interleukin-6 on Cav3.2 T-type calcium channels in dorsal root ganglion neurons contributes to neuropathic pain in rats with spinal nerve ligation

The T-type calcium channels Cav3.2, one of the low voltage-activated (LVA) calcium channels, have been found to play important roles in the neuronal excitability. Recently, we and others have demonstrated that accumulation of Cav3.2 channels in the dorsal root ganglion (DRG) neurons and sensory nerves contributes to neuropathic pain after peripheral nerve injury. In the present study, we aimed to further investigate the regulation of Cav3.2 channels by interleukin-6 (IL-6) in DRG neurons in neuropathic pain rats after spinal nerve ligation (SNL). The results showed that Cav3.2 channel protein expression in L5 DRG neurons was upregulated and blockade of this channel decreased the hyperexcitability of DRG neurons and mechanical allodynia in SNL neuropathic pain rats. Furthermore, inhibition of IL-6 trans-signaling reduced the upregulation of Cav3.2 T-type channel induced by FIL-6 (a fusion protein of IL-6 and sIL-6R) in primary cultured DRG neurons in vitro. In vivo, inhibition of IL-6 trans-signaling reversed the upregulation of Cav3.2, reduced the hyperexcitability of L5 DRG neurons and alleviated mechanical allodynia in SNL rats. Our results suggest that IL-6 upregulates Cav3.2 T-type channels expression and function through the IL-6/sIL-6R trans-signaling pathway in DRG neurons, thus contributes to the development of neuropathic pain in SNL rats.

The Association of Neuronal Stress with Activating Transcription Factor 3 in Dorsal Root Ganglion of in vivo and in vitro Models of Bortezomib- Induced Neuropathy.

The notion that proteasome inhibitor bortezomib (BTZ) induced intracellular oxidative stress resulting in peripheral neuropathy has been generally accepted. The association of mitochondrial dysfunction, cell apoptosis, and endoplasmic reticulum (ER) stress with intracellular oxidative stress is ambiguous and still needs to be investigated. The activation of activating transcription factor 3 (ATF3) is a stress-hub gene which was upregulated in dorsal root ganglion (DRG) neurons after different kinds of peripheral nerve injuries. To investigate a mechanism underlying the action of BTZ-induced intracellular oxidative stress, mitochondrial dysfunction, cell apoptosis, and ER stress via activation of ATF3. Primary cultured DRG neurons with BTZ induced neurotoxicity and DRG from BTZ induced painful peripheral neuropathic rats were used to approach these questions. BTZ administration caused the upregulation of ATF3 paralleled with intracellular oxidative stress, mitochondrial dysfunction, cell apoptosis, and ER stress in DRG neurons both in vitro and in vivo. Blocking ATF3 signaling by small interfering RNA (siRNA) gene silencing technology resulted in decreased intracellular oxidative stress, mitochondrial dysfunction, cell apoptosis, and ER stress in DRG neurons after BTZ treatment. This study exhibited important mechanistic insight into how BTZ induces neurotoxicity through the activation of ATF3 resulting in intracellular oxidative stress, mitochondrial dysfunction, cell apoptosis, and ER stress and provided a novel potential therapeutic target by blocking ATF3 signaling.

Neurotropin inhibits neuronal activity through potentiation of sustained Kv currents in primary cultured DRG neurons

Neurotropin (NTP) is a Japanese analgesic agent for treating neuropathic pain; however, its method of action remains unclear. This study examined the effects of NTP on the activity of small dorsal root ganglion (DRG) neurons using whole-cell patch clamp recordings. After 3 days of treatment, NTP decreased current injection-induced firing activity of cultured DRG neurons by raising the current threshold for action potential generation. Additionally, NTP increased the sustained component of voltage-gated potassium (Kv) channel currents without affecting other K+ currents. These results suggest that NTP inhibits the firing activity of DRG neurons through augmentation of sustained Kv current.

Advanced Oxidative Protein Products Cause Pain Hypersensitivity in Rats by Inducing Dorsal Root Ganglion Neurons Apoptosis via NADPH Oxidase 4/c-Jun N-terminal Kinase Pathways

Pain hypersensitivity is the most common category of chronic pain and is difficult to cure. Oxidative stress and certain cells apoptosis, such as dorsal root ganglion (DRG) neurons, play an essential role in the induction and development of pain hypersensitivity. The focus of this study is at a more specific molecular level. We investigated the role of advanced oxidative protein products (AOPPs) in inducing hypersensitivity and the cellular mechanism underlying the proapoptotic effect of AOPPs. Normal rats were injected by AOPPs-Rat serum albumin (AOPPs–RSA) to cause pain hypersensitivity. Primary cultured DRG neurons were treated with increasing concentrations of AOPPs–RSA or for increasing time durations. The MTT, flow cytometry and western blot analyses were performed in the DRG neurons. A loss of mitochondrial membrane potential (MMP) and an increase in intracellular reactive oxygen species (ROS) were observed. We found that AOPPs triggered DRG neurons apoptosis and MMP loss. After AOPPs treatment, intracellular ROS generation increased in a time- and dose-dependent manner, whereas, N-acetyl-L-cysteine (NAC), a specific ROS scavenger could inhibit the ROS generation. Proapoptotic proteins, such as Bax, caspase 9/caspase 3, and PARP-1 were activated, whereas anti-apoptotic Bcl-2 protein was down-regulated. AOPPs also increased Nox4 and JNK expression. Taken together, these findings suggest that AOPPs cause pain hypersensitivity in rats, and extracellular AOPPs accumulation triggered Nox4-dependent ROS production, which activated JNK, and induced DRG neurons apoptosis by activating caspase 3 and PARP-1.

The Protective Effects of IGF-1 on Different Subpopulations of DRG Neurons with Neurotoxicity Induced by gp120 and Dideoxycytidine In Vitro.

Peripheral neuropathy induced by human immunodeficiency virus (HIV) infection and antiretroviral therapy is not only difficult to distinguish in clinical practice, but also difficult to relieve the pain symptoms by analgesics because of the severity of the disease at the later stage. Hence, to explore the mechanisms of HIV-related neuropathy and find new therapeutic options are particularly important for relieving neuropathic pain symptoms of the patients. In the present study, primary cultured embryonic rat dorsal root ganglion (DRG) neurons were used to determine the neurotoxic effects of HIV-gp120 protein and/or antiretroviral drug dideoxycytidine (ddC) and the therapeutic actions of insulin-like growth factor-1 (IGF-1) on gp120- or ddC-induced neurotoxicity. DRG neurons were exposed to gp120 (500 pmol/L), ddC (50 μmol/L), gp120 (500 pmol/L) plus ddC (50 μmol/L), gp120 (500 pmol/L) plus IGF-1 (20 nmol/L), ddC (50 μmol/L) plus IGF-1 (20 nmol/L), gp120 (500 pmol/L) plus ddC (50 μmol/L) plus IGF-1 (20 nmol/L), respectively, for 72 hours. The results showed that gp120 and/or ddC caused neurotoxicity of primary cultured DRG neurons. Interestingly, the severity of neurotoxicity induced by gp120 and ddC was different in different subpopulation of DRG neurons. gp120 mainly affected large diameter DRG neurons (>25 μm), whereas ddC mainly affected small diameter DRG neurons (≤25 μm). IGF-1 could reverse the neurotoxicity induced by gp120 and/or ddC on small, but not large, DRG neurons. These data provide new insights in elucidating the pathogenesis of HIV infection- or antiretroviral therapy-related peripheral neuropathy and facilitating the development of novel treatment strategies.

Insulin-like growth factor-1 prevents dorsal root ganglion neuronal tyrosine kinase receptor expression alterations induced by dideoxycytidine in vitro.

Dideoxycytidine (zalcitabine, ddC) produces neurotoxic effects. It is particularly important to understand the toxic effects of ddC on different subpopulations of dorsal root ganglion (DRG) neurons which express distinct tyrosine kinase receptor (Trk) and to find therapeutic factors for prevention and therapy for ddC-induced peripheral sensory neuropathy. Insulin-like growth factor-1 (IGF-1) has been shown to have neurotrophic effects on DRG sensory neurons. However, little is known about the effects of ddC on distinct Trk (TrkA, TrkB, and TrkC) expression in DRG neurons and the neuroprotective effects of IGF-1 on ddC-induced neurotoxicity. Here, we have tested the extent to which the expression of TrkA, TrkB, and TrkC receptors in primary cultured DRG neurons is affected by ddC in the presence or absence of IGF-1. In this experiment, we found that exposure of 5, 25, and 50μmol/L ddC caused a dose-dependent decrease of the mRNA, protein, and the proportion of TrkA-, TrkB-, and TrkC-expressing neurons. IGF-1 (20nmol/L) could partially reverse the decrease of TrkA and TrkB, but not TrkC, expression with ddC exposure. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (10μmol/L) blocked the effects of IGF-1. These results suggested that the subpopulations of DRG neurons which express distinct TrkA, TrkB, and TrkC receptors were affected by ddC exposure. IGF-1 might relieve the ddC-induced toxicity of TrkA- and TrkB-, but not TrkC-expressing DRG neurons. These data offer new clues for a better understanding of the association of ddC with distinct Trk receptor expression and provide new evidence of the potential therapeutic role of IGF-1 on ddC-induced neurotoxicity.

Enhanced function of TRPV1 via up-regulation by insulin-like growth factor-1 in a rat model of bone cancer pain.

Up-regulation of transient receptor potential vanilloid subfamily, member 1 (TRPV1) is associated with the development and maintenance of cancer pain. The present study aimed to investigate the electrophysiological function of the up-regulated TRPV1 and the potential regulatory effects of insulin-like growth factor-1 (IGF-1) on TRPV1 expression in peripheral nerves in a rat model of bone cancer pain. A bone cancer pain model of rats was established by injecting MRMT-1 (rat mammary gland carcinoma cells) breast cancer cells into the tibia bone cavity. Thermal hyperalgesia was assessed by paw-withdrawal latency to a thermal stimulus, and mechanical allodynia was measured with von Frey monofilaments. TRPV1 and IGF-1 expression were examined with immunohistochemical staining and Western blot. TRPV1 current density of dorsal root ganglion (DRG) neurons was measured with whole-cell patch clamping recording technique. Rats showed thermal hyperalgesia and mechanical allodynia 14-21 days after MRMT-1 inoculation into the tibia bone marrow. TRPV1 protein expression and its current density increased in DRG neurons. At the same time, IGF-1 expression increased in tibia bone cavity, and IGF-1 incubation increased total or membrane TRPV1 protein expression and TRPV1 current in primary cultured DRG neurons. Inhibition of IGF-1 receptors in vivo reversed mechanical allodynia and thermal hyperalgesia in rats with bone cancer pain. Our results provide novel evidence for the increase of IGF-1 in tibia bone marrow, which is responsible for the up-regulation of TRPV1 expression and function in the peripheral nerves of bone cancer pain rats.

Neuroprotective effect of insulin-like growth factor-1: Effects on tyrosine kinase receptor (Trk) expression in dorsal root ganglion neurons with glutamate-induced excitotoxicity in vitro

Insulin-like growth factor-1 (IGF-1) may play an important role in regulating the expression of distinct tyrosine kinase receptor (Trk) in primary sensory dorsal root ganglion (DRG) neurons. Glutamate (Glu) is the main excitatory neurotransmitter and induces neuronal excitotoxicity for primary sensory neurons. It is not known whether IGF-1 influences expression of TrkA, TrkB, and TrkC in DRG neurons with excitotoxicity induced by Glu. In the present study, primary cultured DRG neurons with Glu-induced excitotoxicity were used to determine the effects of IGF-1 on TrkA, TrkB, and TrkC expression. The results showed that IGF-1 increased the expression of TrkA and TrkB and their mRNAs, but not TrkC and its mRNA, in primary cultured DRG neurons with excitotoxicity induced by Glu. Interestingly, neither the extracellular signal-regulated protein kinase (ERK1/2) inhibitor PD98059 nor the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 blocked the effect of IGF-1, but both inhibitors together were effective. IGF-1 may play an important role in regulating different Trk receptor expression in DRG neurons through ERK1/2 and PI3K/Akt signaling pathways. The contribution of distinct Trk receptors might be one of the mechanisms that IGF-1 rescues dying neurons from Glu excitotoxic injury. These data imply that IGF-1 signaling might be a potential target on modifying distinct Trk receptor-mediated biological effects of primary sensory neurons with excitotoxicity.

Different responses of galanin and calcitonin gene-related peptide to capsaicin stimulation on dorsal root ganglion neurons in vitro

Both galanin (Gal) and calcitonin gene-related peptide (CGRP) are sensory neuropeptides which expressed in dorsal root ganglion (DRG) neurons and are involved in nociceptive processing. Capsaicin (CAP) influences nociceptive processing via influencing the expression of sensory neuropeptides in primary sensory neurons. However, little is known about the alterations of Gal and CGRP expression at the same condition stimulated by CAP. In the present study, primary cultured DRG neurons were used to determine the different responses of Gal and CGRP to CAP stimulation. DRG neurons were cultured for 48hours and then exposed to CAP (2μmol/L), capsazepine (CPZ) (2μmol/L) plus CAP (2μmol/L), or extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor PD98059 (10μmol/L) plus CAP (2μmol/L) for an additional 24hours. The DRG neurons were continuously exposed to culture media as a control. After that, the levels of Gal mRNA and CGRP mRNA of DRG neurons were determined using real time-PCR analysis. Gal and CGRP expression in situ was detected by an immunofluorescent labeling technique. The levels of phosphorylated-ERK1/2 (pERK1/2) protein were detected using a Western blot assay. The results showed that CAP evoked increases of Gal and its mRNA and decreases of CGRP and its mRNA in DRG neurons. Administration of either CPZ or PD98059 blocked the effects of CAP. These data indicate that Gal and CGRP shared different responses to CAP stimulation. Gal and CGRP may have different effects in nociceptive processing during neurogenic inflammation.

Alterations in tyrosine kinase receptor (Trk) expression induced by insulin-like growth factor-1 in cultured dorsal root ganglion neurons

Insulin-like growth factor-1 (IGF-1) is a neurotrophic factor expressed in small dorsal root ganglion (DRG) neurons. IGF-1 promotes neuronal survival by activating its receptor (IGF-1R). Whether IGF-1 and its signaling pathways influence the expression of tyrosine kinase receptors TrkA, TrkB and TrkC in DRG neurons remains unknown. In the present study, primary cultured DRG neurons were used to determine the effects of IGF-1 on TrkA, TrkB and TrkC expression. The involvement of extracellular signal-regulated protein kinase (ERK1/2) and the effects of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways on IGF-1 were also evaluated. DRG neurons were cultured for 48h and then exposed to IGF-1, PD98059 plus IGF-1, LY294002 plus IGF-1, and PD98059 plus LY294002 plus IGF-1 for an additional 24h. The DRG neurons were continuously exposed to culture medium as a control. All cultures were then processed for detection of mRNA levels of TrkA, TrkB and TrkC using real-time PCR analysis. Protein levels of TrkA, TrkB and TrkC were detected using a Western blot assay. The expression of TrkA, TrkB and TrkC in situ was determined by a fluorescent labeling technique. The levels of phosphorylated ERK1/2 (pERK1/2) and phosphorylated Akt (pAkt) were detected using a Western blot assay. The results indicated that in primary cultured DRG neurons, IGF-1 increased the expression of TrkA and TrkB and their mRNAs but not TrkC or its mRNA. Neither the ERK1/2 inhibitor PD98059 nor the PI3K inhibitor LY294002 alone blocked the effect of IGF-1, but the use of both inhibitors together was effective. IGF-1 may play an important role in regulating the expression of different Trk receptors in DRG neurons through the ERK1/2 and PI3K/Akt signaling pathways. These results suggest that IGF-1 signaling might be a potential target on modifying distinct Trk receptor-mediated biological effects.

Expression of tyrosine kinase receptors in cultured dorsal root ganglion neurons in the presence of monosialoganglioside and skeletal muscle cells

The neurotrophic factor-like activity of monosialoganglioside (GM1) has been shown to activate tyrosine kinase receptors (Trk). Targets of neuronal innervation play a vital role in regulating the survival and differentiation of innervating neurotrophin-responsive neurons. Both GM1 and target skeletal muscle (SKM) cells are essential for the maintenance of the function of neurons. However, much less is known about the effects of GM1 or/and target SKM cells on the expression of Trk receptors in dorsal root ganglion (DRG) neurons. Here we have tested what extent to the expression of TrkA, TrkB, and TrkC receptors in primary cultured of DRG neurons in absence or presence of GM1 or/and SKM cells. In this experiment, we found that: (1) GM1 promoted expression of TrkA and TrkB but not TrkC in primary cultured DRG neurons; (2) target SKM cells promoted expression of TrkC but not TrkA and TrkB in neuromuscular cocultures without GM1 treatment; and (3) GM1 and target SKM cells had additional effects on expression of these three Trk receptors. The results of the present study offered new clues for a better understanding of the association of GM1 and target SKM on the expression of Trk receptors.