Zhen Liu

Regulatory effect of nerve growth factor on release of substance P in cultured dorsal root ganglion neurons of rat.

To investigate the regulatory effects of nerve growth factor (NGF) on basal and capsaicin-induced release of neuropeptide substance P (SP) in primary cultured embryonic rat dorsal root ganglion (DRG) neurons. DRGs were dissected from 15-day-old embryonic Wistar rats. DRG neurons were dissociated and cultured, and then exposed to different concentrations of NGF (10 ng/mL, 30 ng/mL, or 100 ng/mL) for 72 h. The neurons cultured in media without NGF served as control. RT-PCR were used for detecting the mRNAs of SP and vanilloid receptor 1 (VR1) in the DRG neurons. The SP basal and capsaicin (100 nmol/L)-induced release in the culture were measured by radioimmunoassay (RIA). SP mRNA and VR1 mRNA expression increased in primary cultured DRG neurons in a dose-dependent manner of NGF. Both basal release and capsaicin-evoked release of SP increased in NGF-treated DRG neurons compared with in control group. The capsaicin-evoked release of SP also increased in a dose-dependent manner of NGF. NGF may promote both basal release and capsaicin-evoked release of SP. NGF might increase the sensitivity of nociceptors by increasing the SP mRNA or VR1 mRNA. 观察神经生长因子 (nerve growth factor, NGF) 对原代培养的背根神经节(dorsal root ganglion, DRG)神经元中 P 物质 (substance P, SP) 的基础释放量和辣椒素诱发释放量的调节效应。 将 15 天胚龄的 Wistar 大鼠 DRG 神经元培养于含有不同浓度 NGF 的 DMEM/F12 培养液中, 不含 NGF 的培养液培养的神经元作为对照。 72 小时后, 用RT-PCR检测神经元中 SP mRNA 和辣椒素受体(vanilloid receptor 1, VR1)mRNA 的表达, 用放射免疫分析 (radioimmunoassay, RIA) 法检测 SP 的基础释放量和辣椒素 (100 nmol/L) 刺激 10 min 后的诱发释放量。 SP mRNA 和VR1 mRNA 在 NGF 孵育的标本中表达增加, 并与孵育液中 NGF 的浓度呈剂量依赖关系。 SP 的基础释放量和辣椒素诱发释放量在 NGF 孵育的标本中均增加?而且诱发释放量与 NGF 的浓度呈剂量依赖关系。 NGF 使 DRG 神经元SP的基础释放量和诱发释放量增加, 表明 NGF 能增加初级传入神经元感受伤害刺激的敏感性, 该效应可能与SP 和VR1 的 mRNA 表达增加有关。

Neuregulin-1β regulates outgrowth of neurites and migration of neurofilament 200 neurons from dorsal root ganglial explants in vitro

Neuregulin-1β (NRG-1β) signaling has multiple functions in neurons. To assess NRG-1β on neurite outgrowth and neuronal migration in vitro, organotypic dorsal root ganglion (DRG) neuronal culture model was established. Neurite outgrowth and neuronal migration were evaluated using this culture model in the presence (5nmol/L, 10nmol/L, 20nmol/L) or absence of NRG-1β. Neurofilament 200 (NF-200)-immunoreactive (IR) neurons were determined as the migrating neurons. The number of nerve fiber bundles extended from DRG explant increased significantly in the presence of NRG-1β (5nmol/L, 23.0±2.2, P<0.05; 10nmol/L, 27.0±2.7, P<0.001; 20nmol/L, 30.8±3.7, P<0.001) as compared with that in the absence of NRG-1β (19.0±2.2). The number of neurons migrating from DRG explants increased significantly in the presence of NRG-1β (5nmol/L, 39.6±5.0, P<0.05; 10nmol/L, 54.6±6.7, P<0.001; 20nmol/L, 62.2±5.7, P<0.001) as compared with that in the absence of NRG-1β (31.6±4.0). Moreover, the increase of the number of nerve fiber bundles and the number of migrating NF-200-IR neurons was dose-dependent for NRG-1β addition. The data in this study imply that NRG-1β promotes neurite outgrowth and neuronal migration from DRG explants in vitro.

The effects of galanin on neuropathic pain in streptozotocin-induced diabetic rats.

Diabetic neuropathy is a common complication associated with diabetes and is frequently painful. However, mechanisms responsible for diabetic neuropathic pain are still unclear. Experimental evidence has shown that the galanin and its receptor are involved in pain sensitization. The objective of the present study was to investigate the role of galanin and its receptor antagonist or agonist on neuropathic pain in streptozotocin-induced diabetic rats. The expression of galanin, galanin receptors 1 and 2 in dorsal root ganglion (DRG) and spinal dorsal horn (SDH) in diabetic rats were detected by Western blot assay. The effects of galanin, galanin receptor antagonist M35, galanin receptor 1 agonist M617, and galanin receptor 2 agonist AR-M1896 on neuropathic pain were evaluated by mechanical stimuli. The results showed that (1) the diabetic rats showed a significant mechanical hyperalgesia between 4 and 12weeks; (2) galanin receptor 1 expression decreased in SDH in diabetic rats; (3) galanin receptor 2 expression decreased in DRG and SDH in diabetic rats; (4) intrathecal administration of exogenous galanin attenuated diabetic neuropathic pain, this effect could be blocked by pre-treatment with galanin receptor antagonist M35; and (5) intrathecal administration of galanin receptor 1 agonist M617, but not galanin receptor 2 agonist AR-M1896, attenuated diabetic neuropathic pain. These results imply that galanin acts through receptor 1, but not galanin receptor 2, to exert analgesic effect in diabetic neuropathic pain and is one of the potential therapeutic targets on diabetic neuropathic pain sensitization.

Neuronal phenotype and tyrosine kinase receptor expression in cocultures of dorsal root ganglion and skeletal muscle cells.

The neuropeptide‐immunoreactive (IR) and neurofilament‐IR neurons are two major phenotypical classes in dorsal root ganglion (DRG). Tyrosine kinase receptor (Trk)A, TrkB, and TrkC are three members of the Trk family which may be relevant to neuronal phenotypes. Whether target skeletal muscle cells generate their expression remains unclear. Neurons containing substance P (SP), calcitonin gene‐related peptide (CGRP), neurofilament 200 (NF‐200), TrkA, TrkB, and TrkC were quantified using immunohistochemistry in rat DRG neuronal cultures and cocultures of DRG neurons and skeletal muscle cells. The percentage of NF‐200 and TrkC‐expressing neurons in cocultures of DRG neurons and skeletal muscle cells was significantly higher, 26.86% ± 3.17% (NF‐200) and 27.74% ± 3.63% (TrkC) compared with 20.92% ± 1.98% (NF‐200) and 16.70% ± 3.68% (TrkC) in DRG cultures; whereas the percentage of SP, CGRP, TrkA, and TrkB‐expressing neurons was not changed significantly by the addition of target skeletal muscle cells. Thus, target skeletal muscle cells may influence neurofilament‐phenotype and TrkC receptor but not neuropeptide‐phenotype and TrkA and TrkB receptors. Anat Rec, 2009.

GM1 and NGF modulate Ca2+ homeostasis and GAP43 mRNA expression in cultured dorsal root ganglion neurons with excitotoxicity induced by glutamate

Abstract Monosialoganglioside (GM1) has been considered to have a neurotrophic factor-like activity. Nerve growth factor (NGF), a member of the neurotrophin family, is essential for neuronal survival, differentiation and maturation. The aim of the present study was to investigate whether co-administration of GM1 and NGF reverses glutamate (Glu) neurotoxicity in primary cultured rat embryonic dorsal root ganglion (DRG) neurons. DRG neurons were exposed to Glu (2 mmol/l), Glu (2 mmol/l) plus GM1 (10 μg/ml), Glu (2 mmol/l) plus NGF (10 ng/ml), Glu (2 mmol/l) plus GM1 (5 μg/ml) and NGF (5 ng/ml) and then processed for detecting intracellular concentrations of Ca2+ ([Ca2−] i ) by confocal laser scanning microscopy and growthassociated protein 43 (GAP43) mRNA by RT-PCR. The fluorescent intensity in Glu plus GM1 and NGF incubated neurons was the lowest as compared with that in other groups. The expression of GAP43 mRNA in Glu plus GM1 and NGF incubated neurons was the highest as compared with that in other groups. These results implicated that GM1 and NGF have synergistic neuroprotective effects on DRG neurons with excitotoxicity induced by Glu in vitro.

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.

Co-administration of Monosialoganglioside and Skeletal Muscle Cells on Dorsal Root Ganglion Neuronal Phenotypes In Vitro

The neuropeptide-immunoreactive (IR) and neurofilament-IR neurons are two major phenotypical classes in dorsal root ganglion (DRG). Targets of neuronal innervation play a vital role in regulating the survival and differentiation of innervating neurotrophin-responsive neurons. Monosialoganglioside (GM1) has been considered to have a neurotrophic factor-like activity. Both GM1 and target skeletal muscle (SKM) cells are essential for the maintenance of the function of neurons. However, whether target SKM cells and GM1, alone or associated, generate neuropeptide or neurofilament expression remains unclear. The aim of the present study is to investigate the effects of GM1 or/and SKM on DRG neuronal phenotypes. DRG neurons containing the neuropeptide substance P (SP) and neurofilament 200 (NF-200) were quantified using immunofluorescent labeling in cultures of DRG, which was dissected out at times before (at embryonic days 12.5, E12.5) and after (at E19.5) sensory neurons contact peripheral targets in vivo. DRG neurons were cultured in absence or presence of GM1 or/and SKM cells. In this experiment, we found that: (1) GM1 promoted expression of SP and NF-200 in E12.5 DRG cultures; (2) SKM cells promoted expression of NF-200 but not SP in E12.5 DRG cultures; (3) GM1 and target SKM cells had additive effects on expression of SP and NF-200 in E12.5 DRG cultures; and (4) SKM or/and GM1 did not have effects on expression of SP and NF-200 in E19.5 DRG cultures. These results suggested that GM1 could influence DRG, two major neuronal phenotypes, before sensory neurons contact peripheral targets in vivo. Target SKM cells could only influence neurofilament-expressed neuronal phenotype before sensory neurons contact peripheral targets in vivo. GM1 and SKM cells had the additive effects on two major DRG neuronal classes, which express neuropeptide or neurofilament when DRG cells were harvested before sensory neurons contact peripheral targets in vivo. These results offered new clues for a better understanding of the association of GM1 or/and SKM with neuronal phenotypes.

Erythropoietin attenuates oxidative stress and apoptosis in Schwann cells isolated from streptozotocin-induced diabetic rats.

High glucose‐evoked oxidative stress and apoptosis within Schwann cells (SCs) are mechanisms facilitating the procession of diabetic peripheral neuropathy (DPN). Although erythropoietin (EPO) was demonstrated to have neuroprotective effects in neurodegenerative diseases, the effects of EPO on glucose‐evoked oxidative stress and apoptosis of SCs remain unknown.

Brain-derived neurotrophic factor promotes vesicular glutamate transporter 3 expression and neurite outgrowth of dorsal root ganglion neurons through the activation of the transcription factors Etv4 and Etv5.

Brain-derived neurotrophic factor (BDNF) is critical for sensory neuron survival and is necessary for vesicular glutamate transporter 3 (VGLUT3) expression. Whether the transcription factors Etv4 and Etv5 are involved in these BDNF-induced effects remains unclear. In the present study, primary cultured dorsal root ganglion (DRG) neurons were used to test the link between BDNF and transcription factors Etv4 and Etv5 on VGLUT3 expression and neurite outgrowth. BDNF promoted the mRNA and protein expression of Etv4 and Etv5 in DRG neurons. These effects were blocked by extracellular signal-regulated protein kinase 1/2 (ERK1/2) inhibitor PD98059 but not phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 or phospholipase C-γ (PLC-γ) inhibitor U73122. Etv4 siRNA and Etv5 siRNA effectively blocked the VGLUT3 expression and neurite elongation induced by BNDF. The overexpression of Etv4 or Etv5 potentiated the effects of BNDF-induced neurite elongation and growth-associated protein 43 (GAP-43), medium neurofilament (NF-M), and light neurofilament (NF-L) expression while these effects could be inhibited by Etv4 and Etv5 siRNA. These data imply that Etv4 and Etv5 are essential transcription factors in modulating BDNF/TrkB signaling-mediated VGLUT3 expression and neurite outgrowth. BDNF, through the ERK1/2 signaling pathway, activates Etv4 and Etv5 to initiate GAP-43 expression, promote neurofilament (NF) protein expression, induce neurite outgrowth, and mediate VGLUT3 expression for neuronal function improvement. The biological effects initiated by BDNF/TrkB signaling linked to E26 transformation-specific (ETS) transcription factors are important to elucidate neuronal differentiation, axonal regeneration, and repair in various pathological states.

The effects of neuregulin-1β on neuronal phenotypes of primary cultured dorsal root ganglion neurons by activation of PI3K/Akt

Neuregulin-1β (NRG-1β) signaling has multiple functions in neurons. NRG-1 signaling regulates neuronal development, migration, myelination, and synaptic maintenance. The neuropeptide- and neurofilament (NF)-immunoreactive (IR) neurons are two major phenotypical classes in dorsal root ganglion (DRG). Whether NRG-1β influences DRG neuronal phenotypes remains unknown. To assess the effects of NRG-1β on DRG neuronal phenotypes, dissociated embryonic rat DRG neuronal culture model was established. Primary cultured DRG neurons were exposed to NRG-1β (5nmol/L), NRG-1β (10nmol/L), NRG-1β (20nmol/L), NRG-1β (20nmol/L) plus LY294002 (10μmol/L) for 3 days, respectively. The DRG neurons were continuously exposed to growth media as control. After that, all above cultured DRG neurons were processed for double fluorescent labeling of calcitonin gene-related peptide (CGRP) or neurofilament-200 (NF-200) and microtubule associated protein 2 (MAP2). The percentage of CGRP-IR neurons and NF-200-IR neurons was counted. The expression of CGRP mRNA and NF-200 mRNA was analyzed by real time-PCR analysis. The percentage of CGRP-IR neurons but not NF-200-IR neurons increased significantly in the presence of NRG-1β as compared with that in the absence of NRG-1β. The levels of CGRP mRNA but not NF-200 mRNA increased significantly in the presence of NRG-1β as compared with that in the absence of NRG-1β. PI3K inhibitor LY294002 blocked the effects of NRG-1β. These results support an important role for exogenous NRG-1β in induction of the distinct neuronal phenotype response by activation of PI3K/Akt in sensory neurons.