The Neuroprotective Effects Of NRG-1β On CS₂-induced Neurotoxicity Of Dorsal Root Ganglion Neurons

Carbon disulfide (CS2), a colorless organic industrial solvent, could induce neurotoxicity of peripheral nervous system (PNS). Although the neurotoxicity of CS2has been recognized for over a century, the precise mechanism of neurotoxic action of CS2remains unknown. Neuregulin-1(NRG-1β) signaling has multiple functions in neurons. Whether NRG-1β regulates neurite outgrowth and neuronal migration of dorsal root ganglion (DRG) neurons remains unknown. In the present study, organotypic DRG neuronal culture model was established. A series studies about the neurotoxic effects of CS2and neuroprotective effects of NRG-1β were carried out by using organotypically cultured embryonic rat DRG explants.Part Ⅰ Carbon disulfide inhibits neurite outgrowth and neuronal migration of dorsal root ganglion in vitroTo assess the neurotoxic effects of CS2, organotypic DRG cultures were exposed to different concentrations of CS2(0.01mmol/L,0.1mmol/L,1mmol/L, respectively). The neurite outgrowth of cultured organotypic DRG explants was evaluated under inverted phase contrast microscope. The neuronal migration from cultured organotypic DRG explants was determined by immunofluorescent labeling of microtubule-associated protein2(MAP2). The number of nerve fiber bundles extended from DRG explants decreased significantly in the presence of CS2(0.01mmol/L,15.00±2.61, P<0.05;0.1mmol/L,11.17±1.47, P<0.001;1mmol/L,8.00±1.41, P<0.001) as compared with that in the absence of CS2(17.83±2.48). The number of neurons migrating from DRG explants decreased significantly in the presence of CS2(0.01mmol/L,79.50±9.40, P<0.01;0.1mmol/L,62.50±14.15, P<0.001;1mmol/L,34.67±7.58, P<0.001) as compared with that in the absence of CS2(99.33±15.16). And also, the decreases of the number of nerve fiber bundles and the number of migrating DRG neurons were in a dose-dependent manner of CS2. These data implicate that CS2could inhibit neurite outgrowth and neuronal migration from DRG explants in vitro.Part Ⅱ Neuregulin-1β regulates outgrowth of neurites and migration of neurofilament200neurons from dorsal root ganglial explants in vitroTo assess NRG-1β on neurite outgrowth and neuronal migration in vitro, organotypic DRG neuronal culture model was established. The neurite outgrowth of cultured organotypic DRG explants was evaluated under inverted phase contrast microscope. The neuronal migration from cultured organotypic DRG explants was determined by immunofluorescent labeling of neurofilament200(NF-200). 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. 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 increases of the number of nerve fiber bundles and the number of migrating NF-200-IR neurons were in a dose-dependent manner of NRG-1β. The data in this study implicate that NRG-1β could promote neurite outgrowth and neuronal migration from DRG explants in vitro.Part Ⅲ Neuregulin-1β protects dorsal root ganglion neurons from carbon disulfide-induced neurotoxicity in vitroTo assess the neuroprotective NRG-1β on DRG neurons from CS2-induced neurotoxicity, organotypic DRG cultures were exposed:(1)0.1mmol/L CS2;(2)0.1mmol/L CS2+20nmol/L NRG-1β;(3)0.1mmol/L CS2+10μmol/L LY294002+20nmol/L NRG-1β;(4)0.1mmol/L CS2+10μmol/L PD98059+20nmol/L NRG-1β;(5)0.1mmol/L CS2+10μmol/L LY294002+10μmol/L PD98059+20nmol/L NRG-1β; for3days, respectively. The DRG cultures were continuously exposed to growth media as a control. All above cultures were incubated at37℃in a humidified5%CO2-air atmosphere for3days. The neurite outgrowth of cultured organotypic DRG explants was evaluated under inverted phase contrast microscope. The neuronal migration from cultured organotypic DRG explants was determined by immunofluorescent labeling of NF-200. The number of nerve fiber bundles extended from DRG explant in CS2, CS2+NRG-1β, CS2+LY294002+NRG-1β, CS2+PD98059+NRG-1β, CS2+LY294002+PD98059+NRG-1β treated samples, and control group was11.5±1.5,19.2±1.7,17.0±1.2,15.8±1.2,15.5±1.4, and19.7±1.3, respectively. The number of neurons migrating from DRG explant in CS2, CS2+NRG-1β, CS2+LY294002+NRG-1β, CS2+PD98059+NRG-1β, CS2+LY294002+PD98059+NRG-1β treated samples, and control group was19.9±2.9,33.3±4.1,27.±3.2,28.5±2.8,24.3±2.9, and34.8±4.6, respectively. The number of nerve fiber bundles and migrating neurons decreased in CS2treated cultures. NRG-1β partially rescued the decreases of nerve fiber bundles and migrating neurons caused by CS2exposure. Phosphatidylinositol3-kinase (PI3K) inhibitor LY294002and extracellular signal-regulated protein kinase1/2(ERK1/2) inhibitor PD98059, either alone or in combination, blocked the effects of NRG-1β. These data imply that NRG-1β has neuroprotective effects on DRG neurons from CS2-induced neurotoxicity. PI3K and ERK1/2signaling pathways were involved in the effects of NRG-1β. NRG-1(3and its receptor signaling system might be through direct or indirect effects to regulates the plasticity of neurofilaments or microtubules, then produces elongation of the axons. To investigate the effects of NRG-1β on sensory neurons might provide potential significance for clinical therapeutic strategy.