The Role Of Nav1.7 Sodium Channel In A Rat Model Of Paclitaxel-Induced Neuropathic Pain

Part one The alteration of the electrophysiology characteristics of DRGneurons in a rat model of paclitaxel-induced peripheral neuropathic painObjectiveTo investigate the changes of electrophysiology characteristics of dorsal root ganglion (DRG) neurons in the paclitaxel-induced peripheral neuropathic pain with whole-cell patch clamp.Methods20 SD rats were randomly divided into two groups (n=10):paclitaxel group (group P) and control group (group vehicle). Neuropathic pain model was produced by intraperitoneal injection paclitaxel of 2 mg/kg every other day for 4 times in group P. While the equal volume of cremophor EL:ethanol was given instead of paclitaxel in group vehicle. Then the L4.5 DRG neurons were acutely isolated after decapitation, and the whole cell patch clamp were established in these acute isolated neurons. In the current clamp mode, the action potential was activated and recorded under the step currents stimulation. The spontaneous activity, action potential, resting potential and rheobase were recorded.ResultCompared with group vehicle, the increased incidence of spontaneous activity in DRG neurons was recorded in large and medium sized neurons (P< 0.05). Paclitaxel decreased the rheobase in medium and large sized neurons (P< 0.05). But after paclitaxel treatment, other characteristics of membrane properties in each group remained the same.ConclusionPaclitaxel increased the neuronal excitability in DRG, which may lead to paclitaxel-induced neuropathy.Part two Sodium channel Nav1.7 expression is upregulated in the dorsal root ganglia in a rat model of paclitaxel-induced peripheral neuropathyObjectiveTo investigate the effect of paclitaxel on Navl.7 expression in rat dorsal root ganglion (DRG) neuronsMethodsAdult male SD rats weighting 220-300 g were used.36 rats were randomly divided into 2 groups (n=18 each):paclitaxel group (group P) and control group (group vehicle). Neuropathic pain was produced by intraperitoneal injection paclitaxel of 2 mg/kg every other day for 4 times in group P. While the equal volume of cremophor EL:ethanol was given instead of paclitaxel in group vehicle. Body weight and mechanical paw withdrawal latencies (PWT) were measured before and at 7,14 and 21 d after administration. Then the rats were sacrificed and the L4-5 segments of DRG were removed for determination of Nav1.7 expression by RT-PCR and Western blot method.ResultsCompared with group vehicle, PWT was significantly decreased in animals administered paclitaxel than those administered vehicle at days 7 (P< 0.05),14 (P < 0.01), and 21 (P< 0.01). DRG Nav1.7 mRNA and protein levels were higher in animals administered paclitaxel than those administered vehicle on days 7,14 and 21 (all P< 0.05).ConclusionThe increased DRG Nav1.7 expression may be partially responsible for paclitaxel-induced peripheral neuropathy.Part three Construction of a lentiviral vector expressing shRNA targeting rat Nav1.7 geneObjectiveTo construct an effective lentiviral vector containing short hairpin RNA (shRNA) inhibiting rat Nav1.7 gene by transfecting PC-12 cells.MethodsDesign shRNA expressing sequences for rat Nav1.7 gene. And then clone them into a lentiviral vector GV248, thus shRNA expressing vector was generated. The effectiveness of the vectors was assessed by transfecting PC-12 cells with nerve growth factor treatment in vitro.96 hours after transfection, the cells were harvested for RT-PCR and western blot analysis of the expression of Navl.7.ResultsThe shRNA expression vector could integrate into PC-12 cells with high efficiency. The transfection rate was 80% detected by fluorescence microscope. After ninety-six hours of transfection, the shRNA expressing vector has the interference effect to block Nav1.7 expression (P< 0.05).ConclusionLentiviral interference vector for rat Nav1.7 shRNA has been constructed successfully.Part four Effects of DRG Micro injection of lentivirus expressing shRNA inhibiting Nav1.7 gene on the rat model of paclitaxel-induced peripheral neuropathic painObjectiveTo observe the effects of DRG microinjection injection of lentivirus expressing shRNA inhibiting Nav1.7 gene on the rat model of paclitaxel-induced peripheral neuropathic pain.MethodsAdult male SD rats weighting 220?300 g were used.36 rats were randomly divided into 3 groups (n=12 each):paclitaxel+PBS group (group PBS)? paclitaxel+control lentiviral vector with EGFP (group LV-Con-EGFP) and paclitaxel+Nav1.7 lentiviral vector with EGFP (group LV-Nav1.7-EGFP). Three groups were microinjected 2?l PBS, control lentiviral vector and LV-Nav1.7 vector respectively in each left L4.5 DRG 7 days after microinjection, Neuropathic pain model was produced by described above. Mechanical paw withdrawal latencies (PWT) were measured before microinjection (Baseline),1 day before paclitaxel administration (OD) and 7 day (7D) after administration. Then the rats were sacrificed and the left L4-5 segments of DRG were removed for determination of Nav1.7 expression by RT-PCR and Western blot method.ResultsCompared with group PBS, PWT was increased in group LV-Nav1.7-EGFP on day 7 (P< 0.05). There is no difference between group PBS and group LV-Con-EGFP (P> 0.05). Compared with group PBS, DRG Nav1.7 mRNA and protein levels were decreased in group LV-Nav1.7-EGFP on day 7 (P< 0.05).ConclusionDRG microinjected of Nav1.7 lentiviral vector plasmid inhibit Nav1.7 mRNA and protein levels, and alleviate mechanical hyperalgesia by paclitaxel injection.