Journal: The Journal of Neuroscience

Article Title: Transcriptional Regulation of Voltage-Gated Sodium Channels Contributes to GM-CSF-Induced Pain

doi: 10.1523/JNEUROSCI.2204-18.2019

Figure Lengend Snippet: Effect of GM-CSF on the current amplitude and expression level of Nav1.7, Nav1.8, and Nav1.9 channels. A, Relative mRNA expression of Nav1.7, Nav1.8, Nav1.9, Kv4.2, TMEM16A, P2X3, KCNQ2, and KCNQ3 in cultured DRG cells after incubation of GM-CSF (200 ng/ml) at 24 h (n = 9, unpaired t test, *p < 0.05 compared with the control). B, Relative mRNA expression of Nav1.7, Nav1.8, and Nav1.9 in DRG neurons of bone cancer pain at the seventh day. (n = 6, unpaired t test, *p < 0.05 compared with the control). C, Typical current traces and current density–voltage relationship of total TTX-S, TTX-R, Nav1.8, and Nav1.9 Na+ currents in cultured DRG cells after incubation with GM-CSF (200 ng/ml) for 24 h. D, Western blot analysis of expression levels of Nav1.7, Nav1.8, and Nav1.9 proteins in DRG neurons treated with GM-CSF (200 ng/ml) for 18 h (n = 3, unpaired t test, *p < 0.05 compared with the control).

Article Snippet: Membranes were blocked with 5% nonfat dairy milk and incubated with primary antibodies against Nav1.7 (1:500; Alomone Labs), Nav1.8 (1:500; Abcam), Nav1.9 (1:200; Abcam), phospho (p)-Jak1 (1:1000; Affinity), p-Jak2 (1:1000; Affinity), p-Jak3 (1:1000; Affinity), p-stat3 (1:1000; Epitomics), and p-Stat5a (1:1000; Affinity) at 4°C overnight.

Techniques: Expressing, Cell Culture, Incubation, Western Blot

Journal: The Journal of Neuroscience

Article Title: Transcriptional Regulation of Voltage-Gated Sodium Channels Contributes to GM-CSF-Induced Pain

doi: 10.1523/JNEUROSCI.2204-18.2019

Figure Lengend Snippet: Downregulation of Nav1.7, Nav1.8, and Nav1.9 reverses nociceptive behavior evoked by GM-CSF. A–C, Application of AS ODNs (ASOs) in DRGs against Nav1.7, Nav1.8, and Nav1.9 (each ASO, 12.5 μg/rat, 5 μl) significantly reduced the mRNA expression level of Nav1.7, Nav1.8, and Nav1.9 increased by GM-CSF treatment (A), and alleviated mechanical (B) and thermal hyperalgesia (C) produced by the focal GM-CSF (200 ng) application. For A: n = 6; unpaired t test, *p < 0.05 compared with control; #p < 0.05 with respect to the corresponding GM-CSF. For B, Left, two-way ANOVA followed by Bonferroni post hoc tests revealed a significant effect of treatment (F(3,305) = 109.59, p = 0), but not time (F(4,305) = 0.78, p = 0.54) or interaction between the two (F(12,305) = 0.65, p = 0.80). Middle, There was a significant effect of treatment (F(3,305) = 80.53, p = 0), but not time (F(4,305) = 0.20, p = 0.94) or interaction between the two (F(12,305) = 0.42, p = 0.95). Right, There was a significant effect of treatment (F(3,335) = 109.87, p = 0), but not time (F(4,335) = 0.89, p = 0.47) or interaction between the two (F(12,335) = 0.37, p = 0.97). For C, Left, Two-way ANOVA followed by Bonferroni post hoc tests revealed a significant effect of treatment (F(3,295) = 168.25, p = 0) and interaction between treatment and time (F(12,295) = 3.73, p < 0.0001), but the effect of time was not significant (F(4,295) = 1.34, p = 0.25). Middle, There was a significant effect of treatment (F(3,295) = 336.23, p = 0) and an interaction between treatment and time (F(12,295) = 2.04, p = 0.02), but the effect of time was not significant (F(4,295) = 1.05, p = 0.38). Right, There was a significant effect of treatment (F(3,250) = 274.66, p = 0) and interaction between treatment and time (F(12,335) = 5.38, p < 0.0001), but the effect of time was not significant (F(4,335) = 0.71, p = 0.74). *p < 0.05 compared with the vehicle saline; n = 6, #p < 0.05 with respect to the corresponding GM-CSF.

Article Snippet: Membranes were blocked with 5% nonfat dairy milk and incubated with primary antibodies against Nav1.7 (1:500; Alomone Labs), Nav1.8 (1:500; Abcam), Nav1.9 (1:200; Abcam), phospho (p)-Jak1 (1:1000; Affinity), p-Jak2 (1:1000; Affinity), p-Jak3 (1:1000; Affinity), p-stat3 (1:1000; Epitomics), and p-Stat5a (1:1000; Affinity) at 4°C overnight.

Techniques: Expressing, Produced

Journal: The Journal of Neuroscience

Article Title: Transcriptional Regulation of Voltage-Gated Sodium Channels Contributes to GM-CSF-Induced Pain

doi: 10.1523/JNEUROSCI.2204-18.2019

Figure Lengend Snippet: GM-CSF increases the mRNA expression level of Nav1.7, Nav1.8, and Nav1.9 channels through the Jak2–Stat3 signaling pathway. A, Relative expression of p-Jak1, p-Jak2, p-Jak3, p-stat3, and p-stat5 in DRG neurons after incubation with GM-CSF for 25 min (n = 3; unpaired t test, *p < 0.05 compared with control). B, Relative mRNA expression level of Nav1.7, Nav1.8, and Nav1.9 in DRG neurons incubated with GM-CSF in the absence or presence of AG-490 (10 μm) and stattic (20 μm) for 4 h (n = 4–6; unpaired t test, *p < 0.05 compared with control; #p < 0.05 with respect to the corresponding GM-CSF). C, Relative Luciferase activity in HEK 293 cells transfected with reporter vector containing Nav1.7, Nav1.8, and Nav1.9 promoter regions (pGL3) coexpressed with either pcDNA3.1 (control) or pcDNA3.1-Stat3 cDNA (n = 3; unpaired t test, *p < 0.05 compared with control). D–F, Relative mRNA level of Nav1.7, Nav1.8, and Nav1.9 in ipsilateral DRGs (L5) of rats receiving AS ODNs (ASOs) against different Jak and Stat signaling molecules (12.5 mg/rat, 5 μl; n = 6, unpaired t test, *p < 0.05 compared with control; #p < 0.05 with respect to the corresponding GM-CSF). G–I, Effect of ASOs against Jak and Stat signaling molecules (12.5 mg/rat, 5 μl) on hyperalgesia responses to mechanical and thermal stimuli induced by GM-CSF. ASOs were given through the DRG cannula for 4 d, and then GM-CSF (200 ng) was given. For G, Left, Two-way ANOVA followed by Bonferroni post hoc tests revealed a significant effect of treatment (F(3,415) = 125.38, p = 0), but not time (F(4,415) = 0.54, p = 0.70) or interaction between the two (F(12,415) = 0.73, p = 0.73). Right, There was a significant effect of treatment (F(3,425) = 77.18, p = 0), but not time (F(4,425) = 1.24, p = 0.29) or interaction between the two (F(12,425) = 1.65, p = 0.07). For H, Left, Two-way ANOVA followed by Bonferroni post hoc tests revealed a significant effect of treatment (F(3,415) = 110.97, p = 0), but not time (F(4,415) = 0.38, p = 0.82) or interaction between the two (F(12,415) = 0.43, p = 0.79). Right, There was a significant effect of treatment (F(3,440) = 115.88, p = 0), but not time (F(4,440) = 1.40, p = 0.25) or interaction between the two (F(12,440) = 1.13, p = 0.33). For I, Left, Two-way ANOVA followed by Bonferroni post hoc tests revealed a significant effect of treatment (F(3,345) = 143.47, p = 0), but not time (F(4,415) = 0.74, p = 0.57) or interaction between the two (F(12,415) = 0.45, p = 0.94). Right, There was a significant effect of treatment (F(3,440) = 111.32, p = 0), but not time (F(4,440) = 0.88, p = 0.47) or an interaction between the two (F(12,440) = 0.94, p = 0.50). n = 6–8; *p < 0.05 compared with the vehicle saline; #p < 0.05 with respect to the corresponding GM-CSF.

Article Snippet: Membranes were blocked with 5% nonfat dairy milk and incubated with primary antibodies against Nav1.7 (1:500; Alomone Labs), Nav1.8 (1:500; Abcam), Nav1.9 (1:200; Abcam), phospho (p)-Jak1 (1:1000; Affinity), p-Jak2 (1:1000; Affinity), p-Jak3 (1:1000; Affinity), p-stat3 (1:1000; Epitomics), and p-Stat5a (1:1000; Affinity) at 4°C overnight.

Techniques: Expressing, Incubation, Luciferase, Activity Assay, Transfection, Plasmid Preparation

Journal: Pain

Article Title: Inhibition of the Ubc9 E2 SUMO conjugating enzyme–CRMP2 interaction decreases NaV1.7 currents and reverses experimental neuropathic pain

doi: 10.1097/j.pain.0000000000001294

Figure Lengend Snippet: (A) Voltage protocol for current-voltage relationship: Na+ currents were evoked by 5-ms voltage steps from −70 to +60 mV in 5-mV increments from a holding potential of −80 mV. (B) Na+ current traces were measured from vehicle treated (black) or 20 μM t-CSM treated (red) neurons. (C) Summary current density in the absence or presence of t-CSM (normalized to 34.2 ± 4.7 pF and 28.6 ± 3.3 pF, respectively). Incubation of CAD cells with t-CSM produced a marked decreased in current density. (*p < 0.05, Student’s t-test, n = 6 cells). (D) Representative Boltzmann fits for activation and steady-state inactivation for NaV1.7. No significant differences were detected in half-maximal voltage or slope properties of fast inactivation or activation. Data show means ±SEM (n=6).

Article Snippet: The neurons were washed in ice-cold PBS twice before incubation (40 min) with anti-NaV1.7 antibody (extracellular epitope, dilution 1/40, Cat# ASC-027, Alomone labs, Israel) for ice.

Techniques: Incubation, Produced, Activation Assay

Journal: Pain

Article Title: Inhibition of the Ubc9 E2 SUMO conjugating enzyme–CRMP2 interaction decreases NaV1.7 currents and reverses experimental neuropathic pain

doi: 10.1097/j.pain.0000000000001294

Figure Lengend Snippet: After an overnight treatment with either 0.1% DMSO or 20 μM t-CSM, Nav1.7 membrane localization was detected in sensory neurons. (A) Representative fluorescent micrographs of DRG neurons immunostained with an antibody against an extracellular epitope for NaV1.7. Fluorescent signal can be detected when NaV1.7 was localized at the plasma membrane in live DRG neurons. (B) Bar graph showing the relative surface NaV1.7 signal compared to the levels in the 0.1% DMSO-treated cells. Fluorescent signal from (A) was quantified and normalized to the area of the cell of interest. Treating the cells with 20 μM of t-CSM resulted in decreased membrane localization of NaV1.7. Mean ± SEM, *p<0.05, Student’s t-test (n=30 to 45 cells).

Article Snippet: The neurons were washed in ice-cold PBS twice before incubation (40 min) with anti-NaV1.7 antibody (extracellular epitope, dilution 1/40, Cat# ASC-027, Alomone labs, Israel) for ice.

Techniques:

Journal: Frontiers in Molecular Neuroscience

Article Title: The Human SCN9A R 185 H Point Mutation Induces Pain Hypersensitivity and Spontaneous Pain in Mice

doi: 10.3389/fnmol.2022.913990

Figure Lengend Snippet: Generation of Scn9a R 185 H mutant mice and Scn9a transcript expression in wild-type (wt) and mutant mice. (A) Scheme for Scn9a targeting strategy. The sgRNA86 guide RNA and protospacer adjacent motif (PAM) are shown. The ssODN contained three basic mutations including two silent mutations and one-point mutation. The first silence mutation, C > T was PAM mutation designed to avoid Cas9 recut. The second mutation, C > T, was designed for Bsp HI enzyme restriction site for genotyping. The third G > A mutation was designed to get the R185H found in human patients. (B) Mutant allele sequence characterized through Sanger sequencing of F0 founder DNA. The silence-mutated nucleotides are indicated in green and targeted point mutant nucleotide in red. (C) Scn9a micro-RNA (mRNA) expression in dorsal root ganglia (DRG) and spinal cord, of wt and Scn9a R 185 H mutant mice of both sexes. There was no significant effect of genotype. DRG. females: wt n = 4; Het n = 4; Homo n = 3; males: wt n = 5; Het n = 4; Homo n = 4; Spinal cord. Females: wt n = 5; Het n = 5; Homo n = 6; males: wt n = 5; Het n = 4; Homo n = 5. Scn9a transcript expression was normalized to Hprt expression. Results are shown as means ± SEM. Two-tailed unpaired t -test for wt allele transcript expression in wt mice vs. mutant allele transcript expression in homozygous mutant mice. Refer to for statistics.

Article Snippet: The following primary antibodies (diluted in the blocking solution) were used: Rabbit anti-SCN9A-ATTO Fluor-663 antibody (1:100, ASC-008-FR, Alomone Labs, Jerusalem, Israel), Mouse anti-PGP9.5 antibody (1:200, ab8189, Abcam, Cambridge, United Kingdom), and rabbit anti-PGP9.5 (1:500, ab108986, Abcam).

Techniques: Mutagenesis, Expressing, Genotyping, Sequencing, DNA Sequencing, Two Tailed Test, CRISPR, Real-time Polymerase Chain Reaction

Journal: Frontiers in Molecular Neuroscience

Article Title: The Human SCN9A R 185 H Point Mutation Induces Pain Hypersensitivity and Spontaneous Pain in Mice

doi: 10.3389/fnmol.2022.913990

Figure Lengend Snippet: Nav1.7 protein expression of DRG and sciatic nerve in Scn9a R 185 H mutant mice. (A) Representative images of PGP9.5 (green) and Nav1.7 (red) fluorescent immunostaining for all neurons (PGP9.5-positive) and Nav1.7-positive neurons in lumbar DRG of wt animals. Scale bar 50 μm. (B) Size distribution for Nav1.7-positive (red) and negative (white) neuron cross-sectional areas in wt , het, and homo female and male mice. Females: n = 5/group; male: n = 3/group. Results are shown as means ± SEM. The two-way ANOVAs for genotype and neuron size effects in each sex showed a size effect and no genotype effect. (C) Representative images of fluorescent PGP9.5 (green) and Nav1.7 (red) immunostaining of sciatic nerves of wt animals. Scale bar of 25 μm. (D) Mean fluorescence density Nav1.7 immunostaining of sciatic nerves in wt, het, and homo female and male mice. Females, n = 4–5/group; males, n = 3/group. Results are shown as means ± SEM. The two-way ANOVA for genotype and sex indicated a sex effect and no genotype effect. The P value of 0.009 is for sex effect. See for statistics.

Article Snippet: The following primary antibodies (diluted in the blocking solution) were used: Rabbit anti-SCN9A-ATTO Fluor-663 antibody (1:100, ASC-008-FR, Alomone Labs, Jerusalem, Israel), Mouse anti-PGP9.5 antibody (1:200, ab8189, Abcam, Cambridge, United Kingdom), and rabbit anti-PGP9.5 (1:500, ab108986, Abcam).

Techniques: Expressing, Mutagenesis, Immunostaining, Fluorescence, Immunofluorescence

Journal: Frontiers in Molecular Neuroscience

Article Title: The Human SCN9A R 185 H Point Mutation Induces Pain Hypersensitivity and Spontaneous Pain in Mice

doi: 10.3389/fnmol.2022.913990

Figure Lengend Snippet: Enhanced heat pain sensitivity in Scn9a R 185 H mice. (A) Wild-type and Scn9a R 185 H mice showed comparable first response latency on the hot plate. (B) Homozygous Scn9a R 185 H females had decreased jump latency in 56°C Hot Plate. (C) Homozygous Scn9a R 185 H female mice showed more coping reactions on the 52°C hot plate. (D) Heterozygous and homozygous Scn9a R 185 H females had lower tail latency in the tail flick test. (E) No difference was found between wt and mutant mice in the Hargreaves test. (F) Control wt and mutant mice showed the same temperature preference. Results are shown as means ± SEM. Hot plate, females: wt, n = 16; Het, n = 16; Homo, n = 15; males: wt, n = 13; Het, n = 14; Homo, n = 11. Tail flick: females: wt, n = 15; Het, n = 15; Homo, n = 14; males: wt, n = 14; Het, n = 14; Homo, n = 11. Hargreaves: females: wt, n = 14; Het, n = 13; Homo, n = 11; males: wt, n = 14; Het, n = 14; Homo, n = 11. Temperature preference: females: wt, n = 12; Homo, n = 12; males: wt, n = 11; Homo, n = 11. One-way ANOVA on females and males and Dunnett’s multiple comparison tests for hot plate, tail flick, and Hargreaves tests; two-way ANOVA for the thermal preference ring test. P -values for genotype difference are shown when significant or close to significance. See – for statistics.

Article Snippet: The following primary antibodies (diluted in the blocking solution) were used: Rabbit anti-SCN9A-ATTO Fluor-663 antibody (1:100, ASC-008-FR, Alomone Labs, Jerusalem, Israel), Mouse anti-PGP9.5 antibody (1:200, ab8189, Abcam, Cambridge, United Kingdom), and rabbit anti-PGP9.5 (1:500, ab108986, Abcam).

Techniques: Sensitivity, Tail Flick Test, Mutagenesis, Control, Comparison

Journal: Frontiers in Molecular Neuroscience

Article Title: The Human SCN9A R 185 H Point Mutation Induces Pain Hypersensitivity and Spontaneous Pain in Mice

doi: 10.3389/fnmol.2022.913990

Figure Lengend Snippet: Scn9a R 185 H mice show enhanced sensitivity to cooling and touch stimuli. (A,B). Homozygous mutant males show increased duration and number of paw reactions in the acetone test. (C). Male mutant mice show a trend to reduced number of paw lifts on the cold plate. (D). Mutant mice display lower sensitivity threshold in the von Frey test. (E). Mutant mice show no difference to wt mice in the tail pressure test. Results are shown as means ± SEM. Acetone duration of paw reactions and number of paw reactions: females: wt, n = 15; Het, n = 13; Homo, n = 13; males: wt, n = 14; Het, n = 13; Homo, n = 12. Cold plate: females: wt, n = 15; Het, n = 15; Homo, n = 13; males: wt, n = 15; Het, n = 14; Homo, n = 12. Von Frey: females: wt, n = 13; Het, n = 13; Homo, n = 10; males: wt, n = 14; Het, n = 14; Homo, n = 11. Tail pressure: females: wt, n = 13; Het, n = 13; Homo, n = 11; males: wt, n = 14; Het, n = 14; Homo, n = 11. One-way ANOVA on females and males and Dunnett’s multiple comparison tests. P -values for genotype difference are shown when significant or close to significance. See , for statistics.

Article Snippet: The following primary antibodies (diluted in the blocking solution) were used: Rabbit anti-SCN9A-ATTO Fluor-663 antibody (1:100, ASC-008-FR, Alomone Labs, Jerusalem, Israel), Mouse anti-PGP9.5 antibody (1:200, ab8189, Abcam, Cambridge, United Kingdom), and rabbit anti-PGP9.5 (1:500, ab108986, Abcam).

Techniques: Sensitivity, Mutagenesis, Comparison

Journal: Frontiers in Molecular Neuroscience

Article Title: The Human SCN9A R 185 H Point Mutation Induces Pain Hypersensitivity and Spontaneous Pain in Mice

doi: 10.3389/fnmol.2022.913990

Figure Lengend Snippet: Scn9a R 185 H mutant mice show spontaneous ongoing pain. Ongoing pain was assessed in a conditioned place preference test. After pre-test/habituation for chamber preference on days 1 to 3, conditioning was made on day 4 by intrathecal treatment with either saline solution in the morning or clonidine solution in the afternoon with a restriction in one chamber. On day 5, the animals were free to explore the chambers and the time spent in each chamber was recorded for 30 min. (A) Females. (B) Males. The time spent in each chamber before conditioning (pre-test) and after conditioning with clonidine or saline (test) are shown. Homozygous mice of both sexes spent more time in clonidine-paired chamber while wt mice spent on average equal time in saline-paired and clonidine-paired chambers. Dots represent individual data points. females: wt, n = 18; Homo, n = 18; males: wt, n = 10; Homo, n = 10. Two-way ANOVA for treatment and trial (pretest and test) on each genotype and sex group, Sidak’s multiple comparison test. P = values for comparing pre-test and test values are shown. Refer to for statistics.

Article Snippet: The following primary antibodies (diluted in the blocking solution) were used: Rabbit anti-SCN9A-ATTO Fluor-663 antibody (1:100, ASC-008-FR, Alomone Labs, Jerusalem, Israel), Mouse anti-PGP9.5 antibody (1:200, ab8189, Abcam, Cambridge, United Kingdom), and rabbit anti-PGP9.5 (1:500, ab108986, Abcam).

Techniques: Mutagenesis, Conditioned Place Preference, Saline, Comparison

Journal: Biomolecules

Article Title: Long-Term Blockade of Nociceptive Na v 1.7 Channels Is Analgesic in Rat Models of Knee Arthritis

doi: 10.3390/biom12111571

Figure Lengend Snippet: Acute Effect of anti-Na v 1.7 Treatment on Joint Pain. Local, subcutaneous injection of anti-Na v 1.7 (25 μg in 50 μL) over the knees of MIA ( A , B ), LPA ( C , D ), and MMT ( E , F ) animals had no effect on hindpaw mechanosensitivity nor hindlimb weight bearing over the acute 6 h time-course compared to IgG controls, p > 0.05, two–way RMANOVA. †††† p < 0.0001, paired Student t -test of baseline (BL) vs. t = 0 min. Data are expressed as mean ± S.E.M ( n = 8–9 animals/group).

Article Snippet: Normal goat serum and lysophosphatidic acid (LPA) were obtained from Abcam (Toronto, ON, Canada), PF05089771 from Tocris Bioscience (Toronto, ON, Canada), AM281 from Cayman Chemical Company (Ann Arbor, MI, USA), PEP-1 from Abbiotec Inc. (San Diego, CA, USA), Fluoro-gold from Fluorochrome LLC (Denver, CO, USA), mouse IgG from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA), and guinea pig anti-Na v 1.7 (for IHC) from Alomone Labs (Jerusalem, Israel).

Techniques: Injection

Journal: Biomolecules

Article Title: Long-Term Blockade of Nociceptive Na v 1.7 Channels Is Analgesic in Rat Models of Knee Arthritis

doi: 10.3390/biom12111571

Figure Lengend Snippet: Chronic Effect of anti-Na v 1.7 Treatment on Joint Pain. Following a single, local injection of anti-Na v 1.7 (25 μg in 50 μL), hindpaw mechanosensitivity was significantly reduced over 28 days in MIA ( A ) and LPA ( C ) animals, but not MMT rats ( E ). Prolonged blockade of Na v 1.7 had no effect on hindlimb weight bearing over the 28-day time-course ( B , D , F ). * p < 0.05, ** p < 0.01, two-way RMANOVA with Bonferroni’s post hoc test. Data are expressed as mean ± S.E.M ( n = 8–9 animals/group).

Article Snippet: Normal goat serum and lysophosphatidic acid (LPA) were obtained from Abcam (Toronto, ON, Canada), PF05089771 from Tocris Bioscience (Toronto, ON, Canada), AM281 from Cayman Chemical Company (Ann Arbor, MI, USA), PEP-1 from Abbiotec Inc. (San Diego, CA, USA), Fluoro-gold from Fluorochrome LLC (Denver, CO, USA), mouse IgG from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA), and guinea pig anti-Na v 1.7 (for IHC) from Alomone Labs (Jerusalem, Israel).

Techniques: Injection

Journal: eLife

Article Title: A-type FHFs mediate resurgent currents through TTX-resistant voltage-gated sodium channels

doi: 10.7554/eLife.77558

Figure Lengend Snippet:

Article Snippet: Cell line ( H. sapiens ) , Nav1.7 cells , Icagen LLC. , , .

Techniques: Transfection, Construct, shRNA, Recombinant, Plasmid Preparation, Sequencing, Mutagenesis, Software