Journal: Cell reports

Article Title: Loss of Prdm12 during development, but not in mature nociceptors, causes defects in pain sensation

doi: 10.1016/j.celrep.2021.108913

Figure Lengend Snippet: (A) RNAscope using exon-V-specific probes confirmed knockout of Prdm12 from mutant DRGs. Scale bar, 50 μm. (B) Prdm12 AvilCKO mice have a reduction of Tom + neurons, p = 0.0326. (C and D) The remaining neurons in Prdm12 AvilCKO are larger, on average (C) (p = 0.0466), because of selective loss of cells ≤ 300 μm 2 (D). (E) Representative image showing number of IB4 + (arrows) and CGRP + (arrowheads) nociceptors are reduced in Prdm12 AvilCKO DRGs. Scale bar, 100 μm. (F and G) Quantification of IB4 + (F) (p < 0.0001) and CGRP + (G) (p = 0.027) nociceptors as a percentage of the total Tom + population of sensory neurons. (H) Representative image of TRKA + nociceptors and NF200 + myelinated neurons in Prdm12 AvilCKO and control mice. Scale bar, 100 μm. (I) Quantification showing significant reduction of TRKA + nociceptors, p = 0.0135. (J) Quantification of NF200 + neurons showing a wide range in Prdm12 AvilCKO mice, but no significant change from control littermates, p = 0.278. (K and L) Representative image (K) and quantification (L) of the reduction in number of TRPV1 + nociceptors, p = 0.0025. Scale bar, 100 μm. (M) qRT-PCR results plotted showing —log( p ) versus log 2 (fold change) reveal significant reductions in many nociceptive gene transcripts, including Prdm12 , but a significant increase in Trpm3 , and no change in touch and proprioceptive genes. The dotted line represents p = 0.05; points above it differ significantly between control and mutant mice. Each point represents the analysis of significance and fold change from average values of n = 3 biological replicates. All analysis was completed using DRGs from lumbar levels 2 through 5. Each data point in (B), (C), (F), (G), (I), (J), and (L) represents the average count across three DRGs from control (n = 3) or Prdm12 AvilCKO (n = 4) mice taken after behavior analysis around 10 weeks of age. All quantification analyzed by two-tailed Student’s t test; results are presented as means ± SEM.

Article Snippet: The following primary antibodies and dilutions were used: mouse anti-Islet1/2 (1:20,000; DSHB 39.4D5), goat anti-TRKA (1:20; R&D Systems AF1056), rabbit anti-RUNX3 (1:50,000; gift from Thomas Jessell), rabbit anti-CASP3 (1:50; BD PharMingen 557035), IB4-488 (1:500, Invitrogen I21411), rabbit anti-CGRP (1:1000; Immunostar 24112), rabbit anti-NF200 (1:500; Sigma N4142), rabbit anti-TRPV1 (1:500; Alomone ACC-030).

Techniques: Knock-Out, Mutagenesis, Quantitative RT-PCR, Two Tailed Test

Journal: Cell reports

Article Title: Loss of Prdm12 during development, but not in mature nociceptors, causes defects in pain sensation

doi: 10.1016/j.celrep.2021.108913

Figure Lengend Snippet: (A) Exon-V-specific RNAscope verified loss of mRNA transcript in Prdm12 AvilERT2CKO (adult CKO) mice. Scale bar, 100 μm. Inset arrows indicate mRNA puncta detected by the probe; inset scale bar, 25 μm. (B) qRT-PCR in Prdm12 AvilERT2 mice after tamoxifen injection revealed reductions in expression of exon 5 of Prdm12 , as well as Trpm8 and Ntrk1 ; other genes were unaffected. The dotted line represents p = 0.05; points above it differ significantly between control and mutant mice. Each point represents the analysis of significance and fold change from average values of n = 6 biological replicates. (C) Relative expression of select genes after SNI or sham surgery specifically from L4–5 DRG. Prdm12 is reduced after SNI in control animals (p = 0.0356) and in sham mutants (p = 0.0482). Additionally, Atf3 expression is increased in both control (p = 0.0001) and mutant (p = 0.0010) mice, but Kcnma1 is reduced only in control animals (p = 0.0239). (D) Representative images of lumbar DRGs from control and Prdm12 AvilERT2CKO DRGs contralateral to and ipsilateral to SNI with immunohistochemistry for IB4 and CGRP. Scale bar, 100 μm. (E–I) Quantification of these images revealed no changes in the number of IB4 + (E), CGRP + (F), TRKA + (G), TRPV1 + (H), or NF200 + (I) neurons after SNI in either control or Prdm12 AvilERT2CKO mice or between those two groups. Each data point in (E)–(I) represents the average counts/section across three DRGs taken from the L2–L5 region of n = 3 mice. DRGs were collected after behavior assessment at 18 weeks. Graphs show means ± SEM; statistical analysis two-tailed Student’s t test (B) or 2-way ANOVA with post hoc Tukey tests when a significant difference was detected (C–I).

Article Snippet: The following primary antibodies and dilutions were used: mouse anti-Islet1/2 (1:20,000; DSHB 39.4D5), goat anti-TRKA (1:20; R&D Systems AF1056), rabbit anti-RUNX3 (1:50,000; gift from Thomas Jessell), rabbit anti-CASP3 (1:50; BD PharMingen 557035), IB4-488 (1:500, Invitrogen I21411), rabbit anti-CGRP (1:1000; Immunostar 24112), rabbit anti-NF200 (1:500; Sigma N4142), rabbit anti-TRPV1 (1:500; Alomone ACC-030).

Techniques: Quantitative RT-PCR, Injection, Expressing, Mutagenesis, Immunohistochemistry, Two Tailed Test

Journal: Cell reports

Article Title: Loss of Prdm12 during development, but not in mature nociceptors, causes defects in pain sensation

doi: 10.1016/j.celrep.2021.108913

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following primary antibodies and dilutions were used: mouse anti-Islet1/2 (1:20,000; DSHB 39.4D5), goat anti-TRKA (1:20; R&D Systems AF1056), rabbit anti-RUNX3 (1:50,000; gift from Thomas Jessell), rabbit anti-CASP3 (1:50; BD PharMingen 557035), IB4-488 (1:500, Invitrogen I21411), rabbit anti-CGRP (1:1000; Immunostar 24112), rabbit anti-NF200 (1:500; Sigma N4142), rabbit anti-TRPV1 (1:500; Alomone ACC-030).

Techniques: Recombinant, SYBR Green Assay, Multiplex Assay, Isolation, Software

Journal: medRxiv

Article Title: Machine Learning Reveals Synovial Fibroblast Genes Associated with Pain Affect Sensory Nerve Growth in Rheumatoid Arthritis

doi: 10.1101/2023.08.17.23294232

Figure Lengend Snippet: ( A ) Survival of CGRP+ DRG neurons cultured with media alone, Ngf, or Net4. Survival was measured by the number of Map2b+B3tub+ cells >10μm. Each dot represents the sum of five 10x magnification views from one experiment. Data from 4 experiments are presented. ns indicates not significant in Kruskal-Wallis test. ( B ) Sum of sprouting neurons divided by the total number of neurons cultured with media alone, Ngf, or Net4. Neurons with at least three axon branches greater than two times the size of the soma were classified as sprouting. Each dot represents the sum of five 10x magnification views from one experiment. Data from 4 experiments are presented. ns indicates not significant in Kruskal-Wallis test. ( C ) Representative images of Sholl analysis of branching of DRG neurons cultured with media alone (no treatment), Ngf, or Net4. ( D ) Branching, as measured by the number of shell intersections of neurites, in DRG neurons cultured with media alone (no treatment), Ngf, or Net4. Each dot represents the median with confidence interval of 40 neurons imaged from four experiments (10 neurons per experiment). **** indicates p<0.0001 in two way ANOVA group*radius interaction with post-hoc Dunnett’s multiple comparisons of each treatment group to the no treatment group.

Article Snippet: They were then blocked with 3% BSA for 1 hour at RT, then incubated at 4°C overnight with primary antibodies rabbit anti-CGRP (Immunostar 24112) diluted 1:20,000 and mouse anti-beta III tubulin (abcam ab78078) diluted 1:1000 in 3% BSA.

Techniques: Cell Culture

Journal: The Journal of Headache and Pain

Article Title: Neurogenic inflammation: a study of rat trigeminal ganglion

doi: 10.1007/s10194-010-0260-x

Figure Lengend Snippet: Antibodies used for immunohistochemical experiments including working solutions

Article Snippet: Primary , Rabbit polyclonal anti CGRP , 1:800 , Euro-Diagnostica, Sweden (2263B47-1).

Techniques: Immunohistochemical staining, Concentration Assay

Journal: Molecular Pain

Article Title: Sigma-1 receptors and progesterone metabolizing enzymes in nociceptive sensory neurons of the female rat trigeminal ganglia: A neural substrate for the antinociceptive actions of progesterone

doi: 10.1177/17448069211069255

Figure Lengend Snippet: Antibodies used for western blotting and fluorescent immunohistochemistry.

Article Snippet: Rabbit anti-CGRP , Immunostar , 24,112 , Goat anti-rabbit 568.

Techniques: Western Blot, Immunohistochemistry