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: PLoS ONE

Article Title: Experimental Cancer Cachexia Changes Neuron Numbers and Peptide Levels in the Intestine: Partial Protective Effects after Dietary Supplementation with L-Glutamine

doi: 10.1371/journal.pone.0162998

Figure Lengend Snippet: Characteristics of the primary and secondary antibodies used in immunoreactions.

Article Snippet: Polyclonal Rabbit anti—Calcitonin gene-related peptide (CGRP); AB-15360 , Merck Millipore Corporation, USA , 1:500 , 1:1000.

Techniques: Immunohistochemical staining, Western Blot

Journal: PLoS ONE

Article Title: Experimental Cancer Cachexia Changes Neuron Numbers and Peptide Levels in the Intestine: Partial Protective Effects after Dietary Supplementation with L-Glutamine

doi: 10.1371/journal.pone.0162998

Figure Lengend Snippet: Myenteric VIP-IR and CGRP-IR varicosities of the jejunum: (a-d) VIP-IR nerve fiber and (i-l) CGRP-IR nerve fiber. Ileum: (e-h) VIP-IR nerve fiber and (m-p) CGRP-IR nerve fiber. Experimental groups: control (C); control supplemented with 2% L-glutamine (CG); Walker-256 tumor (TW); and Walker-256 tumor supplemented with 2% L-glutamine (TWG). (q-x) representative images of the submucous CGRP-IR varicosities nerve fibers of the jejunum (q-t) and ileum (u-x) from experimental groups (C, CG, TW and TWG). All enlarge images on the top right of each image with white arrows indicate examples of immunoreactive varicosity (a'-x'). Scale Bar 25 μm.

Article Snippet: Polyclonal Rabbit anti—Calcitonin gene-related peptide (CGRP); AB-15360 , Merck Millipore Corporation, USA , 1:500 , 1:1000.

Techniques:

Journal: PLoS ONE

Article Title: Experimental Cancer Cachexia Changes Neuron Numbers and Peptide Levels in the Intestine: Partial Protective Effects after Dietary Supplementation with L-Glutamine

doi: 10.1371/journal.pone.0162998

Figure Lengend Snippet: Morphometric analysis of VIP-immunoreactive and CGRP-immunoreactive varicosities areas (μm 2 ) in the myenteric plexus in the intestinal regions (jejunum and Ileum). Experimental groups: control (C); control supplemented with 2% L-glutamine (CG); Walker-256 tumor (TW); and Walker-256 tumor supplemented with 2% L-glutamine (TWG).

Article Snippet: Polyclonal Rabbit anti—Calcitonin gene-related peptide (CGRP); AB-15360 , Merck Millipore Corporation, USA , 1:500 , 1:1000.

Techniques:

Journal: PLoS ONE

Article Title: Experimental Cancer Cachexia Changes Neuron Numbers and Peptide Levels in the Intestine: Partial Protective Effects after Dietary Supplementation with L-Glutamine

doi: 10.1371/journal.pone.0162998

Figure Lengend Snippet: Morphometric analysis of CGRP-immunoreactive varicosities areas (μm 2 ) in the submucosal plexus in the jejunum and Ileum from the following groups: control (C); control supplemented with 2% L-glutamine (CG); Walker-256 tumor (TW); and Walker-256 tumor supplemented with 2% L-glutamine (TWG).

Article Snippet: Polyclonal Rabbit anti—Calcitonin gene-related peptide (CGRP); AB-15360 , Merck Millipore Corporation, USA , 1:500 , 1:1000.

Techniques:

Journal: PLoS ONE

Article Title: Experimental Cancer Cachexia Changes Neuron Numbers and Peptide Levels in the Intestine: Partial Protective Effects after Dietary Supplementation with L-Glutamine

doi: 10.1371/journal.pone.0162998

Figure Lengend Snippet: Groups: control (C); control supplemented with 2% L-glutamine (CG); Walker-256 tumor (TW); and Walker-256 tumor supplemented with 2% L-glutamine (TWG). (a) Jejunum CHAT expression and representative bands (on the top). (b) Ileum CHAT expression and representative bands (on the top). (c) Jejunum VIP expression and representative bands (on the top). (d) Ileum VIP expression and representative bands (on the top). (e) Jejunum CGRP expression and representative bands (on the top). (f) Ileum CGRP expression and representative bands (on the top). Bars represent means ± SEM of samples from four animals. The data are presented as percentage arbitrary units (% of control) after normalization (GAPDH). * indicates significant difference ( p < 0.05) versus control group.

Article Snippet: Polyclonal Rabbit anti—Calcitonin gene-related peptide (CGRP); AB-15360 , Merck Millipore Corporation, USA , 1:500 , 1:1000.

Techniques: Expressing

Journal: Journal of hypertension

Article Title: Hypotension Induced by TRPV4 Activation: Role of Ca 2+ -activated K + Channels and Sensory Nerves

doi: 10.1097/HJH.0b013e328332b865

Figure Lengend Snippet: /B> Plasma CGRP and substance P (SP) levels in response to bolus injection 4α-PDD (2.5 mg/kg) with or without indomethacin (10 mg/kg, i.v.), L-NA (20 mg/kg) and apamin (50 μg/kg, i.v.) plus charybdotoxin (50 μg/kg, i.v.). Values are mean ± SE (n=4-7). *P<0.05 compared with the corresponding control group.

Article Snippet: To determine immunoactive CGRP and substance P in plasma, commercially available rabbit anti-rat CGRP radioimmunoassay kit and rabbit anti-rat substance P radioimmunoassay kit (Bachem Americas Inc., Torrance, CA) were used.

Techniques: Clinical Proteomics, Injection, Control

Journal: Journal of hypertension

Article Title: Hypotension Induced by TRPV4 Activation: Role of Ca 2+ -activated K + Channels and Sensory Nerves

doi: 10.1097/HJH.0b013e328332b865

Figure Lengend Snippet: Confocal microscope images of double-immunofluorescence staining of mesenteric resistant arteries (A, B, C, G, H, I) and DRG neurons (D, E, F). A, D and G: FITC-labeled TRPV4 receptor staining (green). B and E: Cy3-labeled CGRP staining (red). C and F: colocalization of TRPV4 and CGRP (yellow). H: Cy3-labeled MaxiК channels staining (red). I: colocalization of TRPV4 and MaxiК channels (yellow). Blue arrows: the colocalized areas. Negative control not shown. Scale bars, 20 μm.

Article Snippet: To determine immunoactive CGRP and substance P in plasma, commercially available rabbit anti-rat CGRP radioimmunoassay kit and rabbit anti-rat substance P radioimmunoassay kit (Bachem Americas Inc., Torrance, CA) were used.

Techniques: Microscopy, Double Immunofluorescence Staining, Labeling, Staining, Negative Control

Journal: Pain

Article Title: Contribution of mechanoreceptors to spinal cord injury–induced mechanical allodynia

doi: 10.1097/j.pain.0000000000003139

Figure Lengend Snippet: Peptidergic nociceptive fiber changes in the dorsal horn after TrkB-expressing mechanoreceptor ablation. Lumbar (L4-L6) spinal cord sections of injured saline control (A and D) and DTX-treated injured (B and E) TrkB DTR mice stained for CGRP (scale bar, 100 μm). Representative images indicate the SCI-induced increase in CGRP-labeling density in deeper laminae (III-IV) of the spinal dorsal horn in all groups. (C and F) Quantification of the CGRP-labeling density in laminae III-IV of the lumbar (L4-L6) dorsal horn shows no difference comparing the injured saline-injected control group with injured DTX-injected animals but a significant increase compared to previous sham animals (dotted line) (mean ± SD) (post-SCI; saline n = 6; DTX n = 7) (pre-SCI; saline n = 7; DTX n = 9). CGRP, Calcitonin gene-related peptide; DTX, diphtheria toxin; SCI, spinal cord injury; TrkB, tropomyosin receptor kinase B.

Article Snippet: The following primary antibodies were used: rabbit anti-calcitonin gene-related peptide (CGRP) (Immunostar, Hudson, WI, 24112; 1:200), rabbit anti-protein kinase Cγ (PKCγ [C19]; Santa Cruz Biotechnology, Dallas, TX, SC-211; 1:200), guinea pig anti-NeuN (Millipore, Darmstadt, Germany, ABN90; 1:2000) and goat anti-human HB-EGF (R&D Systems, Minneapolis, MN, AF-259-NA; 1:40).

Techniques: Expressing, Saline, Staining, Labeling, Injection