Journal: Pain

Article Title: Potentiation of visualized exosomal miR-1306-3p from primary sensory neurons contributes to chronic visceral pain via spinal P2X3 receptors

doi: 10.1097/j.pain.0000000000003537

Figure Lengend Snippet: Sorted PSN-derived exosomal miR-1306-3p activated spinal P2X3R and enhanced synaptic transmission, thus leading to visceral pain. (A) Schematic diagram and representative images of sorting PSN-derived exosomes from spinal dorsal horn. (B) The expressions of miR-1306-3p, miR-1949, miR-185-5p, and miR-324-5p were detected in the PSN-derived exosomes from spinal dorsal horn of CON and NMD mice by Q-PCR (n = 4 mice for each group, *** P < 0.001, 2-way ANOVA followed by Sidak post hoc test). (C) Fluorescent representation of co-localization of GFP (green), P2X3R (red), and DAPI (blue) in the spinal dorsal horn. Scale bar = 50 μm. Representative 3D confocal images on the right was the enlarged view in the white box of merge. Scale bar = 20 μm. (D) Representative traces of sEPSCs were recorded in the T13 to L2 spinal dorsal horn neurons before and after the incubation of miR-1306-3p and Gefapixant. Histogram of the amplitude and frequency of sEPSCs in Pre, miR-1306-3p, and miR-1306-3p+Gefapixant group (n = 10 cells from 4 mice for each group, ** P < 0.01, *** P < 0.001, one-way ANOVA followed by Tukey post hoc test). (E) Statistical diagram of the visceral pain threshold in NMD mice after injection of antagomir-miR-1306-3p or antagomir-negative control (antagomir-NC) at Pre, 0.5, 1, 2, and 4 hours (antagomir-NC: n = 6 mice, antagomir-miR-1306-3p: n = 7 mice, ** P < 0.01, 2-way ANOVA followed by Sidak post hoc test). (F) Statistical diagram of the visceral pain threshold in NMD mice after injection of Gefapixant at Pre, 0.5, 1, 2, 4, and 8 hours (n = 7 mice for each group, * P < 0.05, ** P < 0.01, *** P < 0.001, 2-way ANOVA followed by Dunnett post hoc test). (G) Histogram of the visceral pain thresholds in CON mice treated with agomir-negative control (agomir-NC) or agomir-miR-1306-3p at Pre, 0.5, 1, 2, and 4 hours, respectively (n = 6 mice for each group, *** P < 0.001, 2-way ANOVA followed by Sidak post hoc test). (H) Histogram of the visceral pain thresholds in CON mice treated with agomir-miR-1306-3p+NS or agomir-miR-1306-3p+Gefapixant at Pre, 0.5, 1, 2, and 4 hours, respectively (n = 6 mice for each group, * P < 0.05, *** P < 0.001, 2-way ANOVA followed by Sidak post hoc test). ANOVA, analysis of variance; NMD, neonatal maternal deprivation; PSN, primary sensory neuron; Q-PCR, real-time quantitative polymerase chain reaction; sEPSC, spontaneous excitatory postsynaptic current.

Article Snippet: The primary antibodies were anti-Rab27a (1:200, Cat no. 168013; Synaptic Systems, Göttingen, Germany, RRID: AB_887766), anti-GFP-FITC (1:500, Cat no. ab6662; Abcam, Cambridge, United Kingdom, RRID: AB_305635), anti-CD63 (1:100, Cat no. sc-5275; Santa Cruz Biotechnology, Dallas, TX, RRID: AB_627877), anti-NeuN (1:50, Cat no. MAB377; Merck Millipore, Burlington, MA, RRID: AB_2298772), anti-glutamine synthetase (1:500, Cat no. ab64613; Abcam, RRID: AB_1140869), anti-CGRP (calcitonin gene-related peptide) (1:100, Cat no. C7113; Sigma-Aldrich, St. Louis, MO, RRID: AB_259000), anti-IB4 + -FITC (1:200, Cat no. L-1104; Vector Laboratories, Newark, CA, RRID: AB_2336498), anti-NF200 (1:200, Cat no. ab213128; Abcam, RRID: AB_3073795), anti-GFAP (glial fibrillary acidic protein) (1:100, Cat no. 3670; Cell Signaling Technology, RRID: AB_561049), anti-Iba-1 (1:100, Cat no. ab5076; Abcam, RRID: AB_2224402), and P2X3R (1:200, Cat no. APR-026; Alomone Labs, Jerusalem, Israel, RRID: AB_2341052).

Techniques: Derivative Assay, Transmission Assay, Incubation, Injection, Negative Control, Real-time Polymerase Chain Reaction

Journal: Pain

Article Title: Potentiation of visualized exosomal miR-1306-3p from primary sensory neurons contributes to chronic visceral pain via spinal P2X3 receptors

doi: 10.1097/j.pain.0000000000003537

Figure Lengend Snippet: siR-Rab27a reduced visible PSN-derived exosomes, suppressed spinal synaptic transmission, and alleviated visceral pain. (A and B) Representative images and fluorescence area of GFP were quantified in the left spinal dorsal horn of the NMD mice injected with siR-NC or siR-Rab27a (siR-NC: n = 3 mice, siR-Rab27a: n = 4 mice, * P < 0.05, 2-sample t test). Scale bar = 50 μm. (C) The protein expression of GFP in the left spinal dorsal horn of siR-NC and siR-Rab27a mice (siR-NC: n = 5 mice, siR-Rab27a: n = 4 mice, * P < 0.05, 2 sample t test). (D) Representative images of GFP, NeuN-labeled neurons, and DAPI-labeled nucleus in the left spinal dorsal horn of siR-NC and siR-Rab27a mice. Scale bar = 50 μm. The white box in the lower right corner was the typical GFP + positive neurons, scale bar = 10 μm. (E) Quantification of the percentage of GFP + positive neurons in the spinal dorsal horn neurons of siR-NC and siR-Rab27a mice (siR-NC: n = 4 mice, siR-Rab27a: n = 5 mice, * P < 0.05, Mann–Whitney test). (F) Representative images of GFP, P2X3R-positive neurons and DAPI in the left spinal dorsal horn of siR-NC and siR-Rab27a mice. Scale bar = 50 μm. The white box in the lower right corner was the typical GFP + P2X3R + positive neurons. Scale bar = 10 μm. (G) Quantification of the percentage of GFP + P2X3R + positive neurons in spinal P2X3R-positive neurons of siR-NC and siR-Rab27a mice (n = 4 mice for each group, * P < 0.05, Mann–Whitney test). (H) Representative traces of sEPSCs were recorded in the T13 to L2 spinal dorsal horn neurons of siR-NC and siR-Rab27a mice. Histogram and cumulative probability distributions of the amplitude and frequency of sEPSCs in siR-NC and siR-Rab27a mice (siR-NC: n = 8 cells from 3 mice, siR-Rab27a: n = 6 cells from 3 mice, * P < 0.05, 2-sample t test). (I) The visceral pain threshold at Pre, 1, 2, 3, 4, and 5 days in NMD mice injected with siR-NC or siR-Rab27a, respectively (n = 8 mice for each group, ** P < 0.01, *** P < 0.001, 2-way ANOVA followed by Sidak post hoc test). (J) Statistical diagram of the time on the rod in the siR-NC or siR-Rab27a group at Pre, 1, 2, 3, 4, and 5 days, respectively (n = 8 mice for each group, P > 0.05, 2-way ANOVA followed by Sidak post hoc test). ANOVA, analysis of variance; GFP, green fluorescent protein; NMD, neonatal maternal deprivation; PSN, primary sensory neuron; sEPSC, spontaneous excitatory postsynaptic current.

Article Snippet: The primary antibodies were anti-Rab27a (1:200, Cat no. 168013; Synaptic Systems, Göttingen, Germany, RRID: AB_887766), anti-GFP-FITC (1:500, Cat no. ab6662; Abcam, Cambridge, United Kingdom, RRID: AB_305635), anti-CD63 (1:100, Cat no. sc-5275; Santa Cruz Biotechnology, Dallas, TX, RRID: AB_627877), anti-NeuN (1:50, Cat no. MAB377; Merck Millipore, Burlington, MA, RRID: AB_2298772), anti-glutamine synthetase (1:500, Cat no. ab64613; Abcam, RRID: AB_1140869), anti-CGRP (calcitonin gene-related peptide) (1:100, Cat no. C7113; Sigma-Aldrich, St. Louis, MO, RRID: AB_259000), anti-IB4 + -FITC (1:200, Cat no. L-1104; Vector Laboratories, Newark, CA, RRID: AB_2336498), anti-NF200 (1:200, Cat no. ab213128; Abcam, RRID: AB_3073795), anti-GFAP (glial fibrillary acidic protein) (1:100, Cat no. 3670; Cell Signaling Technology, RRID: AB_561049), anti-Iba-1 (1:100, Cat no. ab5076; Abcam, RRID: AB_2224402), and P2X3R (1:200, Cat no. APR-026; Alomone Labs, Jerusalem, Israel, RRID: AB_2341052).

Techniques: Derivative Assay, Transmission Assay, Fluorescence, Injection, Expressing, Labeling, MANN-WHITNEY

Journal: Pain

Article Title: Potentiation of visualized exosomal miR-1306-3p from primary sensory neurons contributes to chronic visceral pain via spinal P2X3 receptors

doi: 10.1097/j.pain.0000000000003537

Figure Lengend Snippet: A working model showing that exosomes in the circuit of DRG-spinal cord leading to chronic visceral pain by exosome visualization technologies. The upregulation of Rab27a in colon-related DRGs promoted the release of exosomal miR-1306-3p, which activated P2X3R in the spinal dorsal horn neurons and enhanced synaptic transmission, thereby contributing to chronic visceral pain. Meanwhile, intrathecal injection of GW4869 or siR-Rab27a reduced visible PSN-derived exosomes in spinal cord, suppressed spinal synaptic transmission, and alleviated visceral pain in NMD mice. Inhibition of P2X3R also alleviated visceral pain by intrathecal injection of Gefapixant. DRG, dorsal root ganglia; EPSC, excitatory postsynaptic currents; MVB, multivesicular bodies; NMD, neonatal maternal deprivation; PSN, primary sensory neuron.

Article Snippet: The primary antibodies were anti-Rab27a (1:200, Cat no. 168013; Synaptic Systems, Göttingen, Germany, RRID: AB_887766), anti-GFP-FITC (1:500, Cat no. ab6662; Abcam, Cambridge, United Kingdom, RRID: AB_305635), anti-CD63 (1:100, Cat no. sc-5275; Santa Cruz Biotechnology, Dallas, TX, RRID: AB_627877), anti-NeuN (1:50, Cat no. MAB377; Merck Millipore, Burlington, MA, RRID: AB_2298772), anti-glutamine synthetase (1:500, Cat no. ab64613; Abcam, RRID: AB_1140869), anti-CGRP (calcitonin gene-related peptide) (1:100, Cat no. C7113; Sigma-Aldrich, St. Louis, MO, RRID: AB_259000), anti-IB4 + -FITC (1:200, Cat no. L-1104; Vector Laboratories, Newark, CA, RRID: AB_2336498), anti-NF200 (1:200, Cat no. ab213128; Abcam, RRID: AB_3073795), anti-GFAP (glial fibrillary acidic protein) (1:100, Cat no. 3670; Cell Signaling Technology, RRID: AB_561049), anti-Iba-1 (1:100, Cat no. ab5076; Abcam, RRID: AB_2224402), and P2X3R (1:200, Cat no. APR-026; Alomone Labs, Jerusalem, Israel, RRID: AB_2341052).

Techniques: Transmission Assay, Injection, Derivative Assay, Inhibition

Journal: Purinergic Signalling

Article Title: DT-0111: a novel P2X3 receptor antagonist

doi: 10.1007/s11302-023-09930-5

Figure Lengend Snippet: A, B: Representative traces of P2X3R-current and P2X2/3R-current evoked by increasing concentrations of the reference agonist α,β-methylene ATP. C: Fitting of the average (mean ± SE) of the dose response curves of α,β-methylene ATP for P2X3R (LogEC50 6.13 ± 0.05, n = 28). D: Fitting of the average (mean ± SE) of the dose response curves of α,β-methylene ATP for P2X2/3R (LogEC50 5.11 ± 0.11, n = 36)

Article Snippet: P2X3R VS. P2X2/3R selectivity This study was performed at Axxam S.p.A. (Milan, Italy).

Techniques:

Journal: Purinergic Signalling

Article Title: DT-0111: a novel P2X3 receptor antagonist

doi: 10.1007/s11302-023-09930-5

Figure Lengend Snippet: A: Representative traces of P2X3R-current evoked by 1 µM α,β-methylene ATP in control (black line) and in the presence of increasing concentrations of reference compound AF353. B: Representative traces of P2X2/3R-current evoked by 1 µM α,β-methylene ATP in control (black line) and in the presence of increasing concentrations of reference compound AF353. C: Fitting of average (mean ± SE) of the dose response curves of AF353 on P2X3R peak currents (IC50 52.5 nM, n = 7). D: Fitting of average (mean ± SE) of the dose response curves of AF353 on both P2X2/3R-peak and sustained currents (IC50 59.9 and 68.6 nM, n = 10, respectively)

Article Snippet: P2X3R VS. P2X2/3R selectivity This study was performed at Axxam S.p.A. (Milan, Italy).

Techniques: Control

Journal: Purinergic Signalling

Article Title: DT-0111: a novel P2X3 receptor antagonist

doi: 10.1007/s11302-023-09930-5

Figure Lengend Snippet: A: Representative traces of P2X3R-current evoked by 1 µM α,β-methylene ATP in control (black line) and in the presence of increasing concentrations of compound DT-0111. B: Representative traces of P2X2/3R-current evoked by 8 µM α,β-methylene ATP in control (black line) and in the presence of increasing concentrations of compound DT-0111. C: Fitting of average (mean ± SE) of the dose response curves of compound DT-0111 (IC50 31.2 ± 2.15 nM, n = 6). D: Fitting of average (mean ± SE) of the dose response curves of compound DT-0111 (n = 5)

Article Snippet: P2X3R VS. P2X2/3R selectivity This study was performed at Axxam S.p.A. (Milan, Italy).

Techniques: Control