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

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The exosome visualization technology revealed that upregulation of Rab27a increased primary sensory neurons-derived exosomal miR-1306-3p, activated spinal P2X3R, thus contributing to chronic visceral pain.

Abstract

Exosomes served as “communicators” to exchange information among different cells in the nervous system. Our previous study demonstrated that the enhanced spinal synaptic transmission contributed to chronic visceral pain in irritable bowel syndrome. However, the underlying mechanism of primary sensory neuron (PSN)-derived exosomes on spinal transmission remains unclear. In this study, an exosome visualization method was established to specifically track exosomes derived from PSNs in CD63-GFP^f/+ (green fluorescent protein) mice. Neonatal maternal deprivation (NMD) was adopted to induce chronic visceral pain in CD63-GFP^f/+ male mice. The exosome visualization technology demonstrated that NMD increased visible PSN-derived exosomes in the spinal dorsal horn, enhanced spinal synaptic transmission, and led to visceral pain in CD63-GFP^f/+ male mice. The PSN-derived exosomal miR-1306-3p sorted from spinal dorsal horn activated P2X3R, enhanced spinal synaptic transmission, and led to visceral pain in NMD mice. Moreover, upregulation of Rab27a in dorsal root ganglia mediated the increased release of PSN-derived exosomes, and intrathecal injection of siR-Rab27a reduced visible PSN-derived exosomes in spinal cord, suppressed spinal synaptic transmission, and alleviated visceral pain in NMD mice. This and future studies would reveal the detailed mechanisms of PSN-derived exosomes and provide a potential target for clinical treatment of chronic visceral pain in patients with irritable bowel syndrome.

1. Introduction

Irritable bowel syndrome (IBS) is a prevalent functional gastrointestinal disorder characterized by abdominal pain, etc.^7,9 Given its complex pathogenesis, a significant proportion of patients with IBS experience chronic visceral pain.⁴ Recent studies have primarily focused on peripheral and brain mechanisms in visceral pain,^28,42,44 with less attention given to the spinal cord. The plasticity of primary afferent synapses is crucial in chronic pain, involving glutamatergic receptor activation and increased neurotransmitter release.^17,18,48 Despite efforts to targeting these mechanisms in drug design, clinical trials have not consistently achieved significant therapeutic efficacy, suggesting the presence of other critical signaling pathways in spinal synaptic transmission.

Exosomes, the small extracellular vesicles, participated in intercellular communication by transporting bioactive molecules.⁶ It was reported that cerebral cortical neurons mainly released exosomes by dendrites or cell bodies in situ,^23,30 with minimal release from axons.² But the mechanism of exosomes from primary sensory neurons (PSNs) in dorsal root ganglia (DRG), which is pseudounipolar without dendrites, remains unclear. Previous studies had showed exosomal microRNA (miRNA) was related to pain.^24,31,47,50 MicroRNAs not only silence target gene expression^13,45 but also function as novel endogenous ligands for some ion channels.^3,10,27,42 Our previous study demonstrated that exosomal miR-1306-3p activated the purinergic ion channel P2X3R in PSNs, thus leading to visceral pain.⁴² However, whether exosomal miRNAs from PSNs are involved in spinal synaptic transmission remains unclear. The sources of exosomes in the spinal dorsal horn are multifaceted, including those from cells within the spinal cord, peripheral tissues, and the descending fibers from the brain. Traditionally, exosomes were extracted from tissues using ultracentrifugation, which poses challenges in distinguishing their specific origins. One notable study showed the construction of transgenic exosome reporter mice (CD63-GFP^f/+[green fluorescent protein]) could represent a potent tool for tracking endogenous exosomes from different cells in the brain regions.^15,23,35

Rab GTPase family members (Rab27 and 35, etc.) played crucial roles in the biogenesis of exosomes, specifically in synthesis and release processes.¹⁶ Among them, the role of Rab27a is to transport and dock multivesicular bodies (MVBs) to the plasma membrane, thereby regulating the release of exosomes.²⁵ Our previous study has proved that upregulation of Rab27a in the anterior cingulate cortex led to visceral pain.³² Therefore, we speculated that Rab27a regulated the release of PSN-derived exosomes and led to visceral pain.

In this study, we proposed the hypothesis that the upregulation of Rab27a in PSNs increased the release of PSN-derived exosomal miR-1306-3p in the spinal dorsal horn, enhanced spinal synaptic transmission, thus led to visceral pain. To test this hypothesis, a novel exosome visualization method was developed in the present study. Visceral pain was first successfully induced in CD63-GFP^f/+ mice by neonatal maternal deprivation (NMD).^21,44 This study would clarify the specific mechanism of PSN-derived exosomal miRNAs involved in visceral pain, hoping to provide a new direction and target for the clinical treatment of visceral pain in IBS.

2. Materials and methods

2.1. Animals

All mice in this study were housed in the specific pathogen-free animal room, and operation of experiments met standards of the Institutional Animal Care and Use Committee of Soochow University and the International Association for the Study of Pain. CD63-GFP^f/+ mice were established to specifically track exosomes derived from different tissues or cells.²³ In brief, CRISPR/Cas9 technology was used to insert CAG-LSL-hCD63-copGFP-6xHis-WPRE-pA at Rosa26 gene locus by homologous recombination. To obtain CD63-GFP^f/+ mice (heterozygotes) with stable genetic ability, the breeding method selected in this experiment was heterozygous male and female mice were bred to obtain offspring mice, and the offspring CD63-GFP^f/+mice were selected through genotype identification for subsequent research. The model of chronic visceral pain in CD63-GFP^f/+ mice was induced by NMD.^32,44 CD63-GFP^f/+ offspring mice in NMD group (NMD mice) were kept separated from their mothers for 3 hours per day, lasting for 14 days. CD63-GFP^f/+ offspring mice in the CON group (CON mice) received no treatment. To avoid the periodic effects of estrogen on pain,^26,49 the CD63-GFP^f/+ male pups were used for experiments when they were at the age of 6 weeks or older.

2.2. Behavioral test of visceral pain

Colorectal distention was adopted as described previously.^32,44 Briefly, the balloon (length: 2 cm) was softly pushed into the colorectal site, and the outside tube was fixed to the tail of mouse. After 10 to 20 minutes of acclimation, the tube was connected to the sphygmomanometer. The balloon was expanded at a rate of 3 mm Hg/s until the mouse lifted abdomen or arched body, and the pressure value was recorded. The measurement was repeated for 5 times on each mouse and averaged as the threshold of visceral pain.

2.3. Behavioral test of somatic pain

Paw withdrawal threshold and paw withdrawal latency were measured by Von Frey filaments and thermal radiation instrument.⁴⁶ In short, the left hind paw was stimulated with von Frey filaments according to up–down method, and 50% threshold was recorded. The left hind paw was stimulated by thermal radiation until the positive reactions (lifting or licking paw) occurred, and the time value was record. This test repeated for 3 times on each mouse. The values were averaged as paw withdrawal latency. All behavior tests were operated in a blinded manner.

2.4. Ongoing pain-relevant behavioral tests

To adapt to the environment and the experimenter,^37,39 the mice were placed in the behavior room for 30 minutes each day for 7 consecutive days. During the 30-minute period, the experimenter spent 10 minutes gently touching their fur. After the test of each mouse, the instruments were wiped with diluted alcohol to prevent the odor left. The tested mice were not put back into the original cage to prevent affecting the untested mice. Open field test: As described previously,²² the mouse was placed in the center of the open field box (40 × 40 × 40 cm), and let it explore freely for 10 minutes. The time of mice entering the middle area were tracked and recorded by ANY-maze 2024 software (Stoelting, Wood Dale, IL). Elevated plus-maze: The mice were placed in the middle of the cross maze (5 × 5 cm), with their heads toward the closed arm (15 × 30 × 5 cm). The time of mice entering the open arm (30 × 5 cm) within 5 minutes were tracked and recorded by ANY-maze 2024 software (Stoelting), as described previously.³⁸ Tail suspension test: Briefly, the adhesive tape was tied at 1 cm of the mouse's tail tip and firmly adhered to the cross bar of the tail suspension instrument to make the mouse hang upside down. The immobility time (no limb or body movements) of the mouse was record from the second to sixth minutes by camera. Forced swimming test: The mouse was put into glass cylinder (water depth: 15 cm) and recorded by camera within 6 minutes. The immobility time (body floating without struggle) was counted from the second to sixth minute, as described previously.⁴⁰

2.5. Rotarod tests

The motor function of mice was detected by rotarod instrument, as described previously.³³ In short, the mice were given uniform speed (10, 20, and 30 r/min, 5 minutes respectively) for training every day. When the test began, the mice were given a constant speed of 30 r/min, and the time of mice on rod was recorded, so as to evaluate the motor function.

2.6. Viruses

The viruses were injected into DRG by microinjection pump.⁵¹ Briefly, after anesthesia, the mice were fixed to expose the left T13 DRG at vertebrae corresponding to the last rib, which was easy to locate and minimally invasive. Then AAV2/9-hsyn-Cre or AAV2/9-hsyn-Cre-mCherry (1.3 × 10¹³ V.G./mL, 600 nL) viruses were slowly injected at the rate of 0.03 μL/min by microinjection pump. After the injection, the needle was retained for 10 minutes, and the wound was sutured and disinfected.

2.7. Drugs

GW4869 (30 μM, 10 μL, single), Gefapixant (10, 30, and 100 μM, 10 μL, single), agomir/antagomir (20 μM, 10 μL, single), or siRNA (1 nmol, 10 μL, twice) were administered by intrathecal injection, as described previously.⁴¹ Briefly, the mice were fixed by anesthesia, and the drugs were injected into the subarachnoid space between the lumbar vertebrae (L4–L5). The sequences of siRNA in this study were siRNA-Rab27a: 5′-GCC​AAC​GGG​ACA​AAC​ATA​A-3′ (siM210409100739) and siRNA-NC: 5′-GGC​TCT​AGA​AAA​GCC​TAT​GC-3′ (siB161011044323), which were provided by RiboBio Co, Ltd, Guangzhou, China.

2.8. Separation technology of primary sensory neuron–derived exosomes

Anti-GFP magnetic beads (YJ010; Epizyme Biotech, Shanghai, China) were adopted to sort PSN-derived exosomes from the spinal dorsal horn. In short, AAV2/9-hsyn-Cre-mcherry was injected into the left T13 DRG in CD63-GFP^f/+ mice at 6 w, exosomes were extracted from the spinal dorsal horn by ultracentrifugation at 9 w, as described previously.³² Then the exosome suspension was added to the pretreated anti-GFP magnetic beads and incubated on a flip mixer at room temperature for 2 hours. The above mixture was placed on the magnetic frame for 1 minute, and the supernatant was removed. The precipitate was gently resuspended in the binding or washing buffer, placed on the magnetic frame for 1 minute to remove the supernatant, and repeated for 3 times. The remaining in the original centrifuge tube is the complex of PSN-derived exosomes and magnetic bead.

2.9. Real-time quantitative polymerase chain reaction

The mRNA levels of miRNA and molecules involved in exosomes formation and release were detected in CON and NMD mice by real-time quantitative polymerase chain reaction (Q-PCR).^32,41 Briefly, total RNA was extracted from the DRG or PSN-derived exosomes in spinal dorsal horn, the cDNA of miRNA and U6 were obtained by a reverse transcription kit (Applied Biosystems, Waltham, MA, Thermo Fisher Scientific, Shanghai, China), and other cDNAs were amplified by the One-step reverse transcription kit (Vazyme, Nanjing, China). The primer sequences used in Q-PCR are shown in Table 1. The obtained Ct values were statistically analyzed, and relative expressions of miRNAs were quantified relative to U6. glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used loading control.

Table 1.

Primer sequences used in the present study.

Primers	Sequence (5′ to 3′)
Rab27a-F	GAC​GCT​ATG​GGT​TTC​CTG​CT
Rab27a-R	TCC​AGG​AGC​ATC​TCA​ATC​GC
Rab27b-F	ACA​TCT​GCA​GCT​TTG​GGA​CA
Rab27b-R	TGA​CTT​CCC​TTT​GGT​CTG​GC
Rab2b-F	AAA​TCT​GGG​ATA​CGG​CTG​GG
Rab2b-R	GTT​GAA​GGT​TTC​GCG​TCT​CG
Rab5-F	CTG​GAG​CCC​GAG​TGT​TTG​TGT
Rab5-R	GCT​CGC​TCC​TTC​TTC​TCA​CCC
Rab35-F	GAC​AAC​TTG​GCG​AAA​CAG​CA
Rab35-R	AAC​TGA​GAC​TGT​CCC​CCG​AG
GAPDH-F	GAA​GGT​CGG​TGT​GAA​CGG​AT
GAPDH-R	AAT​CTC​CAC​TTT​GCC​ACT​GC
miR-1306-3p-RT	GTC​GTA​TCC​AGT​GCA​GGG​TCC​GAG​GTA​TTC​GCA​CTG​GAT​ACG​ACC​ATC​AC
miR-1306-3p-F	ACG​GCT​ACG​TTG​GCT​CTG​GT
miR-1306-3p-R	ATC​CAG​TGC​AGG​GTC​CGA​GG
miR-1949-RT	GTC​GTA​TCC​AGT​GCA​GGG​TCC​GAG​GTA​TTC​GCA​CTG​GAT​ACG​ACA​ACT​AT
miR-1949-F	AAC​ACG​TGC​TAT​ACC​AGG​ATG​TCA
miR-1949-R	ATC​CAG​TGC​AGG​GTC​CGA​GG
miR-185-5p-RT	GTC​GTA​TCC​AGT​GCA​GGG​TCC​GAG​GTA​TTC​GCA​CTG​GAT​ACG​ACT​CAG​GA
miR-185-5p-F	AAG​CGG​ATG​GAG​AGA​AAG​GCA​G
miR-185-5p-R	ATC​CAG​TGC​AGG​GTC​CGA​GG
miR-324-5p-RT	GTC​GTA​TCC​AGT​GCA​GGG​TCC​GAG​GTA​TTC​GCA​CTG​GAT​ACG​ACA​CAC​CA
miR-324-5p-F	AAT​TGC​TGC​GCA​TCC​CCT​AGG
miR-324-5p-R	ATC​CAG​TGC​AGG​GTC​CGA​GG
U6-RT	GTC​GTA​TCC​AGT​GCA​GGG​TCC​GAG​GTA​TTC​GCA​CTG​GAT​ACG​ACA​AAA​TA
U6-F	AGA​GAA​GAT​TAG​CAT​GGC​CCC​TG
U6-R	ATC​CAG​TGC​AGG​GTC​CGA​GG

GAPDH, glyceraldehyde-3-phosphate dehydrogenase

2.10. Western blotting

Western blotting was used to detect the expressions of GFP in spinal dorsal horn, and Rab27a and Rab27b in DRG, as described previously.³² Primary antibodies included anti-Rab27a (1:1000, Cat no. 69295; Cell Signaling Technology, Boston, MA, RRID: AB_2799759), anti-Rab27b (1:200, Cat no. 13412-1-AP; Proteintech, Chicago, IL, RRID: AB_2176732), anti-GFP (1:2000, Cat no. P30010; Abmart, Shanghai, China, RRID: AB_2936507), and anti-GAPDH (1:1000, Cat no. AB-P-R001; GoodHere, Auckland, New Zealand, RRID: AB_3096355). Secondary antibodies included goat anti-rabbit immunoglobulin G (IgG) (1:50000, Cat no. 111-035-003; Jackson ImmunoResearch Labs, West Grove, PA, RRID: AB_2313567) and goat anti-mouse IgG (1:50000, Cat# 115-035-003; Jackson ImmunoResearch Labs, RRID: AB_10015289). All blots were analyzed with Image J, and the expressions of GFP, Rab27a, and Rab27b were normalized to GAPDH.

2.11. Immunofluorescence assay

The expressions of Rab27a in DRG and GFP in spinal cord were measured by immunofluorescence staining technique, as described previously.³² All images were obtained by Axio Scope A1 microscope and confocal microscope (40× and 63× oil immersion lenses; Zeiss, Oberkochen, Germany), and one data point for each mouse was analyzed and averaged by 3 images from nonconsecutive sections. 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). The secondary antibodies were Alexa Fluor 488 donkey anti-rabbit lgG (H + L) (1:500, Cat no. A-21206; Thermo Fisher Scientific, RRID: AB_2535792), Alexa Fluor 488 donkey anti-goat lgG (H + L) (1:500, Cat no. A-11055; Thermo Fisher Scientific, RRID: AB_2534102), Alexa Fluor 568 donkey anti-rabbit lgG (H + L) (1:100, Cat no. A-10042; Thermo Fisher Scientific, RRID: AB_2534017), and Alexa Fluor 555 donkey anti-mouse lgG (H + L) (1:100, Cat no. A-31570; Thermo Fisher Scientific, RRID: AB_2536180).

2.12. Patch-clamp recordings

The synaptic transmission in spinal dorsal horn neurons was recorded by patch‐clamp, as described previously.^20,45 In brief, after anesthesia, the T13 to L2 spinal cord was exposed rapidly and immersed in preoxidized (95% O₂ and 5% CO₂) artificial cerebrospinal fluid. The soft spinal arachnoid was removed. Then the spinal cord slices (300 μm thickness) were prepared by vibrating microtome and transferred to the oxygenated artificial cerebrospinal fluid at 32°C for 30 minutes. Spontaneous excitatory postsynaptic currents (spontaneous excitatory postsynaptic currents [sEPSCs]) of spinal dorsal horn neurons were recorded.

2.13. Data analysis

All data in this study were shown as mean ± SEM. The data were analyzed with Graphpad prism 8.0. All data were analyzed by normal distribution test, 2-sample t test, Mann–Whitney test, paired t test, Wilcoxon matched pairs signed rank test, one-way analysis of variance followed by Tukey post hoc test, and 2-way analysis of variance followed by Sidak post hoc test or Dunnett post hoc test. When P < 0.05, the results were statistically significant.

3. Results

3.1. Neonatal maternal deprivation increased visible primary sensory neuron–derived exosomes in spinal dorsal horn of CD63-GFP^f/+ mice

To specifically visualize endogenous exosomes from different tissues or cells, Cre-dependent CD63-GFP^f/+ mice were engineered with the insertion of the -CAG-LSL-hCD63-copGFP-6xHis-WPRE-polyA gene sequence at Rosa26 (Fig. 1A), without affecting physiological function (Fig. S1, http://links.lww.com/PAIN/C221). Neonatal maternal deprivation was used to induce visceral pain and enhanced synaptic transmission in CD63-GFP^f/+ mice (Fig. 1A and Fig. S2, http://links.lww.com/PAIN/C221), which were consistent with our previous study.²⁰ In addition, NMD did not induce somatic pain, anxiety, and depressive-like behaviors in CD63-GFP^f/+ mice (Fig. S3, http://links.lww.com/PAIN/C221). To precise tracking of PSN-derived exosomes, AAV2/9-hsyn-Cre-mCherry viruses were injected into the left T13 DRG of CD63-GFP^f/+ mice at the age of 6 weeks and successfully expressed at 9 weeks (Fig. 1B). We observed that GFP signals were expressed in the ipsilateral spinal dorsal horn, which was basically co-expressed with the exosome marker CD63 (Fig. S4, http://links.lww.com/PAIN/C221). Further quantitative analysis showed that the percentage of GFP fluorescence area in total area of spinal dorsal horn (Figs. 1C and D) and protein expression of GFP in the spinal dorsal horn (Fig. 1E) were significantly increased of CD63-GFP^f/+ mice with NMD. To investigate the function of PSN-derived exosomes in spinal dorsal horn, the co-localization of PSN-derived exosomes with different types of cells was identified by immunofluorescence staining. The results of 3-dimensional confocal images showed that GFP was mainly co-localized with NeuN-labeled neurons in the spinal dorsal horn, but barely co-localized with the GFAP-labeled astrocyte and the Iba-1-labeled microglia (Figs. 1F and G). Statistics analysis showed that NMD significantly increased the percentage of neurons co-localized with GFP in total spinal dorsal horn neurons in CD63-GFP^f/+ mice (Fig. 1H). These results indicated that NMD increased the visible PSN-derived exosomes in the spinal dorsal horn and that PSN-derived exosomes most likely acted on neurons but not glial cells.

Figure 1.

Neonatal maternal deprivation increased visible PSN-derived exosomes in spinal dorsal horn of CD63-GFP^f/+ mice. (A) Schematic diagram of gene recombination. Insertion of CAG-LSL-hCD63-copGFP-6xHis-WPRE-polyA gene sequence at Rosa26 of mice to generate CD63-GFP^f/+ Cre-dependent mice. The time course and pattern diagram of NMD model and virus injection in CD63-GFP^f/+ mice. (B) The time course and pattern diagram of injection of AAV2/9-hsyn-Cre-mCherry viruses into DRG, and the representative diagram of viruses and GFP fluorescence expression. Red fluorescence: mCherry, green fluorescence: CD63-GFP, blue fluorescence: DAPI. Scale bar = 100 μm. (C) Distribution of GFP in the spinal cord of CON and NMD mice. Ipsi, ipsilateral, Contra, contralateral. Scale bar = 100 μm. (D) Quantification of GFP fluorescence area in the left spinal dorsal horn (layers Ⅰ–Ⅲ) of CON and NMD mice (n = 4 mice for each group, **P < 0.01, 2-sample t test). (E) The protein expression of GFP in the left spinal dorsal horn of CON and NMD mice (n = 8 mice for each group, *P < 0.05, Mann–Whitney test). (F) Fluorescent representation of co-localization of GFP (green) and NeuN-labeled neurons (red), GFAP-labeled astrocytes (red) and Iba-1-labeled microglia (red) in the spinal dorsal horn of CON and NMD mice. DAPI-labeled the nucleus (blue). Scale bar = 50 μm. The white box in the lower right corner of merged image showed the typical cells of each group (scale bar = 10 μm). (G) Representative 3D confocal images of the co-localization of GFP and NeuN, GFAP, and Iba-1 in the spinal dorsal horn, which was enlarged view in the white dotted box of (F). Scale bar = 20 μm. (H) Quantification of the percentage of GFP⁺ positive cells in the spinal dorsal horn neurons of CON and NMD mice (n = 3 mice for each group, **P < 0.01, 2-way ANOVA followed by Sidak post hoc test). ANOVA, analysis of variance; DRG, dorsal root ganglia; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; NMD, neonatal maternal deprivation; PSN, primary sensory neuron.

3.2. Intrathecal injection of GW4869 reduced visible primary sensory neuron–derived exosomes, suppressed spinal synaptic transmission, and alleviated visceral pain

To determine whether the increased release of PSN-derived exosomes was involved in visceral pain, AAV2/9-hsyn-Cre-mCherry viruses were injected into the left T13 DRG of NMD CD63-GFP^f/+ mice at 6 w, and the exosome synthesis/release inhibitor GW4869 (30 μM, 10 μL)³² was intrathecally injected into NMD mice at 9 w (Fig. 2A). The results showed that GW4869 significantly reduced the percentage of GFP fluorescence area in total area of spinal dorsal horn (Figs. 2B and C) and protein expression of GFP in the spinal dorsal horn (Fig. 2D). The percentage of neurons co-localized with GFP in total neurons from spinal dorsal horn of GW4869 group was also significantly less than that in normal saline group (Figs. 2E and F). In vitro spinal cord slice recording experiment showed that the frequency of sEPSCs in post-GW4869 incubation (30 μM) was significantly decreased although the amplitude was not altered (Fig. 2G). Furthermore, intrathecal injection of GW4869 (30 μM, 10 μL) alleviated the visceral pain in CD63-GFP^f/+ mice with NMD at 0.5 hour, and lasted for 8 hours (Fig. 2H). We also designed 2 additional experiments to exclude the toxic side effects of GW4869. Intrathecal injection of same dose of GW4869 did not affect visceral pain threshold in CON CD63-GFP^f/+ mice (Fig. 2I) nor affect motor function of CD63-GFP^f/+ mice with NMD (Fig. 2J). These results suggested that intrathecal injection of GW4869 blocked the release of PSN-derived exosomes, decreased spinal synaptic transmission, and alleviated visceral pain in CD63-GFP^f/+ mice with NMD.

Figure 2.

Intrathecal injection of GW4869 reduced visible PSN-derived exosomes, suppressed spinal synaptic transmission, and alleviated visceral pain. (A) Schematic diagram of virus injection into the left T13 DRG and intrathecal injection of NS or GW4869 in NMD mice. (B) Representative images of GFP in the left spinal dorsal horn of NS and GW4869 groups, scale bar = 50 μm. (C and D) Quantification of GFP fluorescence area (layers I to III, n = 3 mice for each group, **P < 0.01, 2-sample t test) and the protein levels (NS: n = 4 mice, GW4869: n = 3 mice, *P < 0.05, 2-sample t test) in the left spinal dorsal horn of NS and GW4869 mice. (E) Representative images of GFP, NeuN-labeled neurons and DAPI-labeled nucleus in the left spinal dorsal horn of NS and GW4869 mice, scale bar = 50 μm. The white box in the lower right corner was the typical GFP⁺ positive neurons, scale bar = 10 μm. (F) Quantification of the percentage of GFP⁺ positive cells in the spinal dorsal horn neurons of NS and GW4869 mice (NS: n = 4 mice, GW4869: n = 3 mice, ***P < 0.001, 2-sample t test). (G) Representative traces of sEPSCs were recorded in the T13 to L2 spinal dorsal horn neurons before (Pre group) and after incubation of GW4869 (30 μM, Post group). Histogram and cumulative probability distributions of the amplitude (n = 7 cells from 3 mice for each group, P > 0.05, paired t test) and frequency (n = 7 cells from 3 mice for each group, *P < 0.05, Wilcoxon matched pairs signed rank test) of sEPSCs in Pre and Post group. (H) Statistical diagram of the visceral pain threshold in NMD mice treated with NS or GW4869 at Pre, 0.5, 1, 2, 4, 8, and 12 hours (n = 8 mice for each group, **P < 0.01, ***P < 0.001, 2-way ANOVA followed by Sidak post hoc test). (I) Statistical diagram of visceral pain threshold in CON mice treated with NS or GW4869 at Pre, 0.5, 1, 2, 4, and 8 hours (n = 6 mice for each group, P > 0.05, 2-way ANOVA followed by Sidak post hoc test). (J) Statistical diagram of the mice' time on the rod in NMD mice treated with NS or GW4869 at Pre, 0.5, 1, 2, 4, 8, and 12 hours (n = 8 mice for each group, P > 0.05, 2-way ANOVA followed by Sidak post hoc test). ANOVA, analysis of variance; DRG, dorsal root ganglia; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; NMD, neonatal maternal deprivation; PSN, primary sensory neuron; sEPSC, spontaneous excitatory postsynaptic current.

3.3. Sorted primary sensory neuron–derived exosomal miR-1306-3p activated spinal P2X3R and enhanced synaptic transmission, thus leading to visceral pain

To further elucidate the specific mechanism by which PSN-derived exosomes contribute to visceral pain, we developed a novel method for isolating PSN-derived exosomes at the spinal dorsal horn. AAV2/9-hsyn-Cre-mcherry was injected into the left T13 DRG at 6 w in CD63-GFP^f/+ mice. Anti-GFP magnetic beads were used to sort the PSN-derived exosomes from total exosomes in the spinal dorsal horn (Fig. 3A). The expression of PSN-derived exosomal miR-1306-3p was significantly increased in the spinal dorsal horn of CD63-GFP^f/+ mice with NMD (Fig. 3B), whereas no differences were detected in the total exosomes from the spinal dorsal horn without sorting (Fig. S5, http://links.lww.com/PAIN/C221). Surprisingly, we observed that the PSN-derived exosomes were co-localized with P2X3R in the spinal dorsal horn neurons (Fig. 3C). To verify whether exosomal miR-1306-3p activated P2X3R in the spinal dorsal horn, the patch clamp recording experiment was performed in vitro. The recording showed incubation of miR-1306-3p (20 μM) significantly increased the frequency of sEPSCs, and Gefapixant (30 μM) reversed the effect in the spinal dorsal horn neurons of control mice (Fig. 3D). Furthermore, intrathecal injection of miR-1306-3p antagomir (Fig. 3E) or Gefapixant (Fig. 3F), the specific antagonist of P2X3R, obviously alleviated visceral pain in NMD mice. Conversely, intrathecal injection of miR-1306-3p agomir induced visceral pain at 0.5 hour, lasted for 2 hours (Fig. 3G), and this effect was reversed by Gefapixant (Fig. 3H) in control CD63-GFP^f/+ mice. These data indicated that increased PSN-derived exosomal miR-1306-3p activated P2X3Rs in the spinal dorsal horn, enhanced spinal synaptic transmission, and led to visceral pain in CD63-GFP^f/+ mice with NMD.

Figure 3.

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.

3.4. Expression of Rab27a was significantly increased and co-localized in GFP-positive dorsal root ganglia neurons

To further investigate the molecular mechanism underlying the enhanced release of PSN-derived exosomes in the spinal dorsal horn, the real-time quantitative PCR and western blotting were performed to detect the expression of Rab GTPase.²⁵ The results showed that NMD significantly increased the mRNA level of Rab27a and did not change the mRNA levels of Rab27b, Rab2b, Rab5, and Rab35 in the T13 to L2 DRGs of CD63-GFP^f/+ mice (Fig. 4A). The protein expression of Rab27a in NMD mice was higher than that in CON mice (Fig. 4B). The protein expression of Rab27b (another subtype of Rab27) remained unchanged, which was constant with mRNA levels in CD63-GFP^f/+ mice (Fig. 4C). The immunofluorescence staining showed that Rab27a was mainly expressed in neurons but hardly in glutamine synthetase–labeled satellite glial cells of the DRGs. Statistics analysis showed that NMD did not alter the proportion of Rab27a-positive neurons in CGRP-positive peptidergic neurons, Ib4⁺ positive nonpeptidergic neurons, and NF200-positive large neurons in CD63-GFP^f/+ mice (Figs. 4D and E). Furthermore, Rab27a was predominantly expressed in GFP-positive DRG neurons using AAV2/9-hsyn-Cre system (Fig. 4F), indicating that Rab27a might be involved in the release of PSN-derived exosomes.

Figure 4.

Expression of Rab27a was significantly increased and co-localized in GFP-positive DRG neurons. (A) The mRNA expressions of Rab27a, Rab27b, Rab2b, Rab5, and Rab35 were detected in the T13 to L2 DRG 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). (B) The protein expression of Rab27a (CON: n = 4 mice; NMD: n = 5 mice, *P < 0.05, 2 sample t test) in the T13 to L2 DRG of CON and NMD mice. (C) The protein expression of Rab27b (CON: n = 4 mice, NMD: n = 5 mice, P > 0.05, 2-sample t test) in the T13 to L2 DRG of CON and NMD mice. (D) Representative images of Rab27a co-localized with NeuN-positive neurons, GS-positive satellite glial cells, CGRP-positive small- and medium-sized peptidergic neurons, Ib4⁺ positive small- and medium-sized nonpeptidergic neurons, and NF200-positive large neurons. Scale bar = 50 μm. (E) Quantification of the proportion of Rab27a-positive cells in the 3 types of neurons and satellite glial cells both CON and NMD group (n = 3 mice for each group, P > 0.05, 2-way ANOVA followed by Sidak post hoc test). (F) Representative images of Rab27a co-localized in GFP-positive T13 to L2 DRG neurons, scale bar = 50 μm. The image of typical co-localization on the right was the enlarged view in the white box of merge. Scale bar = 10 μm. ANOVA, analysis of variance; CGRP, calcitonin gene-related peptide; DRG, dorsal root ganglia; GFP, green fluorescent protein; GS, glutamine synthetase; NMD, neonatal maternal deprivation; Q-PCR, real-time quantitative polymerase chain reaction.

3.5. siR-Rab27a reduced visible primary sensory neuron–derived exosomes, suppressed spinal synaptic transmission, and alleviated visceral pain

To verify whether Rab27a regulated the release of PSN-derived exosomes, AAV2/9-hsyn-Cre-mcherry was injected into the left T13 DRG in CD63-GFP^f/+ mice with NMD at 6 w. siRNA-Rab27a (siR-Rab27a, 1 nmol, 10 μL, twice) was injected intrathecally at 9 w (Fig. S6A, http://links.lww.com/PAIN/C221). The Rab27a protein expression in DRG was significantly knocked down by injection of siR-Rab27a in CD63-GFP^f/+ mice (Fig. S6B, http://links.lww.com/PAIN/C221) and did not affect the expression of Rab27a in the spinal cord (Fig. S6C, http://links.lww.com/PAIN/C221). Intrathecal injection of siR-Rab27a significantly reduced the percentage of GFP fluorescence area in total area of spinal dorsal horn (Figs. 5A and B) and protein expression of GFP in the spinal dorsal horn (Fig. 5C). Immunofluorescence staining showed that the percentage of GFP⁺ positive neurons (Figs. 5D and E) and GFP⁺ P2X3R⁺ positive neurons (Figs. 5F and G) in total spinal dorsal horn neurons in siR-Rab27a-treated mice were significantly less than that in siR-NC-treated mice. Intrathecal injection of siR-Rab27a (1 nmol, 10 μL, twice) obviously decreased the frequency of sEPSCs (Fig. 5H) and alleviated visceral pain (Fig. 5I) in CD63-GFP^f/+ mice with NMD. Intrathecal injection of siR-Rab27a did not affect the motor function (Fig. 5J). These results suggested that intrathecal injection of siR-Rab27a reduced the PSN-derived exosomes in the spinal dorsal horn, suppress spinal synaptic transmission, and alleviate visceral pain in CD63-GFP^f/+ mice with NMD.

Figure 5.

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.

4. Discussion

The pathological mechanisms and triggers of chronic visceral pain in patients with IBS are intricate and varied, and the limited clinical treatment options significantly affect patients' quality of life and mental health.⁸ The present study used a novel exosome visualization technique to reveal that the PSN-derived exosomal-miR-1306-3p levels were significantly enhanced in the spinal dorsal horn, which was likely mediated by upregulation of Rab27a expression in DRG neurons. Further experiments showed that exosomal-miR-1306-3p activated spinal P2X3Rs, thus enhancing synaptic transmission, eventually exacerbating visceral pain in CD63-GFP^f/+ male mice with NMD (Fig. 6). Although sex differences were not specifically explored, this study delves into a role of PSN-derived exosomes, offering a novel perspective and theoretical framework for understanding visceral pain mechanisms in patients with IBS.

Figure 6.

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.

Exosomes have been reported to participate in many physiological and pathophysiologic functions.^11,12,36 However, identifying the origins of exosome release has been a huge challenge. The present study established a novel approach to exhibit the origin of exosome release at spinal cord level. The spinal exosomes may be released not only from the intrinsic spinal cord cells but also from peripheral tissues or the descending terminals of supraspinal brain regions as well. The complexity of exosome release posed a challenge for conventional ultracentrifugation techniques, which could not differentiate these exosomes from different origins. To identify the precise origin of spinal exosomal release, exosome visualization technique was first generated in CD63-GFP^f/+ Cre-dependent mice. This is a breakthrough for investigating the mechanisms and for the precise treatment of visceral pain by targeting the specific exosomes at their origin. Two key experiments were conducted to validate the utility of CD63-GFP^f/+ mice. First, the specificity was confirmed by observing GFP colocalization with the exosome marker CD63 in the spinal dorsal horn of CD63-GFP^f/+ mice by injection of AAV2/9-hsyn-Cre-mCherry into the DRG. The exosomes were indeed tracked by GFP expression. Second, it was verified that gene editing did not affect the physiological functions of CD63-GFP^f/+ mice. This ensures the authenticity of subsequent experiments. The CD63-GFP^f/+ mice exhibited visceral pain behaviors after NMD as described previously in wild-type NMD mice,^20,29,32,44 confirming the successful establishment of a visceral pain model in CD63-GFP^f/+ mice. Together, these data demonstrated that the exosome visualization technique can be used to determine the origin of exosome release at spinal cord level to understand the pain transmission. Importantly, the versatility of exosome visualization technique with CD63-GFP^f/+ mice extends beyond visceral pain research field, promising applications in other neurological disorders.

The exosomes visualization technology was not only used for identifying the origin of exosome release but also for sorting the targeted exosomes for gene sequencing as well. By using the newly established exosomes visualization technology, the present study showed that miR-1306-3p levels in PSN-derived exosomes were significantly increased in spinal dorsal horn of CD63-GFP^f/+ mice with NMD. In CD63-GFP^f/+ mice with injection of AAV2/9-hsyn-Cre-mCherry into the DRG, PSN-derived exosomes were isolated from total spinal exosomes using anti-GFP magnetic beads, revealing a significant increase in miR-1306-3p levels of PSN-derived exosomes in CD63-GFP^f/+ mice with NMD when compared with control mice. However, there was no significant difference in the expression of miR-1306-3p in total nonvisualized exosomes extracted from the spinal dorsal horn between CON and NMD mice, indicating one of many advantages by using exosome visualization techniques.

A surprising discovery is that the PSN-derived exosomal miR-1306-3p activated P2X3R in the spinal dorsal horn neurons. This conclusion is supported by the following observations. Primary sensory neuron–derived exosomes were colocalized with P2X3R-positive neurons in the spinal dorsal horn. Intrathecal injection of miR-1306-3p antagomir or Gefapixant, a specific P2X3R antagonist, markedly alleviated visceral pain in CD63-GFP^f/+ mice with NMD. Furthermore, administration of miR-1306-3p increased the frequency of sEPSCs and induced visceral pain in CD63-GFP^f/+ control mice, whereas Gefapixant reversed these effects. Although the reason why the postsynaptic mechanisms were not involved was not clear, one potential explanation for this was that the expression of P2X3Rs in the spinal dorsal horn was not increased. Our previous study confirmed that miR-1306-3p could function as a novel ligand for P2X3R,⁴² which further strengthens the findings of the current study. However, the specific mechanism by which miRNAs were released from exosomes into the extracellular space is not yet clear. It was reported that there may be 2 possible pathways.¹ First, exosomes could be cleaved by proteases and phospholipases in the synaptic cleft, leading to the release of their cargo, such as miRNAs. Second, exosomes needed to rely on the endocytosis of recipient cells to cleave their bilayer membrane and release their cargo back into the extracellular space. In this study, we also found that exosomes were co-localized with neurons. These data indicated that exosomes may release miR-1306-3p through endocytosis and cleavage by spinal neurons, thereby mediating visceral pain. Of note is that because P2X3Rs were also expressed on the presynaptic membrane, it was difficult to rule out the influence of PSN-derived exosomes on presynaptic activity. In addition, exosomes are known to transport various substances such as nucleic acids, proteins, and lipids, facilitating intercellular communication.^6,11 Although our study focused on miRNA function, the contribution of other exosomal cargos to visceral pain could not be disregarded since a recent review pointed out that exosomes may also function like a neurotransmitter by cargo of the neuropeptide.⁴³ Nevertheless, the exosomes visualization technology reveals that PSN-derived exosomal miR-1306-3p activated P2X3Rs in the spinal dorsal horn, thus enhancing synaptic transmission and leading to visceral pain.

Another important finding is that DRG neurons released exosomes, named PSN-derived exosomes, to the spinal dorsal horn using afferent fibers, introducing an alternative mechanism for neuronal exosome release. The release of exosomes from neurons is primarily observed in neuronal soma or dendrites in situ, rarely in axons.^2,23 The PSNs are pseudounipolar neurons without dendrites. The release of exosomes in the peripheral nervous system remains unclear. In this study, we observed for the first time that DRG neurons could release exosomes to the spinal dorsal horn using afferent fibers. This finding further underscores variations in exosome release across different synaptic types or neuron types.^14,23 Traditionally, synaptic transmission in the nervous system mainly relies on neurotransmitter release.^5,19 However, our study revealed a novel mechanism that increased PSN-derived exosomes enhanced the spinal synaptic transmission, thus leading to visceral pain in CD63-GFP^f/+ mice with NMD by exosome visualization technology. This significant discovery was supported by several experiments. First, injection of AAV2/9-hsyn-Cre-mCherry into DRG could visualize the PSN-derived exosomes in the spinal dorsal horn, and NMD obviously increased the PSN-derived exosomes in CD63-GFP^f/+ mice. Second, intrathecal injection of GW4869, an inhibitor of exosome biosynthesis/release, blocked the release of PSN-derived exosomes, reduced spinal synaptic transmission, and alleviated visceral pain in CD63-GFP^f/+ mice with NMD. These data provided important evidence that PSN-derived exosomes also played an important role in synaptic transmission and visceral pain in IBS.

Mechanisms underlying the enhanced release of exosomes remains largely unknown. The present study demonstrated that upregulation of Rab27a in DRG contributed to the increase of the PSN-derived exosomes in the spinal dorsal horn of CD63-GFP^f/+ mice with NMD. Previous studies have shown that Rab27a is one of the important Rab GTPase family members that mediated the transport and docking of MVBs to the plasma membrane, thus regulating the release of exosomes.^25,34 The present study demonstrated that expression of Rab27a in DRG was significantly increased and co-localized with MVBs-GFP in DRG neurons in CD63-GFP^f/+ mice with NMD. Intrathecal injection of siR-Rab27a significantly reduced the release of PSN-derived exosomes and the proportion of colocalization with P2X3R-positive neurons in the spinal dorsal horn, decreased synaptic transmission, and alleviated visceral pain in CD63-GFP^f/+ mice with NMD. These findings provided a new target for the clearance of PSN-derived exosomes.

In summary, the present study demonstrated that the upregulation of Rab27a in DRG neurons increased the release of PSN-derived exosomal-miR-1306-3p. The exosomal-miR-1306-3p activated P2X3Rs in the spinal dorsal horn and enhanced synaptic transmission, thus leading to visceral pain in CD63-GFP^f/+ mice with NMD (Fig. 6). These findings confirmed a new signal pathway about exosomes in visceral pain and provided a potential target and theoretical basis for the clinical treatment in patients with IBS.

Conflict of interest statement

The authors have no conflicts of interest to declare.

Supplemental digital content

Supplemental digital content associated with this article can be found online at http://links.lww.com/PAIN/C221.

Supplementary Material

jop-166-2054-s001.pdf ^{(1.2MB, pdf)}

jop-166-2054-s002.pdf ^{(283.6KB, pdf)}