Sprouting sympathetic fibres release CXCL16 and norepinephrine to synergistically mediate sensory neuronal hyperexcitability in a rodent model of neuropathic pain

Abstract

Background: Chronic neuropathic pain generally has a poor response to treatment with conventional drugs. Sympathectomy can alleviate neuropathic pain in some patients, suggesting that abnormal sympathetic-somatosensory signaling interactions might underlie some forms of neuropathic pain. The molecular mechanisms underlying sympathetic-somatosensory interactions in neuropathic pain remain obscure.

Methods: Lumbar sympathectomy was performed in spared nerve injury (SNI) mice or rats, and the up-down method was used to measure the mechanical paw withdrawal threshold. Dorsal root ganglia (DRG) injection and perfusion were used to deliver virus or drugs. Methylated RNA immunoprecipitation sequencing, RNA-sequencing, and immunoelectron microscopy were used to identify neurotransmitters.

Results: We found that sprouting tyrosine hydroxylase-positive sympathetic fibres in DRG mediated the maintenance of mechanical allodynia after SNI (day 28, P<0.001). We further found that SNI significantly increased the N⁶-methyladenosine level of CXCL16 messenger RNA (day 28, P<0.001), which was attributable to the reduced N⁶-methyladenosine demethylase fat mass and obesity-associated protein (P=0.002) and increased interaction with YTHDF1 (P=0.013) in the sympathetic ganglion. Enhanced expression of CXCL16 in the sympathetic ganglia can lead to increases release into the DRG and act synergistically with norepinephrine from sympathetic terminals to enhance DRG neuronal excitability.

Conclusions: Norepinephrine and CXCL16 co-released from sympathetic nerve terminals in the DRG synergistically contribute to maintenance of neuropathic pain in a rodent model.

Keywords: CXC motif chemokine ligand 16; N(6)-methyladenosine; chronic neuropathic pain; norepinephrine; sympathetic ganglion.

Fig 1

Sympathetic nerve fibres that sprout into DRG contribute to mechanical allodynia maintenance after spared nerve injury. (a) Mechanical allodynia was induced after SNI (∗∗P<0.01 vs the corresponding sham group using the Mann–Whitney test. Day 3, Z=−3.047, P=0.002; day 7: Z=−2.908, P=0.004; day 14: Z=−2.989, P=0.003; day 21: Z=−2.961, P=0.003; day 28: Z=−2.994, P=0.003, n=6 rats in each group). (b) The sprouting sympathetic nerve fibres in DRG (n=3 in each group). Scale bar, 50 μm. (c) Scheme and typical image for DRG injection with CTB555 in rats. (d) Typical images of CTB in the DRG of rats and representative images in which CTB+ signals were expressed in TH-positive cells in the SG (n=3 in each group). Scale bar, 50 μm. (e) The TH-positive fibres in DRG were not present on day 14 after SYT (n=3). (f) SYT alleviated SNI-induced mechanical allodynia in the maintenance phase (∗∗P<0.01 vs corresponding SNI group, day 15: Z=−2.730, P=0.004; day 18: Z=−3.784, P<0.001; day 21: Z=−3.523, P<0.001; day 24: Z=−3.788, P<0.001; day 28: Z=−3.784, P<0.001, n=6, 8, or 13 rats in each group). CTB, cholera toxin B subunit; DAPI, 4',6-diamidino-2-phenylindole; DRG, dorsal root ganglia; IF, immunofluorescence; PWT, paw withdrawal threshold; SG, sympathetic ganglia; SNI, spared nerve injury; SYT, sympathectomy; TH, tyrosine hydroxylase.

Fig 2

Neurotransmitters released from the sprouting sympathetic nerve into DRG participate in mechanical allodynia maintenance. (a) The expression of Sy38 was co-localised with the TH-positive fibres in DRG (n=3 in each group), scale bar, 50 μm. (b) A synaptic-like structure was revealed between the sympathetic nerve endings (TH-positive signal, blue arrow) and DRG neurones (Na1.7 positive signal, red arrow). (c) Norepinephrine (red arrow) was expressed in the presynaptic terminal of sympathetic nerve fibres (blue arrow indicated the TH-positive signal) in DRG on day 28 after SNI (n=2 in each group). (d) Representative traces of action potentials evoked by 440 pA current injection (above). A line chart showed the number of action potentials evoked by inward currents after incubation of NE or saline (below). (∗P<0.05 relative to the corresponding control group by using the repeated measurement analysis of variance, F=4.843, P=0.042, n=8 or 11 for each group). (e) Intra-DRG injection of NE induced the acute mechanical allodynia in naive rats (∗∗P<0.01 relative to the corresponding saline group using the Mann–Whitney U-test. Day 1: Z=−2.863, P=0.004; day 3: Z=−2.903, P=0.004; day 5: Z=−2.939, P=0.003; n=6 rats for each group). (f) Intra-DRG injection of AAV-TH-TeNT or yohimbine relieved the mechanical allodynia, and the analgesic effect of yohimbine was much lower than that of AAV-TH-TeNT (∗∗P<0.01 relative to the corresponding scramble group using the Mann–Whitney test. In the AAV-TeNT group, day 35: Z=−3.288, P<0.001; day 42: Z=−3.283, P<0.001. In the yohimbine group, day 35: Z=−3.305, P<0.001; day 42: Z=−3.286, P<0.001. ^#P<0.05 relative to the SNI + AAV-TH-TeNT group using the Mann–Whitney test, day 35: Z=−2.129, P=0.035; day 42: Z=−2.010, P=0.043, n=10 or 6 for each group). (g) Scheme and typical images for DRG injection with retro-AAV-TH-TeNT-mCherry and the expression of mCherry in SG in rats. Scale bar, 50 μm. AAV, adeno-associated virus; AP, action potential; DRG, dorsal root ganglia; NE, norepinephrine; PWT, paw withdrawal threshold; SG, sympathetic ganglia; SNI, spared nerve injury; TeNT, tetanus toxin; TH, tyrosine hydroxylase.

Fig 3

The m6A level of CXCL16 mRNA was increased in sympathetic ganglion neurones after spared nerve injury. (a) Dot blotting showed that the level of total m6A modification increased on day 28 after SNI in sympathetic ganglion tissue. Methylene blue (MB) staining was used as a loading control (∗P<0.05 vs corresponding sham group using t-test, F=24.151, t=−3.492, df=10, P=0.017, n=6 rats for each group). (b) Volcano plot showed the number of differential m6A peaks in sympathetic ganglion tissues on day 28 after SNI with a criterion of P<0.01 and |FC|>1.5. (c, d) The distribution of the m6A modification in mRNA in the sham group and SNI group. (e) The motif of m6A modification in the sham group and SNI group was identified by HOMER. (f) RNA-seq was performed to examine the altered gene in sympathetic ganglion on day 28 after SNI. (g) The intersection of MeRIP-seq and RNA-seq data showed that four genes may be regulated by m6A modification in sympathetic ganglion neurones in neuropathic pain. (h) RT–PCR showed that SLFN4 and CXCL16 mRNA were significantly increased in sympathetic ganglion on day 28 after SNI (∗P<0.05, ∗∗P<0.01 vs corresponding sham group using t-test. For SLFN4, F=5.816, t=−3.139, df=4, P=0.035. For CXCL16, F=0.585 t=−6.547, df=4, P=0.003 (n=3 for each group). (i) Me-RIP assay revealed that the level of CXCL16 mRNA, but not SLFN4 mRNA, precipitated by m6A antibody was significantly increased on day 28 after SNI (∗P<0.05 vs corresponding sham group using t-test, F=6.062 t=−3.795, df=4, P=0.019, n=3 for each group). CDS, coding sequence; IgG, immunoglobulin G; M6A, N6-methyladenosine; mRNA, messenger RNA; RNA-seq, RNA-sequencing; RT–PCR, reverse transcription–polymerase chain reaction; SNI, spared nerve injury.

Fig 4

SNI-induced FTO downregulation is involved in CXCL16 upregulation by increasing m6A levels in sympathetic ganglia. (a) The level of FTO mRNA was decreased in sympathetic ganglia on day 28 after SNI (∗P<0.05 vs corresponding sham group using the Mann–Whitney test, Z=−2.562, P=0.009, n=6 rats for each group). (b) FTO protein was downregulated in sympathetic ganglia on day 28 after SNI (∗P<0.05 vs corresponding sham group using t-test, F=3.948, t=7.036, df=4, P=0.002, n=3 rats for each group). (c) Immunostaining assay showed that FTO was fully expressed in the TH-positive cells and co-localised with CXCL16-positive cells in sympathetic ganglia (n=3 rats for each group). (d) Scheme and typical injection position images for AAV-TH-FTO-mCherry injected into DRG in rats and the expression of mCherry in SG in rats. Scale bar, 50 μm. (e) Injecting retro-AAV-TH-FTO into DRG continuously decreased the m6A level on CXCL16 mRNA (∗∗P<0.01 vs corresponding control group using t-test, day 35, F=2.272, t=4.858, df=4, P=0.008; day 42, F=1.675, t=4.837, df=4, P=0.008; day 49, F=3.439, t=5.473, df=4, P=0.005; day 56, F=3.065, t=5.930, df=4, P=0.004, n=3 for each group). (f) Overexpression of FTO decreased expression of CXCL16 mRNA in sympathetic ganglia after SNI (∗∗P<0.01 vs corresponding control group using t-test, day 35, F=3.532, t=4.261, df=10, P=0.002; day 42, F=1.794 t=4.263, df=10, P=0.002; day 49, F=1.794, t=4.263, df=10, P=0.002; day 56, F=4.686, t=5.129, df=10, P<0.001, n=6 rats for each group). (g) Overexpression of FTO decreased expression of CXCL16 protein in sympathetic ganglia after SNI (∗∗∗P<0.001 vs corresponding control group using t-test, day 35, F=6.398, t=10.628, df=4, P<0.001; day 42, F=2.960, t=39.699, df=4, P<0.001; day 49, F=1.880, t=37.784, df=4, P<0.001; day 56, F=0.022, t=31.190, df=4, P<0.001, n=3 for each group). (h) Overexpression of FTO in sympathetic ganglia attenuated mechanical allodynia after intra-DRG injection of retro-AAV-TH-FTO (∗∗∗P<0.001 vs corresponding control group using the Mann–Whitney test, day 35, Z=−4.073, P<0.001; day 42, Z=−3.638, P<0.001; day 49, Z=−3.759, P<0.001; day 56, Z=−4.065, P<0.001, n=11 or 12 rats for each group). AAV, adeno-associated virus; ALKBH5, AlkB homolog 5; DAPI, 4', 6-diamidino-2-phenylindole; DRG, dorsal root ganglia; FTO, fat mass and obesity-associated protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; m6A, N6-methyladenosine; METTL3, methyltransferase-like 3; METTL14, methyltransferase-like 14; mRNA, messenger RNA; PWT, paw withdrawal threshold; SG, sympathetic ganglia; SNI, spared nerve injury; TH, tyrosine hydroxylase; WTAP, Wilms' tumor 1-associating protein.

Fig 5

CXCL16 upregulation mediated by YTHDF1 in sympathetic ganglia enhanced DRG neurone excitability and participated in chronic mechanical allodynia after SNI. (a) Expression of CXCL16 mRNA was examined on days 14, 21, and 28 after SNI in sympathetic ganglia (∗∗P<0.01 vs corresponding sham group using one-way anova of post hoc test, day 14: P=0.002; day 21: P=0.001; day 28: P=0.001, n=6 rats for each group). (b) SNI upregulated the expression of CXCL16 protein on days 14, 21, and 28 in sympathetic ganglia (∗∗P<0.01 vs a corresponding sham group using one-way anova of post hoc test, day 14: P<0.001; day 21: P<0.001; day 28: P<0.001, n=3 rats for each group). (c) A significant amount of CXCL16 (red arrow) was expressed in presynaptic sympathetic nerve fibres (blue arrow indicated the TH-positive signal) in DRG on day 28 after SNI (n=2). (d) Immunostaining showed that CXCL16 receptor CXCR6 was expressed in neurones in DRG (n=3). (e) Incubation with CXCL16 increased the number of action potentials in neurones of naive DRG slices (∗P<0.05 relative to the corresponding control group using repeated measurement anova, F=4.358, P=0.049, n=10 or 13 rats for each group). (f) Intra-DRG injection of CXCL16 induced mechanical allodynia in naive rats (∗∗P<0.01 relative to the corresponding control group using the Mann–Whitney U-test, day 3: Z=−3.514, P<0.001; day 7: Z=−3.456, P<0.001; day 10: Z=−3.583, P<0.001; day 14: Z=−3.514, P<0.001; day 28: Z=−3.459, P<0.001, n=8 rats for each group). (g) Scheme and typical images for DRG after AAV-TH-Cre injected into DRG in CXCL16-flox-mice. Scale bar, 50 μm. (h) Knockout CXCL16 by injected retro-AAV-TH-Cre into the DRG of CXCL16^flox mice alleviated the maintenance of SNI-induced mechanical allodynia (∗∗P<0.01 relative to the corresponding SNI + scramble group using the Mann–Whitney U-test, day 35: Z=−3.148, P=0.002; day 42: Z=−3.137, P=0.002; day 49: Z=−3.137, P=0.002; day 56: Z=−3.084, P=0.002, n=8 rats for each group). (i, j) SNI did not change the expression of CXCL16 mRNA and protein on days 3 and 7 after SNI in DRG (n=4). (k–m) The interaction between CXCL16 mRNA and YTHDF1 (k), YTHDF2 (l), or YTHDF3 (m) was examined on day 28 after SNI (∗P<0.05 relative to the corresponding sham group using t-test, F=1.801, t=−4.340, df=4, P=0.012, n=3 for each group). (n) Scheme and typical images for DRG after AAV-TH-Cre injected into DRG in YTHDF1-flox mice. Scale bar, 50 μm. (o) Knockout YTHDF1 by injected retro-AAV-TH-Cre into the DRG of YTHDF1^flox mice decreased SNI-induced CXCL16 upregulation in sympathetic ganglia (∗∗∗P<0.001 relative to the corresponding sham group SNI + scramble group using t-test, F=0.919, t=41.860, df=4, P<0.001, n=3 for each group). (p) Knockout YTHDF1 of sympathetic ganglia alleviated mechanical allodynia induced by SNI (∗∗P<0.01 relative to the corresponding SNI + scrambled group using the Mann–Whitney U-test, day 35: Z=−3.165, P=0.002; day 42: Z=−3.151, P=0.002; day 49: Z=−3.137, P=0.002; day 56: Z=−3.169, P=0.002, n=7 rats for each group). AAV, adeno-associated virus; anova, analysis of variance; AP, action potential; DRG, dorsal root ganglia; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA; PWT, paw withdrawal threshold; SG, sympathetic ganglia; SNI, spared nerve injury; TH, tyrosine hydroxylase.

Fig 6

Synergy between CXCL16 and norepinephrine from sympathetic ganglia enhances the excitability of DRG neurones and contributes to neuropathic pain maintenance after SNI. (a) CXCL16 was expressed in TH-positive cells in sympathetic ganglia (n=3). (b) Immunoelectron microscopy showed that NE and CXCL16 were co-expressed in sympathetic fibre terminals in DRG on day 28 after SNI (n=2). (c) Compared with the NE group or CXCL16 group, incubation with NE and CXCL16 increased the number of action potentials in naive DRG neurones (∗∗P<0.01 relative to the corresponding CXCL16 group using repeated measurement analysis of variance, F=7.845, P=0.008, n=22 rats for each group. ^#P<0.05 relative to the NE group, F=6.969, P=0.013, n=9 or 22 rats for each group). (d) Compared with the NE or CXCL16 group, intra-DRG injection with NE and CXCL16 induced mechanical allodynia (∗∗P<0.01 relative to the corresponding CXCL16 group using t-test, day 1: F=1.014, t=4.571, df=10, P=0.001; day 3: F=9.055, t=4.120, df=10, P=0.002; day 5: F=6.276, t=4.157, df=10, P=0.002. ^##P<0.01 relative to the yohimbine group using t-test, day 1: F=0.153 t=4.750, df=10, P<0.001; day 3: F=4.617, t=6.174, df=10, P<0.001; day 5: F=0.044, t=7.266, df=10, P<0.001, n=6 rats for each group). (e) Hybrid simulation diagram of the Phox2b-Cre mice and CXCL16^flox mice, and F4 mice with CXCL16^-/- in Phox2b-positive neurones used for behavioural tests. (f and g) CXCL16 mRNA and protein in sympathetic ganglia were significantly decreased in the newborn F4 mice (∗∗P<0.01 relative to the corresponding SNI group using t-test, F=1.236, t=2.949, df=10, P=0.015, n=6 rats for each group). (h) Compared with the yohimbine group or CXCL16^flox mice with injection of retro-AAV-TH-Cre, application of yohimbine in F4 mice relieved mechanical allodynia maintenance induced by SNI (∗∗P<0.01 relative to the corresponding CXCL16 group using the Mann–Whitney U-test, day 28: Z=−3.137, P=0.002; day 35: Z=−3.155, P=0.002; day 42: Z=−3.036, P=0.002. ^##P<0.01 relative to the yohimbine group using the Mann–Whitney U-test, day 28: Z=−2.390, P=0.017; day 35: Z=−2.769, P=0.006; day 42: Z=−2.674, P=0.007, n=7 rats for each group). AAV, adeno-associated virus; AP, action potential ; DAPI, 4',6-diamidino-2-phenylindole; DRG, dorsal root ganglia; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA; NE, norepinephrine; PWT, paw withdrawal threshold; SNI, spared nerve injury; TH, tyrosine hydroxylase.