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. 2018 May 23;38(21):4899-4911.
doi: 10.1523/JNEUROSCI.0421-18.2018. Epub 2018 Apr 30.

GDNF, Neurturin, and Artemin Activate and Sensitize Bone Afferent Neurons and Contribute to Inflammatory Bone Pain

Sara Nencini  1 , Mitchell Ringuet  1 , Dong-Hyun Kim  1 , Claire Greenhill  1 , Jason J Ivanusic  2
Affiliations

GDNF, Neurturin, and Artemin Activate and Sensitize Bone Afferent Neurons and Contribute to Inflammatory Bone Pain

Sara Nencini et al. J Neurosci. .

Abstract

Pain associated with skeletal pathology or disease is a significant clinical problem, but the mechanisms that generate and/or maintain it remain poorly understood. In this study, we explored roles for GDNF, neurturin, and artemin signaling in bone pain using male Sprague Dawley rats. We have shown that inflammatory bone pain involves activation and sensitization of peptidergic, NGF-sensitive neurons via artemin/GDNF family receptor α-3 (GFRα3) signaling pathways, and that sequestering artemin might be useful to prevent inflammatory bone pain derived from activation of NGF-sensitive bone afferent neurons. In addition, we have shown that inflammatory bone pain also involves activation and sensitization of nonpeptidergic neurons via GDNF/GFRα1 and neurturin/GFRα2 signaling pathways, and that sequestration of neurturin, but not GDNF, might be useful to treat inflammatory bone pain derived from activation of nonpeptidergic bone afferent neurons. Our findings suggest that GDNF family ligand signaling pathways are involved in the pathogenesis of bone pain and could be targets for pharmacological manipulations to treat it.SIGNIFICANCE STATEMENT Pain associated with skeletal pathology, including bone cancer, bone marrow edema syndromes, osteomyelitis, osteoarthritis, and fractures causes a major burden (both in terms of quality of life and cost) on individuals and health care systems worldwide. We have shown the first evidence of a role for GDNF, neurturin, and artemin in the activation and sensitization of bone afferent neurons, and that sequestering these ligands reduces pain behavior in a model of inflammatory bone pain. Thus, GDNF family ligand signaling pathways are involved in the pathogenesis of bone pain and could be targets for pharmacological manipulations to treat it.

Keywords: GDNF; artemin; bone pain; neurturin; pain; skeletal pain.

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Figures

Figure 1.
Figure 1.
Experimental setup of the in vivo bone–nerve preparation and spike discrimination. A, Schematic of the electrophysiological setup. B, Whole-nerve recording and rasters of single-unit activity in response to a ramp-and-hold pressure stimulus applied to the marrow cavity. Action potentials from single mechanically activated units were discriminated by their amplitude and duration using Spike Histogram software. An example of a single spike for each unit is at the left of each raster. C, Relationship between conduction velocity and amplitude of action potentials. Action potential amplitude (in μV; peak-to-peak) was plotted against conduction velocity (in m/s) for 51 single units activated by high-intensity intraosseous pressure stimuli (≥300 mmHg) during recordings made from seven animals. Units that also responded to stimulation of surrounding tissues (white circles) had very large action potential amplitudes (>120 μV) and conducted in the Aβ range (≥14.3 m/s). Units with conduction velocities in the C-fiber range (<2.5 m/s) had the smallest action potential amplitudes (<40 μV peak-to-peak; gray circles). All other units, with action potential amplitudes of >40 μV peak-to-peak that did not respond to the stimulation of surrounding tissues, had Aδ conduction velocities (black circles). A/D, Analog to digital.
Figure 2.
Figure 2.
Constitutive expression of GFRα1 to GFRα3 in bone afferent neurons. A, Schematic representation of the retrograde tracing approach used in this study. FB was injected into the marrow cavity (MC) of the rat tibia. The tracer was taken up by nerve terminals and transported back to their soma in the DRG, permitting the identification of sensory neurons that innervate the rat tibia. B, Size/frequency distribution of all retrograde-labeled bone afferent neurons analyzed in this study. Greater than 95% of these were small- or medium-sized sensory neurons (<1800 μm2). C, Images of retrograde-labeled and immunolabeled bone afferent neurons in sections through the DRG. Each horizontal set of panels shows the same field of a single section. Left, Incorporation of the retrograde tracer FB. Middle, GFRα1–3 immunolabeling. Right, A merged image. Arrowheads identify retrograde-labeled bone afferent neurons throughout. Asterisks (*) indicate bone afferent neurons that express GFRα1, GFRα2, or GFRα3. Scale bars, 50 μm. D, Size/frequency distributions of retrograde labeled bone afferent neurons (black) and those that that also express GFRα1 (blue), GFRα2 (green), or GFRα3 (red). SC, Spinal cord.
Figure 3.
Figure 3.
GDNF, neurturin, and artemin applied directly to the marrow cavity activates bone afferent neurons. A, Frequency histograms of the total number of spikes isolated from whole-nerve recordings before and after the application of GDNF, neurturin, or artemin (n = 5 animals each). Bin width, 30 s. Error bars indicate the SEM. B, Group data showing the number of spikes recorded (mean ± SEM) before and 5, 15, 30, 45, and 60 min after the application of GDNF, neurturin, artemin, or saline (n = 5 animals each). At each time-point, the number of spikes has been determined over five consecutive minutes and is represented as the mean ± SEM. Application of GDNF and neurturin resulted in an increase in whole-nerve activity, relative to saline, from the 15 and 30 min time-points, respectively (Bonferroni's post hoc test, *p < 0.05). The application of artemin resulted in a significant increase in whole-nerve activity, relative to saline, at the 5 and 15 min time-points, but not at the later time-points (Bonferroni's post hoc test, *p < 0.05). C, Frequency histograms showing the number of spikes with amplitudes consistent with Aδ- or C-fiber conduction velocities isolated from the same whole-nerve recordings. C-fiber spikes contributed more than Aδ-fiber spikes to the prolonged changes in activity after the application of GDNF, neurturin, and artemin, whereas Aδ-fiber spikes contributed to early activity induced by artemin.
Figure 4.
Figure 4.
GDNF, neurturin, and artemin applied directly to the marrow cavity sensitizes mechanically activated bone afferent neurons. A, Example of a whole-nerve recording and rasters of single-unit activity in response to a 300 mmHg ramp-and-hold pressure stimulus before (left), and 15 min (middle) and 30 min (right) after the application of artemin to the marrow cavity. There was a clear reduction in threshold for activation and an increase in discharge frequency in single units isolated from this recording after the application of artemin. B, Pie charts representing the proportion of units that were sensitized by each of the GFLs. Sensitization was defined as a 20% increase in discharge frequency, as outlined in Materials and Methods. GDNF and neurturin each sensitized 4 of 12 single units tested (n = 5 animals; N = 4 of 12 units), and artemin sensitized 8 of 12 units tested (n = 4 animals; N = 8 of 12 units). C, Threshold for activation (left panels) and the discharge frequency (right panels) of single mechanically activated units expressed as a percentage of preinjection values at 15 min after the injection of each GFL (n = 12 units for each) or saline (n = 4 animals; N = 10 units). At 15 min, the threshold for activation was significantly decreased and discharge frequency was significantly increased in GDNF-, neurturin-, or artemin-sensitized units, relative to nonsensitized units and the saline control (Bonferroni's post hoc test, *p < 0.05). D, Time course of GFL-induced sensitization. GDNF-sensitized units had significantly increased discharge frequency only at 15 min (Bonferroni's post hoc test, *p < 0.05), neurturin-sensitized units had significantly increased discharge frequency at each of the time-points tested (Bonferroni's post hoc test, *p < 0.05), and artemin-sensitized units had increased discharge frequency at 15 and 30 min (Bonferroni's post hoc test, *p < 0.05). Error bars in C and D represent the mean ± SEM.
Figure 5.
Figure 5.
GDNF, neurturin, and artemin applied directly to the marrow cavity results in altered weight bearing. A, Weight bearing was assessed using an incapacitance meter that measures the distribution of weight bearing across each hindlimb. B–D, There was a significant reduction in weight bearing on the injected hindlimb, relative to the uninjected hindlimb, at 15 min and 5 h after application GDNF (B; Bonferroni's post hoc test, *p < 0.05; n = 6 animals) and for up to 1 h after the application of neurturin (C; Bonferroni's post hoc test, *p < 0.05; n = 6 animals) and artemin (D; Bonferroni's post hoc test, *p < 0.05; n = 6 animals).
Figure 6.
Figure 6.
Sequestration of GDNF, neurturin, and artemin with antibodies applied to the marrow cavity abrogates altered weight-bearing behavior in an animal model of CFA-induced inflammatory bone pain. A, Time course of CFA-induced inflammatory bone pain. The percentage of ipsilateral hindlimb weight bearing at the peak of CFA-induced pain (day 4; n = 14 animals) was significantly lower than the values for saline-injected controls at this time-point (Bonferroni's post hoc test *p < 0.05; n = 10 animals). B–D, Sequestration of artemin and neurturin, but not GDNF, completely abrogated the CFA-induced pain-like behavior. B, There was no difference in CFA-induced pain behavior in animals injected with the anti-GDNF sequestering antibody (n = 12 animals) relative to those injected with its isotype control antibody (n = 14 animals). C, There was a significant reduction in pain behavior in animals injected with the neurturin-sequestering antibody (n = 10 animals) relative to those injected with its isotype control antibody (n = 14 animals) at days 4 and 7 (Bonferroni's post hoc test, *p < 0.05). D, There was a significant reduction in pain behavior in animals injected with the artemin-sequestering antibody (n = 12 animals) relative to those injected with its isotype control antibody (n = 14 animals) at day 4 (Bonferroni's post hoc test, *p < 0.05).
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References

    1. Alamri A, Bron R, Brock JA, Ivanusic JJ (2015) Transient receptor potential cation channel subfamily V member 1 expressing corneal sensory neurons can be subdivided into at least three subpopulations. Front Neuroanat 9:71. 10.3389/fnana.2015.00071 - DOI - PMC - PubMed
    1. Alvarez P, Chen X, Bogen O, Green PG, Levine JD (2012) IB4(+) nociceptors mediate persistent muscle pain induced by GDNF. J Neurophysiol 108:2545–2553. 10.1152/jn.00576.2012 - DOI - PMC - PubMed
    1. Amaya F, Shimosato G, Nagano M, Ueda M, Hashimoto S, Tanaka Y, Suzuki H, Tanaka M (2004) NGF and GDNF differentially regulate TRPV1 expression that contributes to development of inflammatory thermal hyperalgesia. Eur J Neurosci 20:2303–2310. 10.1111/j.1460-9568.2004.03701.x - DOI - PubMed
    1. Bennett A. (1988) The role of biochemical mediators in peripheral nociception and bone pain. Cancer Surv 7:55–67. - PubMed
    1. Bennett DL, Michael GJ, Ramachandran N, Munson JB, Averill S, Yan Q, McMahon SB, Priestley JV (1998) A distinct subgroup of small DRG cells express GDNF receptor components and GDNF is protective for these neurons after nerve injury. J Neurosci 18:3059–3072. 10.1523/JNEUROSCI.18-08-03059.1998 - DOI - PMC - PubMed

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