Intra-articular nerve growth factor regulates development, but not maintenance, of injury-induced facet joint pain & spinal neuronal hypersensitivity

Abstract

Objective: The objective of the current study is to define whether intra-articular nerve growth factor (NGF), an inflammatory mediator that contributes to osteoarthritic pain, is necessary and sufficient for the development or maintenance of injury-induced facet joint pain and its concomitant spinal neuronal hyperexcitability.

Method: Male Holtzman rats underwent painful cervical facet joint distraction (FJD) or sham procedures. Mechanical hyperalgesia was assessed in the forepaws, and NGF expression was quantified in the C6/C7 facet joint. An anti-NGF antibody was administered intra-articularly in additional rats immediately or 1 day following facet distraction or sham procedures to block intra-articular NGF and test its contribution to initiation and/or maintenance of facet joint pain and spinal neuronal hyperexcitability. NGF was injected into the bilateral C6/C7 facet joints in separate rats to determine if NGF alone is sufficient to induce these behavioral and neuronal responses.

Results: NGF expression increases in the cervical facet joint in association with behavioral sensitivity after that joint's mechanical injury. Intra-articular application of anti-NGF immediately after a joint distraction prevents the development of both injury-induced pain and hyperexcitability of spinal neurons. Yet, intra-articular anti-NGF applied after pain has developed does not attenuate either behavioral or neuronal hyperexcitability. Intra-articular NGF administered to the facet in naïve rats also induces behavioral hypersensitivity and spinal neuronal hyperexcitability.

Conclusion: Findings demonstrate that NGF in the facet joint contributes to the development of injury-induced joint pain. Localized blocking of NGF signaling in the joint may provide potential treatment for joint pain.

Keywords: Facet joint; NGF; Neuronal hyperexcitability; Pain; Trauma.

Conflict of interest statement

Competing Interest Statement: The authors declare no conflicts of interest.

Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Figures

Figure 1

Facet joint distraction (FJD) induced pain associated with increased NGF expression in the joint. (A) FJD reduced the forepaw withdrawal threshold to mechanical stimulation at day 1 compared to baseline (#p<0.001) and sham procedures (*p<0.001). (B) Representative Western blots show NGF (top) and β-tubulin (bottom) expression in the joint tissue. (C) FJD significantly increased NGF in the joint tissue (*p=0.031) over levels in sham at day 1. Immunolabeling for NGF increased in the soft tissues surrounding the joint (solid arrows), including the capsular ligament, at day 1 after FJD (D) compared to labeling in shams (E). Scale bar in (D) is 50µm and applies to (D) and (E). The amplified inset boxes in (D) and (E) are 50µm wide. CL: capsular ligament; F: inferior facet of the superior vertebra; M: muscle (dashed arrow); NGF: nerve growth factor.

Figure 2

Intra-articular anti-NGF immediately after FJD prevented the development of FJD-induced pain and spinal neuronal hyperexcitability. (A) The forepaw mechanical withdrawal threshold significantly decreased from baseline for FJD+veh at all times (p<0.001), with no change from baseline at any time in the FJD+anti-NGF, sham+veh, or sham+anti-NGF groups. The withdrawal threshold decreased for FJD+veh compared to FJD+anti-NGF (*p<0.015), sham+veh (#p<0.001), and sham+anti-NGF (‡p<0.001) at all days. (B) At day 7, the number of spikes evoked in spinal neurons by forepaw stimulation with 10g and 26g von Frey filaments significantly increased in the FJD+veh group compared to all other groups (*#‡p<0.012). There were no differences between FJD+anti-NGF, sham+veh, and sham+anti-NGF for any filament. (C) The pinch, but not brush, stimulus evoked significantly more spikes in the FJD+veh group than all others (*#‡p<0.001). (D) There was a significant effect of group on the proportion of WDR neurons (+p<0.005), with the largest number in the FJD+veh group. (E) Representative recordings show increased spikes evoked by the 10g, 26g, and pinch stimuli in the FJD+veh group compared to the other groups. FJD: facet joint distraction; NGF: nerve growth factor; WDR: wide dynamic range.

Figure 3

Inhibiting intra-articular NGF signaling at day 1 after FJD did not alter pain or spinal neuronal hyperexcitability. (A) The forepaw withdrawal threshold decreased at all days after FJD+veh compared to FJD+anti-NGF (*p<0.041). FJD+anti-NGFD1 also exhibited decreased thresholds at all days compared to FJD+anti-NGF (#p<0.043). (B) At day 7, there were fewer spikes evoked by noxious von Frey stimulation (26g) for FJD+anti-NGF relative to FJD+veh (*p<0.002) and FJD+anti-NGFD1 (#p<0.022), but both FJD+anti-NGF (*p<0.001) and FJD+anti-NGFD1 (+p<0.001) exhibited fewer evoked spikes than FJD+veh for a noxious pinch. (C–E) Representative spikes are shown for the 26g and pinch stimuli for each group. Datasets for the FJD+veh and FJD+anti-NGF groups are reproduced from Figure 2. FJD: facet joint distraction; NGF: nerve growth factor; d: day.

Figure 4

Intra-articular NGF induced transient behavioral sensitivity that was associated with spinal neuronal hyperexcitability. (A) Intra-articular NGF significantly reduced the withdrawal threshold from baseline at day 1 (*p<0.010), but it returned to baseline by day 3. Injection of PBS vehicle did not alter the withdrawal threshold from baseline at any day. (B) At 1 day after intra-articular NGF the withdrawal threshold significantly decreased relative to baseline (*p<0.001) and to vehicle injection (‡p<0.001). (C) Representative extracellular recordings in the spinal cord at day 1 demonstrated increased evoked neuronal firing after NGF. (D) The number of evoked spikes significantly increased (‡p<0.040) for the noxious 26g filament after NGF injection. (E) Intra-articular NGF increased the number of spinal neurons classified as WDR neurons compared to the number identified after intra-articular vehicle administration (‡p=0.016). NGF: nerve growth factor; WDR: wide dynamic range.