Early afferent activity from the facet joint after painful trauma to its capsule potentiates neuronal excitability and glutamate signaling in the spinal cord

Abstract

Cervical facet joint injury induces persistent pain and central sensitization. Preventing the peripheral neuronal signals that initiate sensitization attenuates neuropathic pain. Yet, there is no clear relationship among facet joint afferent activity, development of central sensitization, and pain, which may be hindering effective treatments for this pain syndrome. This study investigates how afferent activity from the injured cervical facet joint affects induction of behavioral sensitivity and central sensitization. Intra-articular bupivacaine was administered to transiently suppress afferent activity immediately or 4 days after facet injury. Mechanical hyperalgesia was monitored after injury, and spinal neuronal hyperexcitability and spinal expression of proteins that promote neuronal excitability were measured on day 7. Facet injury with saline vehicle treatment induced significant mechanical hyperalgesia (P<.027), dorsal horn neuronal hyperexcitability (P<.026), upregulation of pERK1/2, pNR1, mGluR5, GLAST, and GFAP, and downregulation of GLT1 (P<.032). However, intra-articular bupivacaine immediately after injury significantly attenuated hyperalgesia (P<.0001), neuronal hyperexcitability (P<.004), and dysregulation of excitatory signaling proteins (P<.049). In contrast, intra-articular bupivacaine at day 4 had no effect on these outcomes. Silencing afferent activity during the development of neuronal hyperexcitability (4 hours, 8 hours, 1 day) attenuated hyperalgesia and neuronal hyperexcitability (P<.045) only for the treatment given 4 hours after injury. This study suggests that early afferent activity from the injured facet induces development of spinal sensitization via spinal excitatory glutamatergic signaling. Peripheral intervention blocking afferent activity is effective only over a short period of time early after injury and before spinal modifications develop, and is independent of modulating spinal glial activation.

Keywords: Bupivacaine; Facet joint; Glutamate; Hyperalgesia; Neuronal hyperexcitability; Pain.

Copyright © 2014 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Figures

Figure 1

Behavioral sensitivity after intra-articular bupivacaine given either at injury or 4 days later. (a) Paw withdrawal threshold (PWT) decreases from baseline on all days (*p<0.027) after injury with immediate intra-articular saline (inj-VEH0h), but PWT after bupivacaine treatment at the time of injury (inj-BP0h) is not different from baseline on all days, as is observed with sham (sham-VEH0h). (b) In contrast, intra-articular bupivacaine given at 4 days after injury (inj-BPd4) has no effect on PWT, with decreased PWT compared to baseline on all days (*p<0.003), similar to the injury with a saline vehicle injection (inj-VEHd4) (^p<0.0001).

Figure 2

Extracellular spike activity in the spinal dorsal horn 7 days after facet capsule injury. (a) Traces indicate the filament application, raw extracellular voltage recording, and neuron identification and spike counts. Neuronal firing was sorted and spikes were counted during a baseline period and mechanical stimulation of the forepaw; a representative response to five applications of a 26g von Frey filament 7 days after facet capsule injury is shown. (b) Firing increases after injury (inj-VEH0h) over sham (sham-VEH0h) during light brush, 4g, 10g, and 26g von Frey filament stimulation (*p<0.026). Immediate bupivacaine attenuates firing in response to all stimuli after injury (#p≤0.004). Bupivacaine treatment (inj-BP0h) also reduces activity below that of sham for brush, pinch, and the 1.4g von Frey filament (^p<0.0007). (c) During all stimuli, for injections at day 4, neuronal firing is increased over sham (sham-VEHd4) after injury with either vehicle injection (inj-VEHd4) (*p<0.045) and after bupivacaine injection (inj-BPd4) (^p<0.035).

Figure 3

Western blot of spinal cord at day 7. (a,b) Facet capsule injury (inj-VEH0h) increases pERK1/2, pNR1, mGluR5, GLAST and GFAP, and decreases expression of GLT1 (*p≤0.032). Immediate bupivacaine treatment (inj-BP0h) prevents such increases, with significantly lower expression (#p<0.029) of pERK1/2, pNR1, mGluR5, and GLAST than inj-VEH0h and no differences from sham (sham-VEH0h). Immediate bupivacaine also prevents the decrease in GLT1 expression that is evident after injury (##p=0.049). However, GFAP expression remains significantly elevated over sham (*) and is not different from the inj-VEH0h group. (c,d) Injury with vehicle treatment at day 4 (inj-VEHd4) induces the same changes relative to sham as does injury with vehicle treatment at the time of injury (inj-VEH0h) (*p≤0.046). However, pERK1/2, pNR1, mGluR5, GLT1, and GLAST remain at injury levels with bupivacaine treatment at day 4 (inj-BPd4), and are significantly elevated over sham (^p≤0.048). Total ERK1/2 and NR1 expression is unchanged in all groups.

Figure 4

Immunolabeling of phosphorylated NR1 (pNR1), mGluR5, and GFAP in the spinal dorsal horn at day 7. Representative images are shown for (a-d) pNR1 (red) and (e-h) mGluR5 (red) and GFAP (green) after injury, sham, and injury with immediate or day 4 bupivacaine treatment. (i) The percentage of pNR1- and mGluR5-positive pixels normalized to sham levels increases in the dorsal horn after injury with immediate vehicle treatment (inj-VEH0h) (*p 0.046). Immediate bupivacaine treatment (inj-BP0h) prevents those increases in both mGluR5 and pNR1 (#p<0.0001), although GFAP remains increased (^p<0.0001). (j) The normalized percentages of pNR1-, mGluR5-, and GFAP-positive pixels increase after injury with day 4 vehicle treatment, relative to sham levels (*p≤0.003). However, bupivacaine administered 4 days after injury (inj-BPd4) does not prevent the increases in pNR1, mGluR5, and GFAP (^p≤0.024). Scale bar=100μm.

Figure 5

Bupivacaine administered immediately (0h) or 4 hours (4h) after facet capsule injury prevents any change from baseline paw withdrawal threshold (PWT) at day 1 and day 7 after injury. However, when bupivacaine treatment is given at 8 hours (8h), 1 day (d1), or 4 days (d4) after injury the PWT is significantly decreased from baseline levels on day 1 (*p

Figure 6

Evoked spike activity in the dorsal horn on day 7 after facet capsule injury varies with timing of intra-articular bupivacaine. (a) Neuronal firing evoked by light brush is significantly higher when bupivacaine is administered at time later than 4 hours after injury than when it is given immediately (inj-BP0h) (*p<0.0001) or at 4 hours (inj-BP4h) (#p<0.0001). Firing during a noxious pinch is increased in the inj-BP8h and inj-BPd1 groups compared to the inj-BP0h (*p<0.0001), inj-BP4h (#p<0.002), and inj-BPd4 (^p<0.018) groups. (b) Similarly, firing evoked by stimulation by all of the magnitudes of von Frey filaments is significantly greater when bupivacaine is administered 8 hours (inj-BP8h), 1 day (inj-BPd1), or 4 days after injury (inj-BPd4) than when it is given immediately (inj-BP0h) (*p<0.0001) or at 4 hours (inj-BP4h) (#p<0.045). Firing is also greater after treatment given at 4 hours (inj-BP4h) than it is when given at the time of injury (inj-BP0h) for all von Frey filament stimuli (*p<0.0001). There is also significantly more evoked firing in the inj-BP8h group than the inj-BPd1 group for stimulation by the 1.4g and 26g filaments (†p<0.0003) and the inj-BPd4 group for the 4g, 10g, and 26 filaments (^p<0.0001).