Adjacent intact nociceptive neurons drive the acute outburst of pain following peripheral axotomy

Abstract

Injury of peripheral nerves may quickly induce severe pain, but the mechanism remains obscure. We observed a rapid onset of spontaneous pain and evoked pain hypersensitivity after acute transection of the L5 spinal nerve (SNT) in awake rats. The outburst of pain was associated with a rapid development of spontaneous activities and hyperexcitability of nociceptive neurons in the adjacent uninjured L4 dorsal root ganglion (DRG), as revealed by both in vivo electrophysiological recording and high-throughput calcium imaging in vivo. Transection of the L4 dorsal root or intrathecal infusion of aminobutyrate aminotransferase inhibitor attenuated the spontaneous activity, suggesting that retrograde signals from the spinal cord may contribute to the sensitization of L4 DRG neurons after L5 SNT. Electrical stimulation of low-threshold afferents proximal to the axotomized L5 spinal nerve attenuated the spontaneous activities in L4 DRG and pain behavior. These findings suggest that peripheral axotomy may quickly induce hyperexcitability of uninjured nociceptors in the adjacent DRG that drives an outburst of pain.

Figure 1

Neuropathic pain-related behavior and neurogenic inflammation after acute L5 SNT in rats. (a) Frequency of spontaneous foot lifting at 0 to 14 days after acute L5 SNT (n = 8/group). SNT vs Sham: F_{(1, 14)} = 24.62, ***P = 0.0002; 10 min: P < 0.0001; 1 d: P < 0.0001; 4 d: P = 0.0286. (b) PWT of the hind paw ipsilateral to the side of injury from 10 min to 14 days after SNT (n = 8/group). SNT vs Sham: F_{(1, 14)} = 35.41, ***P < 0.0001; 10 min: P = 0.0021; 1 h: P = 0.0003; 4 h: P = 0.0006; 12 h: P = 0.0007; 1d: P = 0.0007; 7d: P = 0.0003; 14d: P < 0.0001. (c) Changes in the ipsilateral paw withdrawal latency to heat stimuli from 10 min to 14 days after SNT (n = 8/group). SNT vs Sham: F_{(1, 14)} = 9.140, **P = 0.0091; 10 min: P = 0.0282; 1d: P = 0.0011; 7d: P = 0.0196, 14 d: P = 0.3120. (d) Frequency of licking/biting of the ipsilateral hind paw within 3 min after acetone stimulus (n = 8/group). SNT vs Sham: F_{(1, 14)} = 16.75, **P = 0.0011; 10 min: P = 0.0211; 1d: P = 0.0035; 7d: P = 0.0013; 14 d: P = 0.0035. (e) The ipsilateral PWTs to stimulation applied to the L3, L4, and L5 dermatomes after L5 SNT (n = 8/Group). Two-way mixed model ANOVA with Tukey’s multiple comparisons test, F_{(2, 21)} = 63.09, P < 0.0001. (L5 vs. L4, 10 min: P < 0.0001; 1 h: P < 0.0001; 4 h: P < 0.0001; 12 h: P = 0.0002; 1d: P = 0.0003; 3d: P < 0.0001; 7d: P < 0.0001; 14d: P = 0.0009. L5 vs. L3, 10 min: P < 0.0001; 1 h: P < 0.0001; 4 h: P < 0.0001; 12 h: P < 0.0001; 1d: P < 0.0001; 3d: P < 0.0001; 7d: P < 0.0001; 14d: P < 0.0001). (f) Quantitative measurement of Evans blue extravasation in sham-operated (n = 3/time point) and SNT rats (n = 4/time point). SNT vs Sham: F_{(1, 5)} = 257.0, ***P < 0.001; 10 min: P < 0.0001; 1 h: P < 0.0001; 4 h: P < 0.0001; 1d: P < 0.0001; 7d: P < 0.0001; 14d: P = 0.0004. (g) Cutaneous temperature (footpad) in the L4 dermatome of the SNT ipsilateral, contralateral paws and core (rectal) temperature after L5 SNT (n = 5/group). SNT side vs Contralateral side: F_{(1, 8)} = 24.02, **P = 0.0012; 10 min: P < 0.0001; 20 min: P < 0.0001; 30 min: P < 0.0001; 60 min: P < 0.0001; 120 min: P < 0.0001; 240 min: P < 0.0001. (h) Cutaneous temperature (footpad) in the L4 dermatome of the ipsilateral and contralateral paws and core (rectal) temperature after application of 2% lidocaine quickly around L5 spinal nerve for 1 min (n = 3/group). Contralateral side vs Lidocaine L5: F_{(1, 4)} = 87.57, P = 0.0007; 10 min: P = 0.0002; 20 min: P < 0.0001; 30 min: P < 0.0001; 60 min: P < 0.0001. *P < 0.05, **P < 0.01, ***P < 0.001, ^#P < 0.001. (a–d,f–h) Two-way mixed model ANOVA with sidak’s multiple comparisons test. Data are expressed as mean ± SEM.

Figure 2

Spontaneous activity in L4 DRG neurons after acute L5 SNT in rats. (a) Left: Schematic diagram of the experimental setup. Right: Representative example of an initially quiescent C-nociceptive neuron in L4 DRG that quickly developed spontaneous activity after acute L5 SNT. CV, conduction velocity. (b) Mean discharge rates (spikes/min) of C neurons in L4 DRG after acute L5 SNT. (c) Left and middle: Percentage of C neurons in L4 DRG that showed SA at different time points after L5 SNT with or without transection of L4 dorsal root. Right: Percentage of C-neurons in L5 DRG that showed SA after L5 SNT without transection of L4 dorsal root. (L4 DR Intact vs L4 DR Transect, 0–4 h: P < 0.0001; 1d: P = 0.1504; 7d: P = 0.0104. L4 DR Intact vs L5 DRG, 0–4 h: P < 0.0001; 1d: P = 0.0244; 7d: P = 0.0405. L4 DR Intact SNT vs Sham, 0–4 h: ^#P < 0.0001, Chi-square test). (d) Percent of each subtype of neuron in L4 DRG that showed SA during the first 4 h after L5 SNT or sham operation. (CM: P = 0.0099; CMH: P = 0.0010; CMC: P = 0.0191, Chi-square test). (e) Schematic diagram of the recording setup and intrathecal infusion of aminooxyacetic acid (AOAA) at lumbar spinal cord. (f) SA of C-nociceptive neurons in L4 DRG after acute L5 SNT followed by intrathecal infusion of vehicle or AOAA (10 μl, 100 mmol, n = 3). One-way ANOVA, F _{(1.007, 2.013)} = 770.5, ***P = 0.0013; Dunnett’s multiple comparisons test, Pre-drug vs after AOAA: P = 0.0026; Vehicle vs after AOAA: P = 0.0011. *P < 0.05, **P < 0.01, ***P < 0.001, ^#P < 0.001, ns, not significant. Data are expressed as mean ± SEM. CM, C-mechano-sensitive; CMH, C-mechano-heat-sensitive; CMHC, C-mechano-heat-cold-sensitive; CMC, C-mechano-cold-sensitive; APs, action potentials.

Figure 3

Calcium imaging of activity in L4 DRG neurons after acute L5 SNT in pirt-GCaMP6s mice. (a) Representative images of spontaneous activity (SA) in L4 DRG before and at 2 and 30 min after L5 SNT in pirt-GCaMP6s mice. (b) Setup for in vivo calcium imaging of DRG neurons in pirt-GCaMP6 mice. (c) Quantification of number of L4 DRG neurons that showed SA before and 0–30 min after L5 SNT (n = 4). DRG neurons were categorized into three sized-based subgroups with somal areas of < 450 μm² (small), 450–700 μm² (medium), and >700 μm² (large). Two-way ANOVA with Tukey’s multiple comparisons test, F_{(2, 6)} = 98.54, ***P < 0.0001. Pre-SNT vs 2 min Post-SNT, P < 0.0001; Pre-SNT vs 30 min Post-SNT, P = 0.0118. *P < 0.05, ***P < 0.001. Data are expressed as mean ± SEM.

Figure 4

Changes in the excitability of L4 DRG neurons after acute L5 SNT. (a) Bright-field image of L4 DRG showing a small-diameter neuron (arrow) and an extracellular electrophysiology recording electrode (dashed yellow lines). (b) Conduction velocity (CV; 0.42 m/s) was measured by electrically stimulating the peripheral receptive field (RF, red arrow). (c) Action potentials evoked by heat stimulation (51 °C, 5 s) were recorded from the neuronal soma. (d) This neuron did not respond to cold stimulus (0 °C, 20 s) and hence was classified as C-mechano-heat-sensitive (CMH). (e) Responses of a CMH neuron in L4 DRG to different mechanical stimuli, including a cotton-tipped swab, light brush, and von Frey filaments of multiple bending forces (5, 10, 30, and 50 mN) before and after L5 SNT. (f) Responses of a CMH neuron in the L4 DRG to different thermal stimuli (41–53 °C) before and after L5 SNT. (g) Schematic diagram of RFs of C neurons that showed spontaneous activity after SNT. LAT: lateral, MED: medial. (h) Mechanical thresholds of different subtypes of C neurons in L4 DRG from sham-operated (n = 7–12) and SNT groups (n = 11–13). CM: t₍₂₁₎ = 4.884, P < 0.001, two-tailed unpaired t-test; CMC: t₍₁₉₎ = 6.538, P < 0.001, two-tailed unpaired t-test; CMH: P < 0.001, Mann-Whitney test; CMHC: P < 0.001, Mann-Whitney test. (i) Heat thresholds of CMH (sham: n = 13; SNT: n = 8) and CMHC (sham: n = 8; SNT: n = 11) neurons. CMH: P < 0.001, Mann-Whitney test; CMHC: t₍₁₇₎ = 4.642, P < 0.001, two-tailed unpaired t-test. (j) Responses of CMC and CMHC neurons to cold stimuli (0 °C, 20 s) in sham-operated (n = 5) and SNT (n = 7) rats. CMC: t₍₁₀₎ = 4.578, P = 0.001; CMHC: t₍₁₀₎ = 3.179, P = 0.0098; two-tailed unpaired t-test. **P < 0.01, ***P < 0.001, SNT vs. sham. Data are expressed as mean ± SEM.

Figure 5

Effects of electrical stimulation of A-fibers on spontaneous activity of L4 DRG neurons and neuropathic pain after acute L5 SNT. (a) Left: Schematic diagram of electrical stimulation (S1) applied to the proximal end of transected L5 spinal nerve. Right: An example of spontaneous activity (SA) that quickly developed in an initially quiescent nociceptive neuron (conduction velocity = 0.69 m/s) in L4 DRG after L5 SNT. The SA was attenuated by electrical stimulation of the injured L5 spinal nerve at a low-intensity that activated low-threshold A-fibers (0.5 mA, 10 Hz, 3 min). This neuron responded to mechanical stimulation (poke) at the end of the experiment. (b) Quantification of SA in L4 DRG neurons before injury, after SNT, and after electrical stimulation of the L5 spinal nerve (n = 7). One-way repeated measures ANOVA, F_{(2, 27)} = 60.68, ***P < 0.0001; Tukey’s multiple comparisons test, Pre-stim vs Pre-SNT: P < 0.0001; Pre-stim vs Post-stim: P < 0.0001. (c) The increased spontaneous foot lifting after L5 SNT was significantly decreased by A-fiber strength electrical stimulation (n = 6–8/group). F_{(2, 19)} = 13.58, ***P = 0.0002; SNT vs SNT-S: 10 min: P = 0.0075; 1d: P < 0.0001. (d) Changes in the ipsilateral paw withdrawal threshold after sham operation (n = 8), and that after L5 SNT with (SNT-S, n = 6) and without (SNT, n = 8) electrical stimulation. F_{(2, 95)} = 61.89, ***P < 0.0001; SNT vs SNT-S: 1 h: P = 0.0003; 12 h: P < 0.0001; 1d: P = 0.0199; 7d: P = 0.0147. *P < 0.05, **P < 0.01, ***P < 0.001. c,d: Two-way mixed model ANOVA with Tukey’s multiple comparisons test. Data are expressed as mean ± SEM.