Spontaneous pain, both neuropathic and inflammatory, is related to frequency of spontaneous firing in intact C-fiber nociceptors

Abstract

Spontaneous pain, a poorly understood aspect of human neuropathic pain, is indicated in animals by spontaneous foot lifting (SFL). To determine whether SFL is caused by spontaneous firing in nociceptive neurons, we studied the following groups of rats: (1) untreated; (2) spinal nerve axotomy (SNA), L5 SNA 1 week earlier; (3) mSNA (modified SNA), SNA plus loose ligation of the adjacent L4 spinal nerve with inflammation-inducing chromic gut; and (4) CFA (complete Freund's adjuvant), intradermal complete Freund's adjuvant-induced hindlimb inflammation 1 and 4 d earlier. In all groups, recordings of SFL and of spontaneous activity (SA) in ipsilateral dorsal root ganglion (DRG) neurons (intracellularly) were made. Evoked pain behaviors were measured in nerve injury (SNA/mSNA) groups. Percentages of nociceptive-type C-fiber neurons (C-nociceptors) with SA increased in intact L4 but not axotomized L5 DRGs in SNA and mSNA (to 35%), and in L4/L5 DRGs 1-4 d after CFA (to 38-25%). SFL occurred in mSNA but not SNA rats. It was not correlated with mechanical allodynia, extent of L4 fiber damage [ATF3 (activation transcription factor 3) immunostaining], or percentage of L4 C-nociceptors with SA. However, L4 C-nociceptors with SA fired faster after mSNA (1.8 Hz) than SNA (0.02 Hz); estimated L4 total firing rates were approximately 5.0 and approximately 0.6 kHz, respectively. Similarly, after CFA, faster L4 C-nociceptor SA after 1 d was associated with SFL, whereas slower SA after 4 d was not. Thus, inflammation causes L4 C-nociceptor SA and SFL. Overall, SFL was related to SA rate in intact C-nociceptors. Both L5 degeneration and chromic gut cause inflammation. Therefore, both SA and SFL/spontaneous pain after nerve injury (mSNA) may result from cumulative neuroinflammation.

Figure 1.

Summary diagram to show recording setup and likely sites of inflammation in different nerve injury models. The L5 SN was tightly ligated (with a silk suture) and transected just peripheral to the ligation, and the L4 SN was loose ligated with a chromic gut suture (see Materials and Methods). Intracellular recordings to determine conduction velocity and spontaneous firing were made from somata of axotomized L5, adjacent spared (intact) L4, or loose-ligated L4 DRG neurons 7 d after surgery. Inflammation 1, Inflammation in both SNA and mSNA rats caused by Wallerian degeneration of axotomized L5 fibers (dotted line) within the same nerve that influences intact L4 fibers (solid line) (see Discussion). Increased NGF availability for intact fibers in the periphery may result from the loss of axotomized fibers (see Discussion), which occurs in both SNA and mSNA rats. Inflammation 2, Chromic gut causes inflammation (see Introduction) to the L4 SN only in mSNA rats. The cumulative effects of inflammations 1 and 2 on intact L4 C-fiber nociceptors appear to be responsible for the relatively high spontaneous firing rate in these neurons, which is accompanied by, and probably causes, SFL/spontaneous pain.

Figure 2.

SFL in nerve injury and CFA-treated rats. Numbers above histogram columns in A and C indicate number of rats. A shows that the percentage of mSNA rats with SFL of >1 s/10 min is significantly greater than that of SNA rats (p < 0.001, Fisher’s exact test). B shows significantly longer SFL duration in mSNA (n = 25) than SNA (n = 17) rats (Mann–Whitney test). C shows that all rats showed SFL 1 d after CFA, declining to no rats by 4 d (χ² test for trend). D shows that the amount of SFL is greater at 1 d, declining to none by 4 d after CFA (Kruskal–Wallis test with Dunn’s post test between all groups). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3.

Correlation between behavioral tests. Relationships between extents of different types of behavior: SFL versus heat hyperalgesia (A), SFL versus mechanical allodynia (B), and heat hyperalgesia versus allodynia (C). The 50% withdrawal threshold (B and C) was determined using von Frey filaments as a measure of mechanical allodynia. The direction of the axes shows more extreme pain-related behaviors toward the top and right of the graphs. The open circles are data from SNA rats, and the filled circles are data from mSNA rats. Linear regression analysis was performed on SNA and mSNA separately in all cases, and on both together for allodynia versus hyperalgesia (C); regression lines (solid for SNA and dotted for SNA plus mSNA) are shown only for significant correlations. Allodynia was not significantly correlated with SFL in SNA or mSNA rats (A); and the extent of hyperalgesia was not correlated with the extent of SFL in mSNA rats (B). The extents of allodynic and hyperalgesic behaviors were correlated, however, for SNA rats alone and for all rats (C).

Figure 4.

Pain-related behaviors versus extent of L4 SN injury (ATF3). A, B, Interference contrast images of examples of ATF3 staining in mSNA rats in L4 DRGs. A, Contralateral with no positive nuclei. B, Ipsilateral with two positive nuclei; overall, >40% nuclei were ATF3 in this DRG. C–E, Relationships between the extent of mechanical damage to the L4 SN (assessed as the percentage of L4 DRG neurons expressing ATF3) and SFL (C), mechanical allodynia (D), or heat hyperalgesia (E). Linear regression analysis was performed on SNA and mSNA data separately in all cases, and on both together in graphs B and C. Allodynia and hyperalgesia but not SFL were positively correlated with the extent of mechanical damage to the L4 SN measured as the percentage L4 neuronal nuclei with ATF3. The dotted arrows (A, B) indicate neurons, the neurons of which were unlabeled with ATF3, whereas the solid arrows (B) indicate two nuclei labeled for ATF3.

Figure 5.

Percentages of C- and A-fiber neurons with SA in different pain models. A, C-fiber neurons. B, Aδ-fiber neurons. C, Aα/β-fiber neurons. The model above the relevant column in A relates to all graphs. SNA means L5 SN axotomy 1 week previously; mSNA means SNA plus L4 loose ligation of the L4 SN 1 week previously; CFA1 and CFA4 mean CFA-induced hindlimb inflammation 1 and 4 d previously. Below each histogram is indicated the DRG from which recordings were made. L4/5Norm, L4 and L5 DRGs in untreated rats; L5axot, L5 DRGs after L5 SN axotomy in mSNA rats; L4Spar, ipsilateral L4 in SNA rats; L4LL, ipsilateral L4 in mSNA (with loose ligation); L4/5 CFA1 and L4/5 CFA4, L4 and L5 DRGs in CFA rats 1 and 4 d, respectively, after CFA injection. The left two columns show all neurons from control L4/5 DRGs (L4/5Norm) and axotomized L5 DRGs (L5Axot) in A and B but exclude muscle spindle afferents (first column, L4/5Norm) and muscle spindle afferent-like units (second column, L5Axot) in C. The five columns in the right-hand block relate to intact neurons; here, only nociceptors/nociceptive-type neurons are shown, because LTMs did not show increased SA (see Results). These columns show neurons from control (L4/5Norm), SNA- (L4Spar), mSNA- (L4LL), and CFA-treated (L4/5CFA1 and CFA4) rats. In C, the question mark indicates that the proportion of Aα/β nociceptors with SA was not recorded. The horizontal lines above the graphs link columns between which there was a significant difference Fisher’s exact test for 2 × 2 contingency tables, followed by Bonferroni’s correction. Levels of significance are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 6.

The rate of spontaneous firing in nociceptors in nerve injury and CFA rats. A, Significantly higher median SA rate in L4 C-fiber nociceptive-type neurons in mSNA than SNA rats (Mann–Whitney test, two-tailed) and significantly higher median SA rate 1 d compared with 4 d after CFA (Mann–Whitney test, one-tailed). B shows that, in L4 A-fiber nociceptors, there was no significant difference between median rate in mSNA rats (n = 7; 2 Aδ and 5 Aαβ cells) compared with SNA rats (n = 4; 1 Aδ and 3 Aα/β neurons). Ipsil means ipsilateral to nerve injury or CFA injection. C and D show example records of two typical L4 C-fiber nociceptors firing spontaneously (top traces) at 0.02 Hz in SNA (C) and 2.25 Hz in mSNA (D) rats and, on bottom traces, show the same neurons responding to noxious pinch (note different timescale). Both neurons had subcutaneous (deep) receptive fields and did not respond to non-nociceptive stimuli including brush, touch, pressure, and tap (data not shown). *p < 0.05; **p < 0.01.

Figure 7.

Relationships between SFL and SA rate in C-nociceptors. A, A scattergraph of individual C-fiber firing rate recorded in different rats (1 rat per column; the number beneath is the rat number), grouped by treatment [SNA, open circles; mSNA, filled circles; CFA (1 d), asterisks]. SFL duration is indicated above the columns. Each symbol is one C-fiber neuron. B, The SFL for each rat in A is plotted against the mean firing rate for C-nociceptor-type neurons (including units with and without SA) in that rat. There is a highly significant linear correlation (p < 0.01; r² = 0.468) between rate and SFL. C, Plot of mean SFL duration in seconds per 10 min for SNA, mSNA, CFA1, and CFA4 rats (from data plotted in Fig. 6A) against mean firing rates (from data plotted in Fig. 2A) of C-fiber nociceptor type neurons with SA for that group. There is a highly significant correlation whether the means (main graph; p < 0.001, r² = 0.998) or medians (inset graph; p < 005, r² = 0.991) are correlated. The symbols below relate to all graphs; the lines indicate significant correlation.