Partial nerve injury induces electrophysiological changes in conducting (uninjured) nociceptive and nonnociceptive DRG neurons: Possible relationships to aspects of peripheral neuropathic pain and paresthesias

Abstract

Partial nerve injury leads to peripheral neuropathic pain. This injury results in conducting/uninterrupted (also called uninjured)sensory fibres, conducting through the damaged nerve alongside axotomised/degenerating fibres. In rats seven days after L5 spinal nerve axotomy (SNA) or modified-SNA (added loose-ligation of L4 spinal nerve with neuroinflammation-inducing chromic-gut),we investigated (a) neuropathic pain behaviours and (b) electrophysiological changes in conducting/uninterrupted L4 dorsal root ganglion (DRG) neurons with receptive fields (called: L4-receptive-field-neurons). Compared to pretreatment, modified-SNA rats showed highly significant increases in spontaneous-foot lifting duration, mechanical-hypersensitivity/allodynia, and heathypersensitivity/hyperalgesia, that were significantly greater than after SNA, especially spontaneous-foot-lifting. We recorded intracellularly in vivo from normal L4/L5 DRG neurons and ipsilateral L4-receptive-field-neurons. After SNA or modified-SNA, L4-receptive-field-neurons showed the following: (a) increased percentages of C-, Aδ-, and Aβ-nociceptors and cutaneous Aα/β-low-thresholdmechanoreceptors with ongoing/spontaneous firing; (b) spontaneous firing in C-nociceptors that originated peripherally; this was ata faster rate in modified-SNA than SNA; (c) decreased electricalthresholds in A-nociceptors after SNA; (d) hyperpolarised membrane potentials in A-nociceptors and Aα/-low-thresholdmechanoreceptors after SNA, but not C-nociceptors; (e) decreased somatic action potential rise times in C- and A-nociceptors, not Aα/β-low-threshold-mechanoreceptors. We suggest that these changes in subtypes of conducting/uninterrupted neurons after partial nerve injury contribute to the different aspects of neuropathic pain as follows: spontaneous firing in nociceptors to ongoing/spontaneous pain; spontaneous firing in Aα/β-low-threshold-mechanoreceptors to dysesthesias/paresthesias; and lowered A-nociceptor electrical thresholds to A-nociceptor sensitization,and greater evoked pain [corrected].

Fig. 1

Diagram of models: The L5 spinal nerve was ligated (with 6.0 silk suture) and transected in both spinal nerve axotomy (SNA) and modified SNA (mSNA) models; the L4 spinal nerve was loose-ligated with chromic gut in mSNA only. The dashed and continuous lines in the sciatic nerve indicate degenerating axotomised L5 fibres and adjacent uninterrupted L4 fibres, respectively. Electrophysiological properties recorded from uninterrupted L4 dorsal root ganglion (DRG) receptive field-neurons in both models 7 days after surgery were compared with those from L4 and L5 DRG neurons in normal rats. Somatic action potentials were evoked by dorsal root electrical stimulation (S).

Fig. 2

Neuropathic pain behaviour for spinal nerve axotomy (SNA) and modified SNA (mSNA): for each behavioural type, comparisons were between preoperative levels (open bars) and 7-day behaviour for SNA (grey) and mSNA (black bars). Tests were paired t-tests for A and B, and Wilcoxon matched-pairs signed rank test for C to F. There was greater spontaneous foot lifting (A), mechanical allodynia (B), and heat hyperalgesia (C) in mSNA than SNA, but no difference between the behaviours presurgery between SNA and mSNA groups (Mann-Whitney tests).

Fig. 3

Spontaneous firing (SF): percentages of uninterrupted receptive field (RF)-neurons that showed SF (A) nociceptors and (B) Aα/β cutaneous low-threshold mechanoreceptors (LTMs). For each data set, the normal group was compared with 1) spinal nerve axotomy (SNA) and 2) modified SNA (mSNA) groups with 2 × 2 contingency tables with Fisher’s exact test. (A) Significant increases occurred in C- and A-fibre nociceptors in both models (includes identified nociceptors with receptive fields from plus novel data, see text). (B) Percentages of all Aα/β cutaneous LTMs with SF increased significantly in both models. They increased in rapidly adapting (RA) units only in SNA, but no G hair/Field (G/F) units had SF in either model. The solid grey and black bars are units with SF action potentials arising from a flat baseline, suggesting a fibre origin for the SF; the cross-hatched areas indicate those with SF arising from a predepolarisation, suggesting a soma origin.

Fig. 4

Representative examples of evoked and spontaneous action potentials (APs) from nociceptors and Aα/β-cutaneous low-threshold mechanoreceptors (LTMs). (A–F) show examples of intracellularly recorded APs. The 4 traces in A, Ba, C, Da, E and F were as follows. Line 1 (top): AP electrically evoked from the dorsal root. Line 2: Spontaneous firing (SF) over an extended time; SF firing rate shown below line for (A–E). Line 3: the first spontaneous AP shown in line 2. Line 4 (bottom): the same trace and same time scale as line 3 to show the leading edge of the AP at 8 times higher vertical resolution, truncating the AP. For all neurons (A–F), unless otherwise labelled, vertical scales are 40 mV for lines 1–3, and 5 mV for line 4. In (Bb) and (Db), the top 2 traces are for averaged SF APs (15 APs for Bb and 8 APs for Db). The time scales are shown to the left of the traces. Neurons were L4 receptive field-neurons in spinal nerve axotomy (SNA) (A, C, and E) and modified SNA (mSNA) (B, D, and F). They were C-nociceptors (A and B), an Aδ-nociceptor (C), an Aβ-nociceptor (D), and an Aα/β LTM slowly adapting (E, F). The conduction velocity (m/s) and membrane potential (Em; −mV) for each neuron are shown. APs were overshooting; the small lines cross the evoked APs (lines 1) at 0 mV. Note the faster SF rate in C-nociceptor in mSNA (B) than in SNA (A). (A–E) show the predominant irregular pattern of SF. (F) is the only neuron with short-bursting irregular discharges. Spontaneous APs arise from a flat baseline in (A–C) and (E), indicative of a fibre origin (see text). This is shown in (Bb), where 15 spontaneous APs are averaged shown at different vertical scales: 40 mV (line 1) and 1 mV (line 2). The bottom set of traces of (Bb) are a waterfall plot of the individual APs (1 line for each of the 15 spontaneous APs averaged above) showing no evidence of Em oscillations in relation to the APs. In the Aβ-nociceptor in (D), the SF APs have a predepolarisation, suggestive of a soma origin; the averaged traces for 8 SF APs in (Db) show baseline oscillations prior to SF APs; in the waterfall plot (bottom traces Db), these are clear for 6 of the 8 APs. In (F), the burst SF in the slowly adapting (SA) neuron has no predepolarisations; and is not associated with membrane oscillations.

Fig. 5

Dorsal root electrical thresholds: each symbol represents one neuron. All neurons had receptive fields (RFs). L4 and L5 DRG neurons in normal and L4 RF-neurons in spinal nerve axotomy (SNA) rats are plotted. Statistical tests were Mann-Whitney tests. The stimulus voltage (0.03 ms) at the dorsal root required to evoke a somatic AP is plotted as the threshold. Neurons with spontaneous firing are indicated as triangles. (A) In A-fibre nociceptors, thresholds were significantly lower than normal in SNA rats for all A-nociceptors, Aδ-nociceptors, and Aβ-nociceptors. (B) In Aα/β cutaneous low-threshold mechanoreceptors (LTMs), thresholds in SNA rats were slightly lower than normals (not significant) in all LTMs and in G hair/Field units. G/F, G hair/Field units; RA, rapidly adapting units; SA, slowly adapting units. Significance indicated as ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001.

Fig. 6

Membrane potential (Em). Scatterplots show distributions of Ems in L4 receptive field-neurons, in spinal nerve axotomy (SNA) and modified SNA (mSNA). (A) C- and A-nociceptors: Em was unchanged in C-nociceptors, but was hyperpolarised in A-nociceptors in SNA (not significant) and mSNA rats (significant). (B) Cutaneous Aα/β-low-threshold mechanoreceptors (LTMs): Em was significantly hyperpolarised in all cutaneous Aα/β LTMs together and in RA units in SNA rats, but no change from normal was seen in mSNA rats. For dorsal root ganglia recorded, statistics, and symbols, see Fig. 5 legend. G/F, G hair/Field units; RA, rapidly adapting units; SA, slowly adapting units.

Fig. 7

Action potential (AP) rise time. Scatterplots show distributions of AP rise time. (A) Nociceptors: compared with normal, AP rise time was significantly shorter in C-nociceptors in modified spinal nerve axotomy (mSNA), in all A-nociceptors (mSNA) and Aδ-nociceptors (SNA and mSNA) and in Aα/β-nociceptors (SNA). (B) Cutaneous Aα/β- low-threshold mechanoreceptors (LTMs): there was no significant change in AP rise time in the cutaneous Aα/β LTMs or their subtypes in SNA or mSNA models. For dorsal root ganglia recorded, statistics, and symbols, see Methods and Fig. 5 legend. G/F, G hair/Field units; RA, rapidly adapting units; SA, slowly adapting units.