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. 2018 Feb 21;119(5):1993–2000. doi: 10.1152/jn.00882.2017

Time course of ongoing activity during neuritis and following axonal transport disruption

Ieva Satkeviciute 1, George Goodwin 1, Geoffrey M Bove 2, Andrew Dilley 1,✉
PMCID: PMC7938769  PMID: 29465329

Abstract

Local nerve inflammation (neuritis) leads to ongoing activity and axonal mechanical sensitivity (AMS) along intact nociceptor axons and disrupts axonal transport. This phenomenon forms the most feasible cause of radiating pain, such as sciatica. We have previously shown that axonal transport disruption without inflammation or degeneration also leads to AMS but does not cause ongoing activity at the time point when AMS occurs, despite causing cutaneous hypersensitivity. However, there have been no systematic studies of ongoing activity during neuritis or noninflammatory axonal transport disruption. In this study, we present the time course of ongoing activity from primary sensory neurons following neuritis and vinblastine-induced axonal transport disruption. Whereas 24% of C/slow Aδ-fiber neurons had ongoing activity during neuritis, few (<10%) A- and C-fiber neurons showed ongoing activity 1–15 days following vinblastine treatment. In contrast, AMS increased transiently at the vinblastine treatment site, peaking on days 4–5 (28% of C/slow Aδ-fiber neurons) and resolved by day 15. Conduction velocities were slowed in all groups. In summary, the disruption of axonal transport without inflammation does not lead to ongoing activity in sensory neurons, including nociceptors, but does cause a rapid and transient development of AMS. Because it is proposed that AMS underlies mechanically induced radiating pain, and a transient disruption of axonal transport (as previously reported) leads to transient AMS, it follows that processes that disrupt axonal transport, such as neuritis, must persist to maintain AMS and the associated symptoms.

NEW & NOTEWORTHY Many patients with radiating pain lack signs of nerve injury on clinical examination but may have neuritis, which disrupts axonal transport. We have shown that axonal transport disruption does not induce ongoing activity in primary sensory neurons but does cause transient axonal mechanical sensitivity. The present data complete a profile of key axonal sensitivities following axonal transport disruption. Collectively, this profile supports that an active peripheral process is necessary for maintained axonal sensitivities.

Keywords: axonal mechanical sensitivity, axonal transport disruption, neuropathic pain, ongoing activity, vinblastine

INTRODUCTION

Conditions that cause radiating pain remain prevalent. Examples of such conditions include nonspecific arm or back pain, sciatica, fibromyalgia, whiplash-associated disorder, diabetes, complex regional pain syndrome, and endometriosis (Dilley and Greening 2013; Dyck et al. 2000; Jänig and Baron 2003; Loeser 1985; Waddell 1987; Woertgen et al. 1998; Zager et al. 1998). Many of these patients lack signs of a nerve injury on clinical examination. Instead, these patients may have inflamed nerves (i.e., neuritis) that are otherwise intact and are considered normal using typical clinical tests. Magnetic resonance imaging studies in some of these patients have identified increases in T2-weighted signal intensity along nerve trunks that are consistent with neuritis (Dilley et al. 2011; Greening et al. 2018).

A rat model of neuritis has provided much of our understanding about the mechanisms that underlie pain in these patients. In this model, animals develop transient behavioral sensory hypersensitivities to tactile and thermal stimuli in the absence of axonal degeneration (Bove et al. 2003; Chacur et al. 2001; Eliav et al. 1999; Pulman et al. 2013). At some time points following neuritis induction, the axons of intact nociceptors fire spontaneously, respond to direct mechanical stimulation at the site of inflammation (Bove et al. 2003; Dilley et al. 2005; Richards and Dilley 2015), and become sensitive to noxious inflammatory chemicals (Govea et al. 2017). Such ectopic activity and sensitivity constitute the most feasible source of afferent nociceptor activity contributing to spontaneous pain and mechanically evoked radiating pain that are reported in patients in the absence of nerve injury (Dilley and Greening 2013). This activity may also contribute to the afferent barrage that is reputed to drive central mechanisms that cause cutaneous hypersensitivities (Campbell and Meyer 2006; Gracely et al. 1992; Woolf 2011; Xie et al. 2005).

We have shown that neuritis disrupts fast axonal transport along intact sensory axons and that such disruption leads to axonal sensitivities (Dilley and Bove 2008a; Dilley et al. 2013). We have hypothesized that the mechanisms underlying these sensitivities involve the accumulation and insertion of ion channels at the site of disruption. The effects of axonal transport disruption along intact axons can be examined by applying low doses of the vinca alkaloid vinblastine to the rat sciatic nerve (Dilley and Bove 2008a; Fitzgerald et al. 1984). At low doses, perineural vinblastine disrupts microtubule polymerization without causing axonal degeneration or inflammation (Dilley and Bove 2008a; Fitzgerald et al. 1984; Kashiba et al. 1992; Katoh et al. 1992; Zhuo et al. 1995), which allows axonal transport disruption to be examined in the absence of these potentially confounding factors. Using this model, we have shown that localized vinblastine treatment causes the development of a transient tactile-evoked cutaneous hypersensitivity (Dilley et al. 2013). At approximately the peak of this pain behavior (days 4–5), intact C-fiber neurons develop axonal mechanical sensitivity (AMS) at the treatment site (Dilley and Bove 2008a; Dilley et al. 2013). In a previous study, ongoing activity from A- or C-fiber axons was not increased at this time point, which contrasts with neuritis and nerve injury models, where it is a major feature (Boucher et al. 2000; Bove and Dilley 2010; Kajander et al. 1992; Tal and Eliav 1996).

Both neuritis and vinblastine-induced axonal transport disruption are short-lived phenomena (Bove et al. 2009; Dilley et al. 2013), but patient symptoms are often chronic. This inconsistency has led to the suggestion that patients may have a persistent peripheral stimulus. To determine whether such altered nociceptor activities require an active stimulus, we have performed a detailed assessment of the time course of ongoing activity in these models. We also present the temporal profile of vinblastine-induced AMS, to compare with the ongoing activity. Together, the data from the present and past experiments complete the profile of ectopic activity and sensitivity, which informs peripheral mechanisms of radiating pain.

METHODS

Animals and surgery.

Experiments were carried out in strict accordance with the UK Animals (Scientific Procedures) Act (1986) and Home Office guidelines. Formal approval was also obtained from the University of Sussex Animal Welfare Ethical Review Board. Adult male Sprague-Dawley rats (n = 62; 250–500 g; Charles River Laboratories, Margate, UK) were used in this study. Rats were given ad libitum access to food and water. Animals were monitored daily.

Vinblastine was applied to the sciatic nerve of adult rats (n = 32) as previously described (Dilley and Bove 2008a). Under general anesthesia (1.75% isoflurane in oxygen), the left sciatic nerve was exposed by blunt dissection. An 8-mm length was cleared from its connective tissue, which allowed a short length of sterile parafilm (6 mm × 20 mm; Sigma Aldrich, Gillingham, UK) to be positioned under the nerve to prevent leakage of vinblastine onto the surrounding tissue. The sciatic nerve was loosely wrapped with a strip of sterile Gelfoam (5 mm × 5 mm × 10 mm; Spongostan; Ferrosan, Søborg, Denmark) saturated in 0.1 mM vinblastine [Sigma Aldrich; diluted in 0.9% (wt/vol) saline]. Both the Gelfoam and parafilm were removed after 15 min. The nerve was thoroughly rinsed with sterile saline, and the muscle and skin were closed using 4-0 monofilament sutures (Vicryl; Ethicon, West London, UK). The saline group (n = 9) underwent identical surgery, but instead the Gelfoam was saturated with 0.9% (wt/vol) saline alone.

Neuritis was induced as previously described (n = 13) (Dilley and Bove 2008b). The left sciatic nerve was exposed by blunt dissection, and an 8-mm length was cleared from the surrounding connective tissue. A sterile strip of Gelfoam (5 mm × 5 mm × 10 mm) saturated in 50% complete Freund’s adjuvant [diluted in sterile 0.9% (wt/vol) saline] was loosely wrapped around the nerve. The muscle and skin were closed using 4-0 monofilament sutures.

Electrophysiology.

Single-unit extracellular electrophysiological recordings were made from A- and C-fiber axons in the L5 dorsal root as previously described (Bove et al. 2003). The L5 dorsal root innervates much of the plantar surface of the foot that was examined during behavioral testing. Experiments were carried out at 2 h to 15 days following vinblastine treatment (n = 32), neuritis (n = 13) and saline treatment (n = 9), and in untreated animals (n = 9). Briefly, animals were deeply anesthetized [1.5 g/kg 25% (wt/vol) urethane intraperitoneally], and the body temperature was maintained at 37°C with the use of a rectal thermistor probe attached to a feedback controlled heat mat (Harvard Apparatus, Edenbridge, UK). A midsagittal skin incision was made in the lumbar region of the back and a laminectomy was performed from the L2 to L5 vertebrae. The surrounding skin was sutured to a metal ring to form a pool. The dura mater was cut to expose the caudal end of the spinal cord and cauda equina, and the pool was filled with mineral oil warmed to 37°C. The temperature of the pool was monitored during each experiment and was topped up with warmed mineral oil when necessary. The ipsilateral L5 dorsal root was identified and cut just before it enters the spinal cord, and loosely placed onto a glass platform (9 mm × 5 mm). Individual fine filaments were teased from the cut end of the nerve with finely sharpened forceps and carefully positioned on a bipolar recording electrode. In the midthigh, the sciatic nerve was exposed and the surrounding skin flaps were stitched to a metal ring to form a second mineral oil pool. Ongoing activity and AMS were assessed in different animals because repeated mechanical stimulation of the sciatic nerve at the treatment site can lead to the development of ongoing activity (Dilley et al. 2005). Receptive fields were also not examined during experiments to assess levels of ongoing activity, because noxious mechanical stimulation that is necessary to activate the terminals of nociceptors can cause ongoing activity (Bove and Dilley 2010; Richards et al. 2011).

To measure C-fiber conduction velocities (<2 m/s), a bipolar stimulating electrode was positioned under the L5 dorsal root and activity was evoked by electrical stimulation using a constant-voltage isolated stimulator (square-wave pulses; duration: 0.5 ms; amplitude: 30–50 V; Digitimer, Welwyn Garden City, UK). We have previously confirmed that the majority of neurons in the L5 dorsal root conduct through the sciatic nerve in the midthigh (i.e., through the treatment site; Dilley et al. 2013). To measure A-fiber conduction velocities (>2 m/s), bipolar stimulating electrodes were positioned under the sciatic nerve immediately distal to the treatment site and activity was evoked by electrical stimulation at this site (square-wave pulses; duration: 0.05 ms; amplitude: 3–10 V). During these stimuli, the limb was held in place by a noose around the foot. It should be noted that muscular contractions of the foot during electrical stimulation at A-fiber strength were only small, whereas stimulation at C-fiber strength would lead to substantial movement artifacts. The long conduction distance (range: 61–88 mm) allowed identification of individual A-fiber neurons. For each recording, the amplitude of the electrical stimulus was increased until the maximum number of waveforms was evoked. Only neurons with easily identifiable waveforms were examined. The number of waveforms on each filament ranged from one to seven. Action potentials were amplified (1–2 K), bandpass filtered (10–5,000 Hz) and monitored with an oscilloscope. Neuronal activity was digitized, recorded, and analyzed with Spike2 software (Cambridge Electronic Designs, Cambridge, UK). At the end of an experiment, the sciatic nerve and L5 dorsal root were removed and the conduction distance was measured. Conduction velocities of each neuron were calculated from the conduction latency and conduction distance.

In experiments to evaluate ongoing activity, identified neurons were recorded for 3 min. Neurons that fired at least once per minute were considered to exhibit ongoing activity (Bove and Dilley 2010). Muscle spindles, identified by high-frequency position- and movement-dependent repetitive firing, were excluded from the data analysis. Neurons with ongoing activity were identified as either A- or C-fiber neurons by spike shape. C-fiber neurons typically had a longer duration and were often biphasic compared with A-fiber neurons, which were monophasic (Dilley et al. 2005).

In the experiments to evaluate AMS, A-fiber neurons were not examined, because AMS in the Aβ population is rarely observed following vinblastine treatment (unpublished observations) or in the neuritis model (Bove et al. 2003). In the thigh, the sciatic nerve was exposed and a plastic platform (9 mm × 5 mm), notched to accommodate the nerve, was placed under the nerve with the treatment site in its center. Receptive fields for isolated neurons were searched below the knee using mechanical stimuli. Most receptive fields were located by squeezing the periphery, using either fingers or forceps. The loose property of the skin was exploited to carefully discriminate cutaneous vs. deep fields (Bove et al. 2003). Cutaneous neurons had receptive fields that remained associated with the skin, whereas deep neurons (i.e., neurons that innervate muscles, tendons, and joints) had receptive fields that remained in the same underlying spot irrespective of the skin position. For example, if a neuron responded to pinching a fold of skin and maintained similar responsiveness when this fold was displaced, it was concluded that its receptive field was cutaneous. In contrast, if the repeated application of the effective stimulus to same underlying point produced a similar neuronal response despite displacement of the overlying skin, it was concluded that the receptive field was deep (e.g., in muscle). After identification of a receptive field, the conduction velocity of the neuron was determined by electrically stimulating the dorsal root while mechanically stimulating the receptive field. If the neuron fired to mechanically evoked stimulation of the receptive field immediately before the electrical stimulation of the nerve, the same neuron evoked by the electrical stimulus would be delayed or would not be initiated (i.e., the electrical stimulus occurred during the refractory period).

Mechanical stimulation of the nerve was manually applied using a silicone probe as previously described (Bove et al. 2003; Dilley and Bove 2008a, 2008b). The tip of the probe was conical shaped and cut at the end to form a flat footprint ~5 mm × 3.5 mm. The mechanical stimulus was applied for 1–2 s successively along the length of nerve that was located on the notched platform (i.e., the treatment site or equivalent length in untreated animals). The force applied with the probe was <20 cN (as measured on an electronic scale). Using the probe in this manner does not interrupt the conduction of action potentials (i.e., cause damage to axons), confirmed by repeated activation of each neuron at its receptive field following axonal mechanical sensitivity testing. The conduction velocity of axons that were mechanically sensitive was determined by electrically stimulating the dorsal root while mechanically stimulating the treatment site as described above.

Data analysis.

Data were tested for normality using Shapiro-Wilk tests. Comparisons between the proportions of neurons with ongoing activity or AMS were made using Fisher’s exact tests. Conduction velocities of A- and C-fiber neurons and rates of ongoing activity from C-fiber neurons were compared between time points and with saline treatment using Kruskal-Wallis tests followed by Dunn’s post hoc tests. Because of the small number of A-fiber neurons with ongoing activity, rates could not be statistically compared. Comparisons of conduction velocities between neurons with and without AMS were made using Mann-Whitney tests. Conduction velocities and rates of ongoing data are presented as medians ± interquartile range (IQR).

RESULTS

Conduction velocities.

Conduction velocity data are summarized in Fig. 1. Recordings were made from a total of 2,124 neurons in the L5 dorsal root following vinblastine, neuritis, and saline treatment and in untreated animals. Histograms of the conduction velocities are shown in Fig. 1, A and B. Based on the conduction velocities, neurons were characterized as either C/slow Aδ- (0.2–2.1 m/s; n = 1,212) or Aα/β-fiber neurons (>10 m/s; n = 912) (Waddell et al. 1989).

Fig. 1.

Fig. 1.

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Conduction velocities of C- and A-fiber neurons. A and B: histograms of the conduction velocities of C/slow Aδ-fiber (A) and Aα/β-fiber (B) neurons for each group. C and D: median conduction velocities of C/slow Aδ-fiber (C) and Aα/β-fiber (D) neurons for each group at different postoperative time points. Saline-treated animals were only examined at 3–4 h and 4–5 days postoperatively. The number of C/slow Aδ- and Aα/β-fiber neurons in each group at each time point ranged from 72 to 147. C/slow Aδ-fiber neurons were characterized by electrical stimulation of the L5 dorsal root (see Fig. 2), whereas Aα/β-fiber neurons were characterized by electrical stimulation of the sciatic nerve (see Fig. 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 compared with untreated. †††P < 0.001; ††††P < 0.0001 compared with saline treatment (Kruskal-Wallis test followed by Dunn’s post hoc test). Error bars are interquartile range (IQR). d, Day.

In the untreated group, the median conduction velocity of the C/slow Aδ-fiber neurons was 0.58 (IQR 0.29) m/s. Following vinblastine treatment, neuritis, and saline treatment, there was a significant slowing of conduction 3–4 h postoperatively compared with the untreated group (P < 0.05, Kruskal-Wallis test; P < 0.001, Dunn’s post hoc tests). In the vinblastine-treated group, conduction was also slowed at 4–5 and 12–15 days following surgery (P < 0.05, Dunn’s post hoc tests). Following neuritis, conduction was slowed at all of the time points examined (P < 0.01, Dunn’s post hoc tests). The maximum slowing was at 3–4 h postoperatively following vinblastine treatment [19.7% compared with untreated; median = 0.47 (IQR = 0.16) m/s] and neuritis [41.2% compared with untreated; median = 0.34 (IQR 0.22) m/s; Fig. 1C].

In the untreated group, the median conduction velocity of the Aα/β-fiber neurons was 33.01 (IQR 12.78) m/s. Following both vinblastine and saline treatment, the conduction velocities of the Aαβ-fiber neurons were comparable to those of the untreated group at the time points examined. Following neuritis, conduction velocities were significantly reduced at 3–4 h and 1–2 days postoperatively (P < 0.05, Kruskal-Wallis test; P < 0.01, Dunn’s post hoc tests). The maximum reduction was at 3–4 h following treatment [20.8% compared with untreated; median = 26.16 (IQR 8.81) m/s; Fig. 1D].

Ongoing activity.

The proportion of C/slow Aδ-fiber neurons in each group with ongoing activity is summarized in Fig. 2A. Ongoing activity developed in 5.3% of C/slow Aδ-fiber neurons in the untreated group, with a median firing rate of 0.60 (IQR 1.03) Hz. Following vinblastine treatment, the proportions of C/slow Aδ-fiber neurons with ongoing activity ranged from 6.0% to 10.8% at the time points examined, which were not significantly different from the untreated group (P > 0.24, comparing individual time points with untreated) or following saline treatment (P > 0.12 at 3–4 h and 4–5 days postoperatively, Fisher’s exact tests). The median rate of ongoing activity in these neurons was <0.35 Hz, which was comparable between time points (P = 0.21, Kruskal-Wallis test; Table 1). In contrast to vinblastine treatment, there was a significant increase in ongoing activity from C/slow Aδ-fiber neurons following neuritis at 4–5 days postoperatively, with 23.9% (21/88) of neurons firing spontaneously (P < 0.05 Fisher’s exact test, compared with untreated and saline-treated groups). The rates of ongoing activity following neuritis at 1–2 days postoperatively were significantly increased compared with 3–4 h and 12–15 days postoperatively (P < 0.05, Kruskal-Wallis test; P < 0.05, Dunn’s post hoc tests). The firing patterns were all irregular (e.g., Fig. 2B).

Fig. 2.

Fig. 2.

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Development of ongoing activity in C/slow Aδ-fiber neurons. A: the percentage of ongoing activity is shown for the untreated, saline-treated, vinblastine-treated, and neuritis groups at different postoperative time points. Numbers of neurons with ongoing activity are given above bars. Inset shows a schematic of the electrophysiology setup. The recording (Rec) and stimulating (Stim) sites at the L5 dorsal root are indicated. The treatment site is shown along the sciatic nerve in the midthigh (shaded area). ***P < 0.001 (Fisher’s exact test). d, Day. B: typical patterns of ongoing activity. Insets are expanded waveforms.

Table 1.

Rates of ongoing activity in C/slow Aδ- and Aα/β- fiber neurons

C/Slow Aδ-Fiber Neurons
 Aα/β-Fiber Neurons
Treatment Time PO Median IQR Median IQR
Untreated 0 0.60 1.03 0.02 0.00
Vinblastine 3–4 h 0.35 0.62 0.14 0.28
1–2 d 0.17 0.28 0.11 0
4–5 d 0.26 0.48 0.04 0.08
12–15 d 0.08 0.54 0.24 0.03
Neuritis 3–4 h 0.11 0.19 0.04 0.01
1–2 d 1.27* 1.31 0.09 0.17
4–5 d 0.55† 1.53 0.11 0.09
12–15 d 0.10 0.13 0.02 0.00
Saline 3–4 h 0.14 0.10 0.03 0.15
4–5 d 0.47 0.56 0.21 0.20
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Values are rates (Hz) of ongoing activity. Median value for neuritis at 4–5 days postoperatively (PO) is based on 20 neurons (rate could not accurately be determined for 1 neuron). d, Day.

*

P < 0.05 compared with 3–4 h and 12–15 days PO.

†

P < 0.05 compared with 3–4 h PO (Kruskal-Wallis test followed by Dunn’s post hoc tests).

The proportion of Aα/β-fiber neurons in each group with ongoing activity is summarized in Fig. 3A. Ongoing activity was present in only one Aα/β-fiber neuron (1.3%; 1/79 neurons) in the untreated group, which had an irregular, slow (0.02 Hz) firing pattern (Fig. 3B). Following vinblastine treatment, ongoing activity was observed in 1.1–5.3% of Aα/β-fiber neurons at the time points examined, which was not significantly different from the untreated group (P > 0.20 comparing individual time points with untreated) or saline treatment (P > 0.68 at 3–4 h and 4–5 days postoperatively, Fisher’s exact tests). Following neuritis, ongoing activity was observed in 1.3–6.5% of Aα/β-fiber neurons, which was also not significantly different from the untreated group (P > 0.14 comparing individual time points to untreated) or saline treatment (P > 0.44 at 3–4 h and 4–5 days postoperatively, Fisher’s exact tests). Median rates of ongoing activity in all Aα/β-fiber neurons were low (median rate < 0.24 Hz; Table 1). Firing patterns were all irregular (e.g., Fig. 3B).

Fig. 3.

Fig. 3.

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Development of ongoing activity in Aα/β-fiber neurons. A: the percentage of ongoing activity is shown for the untreated, saline-treated, vinblastine-treated, and neuritis groups at different postoperative time points. Numbers of ongoing neurons are given above bars. Inset shows a schematic of the electrophysiology setup. The recording site at the L5 dorsal root is indicated (Rec). The nerve was stimulated along the sciatic nerve in the midthigh (Stim), immediately distal to the treatment site (shaded area). d, Day. B: typical patterns of ongoing activity. Ongoing spindles are also present in the untreated and neuritis traces (smaller units). Insets are expanded waveforms.

Axonal mechanical sensitivity.

The proportion of C/slow Aδ-fiber neurons in each group with deep and cutaneous receptive fields that developed AMS is summarized in Fig. 4. All of the neurons had receptive fields below the knee. The receptive fields of each of these neurons had a high mechanical threshold, responding to firm squeezing of the receptive field. Axonal mechanical sensitivity testing was repeatable, and activation of the receptive field before and following testing confirmed that the axons remained intact. The majority of AMS “hotspots” were either immediately proximal to or at the treatment site. No AMS hotspots were found distally. AMS did not develop in the untreated (0/28 neurons) or saline-treated groups (0/23 neurons). Following vinblastine treatment, AMS was detected only in neurons with deep receptive fields (i.e., in muscles or joints); that is, none of the neurons with cutaneous receptive fields exhibited AMS. Axonal mechanical sensitivity developed rapidly, with 15.8% (6/38) of all C-fiber neurons responding to direct mechanical stimulation at the treatment site at postoperative days 1–2 [P < 0.05 compared with untreated (0/28), Fisher’s exact test]. The proportion of neurons with AMS reached a maximum on postoperative days 4–5, with 27.6% (8/29) of all neurons responding to mechanical stimulation [P < 0.01 and P < 0.05 compared with untreated and saline-treated groups on postoperative days 4–5 (0/23), respectively, Fisher’s exact test]. On postoperative days 8–9, 8.3% (2/24) of all neurons developed AMS. By postoperative days 13–15, AMS had completely reversed with 0% (0/26) of neurons being mechanically sensitive. The median conduction velocity of the neurons that developed AMS was 0.55 (IQR 0.26) m/s (n = 16), which was not significantly different from those that were not mechanically sensitive [combined median for 1–2, 4–5, and 8–9 days postoperatively = 0.60 (IQR 0.26) m/s; n = 75; P = 0.34, Mann-Whitney test].

Fig. 4.

Fig. 4.

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Development of axonal mechanical sensitivity in C/slow Aδ- fiber neurons. A: the percentage of axonal mechanical sensitivity (AMS) in deep and cutaneous innervating neurons is shown for the untreated group and the vinblastine-treated groups at different postoperative time points. Horizontal lines indicate the combined percentage of axons with mechanical sensitivity for deep and cutaneous innervating neurons. Numbers of neurons with AMS innervating deep and cutaneous structures are given above bars. Inset shows a schematic of the electrophysiology setup. The recording (Rec) and stimulating (Stim) sites at the L5 dorsal root are indicated. The treatment site is shown along the sciatic nerve in the midthigh (shaded area). A notched platform was positioned under the treatment site to support the nerve during AMS testing. *P < 0.05; **P < 0.01 compared with untreated, for percentage of neurons innervating deep structures (Fisher’s exact test). †P < 0.05; ††P < 0.01 compared with untreated, for all neurons (combined deep + cutaneous). C, cutaneou; D, deep; d, day. B: the response pattern of AMS is linked to the mechanical stimulus. Horizontal lines above traces represent the duration of the mechanical stimuli. Insets are expanded waveforms.

DISCUSSION

Our experiments show that inflammation of the axons of C/slow Aδ-fiber neurons, which are likely to be nociceptors, causes transient ongoing activity that reaches a maximum on days 4–5 following induction and decreases to control levels after 2 wk. The observation that vinblastine treatment does not evoke a statistically significant increase in ongoing activity at any postoperative point supports that inflammation is key to the development of axonal ongoing activity. Combined, these observations support that if undamaged peripheral nociceptors exhibit ongoing activity, there must be a persistent source of inflammation. The findings may be directly applicable to patients with persistent radiating pain, which may be due to focal inflammation anywhere along the clinically implicated peripheral nerve. The results also clearly demonstrate the vulnerability and adaptability of small-diameter unmyelinated axons to subtle changes in their environment.

Because neuritis disrupts axonal transport, we have proposed that the cellular components necessary for the development of ongoing activity are transported by fast axonal transport and accumulate at the inflamed site. These cellular components might include immune receptors and/or ion channels, such as hyperpolarization-activated channels, which also have been implicated in the mechanisms of ongoing activity (Emery et al. 2011; Richards and Dilley 2015; Weng et al. 2012). The lack of an increase in ongoing activity in A- or C-fiber neurons up to 15 days following vinblastine-induced axonal transport disruption is consistent with the necessity for inflammation to activate the altered axons; vinblastine is in fact anti-inflammatory by virtue of its antimitotic properties. Furthermore, the sensitivity of C-fiber axons following the direct application of noxious inflammatory chemicals to the vinblastine treatment site (Govea et al. 2017) is consistent with the immune-mediated activation of accumulated cellular components.

Data from this study add to the “peripheral generator” theory, which proposes that an ongoing or intermittent afferent barrage from nociceptors is required to maintain the cutaneous symptoms associated with neuropathic pain (Campbell and Meyer 2006; Gracely et al. 1992; Woolf 2011; Xie et al. 2005). The lack of increased ongoing activity from Aα/β- or C/slow Aδ-fiber neurons at any of the examined time points following vinblastine treatment suggests that the previously reported tactile-evoked cutaneous hypersensitivity in this model may be driven by AMS, because the time course of this sensitivity is comparable to the hypersensitivity (Dilley et al. 2013).

The appearance and resolution of C-fiber AMS is consistent with the time course of slowed axonal transport that we have shown in this model (Dilley et al. 2013). Axons that were mechanically responsive were most likely nociceptors, because a high mechanical threshold was required to stimulate the receptive fields. The time course of AMS contrasts with that of neuritis, where similar activity persists beyond 1 mo (Dilley and Bove 2008b). This difference most likely reflects the longer duration of axonal transport disruption following neuritis (Dilley et al. 2013).

The transient development of neuritis-induced ongoing activity and AMS, and vinblastine-induced AMS, contrasts with the reported chronicity in patients. In both models, altered nociceptor activity occurs when there is an active peripheral stimulus. When the stimulus is absent, such as when axonal transport has recovered and levels of specific inflammatory chemicals have resided (Pulman et al. 2013), sensitivities resolve. These observations support that in patients, a peripheral stimulus, such as inflammation, must be active to cause persistent and spontaneous pain and movement-evoked radiating pain. This hypothesis is consistent with MRI findings of possible nerve inflammation in patients with chronic whiplash-associated disorder (Greening et al. 2018) and also with the persistent symptoms in a rat model of repetitive motion disorders, where neuritis is a prominent feature (Barbe et al. 2013). Therefore, persistent nerve inflammation, due to injury to surrounding soft tissues or direct irritation of the affected nerve, is a probable cause of prolonged focal axonal transport disruption that may underlie altered nociceptor activity.

In summary, altered nociceptor activity may cause persistent and spontaneous pain (mainly through the development of nociceptor ongoing activity) and radiating pain (through the development of AMS). Many patients with these symptoms lack signs of nerve injury on routine clinical examination. In such patients, the underlying mechanisms may involve inflammation-induced axonal transport disruption. A detailed examination of ongoing activity following neuritis and vinblastine-induced axonal transport disruption has shown that neuritis causes the transient development of ongoing activity in C/slow Aδ-fiber neurons. The lack of ongoing activity in A- or C-fiber neurons at any of the examined time points following vinblastine treatment is consistent with the need for inflammation to drive ongoing activity. It also raises the question as to whether such activity is essential for the development of the associated cutaneous hypersensitivities. The present data also show that AMS develops rapidly following vinblastine treatment but is relatively short-lived. The present work adds to our understanding of the relative contributions of ectopic activity and reinforces the underestimated role of AMS in neuropathic pain mechanisms. It also supports that for pain to persist, an active stimulus that causes altered axonal transport, such as neuritis, must also persist. Concerning animal welfare, treating nerves with low doses of vinblastine provides a less severe model to examine the role of axonal transport disruption in neuropathic pain mechanisms. It avoids cutting nerves, which can lead to adverse effects, and allows axonal transport disruption along intact axons to be explored.

GRANTS

This study was supported by National Centre for the Replacement, Refinement and Reduction of Animals in Research Grant NC/L00156X/1 and the Brighton and Sussex Medical School.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

ENDNOTE

At the request of the authors, readers are herein alerted to the fact that additional materials related to this manuscript may be found at the institutional Web site of the authors, which at the time of publication they indicate is: https://doi.org/10.25377/sussex.5687875. These materials are not a part of this manuscript and have not undergone peer review by the American Physiological Society (APS). APS and the journal editors take no responsibility for these materials, for the Web site address, or for any links to or from it.

AUTHOR CONTRIBUTIONS

I.S., G.G., G.M.B., and A.D. conceived and designed research; I.S., G.G., and A.D. performed experiments; I.S., G.G., G.M.B., and A.D. analyzed data; I.S., G.G., G.M.B., and A.D. interpreted results of experiments; I.S., G.G., and A.D. prepared figures; I.S., G.G., G.M.B., and A.D. drafted manuscript; I.S., G.G., G.M.B., and A.D. edited and revised manuscript; I.S., G.G., G.M.B., and A.D. approved final version of manuscript.

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