Ectopic activity in cutaneous regenerating afferent nerve fibers following nerve lesion in the rat

Nerve injury

Sural nerve lesion was performed on 40 male Wistar rats (body weight 250–370 g) at 4–21 days before the neurophysiological experiments. The surgery was done under general anaesthesia induced by pentobarbital sodium (Narkoren, 60 mg/kg injected, i.p.). Under aseptic conditions the left sural nerve was exposed 10–25 mm proximal to the ankle. In 18 rats the sural nerve was crushed with fine watchmaker's forceps; in 16 rats the sural nerve was sectioned and resutured. For the crush lesion the nerve was squeezed three times at the same location over 1–1.5 mm, leading to transparency of the nerve at the lesion site. For the second type of lesion the nerve was cut with the help of fine scissors and the peripheral stump was re-anastomosed to the proximal nerve end using two epineural fine sutures (Ethicon, Prolene, Germany). Fine nerve surgery was performed under a stereomicroscope; the lesion site was located 15–20 mm proximal to the ankle. The wound was closed in layers and recovery was uneventful. In six control experiments we tested whether nerve fibers can be activated by mechanical stimulation of the perineurium of the acutely sectioned sural nerve.

Anaesthesia and animal maintenance

On the day of the final experiment the rats were re-anaesthetized with pentobarbital sodium (Narkoren, 60 mg/kg i.p.), catheters were inserted into the tail artery for blood pressure recording (transducer LM-22, List, Darmstadt, Germany) and into the jugular vein for administration of fluid and drugs. Additional doses of pentobarbital were given regularly (10–20 mg/kg/h, i.v.) to maintain a sufficient level of anaesthesia as judged from the absence of spontaneous blood pressure fluctuations. Mean arterial blood pressure during the experiments was always above 70 mmHg.

The trachea was cannulated and the animals breathed spontaneously during the initial surgery. After finishing the initial surgical preparations the animals were paralysed with Pancuronium (Organon, initially 1 mg/kg, i.v., maintenance with 0.4 mg/kg) and artificially ventilated at 75 strokes per minute using O₂-enriched room air. Blood gases were measured regularly throughout the experiment (ABL30, Radiometer Copenhagen) and pO₂ always exceeded 70 mmHg. Rectal temperature was monitored and kept close to 37.0 °C by means of a servo-controlled heating blanket.

At the end of the experiments, the animals were killed under deep anaesthesia by an intravenous injection of a saturated potassium chloride solution. All experiments had been approved by the local animal care committee of the state administration and were conducted in accordance with German Federal Law.

Surgical procedure during neurophysiological experiments

The left sural nerve was exposed from the ankle to its junction with the sciatic nerve. Seven to 5 mm distal to the lesion site the sural nerve was isolated from the surrounding tissue and mounted on a pair of platinum electrodes for electrical stimulation. In 13 experiments the sural nerve was isolated from surrounding tissue also proximal (5–7 mm) to the lesion site and put on a second pair of stimulating electrodes (Fig. 1A). At its junction with the sciatic nerve, the sural nerve was cut, isolated from connective tissue and placed on a rigidly fixed small black perspex platform. The exposure was covered with warm (30 °C) paraffin oil in a pool made from the skin flaps.

Recording and electrical stimulation

The proximal cut end of the sural nerve was split with sharpened watchmaker's forceps under a stereomicroscope. The fine filaments were teased out and put on electrodes for recording. Activity of single fibers was recorded unipolarly using a pair of platinum wire electrodes with the indifferent electrode connected to nearby tissue. The signals were differentially amplified (input resistance 10 MΩ), filtered with a bandwidth of 120 Hz to 1.0–1.2 kHz (unmyelinated fibers) or with a bandwidth of 120 Hz to 40 kHz (myelinated fibers), and fed through window discriminators. Spike discrimination was controlled by means of a delay circuit: the action potentials were delayed by 5 ms and displayed on a storage oscilloscope using the output of the window discriminator as a trigger.

The sural nerve was electrically stimulated with square wave pulses of 0.1 ms (A-fibers) or 0.5 ms (C-fibers) duration at a repetition rate of 3 Hz and 0.3 Hz, respectively, with variable intensities up to 40 V. Conduction velocity was calculated from the latency of the responses to electrical stimulation and the distance between recording and stimulation sites (18.1 ± 0.6 mm from the proximal pair of electrodes; 27.3 ± 0.7 mm from the distal pair of electrodes, Fig. 1A). No systematic discrimination of A-fibers into Aβ and Aδ was attempted because: (1) the distances between recording and stimulation sites were short and (2) lesioned afferent fibers shrink leading to a reduction in their conduction velocity (Michaelis et al., 1996, 2000). The afferent fibers were considered to be A- or C-fibers when their conduction velocities were > 2 m/s and ≤2 m/s, respectively. Some 10% of the fibers that could not be electrically identified were offline classified as A- or C-fibers according to the shape and height of their action potentials. This procedure allows us to classify about 95% of the nerve fibers into A- or C-fibers (see Michaelis et al., 1994).

Experimental procedure

Electrophysiological experiments were conducted 4–21 days after sural nerve lesion. We analysed only those filaments that contained at least one A- or C-fiber that exhibited spontaneous activity or/and mechanical or/and thermosensitivity. We first tested whether a filament isolated from the sural nerve contained fibers with at least one of these types of ectopic activity. If a filament contained a spontaneously active fiber, this discharge was recorded first for 3–5 min in order to determine its rate and pattern. Then a series of mechanical and thermal stimuli was applied along the lesioned sural nerve from 10 mm proximal up to 18 mm distal to the lesion site in order to determine the number of ectopically active fibers within the filament tested and to localize the receptive fields of the mechano- and/or thermosensitive afferents distributed along the nerve. The quantitative responses of the fibers to mechanical and thermal stimuli were analysed offline.

Natural stimulation

Thermal stimulation

Thermal stimuli of 3.5–50 °C were applied by a water-perfused thermode (Fig. 2, see legend for characteristics of the thermode). The surface of the thermode tube contacting the nerve was 2 mm long. As a result of thermal conduction the stimulus covered a distance of 4 mm along the nerve. To test the thermosensitivity the thermode was therefore placed at five sites of the nerve: the lesion site, 4–6 mm proximal to the lesion site as well as 4–6 mm, 9–11 mm and 14–16 mm distal to the lesion site. In this way the area from 7 mm proximal to 17 mm distal to the lesion site was covered. For identification of cold-sensitive fibers the thermode was initially perfused with ice water; the lowest temperature reached was in the range 3.5–5 °C. If a fiber was activated before the minimal temperature was reached, a series of testing stimuli at higher temperatures (10–19 °C) was applied. For initial testing of heat-sensitive fibers, stimuli of 47–50 °C were applied (duration, 3–5 s; longer stimuli were avoided to prevent nerve damage). If a fiber was activated before peak temperature was reached, a series of testing stimuli at lower temperatures (30–42 °C) was applied. These temperature stimuli were maintained for approximately 15 s and started from a baseline temperature of about 22–24 °C. A fiber was regarded as thermosensitive if more than three action potentials were generated during increase or decrease of the temperature in silent fibers and fibers with rates of spontaneous activity below 0.5 imp/s or if the activity increased by ≥ 100% in fibers with rates of spontaneous activity > 0.5 imp/s.

Data analysis

Neural activity, temperature of the stimulation thermode and arterial blood pressure were simultaneously fed into a computer (IBM-compatible) with ADC and counter interface (Burr-Brown PCI-20000, data acquisition software CARDS by S. Tiedemann) and stored on a digital tape recorder (DTR-2602, Biologic, Claix, France) for further off-line analysis (custom data acquisition software and template-matching program; Forster & Handwerker, 1990). Data are expressed as means ± SEM. For statistical analysis the chi-squared test or the non-parametric Wilcoxon signed-rank test were used.

Overall description

Spontaneous activity and responses to mechanical, cold and heat stimuli of axotomized afferent nerve fibers in the sural nerve were investigated 4–21 days after crush (n = 18 rats) or after section and resuturing of the nerve (n = 16 rats). The focus of this study was to analyse the ectopic discharges generated in afferent nerve fibers that were in the process of regeneration but had not yet reached the target tissue. In total 177 A-fibers and 169 C-fibers with at least one type of ectopic discharge property were studied (Table 1). Within the second half of the time period chosen (mostly in the third week post lesion) 29 afferent fibers (24 A- and five C-fibers) had already re-innervated either the peripheral targets in the original sural nerve territory or could be excited from the sural nerve branches below the skin by mechanical stimulation. Four of these fibers (three A- and one C-fiber) had mechanosensitive receptive fields both in the nerve and in the peripheral target tissue. Most of these fibers (23/29 fibers) were found in rats with crush lesion. They are not included in this study and will be presented together with other data in a forthcoming paper that describes regenerating afferent nerve fibers >30 days post nerve lesion, many of them having regenerated to their target tissue.

The numbers of afferent A- and C-fibers with at least one ectopic discharge property were almost equally distributed over the three time periods: first, second and third weeks post lesion in both experimental groups (Table 2). The response characteristics of the afferent nerve fibers throughout these three time periods were the same. Furthermore, the incidence of every ectopic discharge characteristic was, with one exception, not statistically significantly different among the three time periods (P > 0.05, χ² test). Only the incidence of C-fibers with thermosensitivity was significantly lower in the first week than in the third week post lesion in rats with sectioned and resutured sural nerve. Therefore, the data obtained in these three weeks were pooled together (Table 1). Differences in results between both types of nerve lesion are described below.

Percentage of nerve fibers exhibiting ectopic activity after nerve lesion

In 20 experiments we investigated the percentages of ectopically active A- or C-fibers among the total number of electrically activated fibers 5–21 days after crush or after section and resuturing of the sural nerve. For this purpose we analysed only those filaments in which the number of electrically activated A- or C-fibers could be determined (Fig. 1). These filaments contained maximally seven A-fibers (mean three) or maximally ten C-fibers (mean five). Strands in which the nerve fibers could not be identified by electrical stimulation were discarded. All electrically excited fibers were tested for their spontaneous activity and whether they could be excited by mechanical and/or thermal stimuli applied to the nerve. In these experiments we did not test whether individual nerve fibers could be excited from both the proximal and the distal pair of stimulaton electrodes or only from the proximal pair of stimulation electrodes.

Overall, 33–40% (mean 36%) of the A-fibers and 21–27% (mean 23%) of the C-fibers could be excited by mechanical or thermal stimulation and/or exhibited spontaneous activity. No significant difference in the percentages of ectopically active A- or C-fibers was found between the two types of nerve lesion (crush vs. section and resuturing) and within the same group of nerve lesion between the populations of fibers activated from the distal vs. proximal pair of stimulation electrodes (Table 3).

A-fibers

Most A-fibers (157/177, 89%) investigated for all properties were either mechanosensitive (n = 146) or spontaneously active (n = 11). Six fibers were both spontaneously active and mechanosensitive, six both heat-sensitive and mechanosensitive, and three fibers had all three ectopic discharge properties. Four A-fibers were only activated by heat stimuli, two of them being spontaneously active. One spontaneously active A-fiber was activated by cooling. Spontaneous activity in A-fibers was not depressed by cooling or heating.

Spontaneous activity

Twenty-three of 177 ectopically active A-fibers investigated were spontaneously active. Twenty of these spontaneously active A-fibers belonged to the crush group (P < 0.001, χ²-test), most of them in the first 2 weeks after nerve lesion. The remaining three spontaneously active fibers were observed after section and resuturing of the nerve. The rate of spontaneous activity measured at a temperature of the nerve of about 22–24 °C ranged from 0.03 to 10.6 imp/s (median 1 imp/s; mean 2.4 imp/s). The pattern of spontaneous activity was either irregular (14/23 fibers) or contained bursts (9/23 fibers) with intraburst frequencies being in the range 7.5–22.5 Hz (Fig. 3A and B). In 11 A-fibers the spontaneous activity was the only ectopic discharge property. In the remaining A-fibers the spontaneous activity was combined with mechano- or thermosensitivity (see Table 1).

Mechanosensitivity

Mechanosensitivity was observed in 161/177 A-fibers. The responses to mechanical probing of the nerve with a fine-tipped blunt glass rod or with von Frey filaments were phasic (Fig. 4A and B); tonic responses to constant force stimuli were absent. The mechanical thresholds ranged from 0.45 to 45 mN (Fig. 4D, mean 5.9 ± 0.9 mN, n = 81). Most mechanical thresholds were in the non-noxious range; only a few fibers had thresholds ≥ 20 mN being probably in the noxious range (Fig. 4D). The maximal discharge rates during trains of phasic mechanical stimulation at 1 Hz intratrain frequency were in the range 42.5 ± 6.2 imp/s (n = 11). The receptive fields of the mechanosensitive A-fibers extended from 6 mm proximal to the lesion site to 15 mm distal to the lesion site depending on the time after nerve lesion and type of nerve injury (Fig. 5A). Following section and resuturing of the sural nerve, 38% of the mechanosensitive A-fibers could be activated from the lesion site. The other fibers could be activated from sites proximal (14%) or distal (48%) to the nerve lesion, the most distal point on the nerve being 9 mm. Following crush lesion only 6% of the mechanosensitive A-fibers were activated from the lesion site. Most other A-fibers were activated from sites distal to the nerve lesion (88%), the most distal point on the nerve being 15 mm. The size of the receptive fields of mechanosensitive A-fibers varied between 1 and 15 mm along the nerve (inset in Fig. 5A). The fields were frequently discontinuous and consisted of punctuate low threshold spots along the nerve. This is demonstrated for one A-fiber in Fig. 6 (large action potential) in which the mechanosensitivity was present 1 mm and 4–6 mm but not 2–3 mm distal to the nerve lesion.

Thermosensitivity

Fourteen (8%) of the A-fibers in both groups of experimental nerve lesion were thermosensitive. Most of them (13/14 fibers) were activated by heat stimuli and had activation thresholds varying between 37 and 42 °C. One fiber was activated by cooling. The responses to heat stimuli were either phasic or phasic–tonic (Fig. 7A).

C-fibers

The pattern of ectopic discharges in afferent C-fibers was significantly different from the pattern in A-fibers as far as the distribution of types of ectopic activity is concerned (see lower part of Table 1). Ninety-seven of 169 fibers (39/79 fibers [49%] in the crush group; 58/90 fibers [64%] in the section and resuturing group) were either spontaneously active or thermosensitive (responsive to cooling or heating) or mechanosensitive. The remaining responsive C-fibers had either two (45/169 fibers, 27%), three (26/169 fibers, 15%) or all four (one fiber) of these ectopic firing properties (Table 1).

Spontaneous activity

Eighty-four of 169 C-fibers (50%) were spontaneously active at a temperature on the nerve surface of about 22–24 °C (Fig. 3C and D). The proportion of spontaneously active C-fibers was significantly higher after crush lesion than after section and resuturing (51/79 vs. 33/90 fibers, P < 0.005; Table 1). The rate of spontaneous activity ranged from 0.01 to about 8 imp/s (median 0.3 imp/s, mean 0.86 ± 0.20, n = 84; Fig. 8). It was significantly higher in C-fibers after crush lesion than in C-fibers after nerve section and resuturing (1.23 ± 0.30 vs. 0.36 ± 0.06 imp/s, P < 0.01; Fig. 8). The rate of spontaneous activity was not significantly changed by the thermode placed on the nerve (see below). The spontaneous activity was irregular in most C-fibers (Fig. 3D, insets in Fig. 10). Some C-fibers showed discrete bursts in their spontaneous activity, occurring irregularly (as shown in 3, 10). In 32/169 of the spontaneously active C-fibers ongoing activity was the only ectopic property; in the remaining spontaneously active C-fibers it was combined with ectopic mechano- and/or thermosensitivity.

The distribution of spontaneous activity of C-fibers exhibited some distinct characteristics (see lower part of Table 1). The proportion of spontaneously active units among cold-sensitive C-fibers was significantly higher than among heat-sensitive C-fibers (27/45 vs. 7/33 fibers, P < 0.005) and significant higher than in mechanosensitive C-fibers (18/33 vs. 10/43 fibers, P < 0.01).

Thermosensitivity

Thermosensitivity was observed in 94/169 (56%) C-fibers. Forty-five of these thermosensitive C-fibers were activated by cooling, 33 fibers by heating and 16 by both cooling and heating. The proportion of C-fibers that were activated by cooling was higher in nerves with crush lesion than in nerves that were sectioned and resutured (29/79 vs. 16/90 nerve fibers, P < 0.01; Table 1).

Most cold-sensitive C-fibers (42/45, 93%) were activated by cooling steps of < 10 °C starting from a baseline temperature of 22–24 °C, some fibers being very sensitive to small temperature changes (see arrow in Fig. 10B). Seventy-one per cent of these cold-sensitive C-fibers (32/45) responded tonically (Fig. 9). The responses of the remaining cold-sensitive C-fibers were phasic, i.e. these fibers were only activated during lowering of temperature. The peak discharge rates during maximal cooling ranged from 2 to 47 imp/s (8.1 ± 1.5 imp/s, n = 39). Some cold-sensitive C-fibers showed bursting in their discharge during cooling. In 11 of 27 spontaneously active cold-sensitive C-fibers the ongoing activity was depressed during heating of the nerve. Fourteen fibers exhibited a rebound excitation after nerve heating starting at temperatures that were in the range 35–40 °C (Fig. 10A). Ten of these were spontaneously active and activated by cooling; three fibers were silent and heat sensitive; one unit had no other ectopic discharge property. This rebound excitation lasted for 2–6 min. In 16 of the spontaneously active cold-sensitive C-fibers the baseline activity did not change during heating.

The activation thresholds of heat-sensitive fibers ranged from 32 to 50 °C. Thirty-seven per cent (15/41) of the heat-sensitive fibers were activated by temperatures of 32–37 °C, 26% (11/41) by 38–42 °C and 37% (15/41) responded when the temperature exceeded 42 °C. The peak discharge rates during heating ranged from 2 to 31 imp/s (8 ± 1.3 imp/s, n = 28).

C-fibers that were activated by both cooling and heating exhibited the same response pattern to cooling or heating as those C-fibers that were only activated by one of these stimuli.

Control experiments

Six control experiments were performed in order to test whether nerve fibers can be mechanically activated from the perineurium of the sural nerve. For this purpose the sural nerve was sectioned acutely about 20–25 mm distal to the recording site. Forty-six strands with 108 A-fiber and 322 C-fibers were isolated within the first 5–6 h after sural nerve transection from the proximal end of the nerve. Mechanical stimuli were systematically applied to the sural nerve using a fine-tipped blunt glas rod from sites 5 mm distal to the recording electrodes up to the distal nerve end. In this time period only a few fibers can be activated mechanically from the distal lesion site (Michaelis et al., 1995). Six unmyelinated fibers (2%) could be excited by mechanical stimulation along the nerve. The responses of these fibers were phasic and they had high mechanical thresholds. No C-fiber exhibited spontaneous activity. Myelinated fibers did not respond to mechanical stimulation of the nerve nor did they exhibit spontaneous activity.

Ectopic properties of lesioned myelinated and unmyelinated afferent fibers

About 22–27% of the C-fibers and 33–40% of the A-fibers exhibited at least one of the tested ectopic discharge properties. These percentages are comparable with those published recently for the ligated and sectioned sural nerve about 20–30 h after nerve lesion (Michaelis et al., 1995, 1999). The ectopic discharges are not generated in perineural nerve fibers. No A-fibers and very few C-fibers were excited from the perineurium of the acutely sectioned sural nerve by mechanical stimulation at high stimulus strength (see also Michaelis et al., 1995). Thermal stimuli applied to the intact sural nerve activated only 4.1% of the C-fibers tested (5/172 fibers by heat stimuli and 2/172 fibers by cold stimuli) and no A-fibers (Blenk et al., 1996). About 15% of the C-fibers in the sural nerve are sympathetic postganglionic (Baron et al., 1988). These fibers are unresponsive to mechanical stimuli at intensities known to excite afferent cutaneous C-fibers, as shown in microneurographic studies on humans (Schmidt et al., 1994) and do not exhibit spontaneous activity generated in the periphery. It is unclear why approximately 60% of the lesioned A- and C-fibers could not be excited by the physiological stimuli and/or did not exhibit spontaneous activity.

Most ectopically active A-fibers were mechanosensitive only; some were spontaneously active or both, most after crush lesion. A few A-fibers were also heat sensitive, most being mechanosensitive and/or spontaneously active (Tables 1 and, Fig. 11). Cold-sensitive A-fibers were virtually absent. Ectopically active afferent C-fibers were either spontaneously active or mechano-, cold or heat sensitive (49% of the C-fibers in the crush lesion model; 64% of the C-fibers in section and resuturing lesion model) or had different combinations of these ectopic discharge properties (Table 1, Fig. 11). Spontaneous activity and cold sensitivity were more prominent after crush lesion; mechanosensitivity was more prominent after section and resuturing; spontaneous activity was more often found in cold-sensitive fibers than in mechanosensitive or heat-sensitive C-fibers (Table 1, Fig. 11). The mechano- and thermosensitivity of the C-fibers were mostly confined distally to the lesion site, but some units could also be excited by stimulation a few millimetres proximal to the lesion site. The proportion of fibers with thermosensitivity and/or spontaneous activity as well as the overlap between the different types of ectopic discharge properties were significantly larger in C-fibers than in A-fibers (Table 1, Fig. 11).

The modality of ectopic discharge properties of regenerating nerve fibers are similar in many aspects to those obtained in lesioned cutaneous afferent nerve fibers, which are trapped at the lesion site and prevented from sprouting into the peripheral nerve, 6–30 h after ligation and section of the sural nerve (Michaelis et al., 1995, 1999; Blenk et al., 1996). Table 4 compares the distribution of the ectopic discharge properties in A- and C-fibers between regenerating afferent fibers (following sural nerve crush or section and resuturing) and non-regenerating fibers trapped in the neuroma (Michaelis et al., 1995, 1999). The table shows that ectopic discharge properties of lesioned afferent fibers trapped in the neuroma are quite similar to those developed by regenerating fibers post section and resuturing of the sural nerve. The only exception found was that 8% of regenerating A-fibers develop thermosensitivity but none in the neuroma group. The proportion of fibers with spontaneous activity and the rate of spontaneous activity are significantly higher in both regenerating A- and C-fibers following crush nerve lesion than in the other two nerve lesion models (Table 4).

The receptive fields of individual mechanosensitive A- and C-fibers are either confined to the lesion site or distributed over 1–12 mm along the nerve. Interestingly, no mechanosensitive receptive fields were found outside of the nerve. Regenerating nerve fibers have several sprouts growing at different speeds and this may explain the extent as well as the discontinuity of their receptive fields.

The rate of spontaneous activity in the regenerating C-fibers of our study was significantly higher (0.86 ± 0.20, n = 80) than the rate of spontaneous activity in intact C-fibers in control nerves and in intact C-fibers in nerves in which many axons undergo Wallerian degeneration post lesion of neighbouring nerves (< 0.1 imp/s; Ali et al., 1999; Wu et al., 2001). In intact afferent C-fibers of unlesioned cutaneous nerves spontaneous activity seems only to be present in cold-sensitive C-fibers (Kress et al., 1992; Leem et al., 1993). The rate of spontaneous activity in regenerating nerve fibers was determined at a nerve temperature of 22–24 °C on the surface of the nerve. Therefore, this rate probably is somewhat lower in the cold-sensitive C-fibers at temperatures of 30–33 °C, i.e. at a nerve temperature at which the skin is closed.

Mechanosensitivity, thermosensitivity and spontaneous activity of C-fibers exhibited some overlap (Fig. 11), resulting in several functional types of lesioned C-fibers as defined by the ectopic discharge properties (Table 1). Some of these types of discharge pattern are also present in intact afferent C-fibers innervating hairy or hairless skin of the rat hind limb (Kress et al., 1992; Leem et al., 1993). For example, Leem et al. (1993) report that about 33% of the cutaneous C-fibers recorded in vivo respond to both mechanical and heat stimulation and Kress et al. (1992) that ≥ 50% of the responsive C-fibers are activated by both stimuli. Only about 10% of the lesioned C-fibers in the three types of nerve lesion models (crush, section and resuturing, neuroma) exhibit this combination of evoked impulse activity (Tables 1 and 4; Fig. 10). The reason for this difference could be the high heat thresholds of regenerating C-fibers, which might originally have been polymodal nociceptive in function. These high thresholds to heat stimulation could be related to the down-regulation of the vanilloid-receptor subtype 1 in the axotomized afferent neurons, which is suggested to be responsible for heat-transduction (Michael & Priestley, 1999). In order to prevent damage to the nerve fibers we avoided direct application of temperatures > 50 °C to the nerve. However, the heat thresholds for the C-fibers identified in the in vitro preparation of Kress et al. (1992) were in the range 34–44 °C and these values are in the range of the heat stimuli we have used.

Molecular mechanisms underlying ectopic activity

Mechano- and thermosensitivity of regenerating nerve fibers probably are intrinsic properties of the afferent neurons. These transduction properties are dependent on distinct proteins in the membranes of the cut axons that are synthesized in the cell bodies and continuously carried into the peripheral terminals of the afferent neurons by the axoplasmic transport (Caterina et al., 1997, 2000; Davis et al., 2000; McKemy et al., 2002). The specific transduction characteristics are qualitatively independent of the interaction with the surrounding milieu. However, they are most likely modulated by interactions with the peripheral target tissue. For example, during inflammation the increased sensitivity of nociceptive neurons to heat stimulation is mediated by transcriptional up-regulation of the vanilloid receptor type 1 (VR1), which is triggered by nerve growth factor (NGF), and its trkA receptor in the peptidergic C-neurons and possibly by glia-cell-line-derived neurotrophic factor (GDNF) in the non-peptidergic IB-4-positive C-neurons (Bennett et al., 1998; Nicholas et al., 1999). Inflammatory mediators can acutely sensitize lesioned afferent C- and A-fibers for mechanical stimulation (Michaelis et al., 1998). It is unclear how the VR1-mediated heat transduction, and those transduction mechanisms in afferent neurons that underlie the responses to cold and mechanical stimulation, does change after axotomy of the neurons. However, axotomy is followed by down-regulation of the VR-1 receptor in small-diameter peptidergic and non-peptidergic (IB-4 positive) dorsal root ganglion cells (Bennett et al., 1998; Michael & Priestley, 1999).

The regenerating nerve fibers are reported to interact with Schwann cells, macrophages, fibroblasts and other cells that are activated during Wallerian degeneration distal to the nerve lesion, but also proximal to the nerve lesion, and with various signalling molecules secreted by these cells, e.g. neurotrophic factors such as NGF and brain-derived neurotrophic factor (BDNF) (Heuman et al., 1987; Brown et al., 1991; Meyer et al., 1992; Funakoshi et al., 1993) and cytokines such as tumour necrosis factor α and interleukin 6 (for review see Sommer 2001). The way in which these various molecules modulate the physiological response properties and the spontaneous activity of the regenerating afferent neurons is unknown.

Independent of the change of transduction mechanisms following nerve lesion, sodium channels, potassium channels and voltage-sensitive calcium channels are up- or down-regulated in axotomized sensory neurons. These changes may be responsible for the increased excitability of the lesioned afferent neurons and therefore also for the generation of the spontaneous ectopic activity in them. For example, tetrodotoxin (TTX)-sensitive sodium channels are up-regulated and TTX-resistant channels down-regulated in C-neurons after axotomy (Zhang et al., 1997), involving decreased uptake of NGF or GDNF by the afferent neurons. Both neurotrophines can prevent or attenuate the down-regulation of the TTX-resistant channels in axotomized C-neurons in vivo and in vitro (Dib-Hajj et al., 1998; Cummins et al., 2000; Sleeper et al., 2000). Overall the excitability of the afferent cell bodies in dorsal root ganglia increases in all groups of afferent neurons after axotomy and practically all currents are affected, the largest increase in excitability occurring in afferent C-neurons and the smallest in afferent A-neurons (Abdulla & Smith 2001a,b).

Ectopic activity as a possible trigger of neuropathic pain

It is generally assumed that the changes occurring in the cell bodies of the afferent neurons after peripheral axotomy are representative of what occurs in the peripheral afferent fibers after axotomy in vivo and therefore also explain the generation of ectopic impulse discharges in these terminals. It is questionable whether the ectopic activity generated in the afferent cell bodies of the dorsal root ganglia following peripheral nerve lesion is as important as commonly assumed in triggering neuropathic pain or the equivalent pain behaviour in animal models with nerve lesions. The ectopic activity originating in the dorsal root ganglion seems to occur, first, mainly in afferent neurons with myelinated axons and not in those with unmyelinated axons (Michaelis et al., 1996; X. G. Liu et al., 2000; C. N. Liu et al., 2000) and, secondly, probably only in muscle afferent neurons (Michaelis et al., 2000). Furthermore, the proportion of dorsal root ganglion cells with ectopic activity appears to be low and probably in the range of a few per cent of all afferent cells. Therefore, it is likely that the lesioned peripheral afferent terminal is the main site of origin of ectopic activity that is involved in triggering neuropathic pain and the equivalent neuropathic pain-like animal behaviour. However, it is still puzzling how the distinct changes measured in the dorsal root ganglion cells are related to the ectopic activity generated by the afferent nerve terminals, and how this ectopic activity is related to neuropathic pain or pain-like behaviour in animals. For example, dorsal root ganglion cells of cutaneous afferent neurons with myelinated and unmyelinated fibers do not generate ectopic spontaneous activity after axotomy in vivo (Michaelis et al., 2000), whereas this ectopic activity is readily generated in some of these afferent neurons at their lesion site as shown in this and other studies.