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Sympathetic Modulation of Activity in A- and C-Primary Nociceptive Afferents After Intradermal Injection of Capsaicin in Rats

¹Department of Neuroscience and Cell Biology and ²Division of Neurosurgery, Department of Surgery, University of Texas Medical Branch, Galveston, Texas

Submitted 5 August 2004; accepted in final form 8 September 2004

	ABSTRACT

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Neuropathic and inflammatory pain can be modulated by the sympathetic nervous system. In some pain models, sympathetic postganglionic efferents are involved in the modulation of nociceptive transmission in the periphery. The purpose of this study is to examine the sensitization of A- and C-primary afferent nociceptors induced by intradermal injection of capsaicin (CAP) to see whether the presence of sympathetic efferents is essential for the sensitization. Single primary afferent discharges were recorded from the tibial nerve after the fiber types were identified by conduction velocity in anesthetized rats. An enhanced response of some A- and most C-primary afferent fibers to mechanical stimuli was seen in sham-sympathectomized rats after CAP (1%, 15 µl) injection, but the enhanced responses of both A- and C-fibers were reduced after sympathetic postganglionic efferents were removed. Peripheral pretreatment with norepinephrine by intraarterial injection could restore and prolong the CAP-induced enhancement of responses under sympathectomized conditions. In sympathetically intact rats, pretreatment with an ₁-adrenergic receptor antagonist (terazosin) blocked completely the enhanced responses of C-fibers after CAP injection in sympathetically intact rats without significantly affecting the enhanced responses of A-fibers. In contrast, a blockade of ₂-adrenergic receptors by yohimbine only slightly reduced the CAP-evoked enhancement of responses. We conclude that the presence of sympathetic efferents is essential for the CAP-induced sensitization of A- and C-primary afferent fibers to mechanical stimuli and that ₁-adrenergic receptors play a major role in the sympathetic modulation of C-nociceptor sensitivity in the periphery.

	INTRODUCTION

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Inflammation that is initiated by release of inflammatory mediators from sensory nerve terminals (mainly nociceptors) is referred to as neurogenic inflammation (Geppetti and Holzer 1996; Holzer 1998; Lynn 1996; Richardson and Vasko 2002). Neurogenic inflammation contributes to numerous pathophysiological states. Clinically relevant examples include arthritis, inflammatory bowel disease, chronic bronchitis, migraine, and interstitial cystitis (Geppetti and Holzer 1996). Experimentally, intradermal capsaicin (CAP) injection has long been established to be an effective means to induce neurogenic inflammation pain (Jancso et al. 1967; Szolcsanyi 1996; Wall 1999) by activation of the transient receptor potential vanilloid-1 (TRPV₁) receptors that are localized in polymodel C- and some A-nociceptive fibers (Caterina et al. 1997; Szallasi and Blumberg 1999; Tominaga et al. 1998). Sensitization of primary afferent nociceptive terminals arising from activation of TRPV₁ receptors is presumed to be an initial step in the process by which neurogenic inflammation occurs (Holzer 1991; Lin 2003; Lynn 1990; Szolcsanyi 1987, 1993), and this sensitization contributes to primary mechanical and heat hyperalgesia.

Many studies have shown that the sympathetic nervous system can be involved in the modulation of pathological pain signaling. Experimentally, the sympathetic efferents may play a role in pathological states associated with pain and hyperalgesia, such as peripheral nerve injury and tissue trauma with inflammation (Heller et al. 1994; Jänig and McLachlan 1994; Jänig et al. 1996; Raja 1995). Clinically, pain sensations are found to be aggravated by sympathomimetic conditions and can often be alleviated by sympathetic block (Schwartzman and McLellan 1987). Furthermore, the pain of some patients who have obtained relief from sympatholytic therapy can be rekindled by cutaneous administration of norepinephrine (NE) (Torebjörk et al. 1995). The studies on humans also indicate that -adrenoreceptors are involved in the development of CAP-induced ongoing pain and hyperalgesia as well as mechanical allodynia (Kinnman et al. 1997; Liu et al. 1996; however, cf. Baron et al. 1999). Multiple sites for an interaction between the sympathetic and sensory nervous systems have been suggested, including the site of nerve or skin injury and the dorsal root ganglion cells supplying the injury site (Jänig and Häbler 2000). Several lines of evidence suggest that the skin is an important site of this interaction: 1) an increased number of adrenergic receptors have been reported in the skin of patients with hyperalgesia (Drummond et al. 1996); 2) administration of adrenergic agonists into the skin produces pain (Torebjörk et al. 1995; Tracey et al. 1995); and 3) direct stimulation of sympathetic efferents or application of NE activates cutaneous nociceptive afferents (Hu and Zhu 1989; Sato and Kumazawa 1996; Sato and Perl 1991).

In the present study, an intradermal injection of CAP was used in vivo to evoke neurogenic inflammation (Lin et al. 1999b) to examine the changes in activity of primary afferent nociceptors after CAP injection. The goal was to evaluate the possibility that this CAP-induced response is sympathetically modulated in the periphery. In addition, the possible adrenergic receptors involved in the sympathetic modulation have been determined pharmacologically by peripheral administration of -adrenergic receptor agonist and antagonists.

Preliminary data have been published in abstract form (Lin et al. 1999a; Ren et al. 2003).

	METHODS

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Animal preparation and anesthesia

Experiments were performed on adult male Sprague–Dawley rats (250–350 g). All experimental protocols were approved by the Institutional Animal Care and Use Committee and were in accordance with the guidelines of the National Institutes of Health and the International Association for the Study of Pain.

Animals were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally [ip]). The trachea and external jugular vein were cannulated for artificial respiration and anesthetic delivery, respectively. Anesthesia was then maintained by intravenous (iv) infusion of sodium pentobarbital (5–8 mg kg^–1 h^–1) in a saline solution. The level of anesthesia was monitored by frequent examination of pupillary size and responses to stimulation, absence of a flexion reflex, and stability of the level of end-tidal CO₂. Once a stable level of surgical anesthesia was reached, the animals were paralyzed with pancuronium (0.3–0.4 mg/h iv) and artificially ventilated. End-tidal CO₂ was kept between 3.5 and 4.5% by adjusting the respiratory parameters. Core body temperature was monitored by a rectal probe and maintained near 37°C by a servo-controlled heating blanket.

Electrophysiological recordings from single A- and C-primary afferent fibers

An incision was made along the posterior surface of one hindlimb from the midthigh to the ankle to expose the tibial nerve (see Fig. 2A). A warm mineral oil pool was formed over the exposed tissue. A platinum unipolar hook electrode was used for the extracellular recordings. A bipolar stimulating electrode was placed on the nerve at a distance of 20–40 mm from the recording site. The tibial nerve was carefully dissected from surrounding tissues and cut proximally. The distal cut end of the tibial nerve was then teased into small filaments with fine-tipped forceps on a small mirror-based platform under an operating microscope until single-fiber activity of afferents from a fine nerve filament could be isolated on the basis of spike amplitude and waveform and the responses to mechanical stimulation of their receptive fields recorded on a digital oscilloscope. Figure 1 illustrates how the fiber types were identified by measuring the conduction velocity (CV), and how the action potentials were recorded and then converted into analog signals or histograms that were available to be quantified by Spike-2 software. From each single unit, an evoked action potential with a fixed latency was recorded when the nerve was stimulated electrically using a suprathreshold stimulus (Fig. 1A). The CV of the recorded fiber was calculated by dividing the conduction distance by the latency of the action potential after electrical stimulation. Consistent with other studies in rats (Handwerker et al. 1991; Leem et al. 1993), the units were classified as A-, A-, or C-fibers based on conduction velocities (A, >19.9 m/s; A, 2–19.9 m/s; and C, <2 m/s). Recorded action potentials and their responses to peripheral mechanical stimuli before and after intradermal injection of 15 µl of 1% CAP were amplified and displayed on a digital oscilloscope (TDS-3012B) that allowed us to monitor the size and shape of action potential throughout the experiment to ensure that the same unit was being recorded (Fig. 1, B and C). The original signals recorded were also led to a data collection system (CED 1401+) and a personal computer to compile wavemark files using Spike-2 software by which the analog signals (nerve spikes) (see Fig. 1D and Lin et al. 2000) were processed and firing rates were counted by histograms (see Fig. 1E and Lin et al. 1999b).

View larger version (30K): [in this window] [in a new window]   FIG. 2. A: experimental setup for recording from a single unit and sites where capsaicin (CAP) was injected intradermally in the plantar skin, as well as the relationship between the injection site and receptive field of the fiber recorded. B: response of a C nociceptive fiber to mechanical stimuli after CAP was injected intradermally at a site beyond the receptive field of the fiber (square 1). C: response of a C nociceptive fiber to mechanical stimuli after CAP was injected at edge of the receptive field of the fiber recorded (square 2). D: response of a C nociceptive fiber to mechanical stimuli after CAP was injected at the center of receptive field of the fiber recorded (square 3).

View larger version (28K): [in this window] [in a new window]   FIG. 1. Traces showing the recording of afferent activity of a single nociceptive C-fiber from the distal stump of the cut tibial nerve and its responses to mechanical stimuli (von Frey filaments applied to the receptive field). A: action potential was evoked by electrical stimulation (arrow indicates stimulus artifact) to identify the fiber type by calculating the conduction velocity. B: responses of the identified fiber to mechanical stimulation with a von Frey filament. C: recorded action potentials and their responses to mechanical stimuli evoked by applying 3 von Frey filaments of increasing bending force to the receptive field. D: recorded action potentials were converted into analog signals (wavemarks) by Spike-2 software that filtered the baseline. E: recorded action potentials were converted into peristimulus time histograms by which firing rates could be counted.

Surgical sympathectomy was performed as described in our recent studies (Lin et al. 2003; Zou et al. 2002). Briefly, sympathetic ganglia and chains were removed bilaterally at the L_2–6 level through a laparotomy. Animals were given postoperative care to allow for recovery from the surgery for at least 1 wk before experiments were performed. A sham operation was done on a separate group of animals as a control for the surgical procedure, in which the same surgery as for sympathectomy was performed but the lumbar sympathetic ganglia and chains were left intact. A morphological study reported by our group in the same model shows that a successful sympathetic denervation of femoral arteries could be confirmed 7–10 days after the surgical sympathectomy was performed (Zou et al. 2002).

Peripheral administration of -adrenergic receptor agonist and antagonists

An oblique cutaneous incision was made at the groin. A fine branch of the femoral artery was exposed and isolated from surrounding connective tissue. The artery was then cannulated distally to the junction with the femoral artery by polyethylene 10 tubing connected with a 0.5-ml U-100 insulin syringe, so the drug could be administered to the periphery in the direction of blood flow, and the circulation of the operated hindpaw would not be blocked when the drug was not being injected. An agonist of -adrenoceptors, NE (0.1 µg, RBI/Sigma), and antagonists of ₁-adrenergic receptors, terazosin (15 µg, RBI/Sigma), or of ₂-adrenergic receptors, yohimbine (25 µg, RBI/Sigma), were administered intraarterially in a volume of 50 µl 5 min before CAP injection. As a control for the drugs, the vasoconstrictor vasopressin (0.1 µg, RBI/Sigma) or saline (50 µl) was administered in a different group of rats using the same procedure.

Experimental protocol

Afferent activity was presumed to include spontaneous and evoked discharges. However, in our initial experiments we found that spontaneous discharges were seen in only 7 (4 A and 3 C) of 42 fibers tested, with an average firing rate of 0.48 ± 0.21 Hz. Intradermal injection of CAP usually produced a short-lasting increase in spontaneous discharge (lasting 3–5 min, 1–5 Hz) right after injection (see Fig. 2, C and D; Lin et al. 1999a; Ren et al. 2003). Statistical analysis made during a period of 10–15 min after CAP injection shows that there was no significant increase in spontaneous activity (0.54 ± 0.19 Hz, P = 0.848) induced by CAP injection. Therefore only evoked afferent activity was analyzed in the present study. Responses of a single afferent fiber were evoked by brisk phasic mechanical stimuli, which were applied using a series of calibrated von Frey filaments having graded bending forces to the center of the receptive field of the fiber on the plantar surface of the hind paw from which the maximal response to a certain force of von Frey hair could be evoked. Because the threshold for evoking responses by mechanically stimulating peripheral afferent terminals varied with the experiment, an appropriate set of von Frey filaments (3 filaments with graded bending forces) was chosen in each experiment. The first filament had the weakest bending force that was sufficient to evoke action potentials, and 2 additional filaments were chosen with ascending graded forces. The range of bending forces applied to the receptive fields to evoke responses was between 7 and 284 mN. Each filament was applied repetitively for 10 s at a frequency of about 2 strokes/s followed by a 10-s pause before the next filament was used (Fig. 1, C and D). 15 µl of CAP, prepared in a solution of Tween 80 (7%) and saline (93%) at a low concentration of 1% (Lin et al. 1999b, 2003, 2004), was injected intradermally at the edge of the receptive field near the site of mechanical stimulation (about 5 mm away, Fig. 2A) to evoke the acute cutaneous inflammation after control responses were recorded. Changes in responses to mechanical stimuli were then tested at 15, 30, 45, and 60 min after CAP injection. The tests were extended to 2 h after CAP injection in some cases. For control purposes, vehicle (Tween 80 and saline) was given in another group of rats in the same volume as the CAP solution.

Changes in activity of single A-, A-, and C-primary afferent fibers after CAP injection were recorded and compared between sympathectomized rats and sympathetically intact rats, including the sham-sympathectomized group, to determine whether the sensitization of primary afferents that followed acute cutaneous tissue inflammation was dependent on the presence of sympathetic efferents.

To examine whether NE was released from sympathetic efferents during the CAP-induced sensitization of primary afferent nociceptors to mechanical stimuli and to determine what types of peripheral adrenergic receptor subtypes were involved, the following pharmacological manipulations were performed. 1) The effects of activation of peripheral -adrenergic receptors under sympathectomized conditions were tested. In one group of sympathectomized rats, NE (0.1 µg) was injected intraarterially 5 min before CAP was injected intradermally. Changes in responses to mechanical stimuli after CAP injection were recorded for 1.0–2.0 h. As controls, vasopressin (0.1 µg) (Lin et al. 2003) or saline was given intraarterially before CAP injection in different groups of sympathectomized rats. 2) Observations were made of the effects of blockade of the peripheral -adrenergic receptors on the CAP-evoked sensitization of primary afferents. In one group of sympathetically intact rats, an ₁- (terazosin, 15 µg) and, in another group, an ₂-adrenergic (yohimbine, 20 µg) receptor antagonist was administered intraarterially in a volume of 50 µl 5 min before CAP injection. Changes in responses to CAP injection were then recorded. Control experiments were performed by intraarterially injecting saline in the same volume in a separate group of sympathetically intact rats.

Statistical analysis

Recorded fiber activity was analyzed off-line from peristimulus time histograms to obtain the average rate of evoked discharges (Fig. 1E). All responses evoked by stimulation using 3 graded von Frey filaments were calculated by subtracting the background discharges during a given period of time from the total number of action potentials that occurred during each stimulus to produce total response values. The responses to CAP injection were expressed as a percentage of baseline, with baseline set at 100%. Statistical significance was tested using ANOVA with repeated measures and differences across time were assessed with post hoc t-test. A grouped t-test was used to compare the difference in responses between groups having different treatments. P < 0.05 was considered significant. Values were expressed as means ± SE.

	RESULTS

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Because an intradermal injection of CAP adjacent to the site within the receptive field where mechanical stimuli are applied results in desensitization of nociceptors (Baumann et al. 1991; LaMotte et al. 1992), preliminary experiments were first done to search for the site where CAP injection produced sensitization of nociceptors within or near the receptive field. Figure 2 shows that if CAP was injected intradermally beyond the receptive field of the fiber recorded, no changes in the sensitivity of the afferent fiber were seen (Fig. 2B). When CAP was injected intradermally at the edge of the receptive field, there was a short-lasting increase in spontaneous activity followed by an obvious increase in response to mechanical stimuli (development of sensitization, Fig. 2C). However, if CAP was injected at the center of the receptive field, a decrease in response to mechanical stimuli took place at 10–15 min after CAP injection (development of desensitization Fig. 2D). Therefore afferent nociceptive fibers could be sensitized by CAP injection if the injection spot was neither close to the center of the receptive field nor distant to the receptive field.

A total of 210 primary A (n = 148) and C (n = 62) afferent fibers were isolated from the tibial nerves of both paws of 122 rats and studied for the effects of intradermal injection of CAP and sympathetic modulation of the CAP-induced sensitization. Of these fibers, the mechanical thresholds of 181 fibers (86.2%, 124 A and 57 C) were tested without CAP injection, and the remaining 29 fibers (13.8%, 24 A and 5 C) were tested about 2 h after CAP had been injected intradermally. The mechanical thresholds of most fibers (179 of 181, 98.9%) that were without CAP injection ranged from 14 to 118 mN. According to the study by Leem et al. (1993), these fibers were considered to be nociceptive units. In those fibers (n = 29) that were tested about 2 h after CAP injection, 24 units (82.8%) had a low mechanical threshold (<14 mN), and were initially thought to be mechano-sensitive (non-nociceptive) fibers. However, because these fibers were tested after CAP had been injected into the same paw, we consider that these fibers were sensitized by the first injection of CAP given before they were recorded. More important, these fibers responded to a later CAP injection. Because low threshold mechano-sensitive fibers are not activated by CAP injection (Fitzgerald 1983; Kenins 1982; Szolcsanyi et al. 1988), we presume that they should be considered as nociceptors whose thresholds had been lowered by the first injection of CAP. These fibers have been used for the following experiments: 1) tests for responses to CAP injection under sympathetically intact conditions (sham-sympathectomy) (n = 14); 2) tests for the effects of NE pretreatment on the CAP-induced sensitization (n = 9); 3) saline pretreatment as controls for terazosin and yohimbine administration (n = 6). Statistical analysis has shown that results were not changed significantly if data obtained from these fibers were excluded.

Changes in responses of primary afferents to mechanical stimuli after CAP injection and effects of sympathectomy

Experiments were initially started to examine the effects of sympathectomy on the CAP-evoked sensitization of primary afferent nociceptive fibers by comparing 2 groups of rats: sham-sympathectomized and sympathectomized rats. Responses of single afferent fibers to graded mechanical stimuli with von Frey hairs increased in a graded manner as increasing forces were applied to the receptive field (Figs. 3–5). Intradermal injection of CAP produced an immediate increase in spontaneous discharge for 3–5 min (data not shown), followed by enhanced responses to the von Frey filaments. This enhancement of mechanically evoked responses after CAP injection could be seen both in A-fibers and in C-fibers, but not in A-fibers in the sham-sympathectomized rats. The left columns of Figs. 3 and 4 are examples of the CAP-evoked increase in responses of an A- and a C-fiber to mechanical stimuli in sham-sympathectomized rats. CAP injection caused an increase in evoked activity, and the peak increase was at 15–30 min after CAP injection. The enhancement of the responses lasted about 1 h. The response pattern was stimulus dependent before and after CAP injection in both groups (see Fig. 5). A total of 8 A-, 29 A-, and 12 C-fibers were recorded to examine the effect of CAP injection on responses of primary afferents to mechanical stimuli in the sham-sympathectomized rats. Fibers were considered to show increased responses when the changes in responses to the same strength of mechanical stimuli after CAP injection were more than 20% of the control value; this value of 20% was based on the consideration that there might be a variation in mechanical responses attributed to sensitization of the skin by repeated mechanical stimulation because a similar study has shown that there was a <20% variation in responses of dorsal horn neurons to repeated mechanical stimuli (Dougherty et al. 1992). According to this criterion, the responses to mechanical stimuli of 23 A-fibers (79.3%) and 11 C-fibers (91.7%), but no A-fibers, increased after CAP injection (Fig. 6, A and B). The total responses of these A- and C-fibers evoked by 3 von Frey hairs before CAP injection ranged from 1.4 to 10.3 and 1.6 to 4.9 Hz, respectively. After CAP injection, responses were 2.1 to 19.7 and 2.3 to 9.5 Hz, respectively. In contrast, the same dose of CAP produced only a slight increase or no change in the responses of both A- and C-fibers after sympathetic efferents were removed surgically. The total evoked responses of A- and C-fibers before CAP injection ranged from 3.4 to 9.1 and 2.4 to 4.6 Hz, respectively. After CAP injection, responses were 3.5 to 9.7 and 2.3 to 4.8 Hz, respectively. The right panels of Figs. 3 and 4 are examples of fiber recordings showing that no obvious increases in responses to mechanical stimuli were seen after CAP injection both in A- and C-fibers after sympathectomy.

View larger version (35K): [in this window] [in a new window]   FIG. 3. Recorded nerve impulses show responses of single A-fibers in the distal stumps of cut tibial nerves to mechanical stimuli after intradermal injection of CAP into the plantar surface of the hind paw in a sham-sympathectomized rat (left) and in a sympathectomized rat (right). Note that the enhanced CAP-induced responses were nearly completely inhibited after sympathetic efferents were surgically removed. Each stimulus consisted of 3 von Frey applications at about 2/s for 10 s, after which there was a 10-s pause before the next filament was used. Horizontal lines above the top rows of histograms indicate times of application of von Frey hairs. Bending forces are shown above the horizontal lines.

View larger version (26K): [in this window] [in a new window]   FIG. 5. Graphs showing the stimulus–response relations of afferent activity of the A (n = 56, A) and C (n = 20, B) primary afferent fibers in sham-sympathectomized and sympathectomized rats. There was a graded increase in responses of the A- and C-primary afferent fibers to graded mechanical stimuli by applying a graded series of von Frey hairs.

View larger version (31K): [in this window] [in a new window]   FIG. 4. Recorded nerve impulses show responses of single C-fibers of the cut tibial nerves to mechanical stimuli after intradermal injection of CAP in sham-sympathectomized rats (left) and in sympathectomized rats (right). Arrangement of the figure is the same as Fig. 3.

View larger version (38K): [in this window] [in a new window]   FIG. 6. A and B: summary of the proportion of enhanced responses of A- and C-primary afferent fibers to mechanical stimuli induced by intradermal injection of CAP under sham-sympathectomized conditions. Enhanced responses to mechanical stimuli induced by CAP injection were seen in 79.3% of A-fibers (A) and 91.7% of C-fibers (B). C and D: grouped data summarize the mean effects of intradermal injection of CAP on responses of single A- and C-fibers to mechanical stimuli. Enhanced responses to mechanical stimuli after CAP injection were seen in A-fibers and in C-fibers, but not in A-fibers in sympathetically intact rats. However, the enhanced responses induced by CAP injection were nearly completely eliminated under sympathectomized conditions. Baseline level (before CAP injection) was set as 100%. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the baseline level in the same group.

View this table: [in this window] [in a new window]   TABLE 1. Reduction in enhanced responses of A- and C-fibers to mechanical stimuli induced by intradermal injection of CAP after sympathectomy (SYMP) and the effects of local injections of norepinephrine

Effects of peripheral administration of -adrenergic receptor agonist under sympathectomized conditions

In sympathectomized rats, we further examined whether activation of peripheral -adrenergic receptors could affect the CAP-evoked sensitization of A- and C-primary nociceptors. NE, a general -adrenergic receptor agonist, was injected intraarterially 5 min before intradermal injection of CAP in sympathectomized rats. Vasopressin or saline was injected intraarterially 5 min before CAP in different groups of sympathectomized rats as controls. Figure 7 shows examples of fiber recordings showing that the CAP-evoked increases in responses of A- and C-fibers to mechanical stimuli were restored when NE was given before CAP under sympathectomized conditions. Local injection of NE slightly enhanced the responses of A- (105.38 ± 2.15, P = 0.016, compared with baseline level) and C-fibers (109.81 ± 3.39, P = 0.012) to mechanical stimuli in sympathectomized rats before CAP injection. However, the presence of NE restored and prolonged significantly the CAP-evoked enhancement of responses to mechanical stimuli of A- and C-fibers. Such an effect was more obvious in C-fibers; the enhancement could last 2 h (189.50 ± 14.9%) after CAP injection (Fig. 8).

View larger version (32K): [in this window] [in a new window]   FIG. 7. Responses of a single A- (left) and a C-fiber (right) to mechanical stimuli after CAP injection and effect of activation of peripheral -adrenoceptors by intraarterial injection of norepinephrine (NE) before CAP injection under sympathectomized conditions. Note that the presence of NE could restore or even increase the enhancement of A- and C-fibers induced by CAP injection under sympathectomized conditions.

View larger version (27K): [in this window] [in a new window]   FIG. 8. Grouped data summarize the effects of peripheral administration of NE on responses of single A- (A) and C-fibers (B) on CAP-induced enhanced responses under sympathectomized conditions. Presence of NE could significantly restore and prolong the CAP-induced enhancement of response both in A- and in C-fibers. Enhancement of the responses was stronger and lasted more than 2 h in C-fibers. Injection of vasopressin before CAP injection was performed in a separate group of sympathectomized rats for control purposes. No enhancement of responses in either A- or C-fibers was seen in the vasopressin-treated group. *P < 0.05 and **P < 0.01 indicate significant differences from saline-treated group at the same time point.

Effects of blockade of peripheral -adrenergic receptors on the CAP-evoked enhancement of responses to mechanical stimuli under sympathetically intact conditions

Our initial observations have shown that removal of postganglionic sympathetic efferents significantly alleviated the sensitization of both A- and C-nociceptive fibers induced by CAP injection. There was a significant difference in mechanically evoked responses after CAP injection between sham-sympathectomized and sympathectomized rats. Sham-sympathectomized animals are believed to be sympathetically intact (see Zou et al. 2002), and statistical analysis using the grouped t-test showed that there was no significant difference in the CAP-evoked enhancement of responses to mechanical stimuli between sham-sympathectomized and sympathetically intact saline pretreated groups at any time points after CAP injection (A-fiber groups: P values of 0.926, 0.802, and 0.905 at 15, 30, and 60 min, respectively; C-fiber groups: P values of 0.556, 0.857, and 0.994 at 15, 30, and 60 min, respectively). Therefore we used sympathetically intact rats to examine further whether blockade of ₁- or ₂-adrenergic receptors by pretreatment of the paw with terazosin or yohimbine affected the CAP-induced sensitization of primary A- and C-afferent fibers. It was shown in the saline-treated group that CAP injection produced an increase in responses of A-fibers (peak increase 137.1 ± 6.4%, P < 0.001 compared with baseline level) and C-fibers (145.8 ± 11.3%, P = 0.001) to mechanical stimuli (Fig. 9, Table 2).

View larger version (28K): [in this window] [in a new window]   FIG. 9. Grouped data summarize the effects of blockade of peripheral 1- or 2-adrenergic receptors on the CAP-induced enhancement of responses of A- and C-primary afferent fibers to mechanical stimuli under sympathetically intact conditions. A: pretreatment with terazosin, an 1-adrenergic receptor antagonist, or yohimbine, an 2-adrenergic receptor antagonist, slightly reduced the CAP-induced enhancement of responses of A-fibers to mechanical stimuli. B: terazosin administration completely blocked the sensitization of C-fibers induced by CAP injection, whereas blockade of 2-adrenergic receptors with yohimbine only slightly reduced the enhanced responses of C-fiber induced by CAP injection without statistical significance. *P < 0.05 and ***P < 0.001 compared with saline-treated group at the same time point.

View this table: [in this window] [in a new window]   TABLE 2. Effects of blockage of peripheral -adrenergic receptors on the CAP-evoked sensitization of primary afferent fibers in sympathetically intact rats

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
A- and C-nociceptive fibers are involved in the development of CAP-induced primary afferent sensitization

The present study has shown that intradermal injection of a solution containing a low concentration of CAP resulted in an immediate short-lasting increase in spontaneous discharge of A- and C-, but not A-, primary afferent nociceptive fibers, which was followed by enhanced responses to mechanical stimuli for 1 h. This phenomenon was seen only when CAP was injected intradermally in a spot that was located at the edge of the receptive field of the fiber tested. If the injection was at the center of the receptive field, CAP produced desensitization of nociceptive fibers. This result is consistent with the finding by LaMotte et al. (1992) that an intradermal CAP injection in the center of the receptive field led to desensitization. However, CAP injection made remote from the testing spot could lead to sensitization of nociceptors. In this situation, the concentration of CAP that reached the nociceptive terminals was presumed to be low because the concentration of CAP decreases gradually with its spread over distance. In addition, the observation that CAP injection produced an immediately short-lasting increase in spontaneous discharge (3–5 min) was consistent with the human studies in which intradermal injection of CAP causes an ongoing pain and ongoing discharge of primary afferent nociceptors, as well as hypersensitivity to thermal and mechanical stimuli that were applied to the receptive field (primary hyperalgesia), even though a desensitization to mechanical stimuli was seen at the site of the CAP injection (Culf et al. 1989; LaMotte et al. 1992).

Because neurogenic inflammation can be evoked by intradermal injection of CAP, the mechanisms underlying sensitization of primary afferent nociceptors should include 1) a direct action of CAP by which TRPV₁ receptors are activated to cause Ca²⁺ influx that produces membrane depolarization (Bean and Szolsanyi 1990); 2) CAP-induced Ca²⁺ influx into nerve terminals through TRPV₁ receptors and voltage-dependent Ca²⁺ channels causes the exocytosis of inflammatory neuropeptides and excitatory amino acids, such as SP, CGRP, and glutamate (deGroot et al. 2000; Go and Yaksh 1987; Szallasi and Blumberg 1999; Zhang et al. 1997). These substances released into the periphery can produce hyperalgesia, sensitization of primary afferent nociceptors, and neurogenic inflammation (Beirith et al. 2002; Du et al. 2001; Pedersen-Bjergaard et al. 1989; Zhou et al. 1996); 3) Ca²⁺ influx also triggers several second-messenger cascades by which the phosphorylation of receptor proteins, including TRPV₁, is catalyzed (Aley et al. 2001; Gold et al. 1998; Jung et al. 2004; Khasar et al. 1999). This positive feedback pathway would then enhance the activity of TRPV₁ receptors by sensitizing the receptors. In addition, our studies on dorsal root reflex-mediated inflammation have indicated that there is release of inflammatory peptides from the primary afferent nociceptors after CAP injection, and these peptides contribute to the induction and maintenance of neurogenic inflammation (Lin 2003). TRPV₁ receptors are located on the small-diameter neurons of dorsal root ganglia with unmyelinated (C) and small myelinated (A) primary afferent nociceptive axons and convey sensitivity to CAP, noxious heat, and acid (Caterina et al. 1997; Ma et al. 2001; Tohda et al. 2001; Tominaga et al. 1998). Therefore neurogenic inflammation is presumed to be initiated by sensitization of TRPV₁ receptors, which would subsequently trigger the above mechanisms to sensitize primary afferent nociceptors.

Sympathetic efferents modulate the sensitization of primary afferent nociceptors induced by intradermal injection of CAP

Several lines of evidence suggest that sympathetic efferents play a role in neurogenic inflammatory responses in damaged or inflamed tissue by interaction with primary afferent terminals (Jänig et al. 1996; Sato and Kumazawa 1996; Sato and Perl 1991). 1) Sympathectomy and sympathetic block are effective in reducing pain behaviors in some neuropathic and inflammatory models (Kim and Chung 1991; Moon et al. 1999; Xie et al. 1995a). 2) Development of inflammation in some inflammatory models can be prevented or alleviated by sympathectomy (Kim and Chung 1991; Xie et al. 1995a). 3) In an acute cutaneous inflammatory model induced by CAP, NE release can produce a prolonged decrease in heat pain threshold at the point where NE was released (Drummond 1995, 1998). 4) In behavioral studies both in humans and rats, hyperalgesia and ongoing pain induced by CAP injection was sympathetically dependent (Kinnman and Levine 1995; Kinnman et al. 1997). 5) Our recent study in rats suggests that CAP-induced neurogenic inflammation is modulated by sympathetic efferents (Lin et al. 2003). Furthermore, we found in the current study that sensitization of primary afferent A- and C-fibers induced by CAP injection can be dramatically reduced by sympathectomy. The results clearly reveal that the presence of sympathetic efferents is essential for the sensitization of primary afferent nociceptors in rats to mechanical stimuli after intradermal injection of CAP. However, human studies on the sympathetic modulation in some pain models, especially the CAP-evoked pain, have been inconsistent (Kinnman et al. 1997; Liu et al. 1996; Schwartzman and McLellan 1987; Torebjörk et al. 1995; however, cf. Baron et al. 1999). One of the major reasons could be that manipulations activating and inhibiting the sympathetic nervous system were different among these studies. It still remains unclear whether a tonic or enhanced sympathetic outflow is critical for the pathological pain, including inflammatory pain.

A further observation from our present study is that activation of -receptors could restore the responses of primary afferent fibers to mechanical stimuli after CAP injection under sympathectomized conditions. Others have reported that NE and sympathetic stimulation can increase the sensitization of primary afferent C nociceptors in inflamed skin (Sato and Kumazawa 1996; Sato and Perl 1991). These data indicate that NE is released from the sympathetic efferent fibers when peripheral tissue is injured, which may modulate the excitability of primary afferent nociceptors, possibly by an interaction between sympathetic efferents and primary afferents in the periphery (Jänig and Häbler 2000). To exclude the possibility that the increase in responses to mechanical stimuli after NE was caused by vasoconstriction, a control experiment was done in which the hindpaw was pretreated with vasopressin at a dose that has been shown to produce vasoconstriction in our previous study (Lin et al. 2003). Vasoconstriction induced by vasopressin did not produce the same effect as NE did. This result was consistent with our recent study (Lin et al. 2003) that vasopressin did not restore the vasodilation induced by CAP injection in sympathectomized rats. We propose that the mechanism of interaction involves, at least, the release of NE and probably nonadrenergic agents, such as ATP and neuropeptide Y (Lin et al. 2004), from the terminals of sympathetic efferents after tissue injury, and that one or more of these substances result in the sensitization of A- and/or C-fibers.

To investigate further what types of -adrenergic receptors are involved, we have tested whether blockade of either ₁- or ₂-adrenergic receptors could affect the sensitization of primary afferent terminals induced by CAP injection. The results show that the CAP-induced sensitization of primary afferent nociceptors could be significantly reduced predominantly by blockade of peripheral ₁-adrenergic receptors, even though a slight reduction in the enhanced responses was also seen after ₂-receptors were blocked. This blocking effect was mainly seen in the responses of unmyelinated C-fibers. One possible explanation of this weak action on the enhanced responses of small myelinated A-fibers after blockade of -adrenoceptors could be that an enhanced mechanical response induced by CAP injection was seen in only around 79% of A-fibers, which resulted in a smaller grouped response (see Fig. 6). We presume that this consideration might also apply to the groups of A-fibers shown in Fig. 9A. Thus a presumed blocking effect on enhanced responses of A-fibers by -receptor antagonists could be counteracted by such a smaller grouped response. A recent study by our laboratory shows that local activation of ₁-adrenergic receptors, but not ₂-adrenergic receptors, can restore the spread of flare induced by CAP injection under sympathectomized conditions, and the same study also demonstrated that blockade of peripheral ₁-adrenergic receptors with terazosin in sympathetically intact rats dramatically reduced the spread of flare induced by CAP injection (Lin et al. 2003). So far, a variety of observations about the subtype of -adrenergic receptors involved in sympathetic modulation of pathological pain transmission have been reported in clinical studies and also in experimental studies, mostly on neuropathic pain models. In contrast, our previous and present studies have been done using the CAP-induced neurogenic inflammatory pain model. There is evidence that painful neuropathy induced by nerve injury produces neurogenic inflammation that may exacerbate the neuropathic pain (Daemen et al. 1998; Yonehara and Yoshimura 2001). Thus neurogenic inflammatory and neuropathic pains are presumed to share some of the same mechanisms. In a behavioral study in rats, hyperalgesia induced by CAP injection was mediated by an ₁-adrenergic receptor (Kinnman and Levine 1995). Lee et al. (1999) demonstrated that the subtype of -adrenergic receptor mediating the reduction of mechanical hypersensitivity is the ₁-adrenergic, not the ₂-adrenergic, receptor in rats. Their group has also shown an increased expression of the _1b-adrenergic receptor subtype in a neuropathic pain model (Xie et al. 2001). Clinically sympathetically maintained pain is suggested to be mediated by ₁-adrenoceptors (Davis et al. 1991). In addition, some other data suggest that ₂- or both ₁- and ₂-receptors are involved in various types of neuropathic pain models (Chen et al. 1996; Hord et al. 2001; Sato and Perl 1991; Xie et al. 1995b). One possible explanation could be that different -adrenergic receptor subtypes might participate in mediation of different types of neuropathic pain. It has been reported that activation of ₂-receptors produced antinociception in neuropathic animals (Wei et al. 2002). However, our study suggests that the CAP-induced sensitization of primary afferent nociceptors that is involved in the development of neurogenic inflammatory pain is mediated mainly by ₁-adrenoceptors.

In summary, our study suggests that the presence of sympathetic efferents is essential for the CAP-induced sensitization of A- and C-primary afferent fibers to mechanical stimuli. The sensitization of A- and C-fibers contributes importantly to the acute cutaneous neurogenic inflammation produced by intradermal injection of CAP. ₁-Adrenergic receptors located mainly on C-fibers play an important role in modulation of neurogenic inflammation.

	GRANTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-40723 to Q. Lin.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
The authors thank Dr. W. D. Willis for helpful comments and advice on the preparation of this manuscript, Drs. J. H. Du and J. L. Zhou for technical help with electrophysiological recordings, and Dr. J. Freeman for assistance with power analysis.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Address for reprint requests and other correspondence: Q. Lin, Department of Neuroscience and Cell Biology, The University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1069 (E-mail: qilin{at}utmb.edu)

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