| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
The Journal of Neurophysiology Vol. 81 No. 2 February 1999, pp. 455-466
Copyright ©1999 by the American Physiological Society
-Adrenergic Sensitivity Following L6 Spinal Nerve Ligation in Monkey
Johns Hopkins University, School of Medicine, Baltimore, Maryland 21218
 |
ABSTRACT |
|---|
 Abstract
 Introduction
Methods
Results
Discussion
References
|
|---|
Ali, Z., M. Ringkamp, T. V. Hartke, H. F. Chien, N. A. Flavahan, J. N. Campbell, and R. A. Meyer. Uninjured cutaneous C-fiber nociceptors develop spontaneous activity and 
-adrenergic sensitivity following L6 spinal nerve ligation in the monkey. J. Neurophysiol. 81: 455-466, 1999. We investigated whether uninjured cutaneous C-fiber nociceptors in primates develop abnormal responses after partial denervation of the skin. Partial denervation was induced by tightly ligating spinal nerve L6 that innervates the dorsum of the foot. Using an in vitro skin-nerve preparation, we recorded from uninjured single afferent nerve fibers in the superficial peroneal nerve. Recordings were made from 32 C-fiber nociceptors 2-3 wk after ligation and from 29 C-fiber nociceptors in control animals. Phenylephrine, a selective 1-adrenergic agonist, and UK14304 (UK), a selective 2-adrenergic agonist, were applied to the receptive field for 5 min in increasing concentrations from 0.1 to 100 µM. Nociceptors from in vitro control experiments were not significantly different from nociceptors recorded by us previously in in vivo experiments. In comparison to in vitro control animals, the afferents found in lesioned animals had 1) a significantly higher incidence of spontaneous activity, 2) a significantly higher incidence of response to phenylephrine, and 3) a higher incidence of response to UK. In lesioned animals, the peak response to phenylephrine was significantly greater than to UK, and the mechanical threshold of phenylephrine-sensitive afferents was significantly lower than for phenylephrine-insensitive afferents. Staining with protein gene product 9.5 revealed an ~55% reduction in the number of unmyelinated terminals in the epidermis of the lesioned limb compared with the contralateral limb. Thus uninjured cutaneous C-fiber nociceptors that innervate skin partially denervated by ligation of a spinal nerve acquire two abnormal properties: spontaneous activity and -adrenergic sensitivity. These abnormalities in nociceptor function may contribute to neuropathic pain.
Peripheral nerve injury often leads to a neuropathic pain condition characterized by ongoing pain and hyperalgesia. In some neuropathic pain patients, an anesthetic block of the sympathetic ganglia leads to a decrease in both ongoing pain and hyperalgesia. Doupe et al. (1944) A nerve injury was induced in monkeys by tight ligation of the L6 spinal nerve, which innervates the dorsum of the foot (Sherrington 1893). This procedure was chosen to induce a partial denervation in the skin area innervated by the superficial peroneal nerve from which final recordings were made. The peroneal nerve arises from spinal nerves L5-L7 (Howell and Strauss 1971 Tight ligation of the left L6 spinal nerve
Monkeys (Macaca fascicularis, 4.5-13.5 kg) were initially sedated by (10 mg/kg im) ketamine and maintained under halothane anesthesia (0.5-1.5% halothane-50% N20) and artificially ventilated (end tidal pCO2 maintained at 32-40 Torr). Adequacy of anesthesia was checked by ensuring the absence of motor responses to noxious mechanical stimuli and by monitoring heart rate with an electrocardiogram. Hydration was maintained by intravenous infusion of 5% dextrose in saline (4-6 ml·kg Harvesting of skin-nerve preparation
To avoid the confounding effects of adrenergic agents on perfusion, the electrophysiological studies were carried out in an in vitro preparation, which also allows better control of drug concentrations. The superficial peroneal nerve and its innervation territory on the top of the foot were harvested in terminal experiments. The monkeys were initially sedated with (10 mg/kg) ketamine and maintained under pentobarbital sodium (continuous intravenous infusion, 6 mg·kg
Control animals
In vitro control experiments were performed on the skin of uninjured monkeys. The protocols were the same as those for the lesioned animals except that approximately one-half of the data from control monkeys was obtained from a preparation of the superficial radial nerve that innervates the dorsum of the hand.
In vitro perfusion and recording chamber
The in vitro chamber was a scaled up version of a chamber previously developed for the rat skin (Reeh et al. 1986). A schematic of the in vitro perfusion and recording chamber is shown in Fig. 1. The skin was placed corium side up into the perfusion chamber and was continuously perfused with fresh, gassed, synthetic interstitial fluid. The synthetic interstitial fluid (SIF) consisted of (in mM) 107.7 NaCl, 3.48 KCl, 0.69 MgSO4, 26.2 NaHCO3, 1.67 NaH2PO4, 1.53 CaCl2, 9.64 sodium gluconate, 5.5 glucose, and 7.6 sucrose (Bretag 1969 Electrophysiological and pharmacological studies
The mechanical receptive fields of single nociceptive afferents in the skin were identified. Nociceptive afferents were initially identified by their preferential and slowly adapting response to firm prodding of the skin. The mechanical threshold for the nociceptive afferents was determined by applying von Frey filaments to the most sensitive part of the receptive field. The smallest von Frey probe that produced a response in Data collection and analysis
The signal from the recording electrode was differentially amplified, filtered, and displayed on an oscilloscope. In addition, the amplified and filtered signal was recorded using a computer-based data acquisition system (Microstar Laboratories data acquisition board, DAP 3200e315). A customized data acquisition and analysis software (DAPSYS) (Turnquist 1995 Criteria for drug responders
The total number of action potentials generated during the 5-min incubation of each dose of a drug was determined by a posthoc analysis of each fiber. A fiber was considered to have given a positive response during drug application only if 1) more than five action potentials above predrug activity were evoked and 2) the magnitude of the response during drug application was Protein gene product 9.5 staining and counting of epidermal nerve fibers
Skin punches (3-mm diam) were obtained from the dorsal surfaces of both feet (lesioned side as well as nonlesioned side) of L6 lesioned monkeys after completion of the electrophysiological experiments. The skin punches were fixed with a cold mixture of 2% paraformaldehyde, lysine, and sodium periodate (McClean and Nakane 1974) and kept in 0.05 M tris(hydroxymethyl)amionomethane-buffered saline (TBS) until sectioning. Before sectioning, the skin was cryoprotected in 10% sucrose in TBS and snap-frozen in melting isopentane that was cooled with liquid nitrogen.
Statistical analysis
A paired t-test was used to compare the number of epidermal nerve fibers on ligated side versus the contralateral side. Conduction velocities and von Frey thresholds were compared between different groups of nociceptors (e.g., lesioned vs. control) using the t-test and Mann-Whitney U tests, respectively. Receptive field areas were compared using a t-test. A The left L6 spinal nerve was tightly ligated in 11 monkeys. An in vitro skin-nerve preparation was used to record from 32 C-fiber nociceptors in the superficial peroneal nerve. Twenty-nine C- fiber nociceptors were also studied from nine nonlesioned (control) monkeys. In nine lesioned monkeys, skin biopsy punches were obtained from the dorsal skin of both feet (ipsilateral and contralateral to the ligation) after completion of the electrophysiological experiments, and the number of nerve fibers in the epidermis stained with protein gene product 9.5 (PGP9.5) was counted.
Epidermal nerve fiber counts
In skin from the right, nonlesioned foot, PGP9.5-stained fibers could be visualized in both epidermal as well as sub-epidermal tissue. The morphological appearance of PGP9.5 labeled fibers in monkey skin (Fig. 2, A and B) was similar to what has previously been described for human skin (McCarthy et al. 1995
Comparison of nociceptors from in vitro and in vivo experiments
The properties of 299 C-fiber nociceptors recorded in vivo from nerves that innervate the hairy skin were obtained from a retrospective analysis of previous data from our laboratory. As indicated in Table 1, most of the properties of the afferents recorded during the control and lesion in vitro experiments were not significantly different from the control in vivo data. However, the conduction velocities of the in vitro afferents (both from control and lesioned monkeys) were significantly lower than those found in vivo (P < 0.01, t-test).
Properties of nociceptors from lesioned and control animals
There was no significant difference between the conduction velocities or von Frey thresholds of C-fibers recorded from L6 lesioned monkeys and C-fibers recorded from nonlesioned control monkeys (Table 1). In addition, the receptive field sizes were not significantly different, which would suggest that sprouting was not substantial or that the sprouts were not mechanically sensitive. The incidence of cold sensitivity was similar in control and lesioned animals. Most fibers tested with heat responded.
Response to the selective LESIONED ANIMALS.
Phenylephrine evoked a response in 9 of 17 C-fiber nociceptors tested in L6 nerve lesioned monkeys. There was a large variability in the magnitude and duration of the phenylephrine evoked response between the different fibers. The responses of two fibers are shown in Fig. 4. For fiber 1 (Fig. 4, A and C) phenylephrine evoked a vigorous response that increased in a dose dependent manner. For fiber 2 (Fig. 4, B and C) phenylephrine evoked a moderate response that did not increase in a dose related manner.
TACHYPHYLAXIS TO REPEATED PHENYLEPHRINE.
Four of nine C-fibers that responded to phenylephrine were subsequently tested with a second application of phenylephrine. The second application elicited a much weaker response (Fig. 6), indicating a marked tachyphylaxis in the phenylephrine-evoked response. This tachyphylaxis prohibited a study of the effects of selective antagonists.
CONTROL ANIMALS.
The incidence of phenylephrine evoked activity was significantly lower in C-fibers of control monkeys (2 of 16) when compared with the C-fibers of L6 nerve lesioned monkeys (9 of 17; P < 0.02, Response to the selective LESIONED ANIMALS.
The
CONTROL ANIMALS.
In control animals only 1 of 14 C-fibers responded to UK14304. Interestingly, this fiber also responded to phenylephrine. There was a tendency of an increased incidence of UK14304 responses in L6 spinal nerve lesioned monkeys compared with control animals (P = 0.08, Comparison of the response to phenylephrine and to UK14304 in lesioned animals
Fifteen fibers were tested with both phenylephrine and UK14304 (Fig. 8). Of these, eight fibers responded to phenylephrine and five responded to UK14304. Four fibers that responded to phenylephrine did not respond to UK14304, whereas only one fiber that responded to UK14304 did not respond to phenylephrine. The response was greater during phenylephrine administration in three of four fibers that responded to both phenylephrine and UK14304. Two of these fibers are illustrated in Fig. 4. The median peak response to phenylephrine [35 (12-133) imp/5 min] was significantly greater than the median peak response to UK14304 [7 (6-13) imp/5 min; P < 0.05, Mann-Whitney U]. In control skin, only 2 of 13 fibers tested with both agonists responded (Fig. 8); one responded to both phenylephrine and UK14304, and the other to just phenylephrine.
Incidence of adrenergic sensitivity across animals
Successful adrenergic studies were performed on a total of 20 afferents from eight lesioned animals. In three animals, none of the 7 fibers tested responded to adrenergic agents, whereas in the remaining five animals, 11 of 13 fibers tested responded to one or both agonists. This suggests that adrenergic sensitivity may vary between animals just as the occurrence of sympathetically maintained pain varies across patients with neuropathic pain.
We demonstrate that spontaneous activity and adrenergic sensitivity develop in uninjured cutaneous C-fiber nociceptors in primate skin following ligation of the L6 spinal nerve. More fibers responded to the selective Comparison of in vivo and in vitro data from normal skin
In vitro recordings were obtained from cutaneous afferents in monkey skin with the use of a modification of the technique developed for rat skin by Reeh (1986) Cutaneous nociceptors exhibit The incidence of adrenergic sensitivity was significantly higher in nociceptive afferents from the L6 lesioned monkeys compared with nociceptive afferents from normal animals. Our data demonstrate that cutaneous nociceptors develop Increased spontaneous activity in lesioned animals
The incidence of spontaneous activity was significantly higher in nociceptive afferents from the L6 lesioned monkeys compared with nociceptive afferents from normal animals. Because we recorded centripetally conducted action potentials, this spontaneous activity must originate along the course of the peripheral axon or at the peripheral cutaneous terminal. Koltzenburg et al. (1994) Adrenergic activation of C-fibers in normal skin
Most previous studies have found little evidence for sympathetic activation of nociceptors in normal skin (e.g., Barasi and Lynn 1986 Role of peripheral generators in neuropathic pain
The pain generator in human cases of nerve injury is generally thought to arise from spontaneous activity at the nerve injury site or perhaps more proximally from the dorsal root ganglion cells of fibers that have undergone axotomy. However, several lines of evidence suggest that pain may also arise from peripheral generators that are distal to the site of injury. 1) Spontaneous activity and catechol sensitivity develop in cutaneous nociceptors following nerve injury (this study; Koltzenburg et al. 1994
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
and Nathan (1947) suggested that this sympathetically maintained pain (SMP) is because of a functional connection between the sensory and sympathetic nervous system such that primary afferent fibers become responsive to ongoing activity in the sympathetic nervous system.
; Häbler et al. 1987; Korenman and Devor 1981; Scadding 1981) and the dorsal root ganglion supplying the injury site (Abulla and Smith 1997; Chung et al. 1993; Devor et al. 1994a; McLachlan et al. 1993; Michaelis et al. 1996; Petersen et al. 1996; Xie et al. 1995; Zhang et al. 1997). Several lines of evidence suggest that the skin may also be an important site. 1) An increase in the number of adrenergic receptors has been reported in the skin of patients with neuropathic pain (Drummond et al. 1996). 2) Administration of adrenergic agonists to the skin of SMP patients produces pain (Davis et al. 1991; Torebjörk et al. 1995; Wallin et al. 1976). 3) In animal models of neuropathic pain, cutaneous nociceptive afferent fibers are activated following stimulation of sympathetic efferent fibers and following application of adrenergic agonists to their receptive fields (Koltzenburg et al. 1994; Sato and Perl 1991).
; Raja et al. 1991), this sympathetic-sensory coupling is likely -adrenergically mediated. Whereas, based on clinical studies, the 1 adrenoceptor appears to be the culprit (e.g., Ali et al. 1996; Campbell et al.1992; Davis et al. 1991; Drummond et al. 1996; Wesselmann et al. 1996), the 2 adrenoceptor appears to be important in subprimate models of neuropathic pain (e.g., Chen et al. 1996; Peterson et al. 1996; Sato and Perl 1991; Tracey et al. 1995; Xie et al. 1995.)
; Kirk and Denny-Brown 1970), 2) a similar injury in rats produced behavioral signs of hyperalgesia that were reported to be reversed by surgical sympathectomy and by systemic administration of phentolamine (Kim and Chung 1991, 1992; Kinnman and Levine 1995; but see Fontana et al. 1996; Lee et al. 1997; Ringkamp et al. 1999a,b), 3) preliminary in vivo experiments in our laboratory indicated that C-fiber nociceptors in monkey responded to close-arterial injection of norepinephrine after a spinal nerve injury (Selig et al. 1993), 4) the procedure to produce a spinal nerve ligation is standardized and thus results in a reproducible lesion, and 5) this lesion enables the investigation of uninjured nerve fibers in peripheral nerves that innervate the tissue via adjacent nerve roots.
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
). Two to three weeks after the ligation, an in vitro skin-nerve preparation was used to test -adrenergic sensitivity of cutaneous C-fiber nociceptors from the innervation territory of the superficial peroneal nerve. The Johns Hopkins University Animal Care and Use Committee approved all procedures in this study.
1·h1). Core temperature was measured using a rectal probe connected to a thermal controller and maintained close to 38°C with the use of circulating water heating pads. At the beginning of the experiment, penicillin G (450,000 U) was administered for prophylaxis against infection.
1·h1). Adequacy of anesthesia and core temperature were checked and maintained in the manner mentioned previously. Animals were paralyzed every 2 h with (0.1 mg/kg) pancuronium bromide and were artificially ventilated. Hydration was maintained by intravenous infusion of 5% dextrose in normal saline (4-6 ml·kg1·h1).
View larger version (32K):
[in a new window]
FIG. 1.
In vitro skin-nerve preparation as adapted for monkeys. Skin was placed into the bath corium side up, and the nerve was passed through a small hole into the recording chamber. Teased fiber techniques were used to record from single C-fiber nociceptors. Mechanical stimuli were used to locate the receptive field. Electrical, mechanical, thermal, and chemical stimuli were applied to the receptive field. The large bath had 2 inlets for the synthetic interstitial fluid. To minimize temperature and pH gradients, a heating system was introduced into the bath, and the bath was continuously bubbled with the 95% O2-5% CO2 mixture.
; Meyer and Campbell 1981; Meyer et al. 1985, 1988; Treede et al. 1990). In these experiments, the animals were maintained under pentobarbital anesthesia, and a cutaneous nerve that innervated hairy skin was dissected for single nerve fiber recordings (Campbell and Meyer 1983).
; Reeh et al. 1986). The reservoir of SIF was continuously bubbled with a 95% O2-5% CO2 mixture to obtain a pH of 7.4. A roller pump (Gilson, model M312) was used to control the flow of SIF at a rate of 750 ml/h from the reservoir through a heat-exchanger to the perfusion chamber. The heat exchanger was used to raise the temperature of the SIF to 33°C and consisted of a straight stainless steel tube through which the SIF passed. The tube was surrounded by a tightly wound coil of flexible tygon tubing through which water from a temperature-controlled water bath flowed. Several bubbling stones were placed within the perfusion chamber to keep the perfusate saturated with the 95% O2-5% CO2 mixture. The stones were arranged to establish a continuous circulation of fluid within the perfusion chamber, which helped to minimize thermal and pH gradients. The temperature of the chamber was maintained between 32 and 34°C by means of a second temperature-controlled water bath that circulated water through a loop of stainless steel tubing within the perfusion chamber.
50% of the trials was taken as threshold. The conduction velocity of the fiber was determined by electrical stimulation of the receptive field with a constant voltage stimulus. The stimulus intensity was increased until an action potential with a similar shape to that generated mechanically could be recorded. The voltage was increased fourfold to verify that no other fibers of the same action potential shape could be stimulated from this site. The conduction velocity was calculated by dividing the conduction distance by the latency of action potential to a suprathreshold stimulus. In all cases, suprathreshold mechanical stimuli were applied throughout the mechanical receptive field to confirm that only one fiber was being recorded. In this study we concentrated on isolating C-fiber nociceptors, and faster conducting fibers were discarded.
1-adrenergic agonist phenylephrine (1, 10, and 100 µM diluted from a 1% phenylephrine solution in NaCl, Elkins-Sinn, Cherry Hill, NJ) and the 2-adrenergic agonist UK14304 (0.1, 1, 10 µM; a gift from Pfizer Pharmaceuticals, Sandwich, UK). UK14304 is also know as brimonidine.
1- and 2-receptors, respectively. The doses of phenylephrine (1-100 µM) and UK14304 (0.1-10 µM) chosen were based on their potency and selectively for activating 1- and 2-adrenergic receptors, respectively (Flavahan et al. 1984).
) was used to digitize and store all action potential waveforms as well as to perform real-time and posthoc spike discrimination based on multiple time-amplitude window criteria. The software was also used to control the timing of the heat stimuli, to record temperatures during the heat stimulus, to control timing of the drug administration, and to time stamp the action potentials relative to stimulus delivery.
30% greater than predrug activity. To avoid counting action potentials evoked by mechanical stimulation rather than due to drug effects, action potentials that were generated during the time of drug exchange were not counted.
80°C for future use. The sections were washed with TBS and treated with 3% hydrogen peroxide in TBS for 30 min and then incubated for 30 min in 10% normal goat serum (Vector Laboratories, Burlingame, CA) and 1% Triton-X100 (Sigma, St Louis, MO) in TBS. The slides were then incubated overnight at 4°C with polyclonal anti-ubiquitin carboxyl terminal hydrolase (protein gene product 9.5, 1:1000) (Wilkinson et al. 1989) antibody to stain epidermal nerve fibers. The sections were then incubated with a biotinylated secondary antibody (Vector, 1:300) followed by streptoavidin-biotin-peroxide solution (Dako, Carpentria, CA) for 30 min. The slides were developed with an SG chromogen substrate kit (Vector) with dark-blue reaction product, and counterstained with Mayer's hematoxylin, dehydrated, and mounted.
2 test was used to compare the incidence of spontaneous activity, heat sensitivity, cold sensitivity, phenylephrine responsiveness, and UK14304 responsiveness in nociceptors recorded from ligated monkeys compared with control monkeys. Nociceptors tested with phenylephrine and UK14304 were separated into responsive and nonresponsive fibers, respectively. Drug effects on the responsive fibers were tested using a Friedman analysis of variance (ANOVA). When the test indicated a significant difference, posthoc analysis was made between the predrug interval and each of the doses of drug using a Wilcoxon matched pairs test with Bonferroni correction. For fibers tested with both phenylephrine and UK14304, the peak responses (minus the baseline) were compared using a Mann-Whitney U test. Data are presented as means ± SE or as medians (quartiles). Significance was accepted at the 0.05 level.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
). Individual fibers and small bundles crossed the dermis to join the subpapillary plexus. The subpapillary fibers, in general, lay parallel to the surface of the skin in bundles. In addition to innervating specialized endings in the dermis and dermal papillae, many subpapillary fibers turned toward the surface and entered the epidermis. In the epidermis, these thin, varicose fibers passed between keratinocytes and often branched in passage. Many fibers reached the stratum corneum.
View larger version (45K):
[in a new window]
FIG. 2.
PGP9.5-stained nerve fibers in the epidermal skin of the dorsal hindpaw of L6 lesioned monkeys. A: photograph of section from skin contralateral to lesion (calibration bar = 50 µm). B: photograph of section from skin ipsilateral to lesion. C: for each animal, a count of epidermal fibers was made on the lesioned foot and the contralateral, control foot. Lines connect data for a given animal. There were significantly fewer PGP9.5-stained fibers on the lesioned side compared with the control side (P < 0.001, paired t-test).
View this table:
TABLE 1.
Comparison of nociceptor properties
2). In addition, the magnitude of spontaneous activity was significantly higher in C-fibers recorded from lesioned monkeys (median = 1 action potentials/5 min; range = 0-147 action potentials/5 min) than in control C-fibers (median = 0 action potentials/5 min; range = 0-4 action potentials/5 min; P < 0.01 Mann-Whitney U test; Fig. 3). However, the mechanical threshold of spontaneously active fibers (2.2 ± 0.4 bar) was not significantly different from fibers without spontaneous activity (2.6 ± 0.9 bar).
View larger version (18K):
[in a new window]
FIG. 3.
Spontaneous activity recorded during the 5-min interval before administration of the first adrenergic agonists. Each point corresponds to a different fiber; incidence of spontaneous activity was significantly higher in nerve fibers recorded from L6 lesioned monkeys than in control monkeys.
1-adrenergic agonist phenylephrine
View larger version (32K):
[in a new window]
FIG. 4.
Responses of 2 different nociceptors from L6 lesioned monkeys to the 1- and 2-adrenergic agonists. A: phenylephrine evoked responses in fiber 1. Each vertical tic corresponds to one action potential and each horizontal line corresponds to the 5-min interval for one drug trial. Drug trials are arranged from vehicle at the bottom to the highest dose (100 µM) at the top. Fiber 1 had a low level of spontaneous activity before the application of phenylephrine. Fiber 1 exhibited a vigorous, dose-dependent response to phenylephrine. B: phenylephrine evoked responses in fiber 2. Fiber 2 also responded to phenylephrine, but the response did not increase in a dose-dependent fashion (format same as in A). C: summary of the phenylephrine evoked response for fibers 1 and 2. Total response during the 5-min drug trial is plotted as a function of dose. D: UK14304 evoked responses in fiber 1. This fiber exhibited a weak response to UK14304. (Format similar to A) E: UK14304 evoked responses in fiber 2. For fiber 2, testing with UK14304 was carried out before the testing with phenylephrine. F: summary of the UK14304 evoked response for fiber 1 and fiber 2 (format same as in C); note that for both fibers 1 and 2, the UK14304 evoked response was smaller than the phenylephrine evoked response.
View larger version (13K):
[in a new window]
FIG. 5.
Dose-response curve for phenylephrine sensitive afferents (n = 9). Median response during the 5-min drug application period is plotted as a function of phenylephrine dose. The responses at all doses were significantly greater than the response during the predrug interval (* P < 0.05; Wilcoxon matched pairs).
View larger version (15K):
[in a new window]
FIG. 6.
Tachyphylaxis to repeated application of phenylephrine. For 4 fibers the complete phenylephrine ascending dose series was administered twice. For each fiber, the total response to each sequence is plotted. In all 4 fibers a substantial decrease in response was observed for the second administration. The tachyphylaxis was not related to the order of UK14304 testing because for 2 of these fibers UK14304 was tested first and for the other 2 fibers UK14304 was tested after the first application of phenylephrine.
2 test). The peak response was 180 action potentials in one fiber and 9 action potentials in the other.
2-adrenergic agonist UK14304
2-adrenergic agonist UK14304 evoked a response in 6 of 18 C-fiber nociceptors recorded from lesioned monkeys. As with the phenylephrine, the duration and magnitude of the response varied between fibers. Figure 4, D and E, illustrates the responses of two fibers to UK14304. These are the same two fibers whose responses to phenylephrine are illustrated in Fig. 4, A and B.
View larger version (13K):
[in a new window]
FIG. 7.
Dose-response curve for UK14304-sensitive afferents. The median response during the 5-min drug administration period is plotted as a function of dose.
2 test).
View larger version (29K):
[in a new window]
FIG. 8.
Responses of afferents tested with both phenylephrine and UK14304. A: incidence of response in lesioned animals (n = 15). B: incidence of response in control animals (n = 13). PE, phenylephrine sensitive; UK, UK14304 sensitive; 
PE, units whose spontaneous activity decreased during PE administration; NR, nonresponsive. C: peak response to each agonist. Peak response and predrug response are plotted for the 8 phenylephrine-sensitive fibers (left) and for the 5 UK14304-sensitive fibers (right). Peak response to phenylephrine was significantly greater than the peak response to UK14304 (P < 0.05, Wilcoxon matched pairs).
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
1-adrenergic agonist phenylephrine than to the selective 2-adrenergic agonist UK14304, and the maximum response to phenylephrine was significantly greater than the maximum response to UK14304. Thus cutaneous nociceptors in monkeys develop 1-adrenergic sensitivity after nerve lesion.
. An in vitro technique was chosen to control better the concentrations of the applied drugs as well as to avoid potential artifacts associated with the vasoactive properties of the adrenergic agents. The properties of afferents from control skin recorded in vitro were similar to those from normal skin recorded in vivo, except that the conduction velocities were slower. We attribute this difference to the cooler temperatures in the in vitro chamber compared with the limb temperature in vivo. Thus the in vitro technique appears to be valid for investigating the properties of primate nociceptors.
1- and 2-adrenergic sensitivity after nerve lesion
1-adrenergic sensitivity after nerve lesion. Several lines of evidence suggest that the 1-adrenoceptor is also a culprit in human patients suffering from SMP. 1) Intradermal injection of phenylephrine can evoke pain in SMP patients (Ali et al. 1996; Davis et al. 1991 ). 2) The number of 1-adrenoceptors in hyperalgesic skin of patients with SMP is significantly greater than in the skin of normal subjects (Drummond et al. 1996). 3) Topical administration of the 2-adrenergic agonist, clonidine, did not evoke pain in patients with sympathetically maintained pain but actually led to a decrease in pain and hyperalgesia (Davis et al. 1991; Wesselmann et al. 1996)
; Manning et al. 1991). This tachyphylaxis prohibited an investigation of the effects of selective adrenergic antagonists.
2-adrenoceptors in primates. Because UK14304 also activates imidazoline receptors, which are in general inhibitory, the low incidence and low response observed by us to UK14304 could be the result of opposing effects of UK14304 at the imidazoline receptor and the 2-adrenoceptor. In addition, studies in subprimates suggest that 2-adrenoceptors contribute to cutaneous adrenergic sensitivity (O'Halloran and Perl 1997; Sato and Perl 1991; Tracey et al. 1995). However, recent studies in rats indicate that 1-adrenoceptors also contribute to adrenergic sensitivity (Chen et al. 1996; Choi et al. 1996; Lee and Chung 1997).
also found a significant increase of spontaneous activity in saphenous nerve fibers after a chronic constriction injury of that nerve; this spontaneous activity could be abolished by application of lidocaine to the cutaneous receptive field. Thus the receptor terminal is likely a site for generation of spontaneous activity.
). The incidence of spontaneous activity in neuroma of cutaneous nerves is relatively low (Blumberg and Jänig 1984; Meyer et al. 1985). The development of spontaneous activity in uninjured C-fiber nociceptors may be specific to primates because spontaneous activity was rarely observed in presumably uninjured C-fiber nociceptors following a partial injury of the auricular nerve in rabbits (O'Halloran and Perl 1997; Sato and Perl 1961).
) might lead to an increased membrane excitability. Our observation of a significantly lower mechanical threshold in phenylephrine sensitive afferents compared with phenylephrine insensitive afferents would be consistent with this possibility. Another possibility is that the enhanced adrenergic sensitivity results in spontaneous activity caused by the local endogenous leakage of norepinephrine. In this case, the unit with the highest spontaneous activity may not have responded to the exogenous agonists because it was already at its peak discharge frequency. A decrease of spontaneous activity was observed in two other units that had relatively high rates of spontaneous activity. The mechanisms involved in such an inhibition are not known, but a similar observation has been reported for cutaneous afferents from animals with a chronic constriction injury (Koltzenburg et al. 1994).
; Roberts and Elardo 1985; Shea and Perl 1985). We found that a small proportion of C-fiber nociceptors in normal skin could be activated by phenylephrine and UK14304. Although intradermal norepinephrine produces little pain in normal human subjects (Ali et al. 1996; Davis et al. 1991), cutaneous administration of adrenergic agonists can produce heat hyperalgesia in human subjects (Drummond 1995; Meyer and Raja 1996) and mechanical hyperalgesia in rats (Khasar et al. 1995). Sympathetic stimulation also appears to alter the propensity for C-fiber nociceptors in rabbits to become sensitized by repeated heating (Sato and Perl 1991). Adrenergic sensitivity has also been observed in nociceptors following acute injury (Roberts and Elardo 1985), injection of inflammatory mediators (Sanjue and Jun 1989), and chronic inflammation (Sato et al. 1993). -adrenergic receptors have been reported in dorsal root ganglion neurons and peripheral nerve fibers (Gold et al. 1997; Nicholas et al. 1993). Thus adrenergic receptors may normally be present in cutaneous afferents and, under certain conditions, have the capacity when activated to initiate responses in nociceptors.
reported an increase in 1-adrenergic receptors in the skin of neuropathic pain patients. This suggests that a nerve injury alters the synthesis of adrenergic receptors in either the dorsal root ganglion of the primary afferents innervating the skin or of other cells in the skin. Nerve injury may also alter the expression and relative distribution of different adrenergic receptor subtypes in dorsal root ganglion neurons (Birder and Perl 1996). Adrenergic supersensitivity could also develop as the result of the sympathetic denervation associated with a nerve injury. Nociceptors have been shown to develop adrenergic sensitivity following surgical sympathectomies (Bossut et al. 1996). However, chronic pain in the limb is rarely a complication of surgical sympathectomies for hyperhidrosis (Drott and Claes 1996; Lai et al. 1997; Zacherl et al. 1998).
; Sato and Perl 1991). 2) In patients with well-documented radiculopathy from lumbar disk herniation, a distal anesthetic block of the sciatic nerve eliminates the sciatic pain (e.g., North et al. 1996). 3) In tic douloureux, surgical lesions of the trigeminal nerve distal to the presumed trigeminal nerve compression near the brain stem abolish the pain (Quinn 1965; Stookey and Ransohoff 1959). 4) Topical capsaicin is efficacious in treatment of neuropathic pain disorders caused by proximal nerve lesions (Ellison et al. 1997). 5) Topical clonidine attenuates hyperalgesia and pain in SMP (Davis et al. 1991). 6) Low-threshold mechanoreceptors are thought to be the peripheral generators for pain to light touch in the zone of secondary hyperalgesia (LaMotte et al. 1991) as well as in patients with neuropathic pain (Campbell et al. 1988). Most likely the ectopic activity originating from the nerve injury site, the dorsal root ganglion, and the intact fibers may all contribute to the development and maintenance of central sensitization and spontaneous pain. The relative importance of each generator site may be influenced by a myriad of variables, including interval of time from the nerve lesion, species as well as individual differences, and type and location of the lesion.
). In contrast to findings in other species, cutaneous C-fiber nociceptors in primates exhibit 1-adrenergic sensitivity after nerve lesion. Whether primate nociceptive neurons at the nerve injury site and at the level of the dorsal root ganglion also develop adrenergic sensitivity after injury and whether this sensitization also fits with an 1-adrenoceptor characterization remains to be determined.
| |
ACKNOWLEDGEMENTS |
|---|
The authors greatly appreciate the many helpful discussions with Prof. Dr. Peter Reeh regarding modifications of rat skin in vitro preparation to work with monkey skin. We also thank R. Thunga, J. Huang, and S. Horasek for technical assistance.
This research was supported by National Institute of Neurological Disorders and Stroke Grant NS-32386.
| |
FOOTNOTES |
|---|
Address for reprint requests: R. A. Meyer, Johns Hopkins Applied Physics Laboratory, Johns Hopkins Rd., Laurel, MD 20723.
Received 29 May 1998; accepted in final form 15 September 1998.
| |
REFERENCES |
|---|
|
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
2-adrenoceptors couple to N-type Ca2+ channels in axotomized rat sensory neurons.
J. Neurosci.
17: 1633-1641, 1997.2A adrenergic receptor subtype after peripheral nerve injury.
Soc. Neurosci. Abstr.
22: 1803, 1996.
mechanical nociceptors.
J. Neurophysiol.
73: 1721-1723, 1995.1-Adrenoceptors in normal and hyperalgesic human skin.
Clin. Sci.
91: 73-77, 1996.[Medline]1 and 2-adrenoceptors activated by exogenous and nerve-released norepinephrine in the canine saphenous vein.
J. Pharmacol. Exp. Ther.
230: 699-705, 1984.2-Adrenergic receptor subtypes in rat dorsal root and superior cervical ganglion neurons.
Pain
69: 179-190, 1997.[Medline]2-adrenergic receptor agonists in the rat.
Neuroscience
66: 427-432, 1995.[Medline]2-adrenoreceptors on post-ganglionic sympathetic terminals.
Pain
60: 317-327, 1995.[Medline]This article has been cited by other articles:
|
|
 |
|
  K. Orstavik, B. Namer, R. Schmidt, M. Schmelz, M. Hilliges, C. Weidner, R. W. Carr, H. Handwerker, E. Jorum, and H. E. Torebjork Abnormal Function of C-Fibers in Patients with Diabetic Neuropathy. J. Neurosci., November 1, 2006; 26(44): 11287 - 11294. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Djouhri, S. Koutsikou, X. Fang, S. McMullan, and S. N. Lawson Spontaneous Pain, Both Neuropathic and Inflammatory, Is Related to Frequency of Spontaneous Firing in Intact C-Fiber Nociceptors J. Neurosci., January 25, 2006; 26(4): 1281 - 1292. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Liu and J. C. Eisenach Hyperexcitability of Axotomized and Neighboring Unaxotomized Sensory Neurons Is Reduced Days After Perineural Clonidine at the Site of Injury J Neurophysiol, November 1, 2005; 94(5): 3159 - 3167. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Obata, H. Yamanaka, K. Kobayashi, Y. Dai, T. Mizushima, H. Katsura, T. Fukuoka, A. Tokunaga, and K. Noguchi Role of Mitogen-Activated Protein Kinase Activation in Injured and Intact Primary Afferent Neurons for Mechanical and Heat Hypersensitivity after Spinal Nerve Ligation J. Neurosci., November 10, 2004; 24(45): 10211 - 10222. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R.-R. Ji and G. Strichartz Cell Signaling and the Genesis of Neuropathic Pain Sci. Signal., September 28, 2004; 2004(252): re14 - re14. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Zahn, S. Leis, C. Schick, M. Schmelz, and F. Birklein No {alpha}-adrenoreceptor-induced C-fiber activation in healthy human skin J Appl Physiol, April 1, 2004; 96(4): 1380 - 1384. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. D. Drummond Involvement of the Sympathetic Nervous System in Complex Regional Pain Syndrome International Journal of Lower Extremity Wounds, March 1, 2004; 3(1): 35 - 42. [Abstract] [PDF]
|
|
|
|
 |
|
 P D Drummond and P M Finch Persistence of pain induced by startle and forehead cooling after sympathetic blockade in patients with complex regional pain syndrome J. Neurol. Neurosurg. Psychiatry, January 1, 2004; 75(1): 98 - 102. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Schafers, D. H. Lee, D. Brors, T. L. Yaksh, and L. S. Sorkin Increased Sensitivity of Injured and Adjacent Uninjured Rat Primary Sensory Neurons to Exogenous Tumor Necrosis Factor-alpha after Spinal Nerve Ligation J. Neurosci., April 1, 2003; 23(7): 3028 - 3038. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Ma, Y. Shu, Z. Zheng, Y. Chen, H. Yao, K. W. Greenquist, F. A. White, and R. H. LaMotte Similar Electrophysiological Changes in Axotomized and Neighboring Intact Dorsal Root Ganglion Neurons J Neurophysiol, March 1, 2003; 89(3): 1588 - 1602. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Lin, X. Zou, L. Fang, and W. D. Willis Sympathetic Modulation of Acute Cutaneous Flare Induced by Intradermal Injection of Capsaicin in Anesthetized Rats J Neurophysiol, February 1, 2003; 89(2): 853 - 861. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Gold, D. Weinreich, C.-S. Kim, R. Wang, J. Treanor, F. Porreca, and J. Lai Redistribution of NaV1.8 in Uninjured Axons Enables Neuropathic Pain J. Neurosci., January 1, 2003; 23(1): 158 - 166. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. R. Watkins and S. F. Maier Beyond Neurons: Evidence That Immune and Glial Cells Contribute to Pathological Pain States Physiol Rev, October 1, 2002; 82(4): 981 - 1011. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Wu, M. Ringkamp, B. B. Murinson, E. M. Pogatzki, T. V. Hartke, H. M. Weerahandi, J. N. Campbell, J. W. Griffin, and R. A. Meyer Degeneration of Myelinated Efferent Fibers Induces Spontaneous Activity in Uninjured C-Fiber Afferents J. Neurosci., September 1, 2002; 22(17): 7746 - 7753. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. M. Pogatzki, G. F. Gebhart, and T. J. Brennan Characterization of Adelta - and C-Fibers Innervating the Plantar Rat Hindpaw One Day After an Incision J Neurophysiol, February 1, 2002; 87(2): 721 - 731. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. M. Cain, P. W. Wacnik, M. Turner, G. Wendelschafer-Crabb, W. R. Kennedy, G. L. Wilcox, and D. A. Simone Functional Interactions between Tumor and Peripheral Nerve: Changes in Excitability and Morphology of Primary Afferent Fibers in a Murine Model of Cancer Pain J. Neurosci., December 1, 2001; 21(23): 9367 - 9376. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. D. Drummond, P. M. Finch, S. Skipworth, and P. Blockey Pain increases during sympathetic arousal in patients with complex regional pain syndrome Neurology, October 9, 2001; 57(7): 1296 - 1303. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. KRESS and H. FICKENSCHER Infection by human varicella-zoster virus confers norepinephrine sensitivity to sensory neurons from rat dorsal root ganglia FASEB J, April 1, 2001; 15(6): 1037 - 1043. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-M. Zhang, H. Li, and S. J. Brull Perfusion of the Mechanically Compressed Lumbar Ganglion With Lidocaine Reduces Mechanical Hyperalgesia and Allodynia in the Rat J Neurophysiol, August 1, 2000; 84(2): 798 - 805. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X.-J. Song, S.-J. Hu, K. W. Greenquist, J.-M. Zhang, and R. H. LaMotte Mechanical and Thermal Hyperalgesia and Ectopic Neuronal Discharge After Chronic Compression of Dorsal Root Ganglia J Neurophysiol, December 1, 1999; 82(6): 3347 - 3358. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. R. Perl Causalgia, pathological pain, and adrenergic receptors PNAS, July 6, 1999; 96(14): 7664 - 7667. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Wu, M. Ringkamp, T. V. Hartke, B. B. Murinson, J. N. Campbell, J. W. Griffin, and R. A. Meyer Early Onset of Spontaneous Activity in Uninjured C-Fiber Nociceptors after Injury to Neighboring Nerve Fibers J. Neurosci., April 15, 2001; 21(8): RC140 - RC140. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
