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Clinical Science  |   June 1997
Alfentanil, but Not Amitriptyline, Reduces Pain, Hyperalgesia, and Allodynia from Intradermal Injection of Capsaicin in Humans 
Author Notes
  • (Eisenach) Professor and Chair for Anesthesia Research.
  • (Hood) Associate Professor of Anesthesia.
  • (Curry) Research Nurse.
  • (Tong) Resident in Anesthesia.
  • Received from the Department of Anesthesia, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, North Carolina. Submitted for publication December 6, 1996. Accepted for publication February 19, 1997. Supported in part by National Institutes of Health grants 48085 and M01 RR07122.
  • Address reprint requests to Dr. Eisenach: Department of Anesthesia, Wake Forest University Medical Center, Medical Center Boulevard, Winston-Salem, North Carolina 27157–1009. Address electronic mail to: eisenach@bgsm.edu.
Article Information
Clinical Science
Clinical Science   |   June 1997
Alfentanil, but Not Amitriptyline, Reduces Pain, Hyperalgesia, and Allodynia from Intradermal Injection of Capsaicin in Humans 
Anesthesiology 6 1997, Vol.86, 1279-1287. doi:
Anesthesiology 6 1997, Vol.86, 1279-1287. doi:
Most pain can be classified according to its three components. Acute noxious stimuli result in well-localized pain. A more sustained noxious stimulus may result in more diffuse pain and generation of increased pain perception from a painful stimulus (hyperalgesia) and pain from a stimulus previously perceived as innocuous (allodynia). Nerve injury may result in chronic pain, hyperalgesia, and allodynia in a dermatomal or peripheral nerve territory. Recent research has focused on the latter components of pain, because these may play important roles in postsurgical pain and in difficult-to-manage chronic pain syndromes.
Intradermal injection of capsaicin results in acute burning pain that is dose-dependent and brief after doses of 100 micro gram or less, followed by development of hyperalgesia and allodynia to mechanical stimuli that lasts several hours. [1 ] Development of hyperalgesia and allodynia can be prevented by peripheral nerve block to prevent perception of the acute pain of injection. [2 ] Animal studies suggest that hyperalgesia and allodynia after intradermal capsaicin injection is mediated via stimulation of N-methyl-D-aspartate (NMDA) and other excitatory receptors in the spinal cord, leading to the development of hypersensitivity of dorsal horn neurons to afferent input. [3,4 ] Thus, this simple model shares many characteristics of animal models of inflammatory pain and has been used experimentally in humans to examine the pharmacology of spinal hypersensitivity.
Systemic administration of the NMDA antagonist ketamine or the opioid alfentanil has been shown to reduce ongoing pain from intradermal injection of a large dose (250 micro gram) of capsaicin and to reduce simultaneously the area of attendant mechanical hyperalgesia in humans. [5 ] However, the relation between plasma opioid concentration and reduction in pain or hyperalgesia/allodynia in humans has not been examined. One purpose of the current study was to determine the relation between plasma opioid concentrations and acute and chronic pain responses to intradermal injection of capsaicin.
Ketamine only reduces pain from intradermal injection of capsaicin after doses that produce unwanted side effects. Recently, amitriptyline and related compounds have been shown to bind to NMDA receptors in vitro in low micromolar concentrations [6 ] and to function as an NMDA antagonist in vivo. [7 ] Intrathecal injection of amitriptyline reduces hyperalgesia after carrageenan inflammation in rats, [8 ] and amitriptyline is frequently administered systemically to manage neuropathic pain. As part of a series of studies examining potential efficacy of systemic and intrathecal amitriptyline for hyperalgesic pain, we tried to determine amitriptyline's efficacy after acute intramuscular injection alone. Because amitriptyline could enhance the opioid's effects by monoamine reuptake inhibition, [9 ] we also examined the effect of amitriptyline plus alfentanil in this model.
Materials and Methods 
The study was divided into two parts: an initial study of six volunteers to test the reproducibility of repeated intradermal capsaicin injections over a short time, and a second study of 40 other volunteers to test the effects of study drugs. Both studies were approved by the Clinical Research Practices Committee, written informed consent was obtained, and volunteers reported to the outpatient General Clinical Research Center at 7:00 AM, having had nothing to eat or drink since midnight. The same schedule of capsaicin injections and sensory testing was used in both studies, and each volunteer received an intradermal capsaicin injection on a day before the study to train them in assessing pain and in determining areas of hyperalgesia and allodynia. Volunteers were trained before this test injection of capsaicin to assess pain numerically in response to controlled noxious heat stimuli using a Peltier device. This training using the Peltier device was repeated on the morning of the study before capsaicin injection.
Capsaicin (Fluka Chemie, Buchs, Switzerland) was dissolved in Tween-80 and passed through a 20-micro Meter filter before injection. Injections were made in less than 2 s on the volar aspect of the forearm or lateral aspect of the calf in a volume of 10 micro liter containing 100 micro gram capsaicin, and then the needle was immediately removed. Volunteers rated their pain numerically at 15-s intervals for 3 min after injection, then at 5, 10, 20, 30, 60, 120, 180, and 240 min after injection. Areas of hyperalgesia were determined by sensation of pain to mechanical testing with a calibrated, 225-mN von Frey filament at 5, 10, 15, 30, 45, and 60 min after each injection, then at hourly intervals. Areas of allodynia were determined at these same times to mechanical testing with a cotton applicator attached to a metal band and calibrated to apply 100 g force. In each case, testing was performed from an area of normal sensation until the affected area was reached. Testing was performed along at least four radial spokes, the skin was marked at these points, and marks were transcribed to a clear plastic sheet, and the area was calculated by trapezoidal approximation.
Four capsaicin injections were performed on the day of each study. Volunteers were randomized to receive the first injection in either the upper or lower extremity. The second injection was performed ipsilateral to the first on the other extremity (for example, the first injection in left arm, second injection in left leg). The site of the third injection was opposite the second, and the fourth injection was opposite the second (in this example, the third injection in right leg, the fourth in right arm). Injections were separated by 60 min. In the first part of the study, six volunteers received this regimen of injections to determine whether there was an effect of injection site or order on pain response.
In the second part of the study, an intravenous catheter was inserted in one antecubital vein to administer drug and in an antecubital vein in the contralateral extremity to sample venous blood. Thirty minutes after the second capsaicin injection, volunteers received an intramuscular injection, and the intravenous infusion was started with rate programmed to achieve the first target plasma concentration. Pain, hyperalgesia, and allodynia from the first two injections were considered control values. Thirty minutes after the intramuscular injection/intravenous infusion, volunteers received the third capsaicin injection. Pain responses from this injection were considered to reflect the effect of the first target plasma concentration and the intramuscular injection. Just before the fourth capsaicin injection, the intravenous infusion was adjusted to achieve the second target plasma concentration. Pain responses from this last injection were considered to reflect the effect of the second target plasma concentration and the intramuscular injection. A different batch of capsaicin was prepared for volunteers in the second part of the study than had been used in the first part.
Volunteers were randomized into four drug treatment groups, and neither the volunteer nor the investigator was aware of the assignment. Volunteers received either 4 mg midazolam given intramuscularly plus intravenous saline (considered as an active placebo), 25 mg amitriptyline given intramuscularly plus intravenous saline, 25 mg amitriptyline given intramuscularly plus intravenous alfentanil, or saline given intramuscularly plus intravenous alfentanil. Alfentanil was given by computer-controlled infusion using the STANPUMP algorithm to achieve target plasma concentrations of 50 ng/ml (first target) and 200 ng/ml (second target).
In addition to assessing their pain, patients rated their nausea and sedation on 10-cm visual analog scales (VAS) at hourly intervals after intramuscular drug injection and initiating the intravenous infusion. Venous blood was drawn 5, 15, 30, 60, 90, 120, 180, and 240 min after drug injection. Samples were stored at -70 degrees Celsius until analysis. At the time of analysis, the code was broken, and only samples in volunteers receiving amitriptyline or alfentanil were analyzed. Amitriptyline plasma concentration was determined by high-pressure liquid chromatography with ultraviolet detection. [10 ] Sensitivity of the method is 3 ng/ml in the original sample. Alfentanil plasma concentration was determined by radioimmunoassay with interassay coefficient of variation of 4% and detection limit of 0.05 ng/ml. [11 ]
Data are presented as means +/- SD or median +/- 25th and 75th percentiles as appropriate. Pain magnitude was estimated by each participant according to their own numeric scale. These estimates were normalized to each person using a ratio whereby 10 reflected the largest pain estimate of that person. This method has been shown to produce linear stimulus response relations to graded pain stimuli in volunteers. [12 ] In the first part of the study (validation of reproducibility of four injections), pain, hyperalgesia, and allodynia were compared according to site of injection (arm vs. leg) and injection order by two-way analysis of variance for repeated measures. In the second part of the study, the effect of drug within each treatment group for pain, hyperalgesia, and allodynia was determined by two-way analysis of variance for repeated measures on ranks, with factors of volunteer and injection order. Groups were compared for amitriptyline and alfentanil plasma concentrations and for VAS sedation and nausea by two-way analysis of variance for repeated measures. Correlation analysis was performed by linear regression. Probability values less than 0.05 were considered significant.
Results 
Part 1: Validation Study 
The average age of volunteers was 31 yr (range, 19–41 yr), and three were women and three were men. Pain to intradermal capsaicin injection was intense but brief, lasting 5–10 min, and did not differ with injection order (Figure 1(A)) by analysis of variance. Similarly, there was no effect on pain report of injection site (mean pain score during the first 3 min was 6.4 +/- 1.8 after injection in the leg vs. 6.4 +/- 1.8 in the arm) or sex (mean pain score over the first 3 min was 5.9 +/- 2.2 in women and 6.9 +/- 1.2 in men). Similarly, areas of hyperalgesia (Figure 1(B)) and allodynia (Figure 1(C)) were unaffected by injection order. As with pain, mean area of hyperalgesia over 60 min was similar after injection in the leg (46 +/- 20 cm2) as in the arm (43 +/- 14 cm2) and was similar in women (49 +/- 15 cm2) and in men (40 +/- 18 cm sup 2). In addition, mean area of allodynia after 60 min was similar after injection in the arm (21 +/- 11 cm2) as in the leg (22 +/- 10 cm2), and was similar in women (22 +/- 8 cm2) and in men (21 +/- 13 cm sup 2). Overall average area of hyperalgesia (47 +/- 9.2 cm2) was larger than allodynia (24 +/- 4.9 cm2) during first hour (P < 0.05) after intradermal capsaicin injection.
Figure 1. Magnitude of pain (4), area of hyperalgesia (B), and allodynia (C) after the first (square), second (open circle), third (filled circle), and fourth (closed inverted triangle) hourly intradermal injection of 100 micro gram capsaicin. For pain, each symbol represents the mean of six volunteers, with +standard deviation and -standard deviation shown for the first and fourth injections, respectively. For hyperalgesia and allodynia, each symbol represents the median of six volunteers, with 75th and 25th percentiles shown for the first and fourth injections, respectively. Injection site was randomly assigned as the right and left upper and lower extremities. No difference was observed in pain response or areas of hyperalgesia or allodynia as a function of injection order.
Figure 1. Magnitude of pain (4), area of hyperalgesia (B), and allodynia (C) after the first (square), second (open circle), third (filled circle), and fourth (closed inverted triangle) hourly intradermal injection of 100 micro gram capsaicin. For pain, each symbol represents the mean of six volunteers, with +standard deviation and -standard deviation shown for the first and fourth injections, respectively. For hyperalgesia and allodynia, each symbol represents the median of six volunteers, with 75th and 25th percentiles shown for the first and fourth injections, respectively. Injection site was randomly assigned as the right and left upper and lower extremities. No difference was observed in pain response or areas of hyperalgesia or allodynia as a function of injection order.
Figure 1. Magnitude of pain (4), area of hyperalgesia (B), and allodynia (C) after the first (square), second (open circle), third (filled circle), and fourth (closed inverted triangle) hourly intradermal injection of 100 micro gram capsaicin. For pain, each symbol represents the mean of six volunteers, with +standard deviation and -standard deviation shown for the first and fourth injections, respectively. For hyperalgesia and allodynia, each symbol represents the median of six volunteers, with 75th and 25th percentiles shown for the first and fourth injections, respectively. Injection site was randomly assigned as the right and left upper and lower extremities. No difference was observed in pain response or areas of hyperalgesia or allodynia as a function of injection order.
×
Part 2: Drug Study 
Volunteers were aged 31 +/- 8 yr, measured 173 +/- 11 cm in height, and weighed 79 +/- 17 kg. Twenty were women, and 20 were men. The computer-controlled infusion rapidly achieved and maintained reasonably stable alfentanil concentrations, and amitriptyline did not affect alfentanil concentrations (Figure 2(A)). The average plasma alfentanil concentrations during the 60 min after the third capsaicin injection were 52 +/- 10 ng/ml in those receiving alfentanil alone and 51 +/- 13 ng/ml in those receiving alfentanil and amitriptyline. Corresponding values during the 60 min after the fourth capsaicin injection were 173 +/- 47 ng/ml and 182 +/- 47 ng/ml. Amitriptyline was rapidly absorbed after intramuscular injection (Figure 2(B)) and peaked in concentration in plasma earlier when given with intravenous injection of alfentanil than when given alone (median of 30 vs. 60 min; P < 0.05). Plasma amitriptyline concentrations averaged 36 +/- 29 ng/ml and 26 +/- 23 ng/ml during the 60 min after the third capsaicin injection in the amitriptyline-alone and amitriptyline-plus-alfentanil treatment groups, respectively. These values do not differ, nor do the average amitriptyline concentrations during the 60 min after the fourth capsaicin injection in these two groups (16 +/- 10 ng/ml and 8 +/- 7 ng/ml, respectively).
Figure 2. Plasma alfentanil (A) or amitriptyline (B) concentrations in volunteers randomized to receive alfentanil alone (filled triangle) or with intramuscular amitriptyline (open circle), or amitriptyline alone (filled circle) or with alfentanil (open circle). Each symbol represents the mean +/- SD of eight to ten volunteers. No differences were observed between groups, except earlier peak amitriptyline concentrations in the amitriptyline-alfentanil group (see text for details).
Figure 2. Plasma alfentanil (A) or amitriptyline (B) concentrations in volunteers randomized to receive alfentanil alone (filled triangle) or with intramuscular amitriptyline (open circle), or amitriptyline alone (filled circle) or with alfentanil (open circle). Each symbol represents the mean +/- SD of eight to ten volunteers. No differences were observed between groups, except earlier peak amitriptyline concentrations in the amitriptyline-alfentanil group (see text for details).
Figure 2. Plasma alfentanil (A) or amitriptyline (B) concentrations in volunteers randomized to receive alfentanil alone (filled triangle) or with intramuscular amitriptyline (open circle), or amitriptyline alone (filled circle) or with alfentanil (open circle). Each symbol represents the mean +/- SD of eight to ten volunteers. No differences were observed between groups, except earlier peak amitriptyline concentrations in the amitriptyline-alfentanil group (see text for details).
×
Pain report after either the third or fourth capsaicin injection was unaffected by intramuscular injection of midazolam or amitriptyline. Figure 3shows the response to the first two capsaicin injections, which did not differ among groups, and the averaged response to the third and fourth injections in the midazolam and amitriptyline groups. Similarly, alfentanil, either alone or with amitriptyline, had no effect on acute pain report to the third capsaicin injection (plasma concentration, 51 or 52 ng/ml, as described before). In contrast, alfentanil alone and with amitriptyline decreased pain report after the fourth capsaicin injection (Figure 3; plasma concentration, 173 to 182 ng/ml, as described before). There was a similar, significant correlation between plasma alfentanil concentration and pain report for alfentanil alone (r =-0.59, P = 0.006) and for alfentanil plus amitriptyline (r =-0.57, P = 0.01).
Figure 3. Magnitude of pain after intradermal injection of capsaicin, averaged for the first two injections (control: open square), or after the fourth capsaicin injection after intramuscular midazolam (open diamond), intramuscular amitriptyline (open triangle), intravenous alfentanil (200 ng/ml targeted plasma concentration: Filled circle), or the combination of amitriptyline and alfentanil (filled inverted triangle). Each symbol represents the mean of ten volunteers, with +standard deviation and -standard deviation shown for the control and amitriptyline-plus-alfentanil groups, respectively. *P < 0.05 for comparison of alfentanil alone and with amitriptyline to control by two-way analysis of variance.
Figure 3. Magnitude of pain after intradermal injection of capsaicin, averaged for the first two injections (control: open square), or after the fourth capsaicin injection after intramuscular midazolam (open diamond), intramuscular amitriptyline (open triangle), intravenous alfentanil (200 ng/ml targeted plasma concentration: Filled circle), or the combination of amitriptyline and alfentanil (filled inverted triangle). Each symbol represents the mean of ten volunteers, with +standard deviation and -standard deviation shown for the control and amitriptyline-plus-alfentanil groups, respectively. *P < 0.05 for comparison of alfentanil alone and with amitriptyline to control by two-way analysis of variance.
Figure 3. Magnitude of pain after intradermal injection of capsaicin, averaged for the first two injections (control: open square), or after the fourth capsaicin injection after intramuscular midazolam (open diamond), intramuscular amitriptyline (open triangle), intravenous alfentanil (200 ng/ml targeted plasma concentration: Filled circle), or the combination of amitriptyline and alfentanil (filled inverted triangle). Each symbol represents the mean of ten volunteers, with +standard deviation and -standard deviation shown for the control and amitriptyline-plus-alfentanil groups, respectively. *P < 0.05 for comparison of alfentanil alone and with amitriptyline to control by two-way analysis of variance.
×
Hyperalgesia did not differ in the first 60 min after the first and second capsaicin injections in volunteers receiving alfentanil or amitriptyline or their combination (Figure 4(A-C)). In contrast, the area of hyperalgesia was less after the second capsaicin injection than after the first in those receiving midazolam (Figure 4(D)). Hyperalgesia after the third and fourth capsaicin injections were unaffected by amitriptyline (Figure 4(B)), whereas hyperalgesia after the fourth injection was similarly reduced in volunteers receiving alfentanil alone (Figure 4(A)) or with amitriptyline (Figure 4(C)). Hyperalgesia after the third and fourth capsaicin injections in volunteers receiving midazolam was similar to that after the second injection. There was a significant correlation between plasma alfentanil concentration and reduction in area of hyperalgesia in the alfentanil-plus-amitriptyline group (r =-0.52, P = 0.02) but not in the alfentanil-alone group.
Figure 4. Area of hyperalgesia after the first (open square), second (open circle), third (filled circle), or fourth (filled inverted triangle) intradermal capsaicin injection in volunteers receiving intravenous alfentanil (A), intramuscular amitriptyline (B), alfentanil plus amitriptyline (C), or intramuscular midazolam (D). Each symbol represents the median of ten volunteers, with 75th and 25th percentiles shown for the first and fourth injections, respectively. *P < 0.05 for comparison of the fourth injection of alfentanil alone and with amitriptyline to the first injection. (tau)P < 0.05 for second, third, and fourth injections compared with the first.
Figure 4. Area of hyperalgesia after the first (open square), second (open circle), third (filled circle), or fourth (filled inverted triangle) intradermal capsaicin injection in volunteers receiving intravenous alfentanil (A), intramuscular amitriptyline (B), alfentanil plus amitriptyline (C), or intramuscular midazolam (D). Each symbol represents the median of ten volunteers, with 75th and 25th percentiles shown for the first and fourth injections, respectively. *P < 0.05 for comparison of the fourth injection of alfentanil alone and with amitriptyline to the first injection. (tau)P < 0.05 for second, third, and fourth injections compared with the first.
Figure 4. Area of hyperalgesia after the first (open square), second (open circle), third (filled circle), or fourth (filled inverted triangle) intradermal capsaicin injection in volunteers receiving intravenous alfentanil (A), intramuscular amitriptyline (B), alfentanil plus amitriptyline (C), or intramuscular midazolam (D). Each symbol represents the median of ten volunteers, with 75th and 25th percentiles shown for the first and fourth injections, respectively. *P < 0.05 for comparison of the fourth injection of alfentanil alone and with amitriptyline to the first injection. (tau)P < 0.05 for second, third, and fourth injections compared with the first.
×
The response of allodynia to treatments was essentially the same as that of hyperalgesia and is depicted in the same manner (Figure 5(A-D)). Similar to hyperalgesia, there was a significant correlation between plasma alfentanil concentration and reduction in area of allodynia in the alfentanil-plus-amitriptyline group (r =-0.52, P = 0.02) but not in the alfentanil-alone group.
Figure 5. Area of allodynia after the first (open square), second (open circle), third (filled circle), or fourth (filled inverted diamond) intradermal capsaicin injection in volunteers receiving intravenous alfentanil (A), intramuscular amitriptyline (B), alfentanil plus amitriptyline (C), or intramuscular midazolam (D). Each symbol represents the median of ten volunteers, with 75th and 25th percentiles shown for the first and fourth injections, respectively. *P < 0.05 for comparison of the fourth injection of alfentanil alone and with amitriptyline with the first injection. (tau)P < 0.05 for the second, third, and fourth injections compared with the first.
Figure 5. Area of allodynia after the first (open square), second (open circle), third (filled circle), or fourth (filled inverted diamond) intradermal capsaicin injection in volunteers receiving intravenous alfentanil (A), intramuscular amitriptyline (B), alfentanil plus amitriptyline (C), or intramuscular midazolam (D). Each symbol represents the median of ten volunteers, with 75th and 25th percentiles shown for the first and fourth injections, respectively. *P < 0.05 for comparison of the fourth injection of alfentanil alone and with amitriptyline with the first injection. (tau)P < 0.05 for the second, third, and fourth injections compared with the first.
Figure 5. Area of allodynia after the first (open square), second (open circle), third (filled circle), or fourth (filled inverted diamond) intradermal capsaicin injection in volunteers receiving intravenous alfentanil (A), intramuscular amitriptyline (B), alfentanil plus amitriptyline (C), or intramuscular midazolam (D). Each symbol represents the median of ten volunteers, with 75th and 25th percentiles shown for the first and fourth injections, respectively. *P < 0.05 for comparison of the fourth injection of alfentanil alone and with amitriptyline with the first injection. (tau)P < 0.05 for the second, third, and fourth injections compared with the first.
×
All drugs produced sedation. Sedation was similar in the active control (midazolam) and amitriptyline groups (Figure 6(A)) and in the alfentanil-alone and alfentanil-plus-amitriptyline groups (Figure 6(B)). No volunteer experienced nausea after receiving midazolam. One volunteer receiving amitriptyline had nausea rated as 4 on a VAS before drug injection, and this nausea resolved in the ensuing 2 h after drug administration. No other volunteer receiving amitriptyline experienced nausea. Nine of ten volunteers receiving alfentanil alone experienced nausea, which peaked at the end of the infusion (VAS for nausea, 2.5 +/- 4.1 cm). In contrast, only three of ten volunteers receiving alfentanil plus amitriptyline experienced nausea (P < 0.05 vs. alfentanil alone), and nausea in the amitriptyline-alfentanil group was milder by VAS (at the time of peak, at the end of infusion, VAS for nausea was 0.1 +/- 0.3 cm).
Figure 6. Visual analog scale sedation in volunteers randomized to receive or amitriptyline alone (filled circle) or intramuscular midazolam alone (open square; A), or alfentanil alone (filled triangle) or with intramuscular amitriptyline (open circle; B). Each symbol represents the mean +/- SD of ten volunteers. There was no difference between groups.
Figure 6. Visual analog scale sedation in volunteers randomized to receive or amitriptyline alone (filled circle) or intramuscular midazolam alone (open square; A), or alfentanil alone (filled triangle) or with intramuscular amitriptyline (open circle; B). Each symbol represents the mean +/- SD of ten volunteers. There was no difference between groups.
Figure 6. Visual analog scale sedation in volunteers randomized to receive or amitriptyline alone (filled circle) or intramuscular midazolam alone (open square; A), or alfentanil alone (filled triangle) or with intramuscular amitriptyline (open circle; B). Each symbol represents the mean +/- SD of ten volunteers. There was no difference between groups.
×
Discussion 
Intradermal capsaicin injection has been described in a series of studies in volunteers as a method to examine central sensitization and to screen drugs for efficacy against hyperalgesic pain states. The current study provides novel information that validates this model and describes the effects of systemically administered drugs on the model.
Intradermal injection of capsaicin produces acute pain by direct stimulation of chemosensitive primary afferents and central release of excitatory neurotransmitters in the spinal cord. In addition, mechanical hyperalgesia and allodynia after intradermal capsaicin results from both peripheral and central sensitization. These pain responses depend on the initial stimulus from the capsaicin injection, because they do not occur when the capsaicin is injected in skin that has been anesthetized by infiltration with local anesthetic or by peripheral nerve block. [2 ] Support for peripheral sensitization comes primarily from observations: increased ongoing activity in a minority of peripheral single units in humans in the area of hyperalgesia, [13 ] reduction in the area of hyperalgesia by cooling the skin in the receptive area, [13 ] and block of spread of the area of hyperalgesia beyond a line of local anesthetic infiltration. [2 ] However, peripheral sensitization alone does not explain the development of hyperalgesia and allodynia, because local cooling reduces but does not abolish these effects, and most primary afferent units fail to exhibit ongoing discharge or enhanced responses to stimuli.
Rather than peripheral sensitization, several lines of evidence support a predominant role of spinal sensitization in the development of hyperalgesia and allodynia after intradermal injection of capsaicin. As discussed already, these effects of capsaicin do not occur when the area of injection is anesthetized. Hyperalgesia crosses a tight arm band that blocks circulation of blood and lymph, arguing against a peripheral effect determined by substances transported in these fluids. [2 ] Mechanical stimulation producing the sensation of hyperalgesia after capsaicin injection did not result in increased activity in the peripheral sensory afferents carrying this information to the spinal cord, which suggests a central sensitization effect. [13 ] Similarly, direct electrical stimulation of sensory afferents eliciting nonpainful tactile sensations in humans before capsaicin injection resulted in the sensation of pain when performed after capsaicin injection if they were in the area of hyperalgesia. [14 ]
These psychophysical studies in humans support a primary central mechanism of hyperalgesia and allodynia from intradermal injection of capsaicin. More precise experiments in animals have shown that capsaicin injected intradermally results in exaggerated responses of dorsal horn neurons to peripheral stimuli (either mechanical stimuli or direct electrical stimulation of primary afferents) and that this sensitization can be abolished by administering NMDA antagonists before and after treatment. [3,4 ] Spinal NMDA receptor stimulation is also thought to be central to the development of neuropathic pain syndromes, [15 ] and thus intradermal capsaicin is considered an appropriate experimental model in humans to examine the pharmacology of pain produced experimentally.
Previous investigators have shown the dose-dependent pain responses from intradermal capsaicin in humans, [1 ] but this is the first validation of four repeated injections in one experimental setting and comparison of effects when the drug is injected in the upper versus lower extremity. We chose this rotating series of injections in all four extremities in one session to subsequently test the dermatomally restricted action of spinally administered drugs. The current results suggest no effect of injection site or order in capsaicin-induced responses, supporting this model for subsequent studies.
Previous reports show that intravenous alfentanil reduces ongoing pain, hyperalgesia, and allodynia in volunteers receiving intradermal capsaicin [5 ] and in patients with chronic pain after trauma. [16 ] However, the previous two studies dosed alfentanil to the onset of side effects in a non-steady-state manner and did not determine the relation between plasma concentration and analgesic effect. In contrast, our study used computer-controlled infusion to rapidly achieve and maintain plasma concentrations of alfentanil in the analgesic range to acute experimental [17 ] or postoperative pain. [18 ] The results fail to show a selective reduction in hyperalgesia and allodynia by systemic alfentanil, because only high plasma concentrations, which also reduced acute pain to injection, reduced these elicited responses. Opioids can reduce spinal glutamate release after capsaicin injection in animals, [19 ] but the results of the current study correspond with the previous human studies in demonstrating that this only occurs in humans receiving large systemic doses accompanied by bothersome side effects. Although we could not determine the mechanism of opioid effect, the simultaneous reduction in acute pain from injection and development of hyperalgesia and allodynia suggest an action in reducing the spinal cord effects of the afferent barrage from the intradermal capsaicin rather than a specific effect on hyperalgesia and allodynia per se.
Amitriptyline produces analgesia in patients with neuropathic pain, [20 ] although its mechanism of action is not understood. Recent studies demonstrating an NMDA antagonist property of amitriptyline [21 ] suggest that amitriptyline may reduce neuropathic pain by this mechanism, and studies in animals show reversal of inflammatory and NMDA-induced hyperalgesia by spinal amitriptyline. [8 ] Other studies have found reductions in ongoing pain, hyperalgesia, and allodynia by systemic administration of NMDA antagonists (ketamine or dextromethorphan) in patients with neuropathic pain [16,22 ] and in volunteers receiving intradermal injections of capsaicin, [5 ] but only at doses that produced symptoms of intoxication. We could not observe any such effect of amitriptyline alone in our study, although the dose administered was sufficient to produce sedation. It is possible, therefore, that amitriptyline fails to act as an NMDA antagonist in this model and in patients with chronic pain, who typically achieve pain relief only after weeks of amitriptyline administration. On the other hand, it could also reflect inability to achieve sufficient concentrations of amitriptyline in the spinal cord after acute systemic administration to affect these elicited responses. Other researchers have suggested that future treatment of neuropathic pain with fewer side effects may be possible with spinal administration of NMDA antagonists, [16,22 ] and we are performing toxicology studies to support investigation of spinal injection of amitriptyline in humans.
Spinal injection of amitriptyline enhances analgesia from systemic opioids in animals, probably because amitriptyline is inhibited of reuptake of serotonin and norepinephrine. [23,24 ] However, systemic administration of monoamine reuptake inhibitors, including amitriptyline, exhibits minimal enhancement of analgesia from systemic opioids in humans. [25–27 ] Similarly, we failed to observe enhancement of alfentanil analgesia to acute or subacute pain responses to intradermal capsaicin in the current study. Again, spinal injection of amitriptyline would be a more logical choice to enhance the effects of opioids. Amitriptyline significantly reduced nausea from alfentanil in our study in volunteers. The mechanism for this effect and its applicability to clinical settings are unclear.
Finally, we could not clearly determine the effect of an active placebo (midazolam) in this, because pain responses to the second control capsaicin injection were reduced compared to the first. Because the last two injections were similar to the second, we could either conclude that midazolam had an analgesic effect (compared with first injection) or that it did not (compared with the second injection). Although not conclusive, a previous study of human volunteers showed no effect of midazolam (mean dose, 3.4 mg) on ongoing pain, hyperalgesia, and allodynia after intradermal injection of capsaicin. [5 ]
In summary, acute pain, hyperalgesia, and allodynia are similar in four repeated intradermal injections of capsaicin during 4 h in the leg and arm of volunteers. Systemically administered alfentanil reduces all these pain responses, but only at high plasma concentrations (more than 150 ng/ml) associated with side effects. Amitriptyline in a dose causing acute sedation did not affect these pain responses, nor did it enhance alfentanil's effects. Ongoing studies are aimed at introducing spinal injection of amitriptyline into clinical trials as a more effective route of administration in the treatment of neuropathic pain.
The authors thank Robert LaMotte, Ph.D., for helping to design the experiments and establishing the intradermal capsaicin model at our institution.
References 
References 
Simone DA, Baumann TK, LaMotte RH: Dose-dependent pain and mechanical hyperalgesia in humans after intradermal injection of capsaicin. Pain 1989; 38:99-107.
LaMotte RH, Shain CN, Simone DA, Tsai E-FP: Neurogenic hyperalgesia: psychophysical studies of underlying mechanisms. J Neurophysiol 1991; 66:190-211.
Dougherty PM, Palecek J, Paleckova V, Sorkin LS, Willis WD: The role of NMDA and non-NMDA excitatory amino acid receptors in the excitation of primate spinothalamic tract neurons by mechanical, chemical, thermal, and electrical stimuli. J Neurosci 1992; 12:3025-41.
Dougherty PM, Willis WD: Enhanced responses of spinothalamic tract neurons to excitatory amino acids accompany capsaicin-induced sensitization in the monkey. J Neurosci 1992; 12:883-94.
Park KM, Max MB, Robinovitz E, Gracely RH, Bennett GJ: Effects of intravenous ketamine, alfentanil, or placebo on pain, pinprick hyperalgesia, and allodynia produced by intradermal capsaicin in human subjects. Pain 1995; 63:163-72.
Bakker MHM, McKernan RM, Wong EHF, Foster AC: [sup 3 H]MK-801 binding to N-methyl-D-aspartate receptors solubilized from rat brain: effects of glycine site ligands, polyamines, ifenprodil, and desipramine. J Neurochem 1991; 57:39-45.
Sernagor E, Kuhn D, Vyklicky L Jr, Mayer ML: Open channel block of NMDA receptor responses evoked by tricyclic antidepressants. Neuron 1989; 2:1221-7.
Eisenach JC, Gebhart GF: Intrathecal amitriptyline acts as an N-methyl-D-asparate receptor antagonist in the presence of inflammatory hyperalgesia in rats. Anesthesiology 1995; 83:1046-54.
Eisenach JC, Gebhart GF: Intrathecal amitriptyline2d-antinociceptive interactions with intravenous morphine and intrathecal clonidine, neostigmine, and carbamylcholine in rats. Anesthesiology 1995; 83:1036-45.
Coudore F, Ardid D, Eschalier A, Lavarenne J, Fialip J: High-performance liquid chromatographic determination of amitriptyline and its main metabolites using a silica column with reversed-phase eluent. Application in mice. J Chromatogr B Biomed Appl 1992; 584:249-55.
Bjorkman S, Aziz N, Stein D, Stanski DR: Determination of alfentanil in serum by radioimmunoassay or capillary column gas-liquid chromatography. A comparison of the assays. Acta Pharm Nord 1989; 1:211-20.
LaMotte RH, Thalhammer JG, Robinson CJ: Peripheral neural correlates of magnitude of cutaneous pain and hyperalgesia: a comparison of neural events in monkey with sensory judgments in human. J Neurophysiol 1983; 50:1-25.
LaMotte RH, Lundberg LER, Torebjork HE: Pain, hyperalgesia and activity in nociceptive C units in humans after intradermal injection of capsaicin. J Physiol 1992; 448:749-64.
Torebjork HE, Lundberg LER, LaMotte RH: Central changes in processing of mechanoreceptive input in capsaicin-induced secondary hyperalgesia in humans. J Physiol 1992; 448:765-80.
Bennett GJ: Animal Models of Neuropathic Pain, Proceedings of the 7th World Congress on Pain. Edited by Gebhart GF, Hammond DL, Jensen TS. Seattle, WA, IASP Press, 1994, pp. 495-510.
Max MB, Byas-Smith MG, Gracely RH, Bennett GJ: Intravenous infusion of the NMDA antagonist, ketamine, in chronic posttraumatic pain with allodynia: a double-blind comparison to alfentanil and placebo. Clin Neuropharmacol 1995; 18:360-8.
Hill HF, Chapman CR, Saeger LS, Bjurstrom R, Walter MH, Schoene RB, Kippes M: Steady-state infusions of opioids in human. II. Concentration-effect relationships and therapeutic margins. Pain 1990; 43:69-79.
Irwin MG, Jones RDM, Visram AR, Kenny GNC: Patient-controlled alfentanil-target-controlled infusion for postoperative analgesia. Anaesthesia 1996; 51:427-30.
Ueda M, Sugimoto K, Oyama T, Kuraishi Y, Satoh M: Opioidergic inhibition of capsaicin-evoked release of glutamate from rat spinal dorsal horn slices. Neuropharmacology 1995; 34:303-8.
Onghena P, Houdenhove BV: Antidepressant-induced analgesia in chronic non-malignant pain: a meta-analysis of 39 placebo-controlled studies. Pain 1992; 49:205-19.
Watanabe Y, Saito H, Abe K: Tricyclic antidepressants block NMDA receptor-mediated synaptic responses and induction of long-term potentiation in rat hippocampal slices. Neuropharmacology 1993; 32:479-86.
Kauppila T, Gronroos M, Pertovaara A: An attempt to attenuate experimental pain in humans by dextromethorphan, an NMDA receptor antagonist. Pharmacol Biochem Behav 1995; 52:641-4.
Botney M, Fields HL: Amitriptyline potentiates morphine analgesia by a direct action on the central nervous system. Ann Neurol 1983; 13:160-4.
Larsen J-J, Arnt J: Spinal 5-HT or NA uptake inhibition potentiates supraspinal morphine antinociception in rats. Acta Pharmacol et Toxicol 1984; 54:72-5.
Kerrick JM, Fine PG, Lipman AG, Love G: Low-dose amitriptyline as an adjunct to opioids for postoperative orthopedic pain: a placebo-controlled trial. Pain 1993; 52:325-30.
Levine JD, Gordon NC, Smith R, McBryde R: Desipramine enhances opiate postoperative analgesia. Pain 1986; 27:45-9.
Gordon NC, Heller PH, Gear RW, Levine JD: Interactions between fluoxetine and opiate analgesia for postoperative dental pain. Pain 1994; 58:85-8.
Figure 1. Magnitude of pain (4), area of hyperalgesia (B), and allodynia (C) after the first (square), second (open circle), third (filled circle), and fourth (closed inverted triangle) hourly intradermal injection of 100 micro gram capsaicin. For pain, each symbol represents the mean of six volunteers, with +standard deviation and -standard deviation shown for the first and fourth injections, respectively. For hyperalgesia and allodynia, each symbol represents the median of six volunteers, with 75th and 25th percentiles shown for the first and fourth injections, respectively. Injection site was randomly assigned as the right and left upper and lower extremities. No difference was observed in pain response or areas of hyperalgesia or allodynia as a function of injection order.
Figure 1. Magnitude of pain (4), area of hyperalgesia (B), and allodynia (C) after the first (square), second (open circle), third (filled circle), and fourth (closed inverted triangle) hourly intradermal injection of 100 micro gram capsaicin. For pain, each symbol represents the mean of six volunteers, with +standard deviation and -standard deviation shown for the first and fourth injections, respectively. For hyperalgesia and allodynia, each symbol represents the median of six volunteers, with 75th and 25th percentiles shown for the first and fourth injections, respectively. Injection site was randomly assigned as the right and left upper and lower extremities. No difference was observed in pain response or areas of hyperalgesia or allodynia as a function of injection order.
Figure 1. Magnitude of pain (4), area of hyperalgesia (B), and allodynia (C) after the first (square), second (open circle), third (filled circle), and fourth (closed inverted triangle) hourly intradermal injection of 100 micro gram capsaicin. For pain, each symbol represents the mean of six volunteers, with +standard deviation and -standard deviation shown for the first and fourth injections, respectively. For hyperalgesia and allodynia, each symbol represents the median of six volunteers, with 75th and 25th percentiles shown for the first and fourth injections, respectively. Injection site was randomly assigned as the right and left upper and lower extremities. No difference was observed in pain response or areas of hyperalgesia or allodynia as a function of injection order.
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Figure 2. Plasma alfentanil (A) or amitriptyline (B) concentrations in volunteers randomized to receive alfentanil alone (filled triangle) or with intramuscular amitriptyline (open circle), or amitriptyline alone (filled circle) or with alfentanil (open circle). Each symbol represents the mean +/- SD of eight to ten volunteers. No differences were observed between groups, except earlier peak amitriptyline concentrations in the amitriptyline-alfentanil group (see text for details).
Figure 2. Plasma alfentanil (A) or amitriptyline (B) concentrations in volunteers randomized to receive alfentanil alone (filled triangle) or with intramuscular amitriptyline (open circle), or amitriptyline alone (filled circle) or with alfentanil (open circle). Each symbol represents the mean +/- SD of eight to ten volunteers. No differences were observed between groups, except earlier peak amitriptyline concentrations in the amitriptyline-alfentanil group (see text for details).
Figure 2. Plasma alfentanil (A) or amitriptyline (B) concentrations in volunteers randomized to receive alfentanil alone (filled triangle) or with intramuscular amitriptyline (open circle), or amitriptyline alone (filled circle) or with alfentanil (open circle). Each symbol represents the mean +/- SD of eight to ten volunteers. No differences were observed between groups, except earlier peak amitriptyline concentrations in the amitriptyline-alfentanil group (see text for details).
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Figure 3. Magnitude of pain after intradermal injection of capsaicin, averaged for the first two injections (control: open square), or after the fourth capsaicin injection after intramuscular midazolam (open diamond), intramuscular amitriptyline (open triangle), intravenous alfentanil (200 ng/ml targeted plasma concentration: Filled circle), or the combination of amitriptyline and alfentanil (filled inverted triangle). Each symbol represents the mean of ten volunteers, with +standard deviation and -standard deviation shown for the control and amitriptyline-plus-alfentanil groups, respectively. *P < 0.05 for comparison of alfentanil alone and with amitriptyline to control by two-way analysis of variance.
Figure 3. Magnitude of pain after intradermal injection of capsaicin, averaged for the first two injections (control: open square), or after the fourth capsaicin injection after intramuscular midazolam (open diamond), intramuscular amitriptyline (open triangle), intravenous alfentanil (200 ng/ml targeted plasma concentration: Filled circle), or the combination of amitriptyline and alfentanil (filled inverted triangle). Each symbol represents the mean of ten volunteers, with +standard deviation and -standard deviation shown for the control and amitriptyline-plus-alfentanil groups, respectively. *P < 0.05 for comparison of alfentanil alone and with amitriptyline to control by two-way analysis of variance.
Figure 3. Magnitude of pain after intradermal injection of capsaicin, averaged for the first two injections (control: open square), or after the fourth capsaicin injection after intramuscular midazolam (open diamond), intramuscular amitriptyline (open triangle), intravenous alfentanil (200 ng/ml targeted plasma concentration: Filled circle), or the combination of amitriptyline and alfentanil (filled inverted triangle). Each symbol represents the mean of ten volunteers, with +standard deviation and -standard deviation shown for the control and amitriptyline-plus-alfentanil groups, respectively. *P < 0.05 for comparison of alfentanil alone and with amitriptyline to control by two-way analysis of variance.
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Figure 4. Area of hyperalgesia after the first (open square), second (open circle), third (filled circle), or fourth (filled inverted triangle) intradermal capsaicin injection in volunteers receiving intravenous alfentanil (A), intramuscular amitriptyline (B), alfentanil plus amitriptyline (C), or intramuscular midazolam (D). Each symbol represents the median of ten volunteers, with 75th and 25th percentiles shown for the first and fourth injections, respectively. *P < 0.05 for comparison of the fourth injection of alfentanil alone and with amitriptyline to the first injection. (tau)P < 0.05 for second, third, and fourth injections compared with the first.
Figure 4. Area of hyperalgesia after the first (open square), second (open circle), third (filled circle), or fourth (filled inverted triangle) intradermal capsaicin injection in volunteers receiving intravenous alfentanil (A), intramuscular amitriptyline (B), alfentanil plus amitriptyline (C), or intramuscular midazolam (D). Each symbol represents the median of ten volunteers, with 75th and 25th percentiles shown for the first and fourth injections, respectively. *P < 0.05 for comparison of the fourth injection of alfentanil alone and with amitriptyline to the first injection. (tau)P < 0.05 for second, third, and fourth injections compared with the first.
Figure 4. Area of hyperalgesia after the first (open square), second (open circle), third (filled circle), or fourth (filled inverted triangle) intradermal capsaicin injection in volunteers receiving intravenous alfentanil (A), intramuscular amitriptyline (B), alfentanil plus amitriptyline (C), or intramuscular midazolam (D). Each symbol represents the median of ten volunteers, with 75th and 25th percentiles shown for the first and fourth injections, respectively. *P < 0.05 for comparison of the fourth injection of alfentanil alone and with amitriptyline to the first injection. (tau)P < 0.05 for second, third, and fourth injections compared with the first.
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Figure 5. Area of allodynia after the first (open square), second (open circle), third (filled circle), or fourth (filled inverted diamond) intradermal capsaicin injection in volunteers receiving intravenous alfentanil (A), intramuscular amitriptyline (B), alfentanil plus amitriptyline (C), or intramuscular midazolam (D). Each symbol represents the median of ten volunteers, with 75th and 25th percentiles shown for the first and fourth injections, respectively. *P < 0.05 for comparison of the fourth injection of alfentanil alone and with amitriptyline with the first injection. (tau)P < 0.05 for the second, third, and fourth injections compared with the first.
Figure 5. Area of allodynia after the first (open square), second (open circle), third (filled circle), or fourth (filled inverted diamond) intradermal capsaicin injection in volunteers receiving intravenous alfentanil (A), intramuscular amitriptyline (B), alfentanil plus amitriptyline (C), or intramuscular midazolam (D). Each symbol represents the median of ten volunteers, with 75th and 25th percentiles shown for the first and fourth injections, respectively. *P < 0.05 for comparison of the fourth injection of alfentanil alone and with amitriptyline with the first injection. (tau)P < 0.05 for the second, third, and fourth injections compared with the first.
Figure 5. Area of allodynia after the first (open square), second (open circle), third (filled circle), or fourth (filled inverted diamond) intradermal capsaicin injection in volunteers receiving intravenous alfentanil (A), intramuscular amitriptyline (B), alfentanil plus amitriptyline (C), or intramuscular midazolam (D). Each symbol represents the median of ten volunteers, with 75th and 25th percentiles shown for the first and fourth injections, respectively. *P < 0.05 for comparison of the fourth injection of alfentanil alone and with amitriptyline with the first injection. (tau)P < 0.05 for the second, third, and fourth injections compared with the first.
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Figure 6. Visual analog scale sedation in volunteers randomized to receive or amitriptyline alone (filled circle) or intramuscular midazolam alone (open square; A), or alfentanil alone (filled triangle) or with intramuscular amitriptyline (open circle; B). Each symbol represents the mean +/- SD of ten volunteers. There was no difference between groups.
Figure 6. Visual analog scale sedation in volunteers randomized to receive or amitriptyline alone (filled circle) or intramuscular midazolam alone (open square; A), or alfentanil alone (filled triangle) or with intramuscular amitriptyline (open circle; B). Each symbol represents the mean +/- SD of ten volunteers. There was no difference between groups.
Figure 6. Visual analog scale sedation in volunteers randomized to receive or amitriptyline alone (filled circle) or intramuscular midazolam alone (open square; A), or alfentanil alone (filled triangle) or with intramuscular amitriptyline (open circle; B). Each symbol represents the mean +/- SD of ten volunteers. There was no difference between groups.
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