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Departments of
*Anesthesiology and
Physiology I, University of Erlangen-Nuremberg, Erlangen, Germany; and
Department of Anesthesiology, LKH Klagenfurt, Austria
Address correspondence and reprint requests to Dr. W. Koppert, Department of Anesthesiology, University of Erlangen-Nuremberg, Germany, Krankenhausstr. 12, D-91054 Erlangen, Germany. Address e-mail to koppert{at}physiologie1.uni-erlangen.de
| Abstract |
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Implications: We investigated the peripheral effects of fentanyl and ketamine on capsaicin-induced hyperalgesia and axon-reflex flare. In large concentrations, the opioid diminished axon-reflex flare without effects on secondary hyperalgesia. We found no evidence for the involvement of endogenous glutamate in secondary hyperalgesia or axon reflex flare.
| Introduction |
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-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid- and the N-methyl-D-aspartate- (NMDA) receptors play an important role in developing central sensitization. Upon spinal and systemic administration of opioids, NMDA-receptor antagonists can prevent central sensitization (46). However, some experimental research suggests that these types of drugs can have direct antinociceptive effects on peripheral nerve endings and may play an important role in the modulation of both primary and secondary hyperalgesia (7,8). We evaluated the peripheral effects of the opioid fentanyl and of the NMDA-receptor antagonist ketamine on capsaicin-induced hyperalgesia. An intradermal injection of a small dose of capsaicin represents a commonly used experimental human pain model, which reliably produces dermal neurogenic inflammation with intense burning pain, secondary brush-evoked allodynia, and secondary hyperalgesia to punctate stimuli (9,10). Besides determination of sensory capacities, the activation of C nociceptors was evaluated. For this purpose, the axon-reflex flare was examined by measuring the superficial blood flow around the injection sites with laser-Doppler imaging. Short accounts of the present work have been published in abstract form. | Methods |
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In two series of experiments, either 200 µL saline 0.9% and 200 µL fentanyl solution (1 µg or 10 µg, respectively) or 200 µL saline 0.9% and 200 µL ketamine solution (100 µg or 1000 µg, respectively) were simultaneously injected intradermally into the central volar forearm 4 cm apart (Fig. 1). Nine minutes later, 20 µL of a capsaicin solution (10 µg in 7% ethanol and H2O) was injected intradermally exactly between the two previous injection sites by using a 100-µL syringe topped with a sterile filter (Fig. 1B). The volunteers rated the pain magnitude at 15-s intervals for 2 min after capsaicin injection. Pain ratings were given on a horizontal visual analog scale; the end points of the scale were defined as "no pain" (0) and "maximum imaginable pain" (10).
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Superficial blood flow around the injection site was repeatedly assessed using a laser-Doppler imager (Moor Instruments Ltd., Devon, UK). An area of 9 x 4.5 cm around the injection sites was scanned with a resolution of 16380 pixels, from which each pixel represented a separate Doppler-flux measurement. Flare profile and mean flux around the injection sites of the drugs were determined and processed with dedicated software (Fig. 1A). Images were taken twice under control conditions, three times after the injection of the drugs, and four times after capsaicin injection (Fig. 1B).
In another series of experiments, direct analgesic effects of peripheral fentanyl and ketamine were investigated comparing saline 0.9% (negative control) and lidocaine 1% (positive control) with fentanyl (1, 5, and 10 µg) and ketamine (100, 500, and 1000 µg). According to the preceding experiment, 200 µL was injected intradermally in the medial aspect of the central volar forearm of the subjects. Calibrated von Frey filaments were used to determine the detection thresholds. The subjects were instructed to close their eyes and report when they felt a touch sensation. Filaments exerting increasing bending forces were applied five times each for 1 s until the subject had correctly sensed at least 3 of 5 trials.
Students t-tests were performed to examine differences between single pain ratings and hyperalgesic as well as allodynic areas after the different treatments. Repetitive pain ratings after capsaicin injection and data obtained from the laser-Doppler imager measurements were statistically evaluated using analysis of variance in a two-way within-subjects (repeated measures) model. Scheffé post hoc tests were performed when suitable. Changes in detection thresholds were evaluated using Wilcoxons signed rank test. Significance levels throughout this study were P < 0.05; all data were expressed as mean ± SD, except for figures (mean ± SEM, median ± 25th/75th percentile). The STATISTICA software package (Statsoft, Tulsa, NC) was used for statistical analyses.
| Results |
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After capsaicin injection, pain ratings induced by mechanical stimulation (pinprick hyperalgesia) around the injection sites of fentanyl and ketamine 100 µg did not differ significantly from the ratings of the saline injection site (Table 1). In contrast, pain ratings induced by stimulation around the injection site of ketamine 1000 µg were significantly reduced during the observation period (Table 1). However, no significant differences were found in the size (radius) of the hyperalgesic and allodynic areas between fentanyl and saline or between ketamine and saline (Table 2).
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Local anesthetic effects on mechanical stimulation were found after ketamine injection (Fig. 4). Ketamine led to a dose-dependent increase of von Frey detection thresholds, whereas fentanyl, even in the largest concentration, did not cause impairment of touch sensation. After the injection of ketamine (1000 µg), von Frey detection thresholds increased approximately four-fold (3.8, 3.68.9; median, 25th75th percentile) (Fig. 4).
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| Discussion |
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In microneurographic experiments in humans, burning pain after capsaicin injection was observed in parallel with continuing discharges in polymodal C fibers (11). Both pain and C fiber discharges disappeared after cooling the skin (11). Therefore, continuous nociceptor discharges near the injection site are most probably the source of burning pain after capsaicin injection. In our study, drugs were injected intradermally 2 cm from the capsaicin injection site and, thus, had no effect on the magnitude or time course of pain.
We observed the well known features of capsaicin-induced secondary hyperalgesia to mechanical stimulation after intradermal injection of capsaicin, including touch-evoked allodynia and pinprick hyperalgesia. There is evidence that allodynia and pinprick hyperalgesia are generated by sensitization of spinal neurons by noxious input of primary nociceptive fibers (12,13). Opioids, as well as NMDA-receptor antagonists, significantly reduced pain and signs of secondary hyperalgesia when administered systemically or spinally, which would also favor a central mechanism (4,5). Fentanyl exerts its effects on spinal cord neurons preferentially via µ-, but also
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-receptors, by blocking voltage-gated calcium channels or by opening potassium channels, resulting in neuronal hyperpolarization. In contrast, ketamine interacts with multiple binding sites, including NMDA and non-NMDA glutamate receptors and cholinergic and opioid receptors, with the latter two having only a minor role in its analgesic effect (14).
However, peripheral mechanisms for the induction of pinprick hyperalgesia have also been postulated. Using infrared thermography, Ochoa et al. (15) found precise matching of punctate mechanical hyperalgesia and heat hyperalgesia with the flare response after intradermal injection of capsaicin. This vascular-sensory matching was described by Lewis (16) and was assumed to be of peripheral origin, because it seems unlikely that central sensitization occurs exactly in those dorsal horn neurons that match a local vascular process in the skin. Polymodal C fibers do not change their properties in the area of secondary hyperalgesia. Thus, Ochoa et al. (15) argued that sensitization of silent C nociceptors in areas of secondary hyperalgesia after capsaicin injection in human skin could be a peripheral mechanism for secondary hyperalgesia. However, this report is in conflict with the unaltered sensitivity of silent nociceptors after capsaicin injection found by an another group (17).
In our study, no effects of peripherally administrated opioids and NMDA-receptor antagonists on the area of secondary hyperalgesia were observed. Opioids produce antinociceptive effects on terminal nerve endings, mainly under inflammatory conditions (18); convincing results were achieved only in reducing primary hyperalgesia to heat and to mechanical stimulation (7,19,20). Kinnman et al. (7) reported that morphine, administered subcutaneously before capsaicin injection, attenuates capsaicin-evoked primary and secondary hyperalgesia to mechanical stimulation. Although no effect on capsaicin-induced continuous pain was observed in their study, the reduction of primary hyperalgesia is most likely mediated by altered responsiveness of polymodal C fibers by morphine. We assumed that the decreased temporal summation of nociceptive input from the primary site contributes to the antihyperalgesic effects of opioids in the secondary zone.
Similar mechanisms were observed for the NMDA-receptor antagonist ketamine. Warncke et al. (8) found local antihyperalgesic effects of ketamine after burn-induced hyperalgesia. Pain thresholds to mechanical and heat stimulation at the site of primary hyperalgesia were elevated and the area of secondary hyperalgesia was diminished after ketamine pretreatment. Again, a combination of antihyperalgesic effects on primary and secondary hyperalgesia was observed.
Thus, the modulation of secondary hyperalgesia by peripherally-applied opioids and NMDA-receptor antagonists seen in previous studies is most likely a spinal effect after a diminished nociceptive input from the area of primary hyperalgesia, because no effect on the area in which secondary hyperalgesia was seen when the primary hyperalgesic zone was unaffected.
The axon-reflex flare (neurogenic flare) mechanism is resolved locally in the skin and spreads through the arborizations of activated polymodal C nociceptor. The activation of terminal nerve endings causes the release of substance P and calcitonin gene-related peptide with subsequent vasodilatation of small arterioles in the receptive fields of activated nociceptors surrounding the injury site. Laser-Doppler imaging provides a rapid, noninvasive, detailed analysis of intensity and spatial pattern of vasodilation in the skin, and in contrast to the analysis of the visible flare, is a more sensitive method for investigating changes of superficial blood flow.
In our study, fentanyl 10 µg and ketamine 1000 µg inhibited the capsaicin-induced axon-reflex flare only around the injection sites. We propose distinct mechanisms for either fentanyl or ketamine based on observations that ketamine 1000 µg did increase mechanical thresholds while fentanyl 10 µg did not.
The inhibition of the axon-reflex flare by fentanyl 10 µg without effecting mechanical thresholds is a result of a peripheral inhibition of neuropeptide release by opioids (21). Polymodal C nociceptors are the most likely the target for the peripheral site of action of opioids (20,22). Some authors have hypothesized that, by preventing neurogenic inflammation, opioids could reduce capsaicin-induced activation of C nociceptors with subsequent central sensitization (7). However, although an inhibition of neuropeptide release by fentanyl was determined in our study, no antihyperalgesic effect was visible. Additionally, our findings do not support the concept of a tight relation between vascular and sensory changes observed after capsaicin injection, which has been discussed as evidence for a peripheral mechanism of secondary mechanical hyperalgesia (15).
In contrast, ketamine in a concentration of 1.8 mM (100 µg in 200 µL) did not significantly reduce capsaicin-induced axon-reflex vasodilation or secondary hyperalgesia. It should be emphasized that the concentration of 1.8 mM is already supramaximal for the blockade of NMDA-receptors by ketamine (half maximal inhibiting concentrations [IC50] in cultured neurons: 19 µM) (23,24).
When the ketamine concentration was even increased to 18 mM (1000 µg in 200 µL), a decrease in capsaicin-induced axon-reflex vasodilation and secondary hyperalgesia was observed at the injection site. In parallel, this concentration of ketamine massively increased mechanical detection threshold, indicating a local anesthetic effect. Laboratory investigations have revealed different target sites for ketamine on peripheral nerves. Besides its effect on NMDA- and opioid-receptors, ketamine also shows local anesthetic properties by blocking Na+ and K+ currents in peripheral nerve preparations (2527). The concentrations necessary, however, were much larger than those reached during systemic administration and could only be achieved by local application. The IC50 for the peak Na+ currents varied from 325 µM to 2 mM, depending on differences in species and experimental conditions (2628). In contrast, IC50 values for blockade of NMDA-receptors have been reported to be in the range of 19 µM for ketamine. The concentrations we used varied from 1.8 to 18 mM. Therefore, we assume that the analgesic effect of peripheral ketamine observed at a concentration of 18 mM is most probably a result of local anesthetic properties rather than blocking NMDA-receptors.
In conclusion, we have demonstrated that peripherally administered fentanyl and ketamine do not affect the area of capsaicin-induced secondary hyperalgesia. In previous studies, antihyperalgesic effects of both drugs in primary and secondary hyperalgesia have been demonstrated when injected into the primary hyperalgesic area. We assume that modulation of secondary hyperalgesia by opioids and NMDA-receptor antagonists seen in these studies is most likely caused by a diminished nociceptive input from the area of primary hyperalgesia, leading to a reduction of spinal secondary hyperalgesia. Our findings do not support a role of opioids or endogenous glutamate in peripheral sensitization of nociceptors in the secondary zone. In contrast, the results are consistent with a spinal origin of secondary hyperalgesia to touch and punctate stimuli.
| Acknowledgments |
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The authors thank Dieter Märkert for his technical assistance.
| Footnotes |
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| References |
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