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Neurosensory changes in a human model of endothelin-1 induced pain: A behavioral study
Neuroscience Letters 418 (2007) 117–121
Neurosensory changes in a human model of endothelin-1 induced pain: A behavioral study Guy Hans a,b,∗ , Kristof Deseure a,b , Dominique Robert a,b , Stefan De Hert a,b a b
Multidisciplinary Pain Center, Antwerp University Hospital, Edegem, Belgium Department of Anesthesiology, Antwerp University Hospital, Edegem, Belgium
Received 23 January 2007; received in revised form 4 March 2007; accepted 5 March 2007
Abstract Although pain is a frequent feature in patients with cancer, its etiology is still poorly understood. In recent years, endothelin-1 (ET-1) has become a major target molecule in the etiology of cancer pain. In this randomised, double-blind study the effects of intradermal injection of ET-1 on spontaneous pain, temperature perception and sensation of punctate stimulation were evaluated. Thirty-five subjects were randomised to receive either placebo or one of four concentrations of ET-1 (ranging from 10−10 to 10−6 M). Besides assessment of spontaneous pain, three neurosensory testings were performed: (1) cold and warm sensation, (2) cold and heat pain, and (3) punctate stimulation using a von Frey monofilament. ET-1 produced a dose-dependent flare zone that was absent after placebo injection. Subjects reported a short-lasting spontaneous pain upon administration of the highest concentrations of ET-1. Injection of ET-1 induced a long-lasting and dose-dependent punctate hyperalgesia in an area around the injection site (secondary hyperalgesia). Thermal testing revealed a short period of hypoesthesia to non-noxious warm and cold stimuli after some doses of ET-1. In addition to the mechanical hyperalgesia, intradermal injection of ET-1 almost instantaneously induced a state of cold hyperalgesia outlasting the study period (120 min). No development of heat hyperalgesia was observed. The observed psychophysical characteristics of this new model of ET-1 induced nociception indicate its potential as a human experimental model for cancer pain. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Endothelin-1; Intradermal injection; Cancer pain; Human
Pain is a frequent and disabling consequence of (metastatic) cancer syndromes in humans [7]. Our understanding of the basic mechanisms that underlie the development of pain associated with malignancy is limited, but may involve mediator-dependent signalling by tumor cells. Tumor cells are known to secrete a variety of different substances, many of which are potential algogens [10,18]. One of these mediators is endothelin-1 (ET-1). This 21-residue peptide, known mainly for its potent vasoconstrictor effects, is secreted in high concentrations by several cancer cell lines, such as metastatic prostate and breast cancer cells [22,27,32]. Increasing data suggest that multiple functions of the endothelin axis have associations with mitogenesis, apoptosis inhibition, and angiogenesis [17,34]. In addition, a role for this peptide in nociception is also envisaged and as such ET-1 is known to induce pain or overt nociception in animals and humans [13,14,29]. ET-1 also seems ∗ Corresponding author at: Antwerp University Hospital, Multidisciplinary Pain Center, Wilrijkstraat 10, 2650 Edegem, Belgium (BE). Tel.: +32 3 8214945; fax: +32 3 8214586. E-mail address: [email protected] (G. Hans).
0304-3940/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2007.03.008
to trigger the development of hyperalgesia to noxious chemical, mechanical and thermal stimuli [8,25,26,28]. A crucial point for supporting the ET-1 role in nociception was the demonstration that ET-1 induced pain does not derive from its vasoconstrictor effect [13]. Furthermore, in vivo and in vitro neurophysiological studies have shown that the nociceptive actions of ET-1 are most likely mediated by direct excitation of nociceptive primary afferents [16,36]. New findings suggest that activation of ETA receptors leads to the onset of nociceptive behaviors in animal pain models [20], whereas ETB receptors seem to play a role in communicating an inhibitory signal. When injected into human skin, ET-1 causes a potent, long-lasting vasoconstriction at the site of the administration, surrounded by a profound, wide-spread vasodilatation [6,11,23]. Previous studies with ET-1 in humans have primarily focused on its vasoconstrictive effects. Nociceptive changes were hereby considered as side effects and never the main focus of interest. Injection of ET-1 into the brachial artery induced severe muscular pain and prolonged, touch-evoked allodynia [12]. Another study reported a burning pruritus after intradermal injection of ET-1 [19].
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Aiming to further characterize the role of ET-1 in human nociception, the present study was designed to elucidate whether intradermal injection of minute doses of ET-1 altered the perception of different sensory modalities. In addition, doseresponsiveness and time course of these sensory alterations were investigated to infer mechanisms of action of ET-1. To our understanding this is the first report targeting the ET-1-induced sensory alterations in humans. Experiments were performed on the volar aspect of the right forearm of 35 healthy male volunteers (mean age 26 ± 4 years, range 21–38). Subjects were asked to refrain from food and caffeine-containing drinks for at least 4 h prior to their participation in the study in order to prevent any interfering vasoconstrictive effects from these substances. All experiments were performed in the supine position in a room at a constant temperature and a relative humidity of 30.2 ± 1.4%. None of the subjects were on any medication. The aim of the study and the nature of the testing were explained to the subjects. The local Ethics Committee of the Antwerp University Hospital approved the study. All volunteers gave written informed consent to participate in the study, which was performed according to the Declaration of Helsinki. A randomized, double-blinded, placebo-controlled design methodology was applied. Volunteer subjects were randomized to one of five experimental groups (n = 7 for each group), receiving either placebo or one of the following concentrations of ET-1: 10−6 , 10−7 , 10−8 and 10−10 M. After rating of spontaneous pain, three neurosensory tests were performed: (a) warm and cold sensation, (b) heat and cold pain, and (c) punctate stimulation. The same order of the stimuli was used in all subjects: punctate stimulation, cool, warm, cold pain and heat pain. This order was chosen because it ranges from the lowest stimulus to the highest stimulus. Following the reading of a standard script of instructions, baseline neurosensory testing was performed on the volar aspect of the subject’s right forearm (half-way between the wrist and antecubital fossa). The skin was prepped with an alcohol pad and 1 min later 40 l of one of four concentrations of ET-1 or 40 l of 0.9% saline placebo was injected intradermally using a micro-volume syringe mounted with a 25-gauge needle (SGE, Australia). To ensure proper intradermal injection, the surface of the skin was stretched and the tip of the needle was inserted, bevel upwards, almost parallel to the skin surface. ET-1 solution was then slowly injected into the uppermost layer of the skin, inducing a raised papule with ‘peau d’orange’ appearance. Solutions of ET-1 (Clinalfa® , Merck Biosciences AG, Switzerland) were prepared by dilution with 0.9% sterile pyrogen-free saline solution (PBS) to give final concentrations of 10−6 , 10−7 , 10−8 and 10−10 M. An equivalent of normal sterile saline in identical vials was prepared for placebo testing. The doses of ET-1 chosen for this experiment represent the lower end of those applied in previous studies on the vasoconstrictive effects of ET-1 and were considered safe for administration to volunteers with minimal risk of side effects and unblinding the study. Pain rating and neurosensory testing was performed before and at 1, 10, 30, 60 and 120 min after ET-1 or placebo injection. To minimize external influences, all testing was performed by the same investigator (GH) and took place at the same time of day. All subjects
and the examiner were blinded with respect to administration of ET-1 or saline. The code was broken after data entry and analysis. At the beginning of each testing session the subjects were asked to report any sensation of pain. Volunteers rated the intensity of spontaneous pain induced by ET-1 using a visual analogue scale (VAS) 10 cm in length and anchored by word descriptors at each end (left-hand end: ‘no pain’ and on the right-hand end: ‘worst imaginable pain’). Subjects marked on the line the point that they felt represented their current state of nociception. Development of secondary hyperalgesia was assessed by punctate mechanical stimulation with a von Frey monofilament applied at 90◦ to the skin surface (bending force of 254.9 mN, Stoelting Co., USA). This von Frey probe, which causes only a slight discomfort sensation in normal skin, was applied along a line that marked the edge of the visual flare. The subjects were instructed to report the occurrence of a definite change in sensation during this stimulation, often to a more intense stinging with a prolonged after-sensation. The hyperalgesic area was defined as the skin region in which punctate stimulation produced a definite change in the quality of the sensation described by the subjects as ‘painful’, ‘burning’, ‘tenderness’, ‘more intense pricking’, ‘more unpleasant’ (from high to low intensity). No numerical scale was used; subjects were asked to describe the qualitative perception of von Frey hair stimulation in the presence or absence of ET-1 treatment to confirm that the descriptors mentioned above were reported after treatment only. These response codes have been used previously by other investigators in order to monitor development of hyperalgesia in humans [5,35]. Perception thresholds for thermal sensation (warm, cold, cold pain and heat pain) were measured by a thermotest device (Medoc TSA 2001, Ramat Yishay, Israel) prior to and after ET-1 application and placebo. A thermode of Peltier elements measuring 32 mm × 32 mm was applied exactly in the flare area surrounding the ET-1 injection site. The temperature of the thermode could either rise or fall depending on the direction and the intensity of the current flow through the Peltier device. The methods of limits was used by applying ramp stimuli with a velocity of 1 ◦ C/s starting from 32 ◦ C (baseline temperature). Warm and cold detection thresholds, cold pain thresholds, and heat pain thresholds (in that order) were recorded. By pressing a button, subjects indicated when the respective thresholds were reached. Thresholds were calculated as the average of four (cold and warm sensation) or three (cold and heat pain) successive measurements with a random interstimulus interval of 3–8 s. All measurements of a given perception were completed before testing the next perception. To protect the skin from possible thermal injury, the increase of the thermode temperature was limited to 50.5 ◦ C. Limit for decreasing temperatures was 0 ◦ C. Prior sample size calculation for differences in spontaneous pain and sensitivity to punctate stimulation, revealed the necessity of a sample size of 7 (double sided population analysis with alpha 0.05 and power 0.9). To compare the data of spontaneous pain measurement and thermal testing, two-way repeated measures ANOVA (two-way RM-ANOVA) was performed, and a
G. Hans et al. / Neuroscience Letters 418 (2007) 117–121
Fig. 1. Effects of ET-1 administration on visual analogue scale (VAS). Saline (䊉) or one of four different dosages of ET-1 were injected: 10−10 M (), 10−8 M (), 10−7 M (♦), and 10−6 M (). Asterisks indicate a significant difference between ET-1 injection and saline (Student–Newman–Keul’s test, p < 0.05). Data are expressed as mean values ± S.E.M.
post-hoc Student–Newman–Keul’s for pair-wise multiple comparisons was made if the ANOVA was significant. Response scores to punctate stimulation were evaluated using nonparametric analysis (Friedman test), and a post-hoc Dunn’s multiple comparisons test was performed if this Friedman test showed significance. The significance level throughout this study was p < 0.05. Analyses were performed using Prism’s Statistical software (version 4.0c for Macintosh). Intradermal injections of ET-1 caused in all subjects the development of a central pale zone (around the injection site), surrounded by a much larger zone of flare. Magnitude and duration of flare seemed dependent on the dose of ET-1, outlasting the study period in the highest doses of the peptide. Spontaneous ongoing pain (Fig. 1) developed rapidly after administration of 10−6 and 10−7 M ET-1 (maximum score 1 min after injection) and decreased gradually to reach pre-injection
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Fig. 2. Time course of changes in response code to punctate stimulation. Saline (䊉) or one of four different dosages of ET-1 were administered: 10−10 M (), 10−8 M (), 10−7 M (♦), and 10−6 M (). The following response codes were applied: normal sensation (indicated by 0 in the X-axis of the graph), ‘more unpleasant’ (1), ‘more intense pricking’ (2), ‘tenderness’ (3), ‘burning’ (4) and ‘painful’ (5). Data are expressed as median values ± interquartile range. Over time 10−7 M and 10−6 M ET-1 induced a significant increase in response scores compared to placebo (Friedman test, Dunn’s post-hoc testing, p < 0.05).
levels, respectively, 30 and 60 min after ET-1 administration. There was a clear dose-dependent effect in induction of spontaneous pain. Pain sensations induced by the two highest doses of ET-1 were not only significantly more pronounced compared to saline (p < 0.05), but also in comparison to injections of lower doses of ET-1 (not shown on graph). The two highest doses of ET-1 led to a significant tenderness to von Frey probe stimulation compared to placebo and lowest doses of ET-1 (p < 0.05). Secondary hyperalgesia in response to punctate mechanical stimuli was observed after 10 min and outlasted the observation period (120 min). Fig. 2 shows a clear dose-responsiveness between the applied dose of ET-1 and the intensity of punctate hyperalgesia.
Fig. 3. Time course of detection thresholds for (A) cold sensation, (B) cold pain, (C) warm sensation and (D) heat pain. Saline (䊉) and one of four different dosages of ET-1 were applied: 10−10 M (), 10−8 M (), 10−7 M (♦), and 10−6 M (). Asterisks indicate a significant difference between ET-1 and placebo (Student–Newman–Keul’s test, p < 0.05). Data are expressed as mean values ± S.E.M.
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No differences in the baseline thermal threshold were noted in the study volunteers. Injection of saline did not induce any significant change in thermal sensation. Detection thresholds for cold sensation were not significantly altered by ET-1 compared to placebo (Fig. 3A), except for the highest dose which induced a significant increase in detection threshold after 60 min and that persisted until the end of the study period (p < 0.05). Volunteer subjects who received the two highest doses of ET-1 displayed significant decreases in detection thresholds for cold pain, compared to placebo (p < 0.05) as well as to the lower ET1 doses (p < 0.05). Cold hyperalgesia developed quickly after injection (between 1 and 10 min) and persisted throughout the entire observation period (Fig. 3B). Significant changes occurred in detection of warmth (Fig. 3C). With exception of the lowest dose, all three higher doses elicited a significant dose-dependent increase in detection threshold for warm sensation (p < 0.05). In comparison to cold sensation, the observed changes in warm detection developed sooner, lasted longer and were more pronounced. Detection thresholds for heat pain (Fig. 3D) were never influenced by intradermal injection of ET-1 (p > 0.05). The present study constitutes to our knowledge the first report on the neurosensory effects of intradermal injection of endothelin-1 (ET-1) in humans. The results demonstrate that intradermal administration of ET-1 elicits spontaneous sensations of pain, as well as cold and punctate hyperalgesia. In addition, ET-1 injection caused a dose-dependent area of pallor that has been shown to be associated with a significant reduction in basal skin blood flow [11]. Besides its vasoconstrictor effects, ET-1 produced a dose-dependent flare that surrounded the area of pallor. This erythema is associated with a significant increase in skin blood flow and has been shown to be a sign of neurogenic inflammation (axon reflex) [6,4,24]. It should be mentioned that the observed responses cannot be attributed to vasoconstrictive effects of ET-1. Animal studies have shown that administration of equipotent doses of epinephrine did not cause any pain or sensory alterations [13]. In addition, adrenergic injections in normal human skin produced a dose-dependant decrease in heat-pain threshold, but failed to show any changes in mechanical thresholds [15]. The potential role of vasoactive substances (e.g. histamine, nitric oxide, cyclooxygenase products) in ET1-induced nociception remains unclear. It has been previously suggested that the flare component is partially histamine dependent [3]. Human data suggest that the mechano-insensitive type of C-nociceptors plays a key role in the generation of axon reflex erythema and development of itch [21]. By contrast, the common polymodal (mechano-sensitive) nociceptors only show spurious histamine responses [31]. Considering these neurophysiological interactions of histamine, the low doses of ET-1 applied in our study and the fact that none of our subjects reported any pruritic symptoms, it is doubtful that histamine plays a major role in the observed hyperalgesic syndrome. In contrast to the pronounced hyperalgesic changes, administration of ET-1 causes moderate and short-lasting spontaneous pain sensations. An interaction between ET-1 and nociception was suggested earlier, although most of these human studies merely provided descriptions of painful and/or itching sensa-
tions without performing any quantitative neurosensory testing [12,19]. Previous reports on the algesic effects of ET-1 in humans situated its peak effect around 2 min after injection—with a total duration of 10 min [19]. It is therefore possible that we missed the peak in spontaneous sensation (if situated between 1 and 10 min after injection). When evaluating the intensity of the non-evoked nociceptive sensations, we should consider the low doses that were applied in this study. We opted to investigate concentrations that were shown to be safe for administration to volunteer subjects. Finally, previous studies have shown that application of very low doses of ET-1 causes minimal nociception, but still could lead to prolonged and robust mechanical allodynia [2]. Previous studies have suggested that ET-1 produces nociception [30], and potentiates various types of inflammatory pain [29]. In addition, electrophysiological experiments have shown that injection of ET-1 into the receptive field causes rapid and long-lasting spontaneous discharges from C fibers [16]. The testing area around the area of flare displayed the occurrence of different evoked pain symptoms. First a pronounced and long-lasting mechanical hyperalgesia was observed (secondary hyperalgesia). As peripheral sensitization does not spread after injury, this secondary hyperalgesia should be considered the result of central sensitization. As it is suggested that nociceptors signal punctate hyperalgesia, one can conclude that ET-1 leads to activation of cutaneous C-fiber nociceptors that sensitizes interneurons in the spinal cord that project high-threshold mechanonociceptor (HTM) input to central pain-signalling neurons (CPSN) [1]. This would be in accordance with the findings of animal electrophysiologic studies where ET-1 was shown to induce selective activation of small fiber afferents [16]. Despite the very short half-life of ET-1, mechanical hyperalgesia outlasted the duration of the observation period. This finding is in accordance with previous animal studies, where longlasting tactile allodynia was observed after administration of low doses of ET-1 [2]. In addition, a recent study has shown that the long-lasting mechanical allodynia that occurs after chronic constriction injury of the rat’s infraorbital nerve (IoN-CCI) results from endothelin receptor-mediated mechanisms [8]. A surprising finding was the development of a diminished threshold to noxious cold (cold hyperalgesia). Hyperalgesia to cold stimuli develops after carrageenan injection [9], as well as after complete Freund’s adjuvant [33]. Recently, investigators have shown that IoN-CCI induces orofacial cold hyperalgesia, which could be reversed by endothelin receptor antagonists [9]. In the present study cold hyperalgesia developed within minutes after ET-1 administration, suggesting that it depends largely on peripheral ET-linked mechanisms. Since cold hyperalgesia is a crucial element of multiple clinical neuropathic syndromes, this element holds promise for further investigations. Some methodological limitations should be considered. The time schedule did not allow a full characterization of the time course of spontaneous pain sensations. Future studies should focus specifically on the development of non-evoked pain during the first minutes after ET-1 administration. In addition, a more detailed evaluation of the area of flare and the resulting changes in sensitivity to mechanical stimulation would have increased the value of our findings, but this was not possible
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Neurosensory changes in a human model of endothelin-1 induced pain: A behavioral study Guy Hans a,b,∗ , Kristof Deseure a,b , Dominique Robert a,b , Stefan De Hert a,b a b
Multidisciplinary Pain Center, Antwerp University Hospital, Edegem, Belgium Department of Anesthesiology, Antwerp University Hospital, Edegem, Belgium
Received 23 January 2007; received in revised form 4 March 2007; accepted 5 March 2007
Abstract Although pain is a frequent feature in patients with cancer, its etiology is still poorly understood. In recent years, endothelin-1 (ET-1) has become a major target molecule in the etiology of cancer pain. In this randomised, double-blind study the effects of intradermal injection of ET-1 on spontaneous pain, temperature perception and sensation of punctate stimulation were evaluated. Thirty-five subjects were randomised to receive either placebo or one of four concentrations of ET-1 (ranging from 10−10 to 10−6 M). Besides assessment of spontaneous pain, three neurosensory testings were performed: (1) cold and warm sensation, (2) cold and heat pain, and (3) punctate stimulation using a von Frey monofilament. ET-1 produced a dose-dependent flare zone that was absent after placebo injection. Subjects reported a short-lasting spontaneous pain upon administration of the highest concentrations of ET-1. Injection of ET-1 induced a long-lasting and dose-dependent punctate hyperalgesia in an area around the injection site (secondary hyperalgesia). Thermal testing revealed a short period of hypoesthesia to non-noxious warm and cold stimuli after some doses of ET-1. In addition to the mechanical hyperalgesia, intradermal injection of ET-1 almost instantaneously induced a state of cold hyperalgesia outlasting the study period (120 min). No development of heat hyperalgesia was observed. The observed psychophysical characteristics of this new model of ET-1 induced nociception indicate its potential as a human experimental model for cancer pain. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Endothelin-1; Intradermal injection; Cancer pain; Human
Pain is a frequent and disabling consequence of (metastatic) cancer syndromes in humans [7]. Our understanding of the basic mechanisms that underlie the development of pain associated with malignancy is limited, but may involve mediator-dependent signalling by tumor cells. Tumor cells are known to secrete a variety of different substances, many of which are potential algogens [10,18]. One of these mediators is endothelin-1 (ET-1). This 21-residue peptide, known mainly for its potent vasoconstrictor effects, is secreted in high concentrations by several cancer cell lines, such as metastatic prostate and breast cancer cells [22,27,32]. Increasing data suggest that multiple functions of the endothelin axis have associations with mitogenesis, apoptosis inhibition, and angiogenesis [17,34]. In addition, a role for this peptide in nociception is also envisaged and as such ET-1 is known to induce pain or overt nociception in animals and humans [13,14,29]. ET-1 also seems ∗ Corresponding author at: Antwerp University Hospital, Multidisciplinary Pain Center, Wilrijkstraat 10, 2650 Edegem, Belgium (BE). Tel.: +32 3 8214945; fax: +32 3 8214586. E-mail address: [email protected] (G. Hans).
0304-3940/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2007.03.008
to trigger the development of hyperalgesia to noxious chemical, mechanical and thermal stimuli [8,25,26,28]. A crucial point for supporting the ET-1 role in nociception was the demonstration that ET-1 induced pain does not derive from its vasoconstrictor effect [13]. Furthermore, in vivo and in vitro neurophysiological studies have shown that the nociceptive actions of ET-1 are most likely mediated by direct excitation of nociceptive primary afferents [16,36]. New findings suggest that activation of ETA receptors leads to the onset of nociceptive behaviors in animal pain models [20], whereas ETB receptors seem to play a role in communicating an inhibitory signal. When injected into human skin, ET-1 causes a potent, long-lasting vasoconstriction at the site of the administration, surrounded by a profound, wide-spread vasodilatation [6,11,23]. Previous studies with ET-1 in humans have primarily focused on its vasoconstrictive effects. Nociceptive changes were hereby considered as side effects and never the main focus of interest. Injection of ET-1 into the brachial artery induced severe muscular pain and prolonged, touch-evoked allodynia [12]. Another study reported a burning pruritus after intradermal injection of ET-1 [19].
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Aiming to further characterize the role of ET-1 in human nociception, the present study was designed to elucidate whether intradermal injection of minute doses of ET-1 altered the perception of different sensory modalities. In addition, doseresponsiveness and time course of these sensory alterations were investigated to infer mechanisms of action of ET-1. To our understanding this is the first report targeting the ET-1-induced sensory alterations in humans. Experiments were performed on the volar aspect of the right forearm of 35 healthy male volunteers (mean age 26 ± 4 years, range 21–38). Subjects were asked to refrain from food and caffeine-containing drinks for at least 4 h prior to their participation in the study in order to prevent any interfering vasoconstrictive effects from these substances. All experiments were performed in the supine position in a room at a constant temperature and a relative humidity of 30.2 ± 1.4%. None of the subjects were on any medication. The aim of the study and the nature of the testing were explained to the subjects. The local Ethics Committee of the Antwerp University Hospital approved the study. All volunteers gave written informed consent to participate in the study, which was performed according to the Declaration of Helsinki. A randomized, double-blinded, placebo-controlled design methodology was applied. Volunteer subjects were randomized to one of five experimental groups (n = 7 for each group), receiving either placebo or one of the following concentrations of ET-1: 10−6 , 10−7 , 10−8 and 10−10 M. After rating of spontaneous pain, three neurosensory tests were performed: (a) warm and cold sensation, (b) heat and cold pain, and (c) punctate stimulation. The same order of the stimuli was used in all subjects: punctate stimulation, cool, warm, cold pain and heat pain. This order was chosen because it ranges from the lowest stimulus to the highest stimulus. Following the reading of a standard script of instructions, baseline neurosensory testing was performed on the volar aspect of the subject’s right forearm (half-way between the wrist and antecubital fossa). The skin was prepped with an alcohol pad and 1 min later 40 l of one of four concentrations of ET-1 or 40 l of 0.9% saline placebo was injected intradermally using a micro-volume syringe mounted with a 25-gauge needle (SGE, Australia). To ensure proper intradermal injection, the surface of the skin was stretched and the tip of the needle was inserted, bevel upwards, almost parallel to the skin surface. ET-1 solution was then slowly injected into the uppermost layer of the skin, inducing a raised papule with ‘peau d’orange’ appearance. Solutions of ET-1 (Clinalfa® , Merck Biosciences AG, Switzerland) were prepared by dilution with 0.9% sterile pyrogen-free saline solution (PBS) to give final concentrations of 10−6 , 10−7 , 10−8 and 10−10 M. An equivalent of normal sterile saline in identical vials was prepared for placebo testing. The doses of ET-1 chosen for this experiment represent the lower end of those applied in previous studies on the vasoconstrictive effects of ET-1 and were considered safe for administration to volunteers with minimal risk of side effects and unblinding the study. Pain rating and neurosensory testing was performed before and at 1, 10, 30, 60 and 120 min after ET-1 or placebo injection. To minimize external influences, all testing was performed by the same investigator (GH) and took place at the same time of day. All subjects
and the examiner were blinded with respect to administration of ET-1 or saline. The code was broken after data entry and analysis. At the beginning of each testing session the subjects were asked to report any sensation of pain. Volunteers rated the intensity of spontaneous pain induced by ET-1 using a visual analogue scale (VAS) 10 cm in length and anchored by word descriptors at each end (left-hand end: ‘no pain’ and on the right-hand end: ‘worst imaginable pain’). Subjects marked on the line the point that they felt represented their current state of nociception. Development of secondary hyperalgesia was assessed by punctate mechanical stimulation with a von Frey monofilament applied at 90◦ to the skin surface (bending force of 254.9 mN, Stoelting Co., USA). This von Frey probe, which causes only a slight discomfort sensation in normal skin, was applied along a line that marked the edge of the visual flare. The subjects were instructed to report the occurrence of a definite change in sensation during this stimulation, often to a more intense stinging with a prolonged after-sensation. The hyperalgesic area was defined as the skin region in which punctate stimulation produced a definite change in the quality of the sensation described by the subjects as ‘painful’, ‘burning’, ‘tenderness’, ‘more intense pricking’, ‘more unpleasant’ (from high to low intensity). No numerical scale was used; subjects were asked to describe the qualitative perception of von Frey hair stimulation in the presence or absence of ET-1 treatment to confirm that the descriptors mentioned above were reported after treatment only. These response codes have been used previously by other investigators in order to monitor development of hyperalgesia in humans [5,35]. Perception thresholds for thermal sensation (warm, cold, cold pain and heat pain) were measured by a thermotest device (Medoc TSA 2001, Ramat Yishay, Israel) prior to and after ET-1 application and placebo. A thermode of Peltier elements measuring 32 mm × 32 mm was applied exactly in the flare area surrounding the ET-1 injection site. The temperature of the thermode could either rise or fall depending on the direction and the intensity of the current flow through the Peltier device. The methods of limits was used by applying ramp stimuli with a velocity of 1 ◦ C/s starting from 32 ◦ C (baseline temperature). Warm and cold detection thresholds, cold pain thresholds, and heat pain thresholds (in that order) were recorded. By pressing a button, subjects indicated when the respective thresholds were reached. Thresholds were calculated as the average of four (cold and warm sensation) or three (cold and heat pain) successive measurements with a random interstimulus interval of 3–8 s. All measurements of a given perception were completed before testing the next perception. To protect the skin from possible thermal injury, the increase of the thermode temperature was limited to 50.5 ◦ C. Limit for decreasing temperatures was 0 ◦ C. Prior sample size calculation for differences in spontaneous pain and sensitivity to punctate stimulation, revealed the necessity of a sample size of 7 (double sided population analysis with alpha 0.05 and power 0.9). To compare the data of spontaneous pain measurement and thermal testing, two-way repeated measures ANOVA (two-way RM-ANOVA) was performed, and a
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Fig. 1. Effects of ET-1 administration on visual analogue scale (VAS). Saline (䊉) or one of four different dosages of ET-1 were injected: 10−10 M (), 10−8 M (), 10−7 M (♦), and 10−6 M (). Asterisks indicate a significant difference between ET-1 injection and saline (Student–Newman–Keul’s test, p < 0.05). Data are expressed as mean values ± S.E.M.
post-hoc Student–Newman–Keul’s for pair-wise multiple comparisons was made if the ANOVA was significant. Response scores to punctate stimulation were evaluated using nonparametric analysis (Friedman test), and a post-hoc Dunn’s multiple comparisons test was performed if this Friedman test showed significance. The significance level throughout this study was p < 0.05. Analyses were performed using Prism’s Statistical software (version 4.0c for Macintosh). Intradermal injections of ET-1 caused in all subjects the development of a central pale zone (around the injection site), surrounded by a much larger zone of flare. Magnitude and duration of flare seemed dependent on the dose of ET-1, outlasting the study period in the highest doses of the peptide. Spontaneous ongoing pain (Fig. 1) developed rapidly after administration of 10−6 and 10−7 M ET-1 (maximum score 1 min after injection) and decreased gradually to reach pre-injection
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Fig. 2. Time course of changes in response code to punctate stimulation. Saline (䊉) or one of four different dosages of ET-1 were administered: 10−10 M (), 10−8 M (), 10−7 M (♦), and 10−6 M (). The following response codes were applied: normal sensation (indicated by 0 in the X-axis of the graph), ‘more unpleasant’ (1), ‘more intense pricking’ (2), ‘tenderness’ (3), ‘burning’ (4) and ‘painful’ (5). Data are expressed as median values ± interquartile range. Over time 10−7 M and 10−6 M ET-1 induced a significant increase in response scores compared to placebo (Friedman test, Dunn’s post-hoc testing, p < 0.05).
levels, respectively, 30 and 60 min after ET-1 administration. There was a clear dose-dependent effect in induction of spontaneous pain. Pain sensations induced by the two highest doses of ET-1 were not only significantly more pronounced compared to saline (p < 0.05), but also in comparison to injections of lower doses of ET-1 (not shown on graph). The two highest doses of ET-1 led to a significant tenderness to von Frey probe stimulation compared to placebo and lowest doses of ET-1 (p < 0.05). Secondary hyperalgesia in response to punctate mechanical stimuli was observed after 10 min and outlasted the observation period (120 min). Fig. 2 shows a clear dose-responsiveness between the applied dose of ET-1 and the intensity of punctate hyperalgesia.
Fig. 3. Time course of detection thresholds for (A) cold sensation, (B) cold pain, (C) warm sensation and (D) heat pain. Saline (䊉) and one of four different dosages of ET-1 were applied: 10−10 M (), 10−8 M (), 10−7 M (♦), and 10−6 M (). Asterisks indicate a significant difference between ET-1 and placebo (Student–Newman–Keul’s test, p < 0.05). Data are expressed as mean values ± S.E.M.
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No differences in the baseline thermal threshold were noted in the study volunteers. Injection of saline did not induce any significant change in thermal sensation. Detection thresholds for cold sensation were not significantly altered by ET-1 compared to placebo (Fig. 3A), except for the highest dose which induced a significant increase in detection threshold after 60 min and that persisted until the end of the study period (p < 0.05). Volunteer subjects who received the two highest doses of ET-1 displayed significant decreases in detection thresholds for cold pain, compared to placebo (p < 0.05) as well as to the lower ET1 doses (p < 0.05). Cold hyperalgesia developed quickly after injection (between 1 and 10 min) and persisted throughout the entire observation period (Fig. 3B). Significant changes occurred in detection of warmth (Fig. 3C). With exception of the lowest dose, all three higher doses elicited a significant dose-dependent increase in detection threshold for warm sensation (p < 0.05). In comparison to cold sensation, the observed changes in warm detection developed sooner, lasted longer and were more pronounced. Detection thresholds for heat pain (Fig. 3D) were never influenced by intradermal injection of ET-1 (p > 0.05). The present study constitutes to our knowledge the first report on the neurosensory effects of intradermal injection of endothelin-1 (ET-1) in humans. The results demonstrate that intradermal administration of ET-1 elicits spontaneous sensations of pain, as well as cold and punctate hyperalgesia. In addition, ET-1 injection caused a dose-dependent area of pallor that has been shown to be associated with a significant reduction in basal skin blood flow [11]. Besides its vasoconstrictor effects, ET-1 produced a dose-dependent flare that surrounded the area of pallor. This erythema is associated with a significant increase in skin blood flow and has been shown to be a sign of neurogenic inflammation (axon reflex) [6,4,24]. It should be mentioned that the observed responses cannot be attributed to vasoconstrictive effects of ET-1. Animal studies have shown that administration of equipotent doses of epinephrine did not cause any pain or sensory alterations [13]. In addition, adrenergic injections in normal human skin produced a dose-dependant decrease in heat-pain threshold, but failed to show any changes in mechanical thresholds [15]. The potential role of vasoactive substances (e.g. histamine, nitric oxide, cyclooxygenase products) in ET1-induced nociception remains unclear. It has been previously suggested that the flare component is partially histamine dependent [3]. Human data suggest that the mechano-insensitive type of C-nociceptors plays a key role in the generation of axon reflex erythema and development of itch [21]. By contrast, the common polymodal (mechano-sensitive) nociceptors only show spurious histamine responses [31]. Considering these neurophysiological interactions of histamine, the low doses of ET-1 applied in our study and the fact that none of our subjects reported any pruritic symptoms, it is doubtful that histamine plays a major role in the observed hyperalgesic syndrome. In contrast to the pronounced hyperalgesic changes, administration of ET-1 causes moderate and short-lasting spontaneous pain sensations. An interaction between ET-1 and nociception was suggested earlier, although most of these human studies merely provided descriptions of painful and/or itching sensa-
tions without performing any quantitative neurosensory testing [12,19]. Previous reports on the algesic effects of ET-1 in humans situated its peak effect around 2 min after injection—with a total duration of 10 min [19]. It is therefore possible that we missed the peak in spontaneous sensation (if situated between 1 and 10 min after injection). When evaluating the intensity of the non-evoked nociceptive sensations, we should consider the low doses that were applied in this study. We opted to investigate concentrations that were shown to be safe for administration to volunteer subjects. Finally, previous studies have shown that application of very low doses of ET-1 causes minimal nociception, but still could lead to prolonged and robust mechanical allodynia [2]. Previous studies have suggested that ET-1 produces nociception [30], and potentiates various types of inflammatory pain [29]. In addition, electrophysiological experiments have shown that injection of ET-1 into the receptive field causes rapid and long-lasting spontaneous discharges from C fibers [16]. The testing area around the area of flare displayed the occurrence of different evoked pain symptoms. First a pronounced and long-lasting mechanical hyperalgesia was observed (secondary hyperalgesia). As peripheral sensitization does not spread after injury, this secondary hyperalgesia should be considered the result of central sensitization. As it is suggested that nociceptors signal punctate hyperalgesia, one can conclude that ET-1 leads to activation of cutaneous C-fiber nociceptors that sensitizes interneurons in the spinal cord that project high-threshold mechanonociceptor (HTM) input to central pain-signalling neurons (CPSN) [1]. This would be in accordance with the findings of animal electrophysiologic studies where ET-1 was shown to induce selective activation of small fiber afferents [16]. Despite the very short half-life of ET-1, mechanical hyperalgesia outlasted the duration of the observation period. This finding is in accordance with previous animal studies, where longlasting tactile allodynia was observed after administration of low doses of ET-1 [2]. In addition, a recent study has shown that the long-lasting mechanical allodynia that occurs after chronic constriction injury of the rat’s infraorbital nerve (IoN-CCI) results from endothelin receptor-mediated mechanisms [8]. A surprising finding was the development of a diminished threshold to noxious cold (cold hyperalgesia). Hyperalgesia to cold stimuli develops after carrageenan injection [9], as well as after complete Freund’s adjuvant [33]. Recently, investigators have shown that IoN-CCI induces orofacial cold hyperalgesia, which could be reversed by endothelin receptor antagonists [9]. In the present study cold hyperalgesia developed within minutes after ET-1 administration, suggesting that it depends largely on peripheral ET-linked mechanisms. Since cold hyperalgesia is a crucial element of multiple clinical neuropathic syndromes, this element holds promise for further investigations. Some methodological limitations should be considered. The time schedule did not allow a full characterization of the time course of spontaneous pain sensations. Future studies should focus specifically on the development of non-evoked pain during the first minutes after ET-1 administration. In addition, a more detailed evaluation of the area of flare and the resulting changes in sensitivity to mechanical stimulation would have increased the value of our findings, but this was not possible
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