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Capsaicin sensitive afferents mediate the development of heat hyperalgesia and hindpaw edema after sciatic section in rats
Neuroscience Letters 318 (2002) 39–43 www.elsevier.com/locate/neulet
Capsaicin sensitive afferents mediate the development of heat hyperalgesia and hindpaw edema after sciatic section in rats Wade S. Kingery a,b,*, Geeta S. Agashe c,d, Tian-Zhi Guo e, M. Frances Davies c,d, J. David Clark c,d, Mervyn Maze e a
Department of Functional Restoration, Stanford University School of Medicine, Stanford, CA, USA Physical Medicine and Rehabilitation Service, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA, USA c Department of Anesthesia, Stanford University School of Medicine, Stanford, CA, USA d Anesthesiology Service, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA, USA e Magill Department of Anaesthetics, Imperial College School of Medicine, London, UK
b
Received 2 October 2001; received in revised form 6 November 2001; accepted 6 November 2001
Abstract Sciatic section in rats evokes chronic hyperalgesia, autotomy pain behavior, and hindpaw edema, a constellation resembling complex regional pain syndrome (CRPS) in man. Glucocorticoid treatment inhibits these sequelae of sciatic section and also blocks neurogenic extravasation. Small diameter afferent neurons release substance P (SP), a mediator of both hyperalgesia and neurogenic extravasation. Now, we show that pre-emptive destruction of the small diameter fibers prevents neurogenic extravasation, and prevented the development of heat hyperalgesia and hindpaw edema after sciatic section. Thus, capsaicin sensitive primary afferent neurons which release SP are required for the development of heat hyperalgesia and hindpaw edema in this animal model of CRPS. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Edema; Neurogenic extravasation; Nerve injury; Complex regional pain syndrome
Nerve injuries can lead to the development of a complex regional pain syndrome (CRPS), including hyperalgesia (increased sensitivity to painful stimuli), spontaneous pain, distal limb edema, and increased spontaneous cutaneous protein extravasation [12]. We have shown that we can develop a model of this syndrome by sciatic nerve transection in rats [13–15]. In both the clinical condition of CRPS, as well as in the rat model, treatment with methylprednisolone can reverse edema and hyperalgesia; it also prevented the development of autotomy pain behavior in the rat model. We posited that the steroids inhibit neurogenic inflammation to produce these actions. Neurogenic inflammation is initiated by activation of small diameter primary afferent neurons through the release of substance P (SP), which binds to NK1 receptors on blood vessels; the resulting increase in vascular permeability causes fluid extravasation. Capsaicin, the pungent ingredient in hot chili peppers, activates the vanilloid receptors * Corresponding author. Physical Medicine and Rehabilitation Service (117), Veterans Affairs Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, CA 94304, USA. Tel.: 11-650493-5000 ext. 64768; fax: 11-650-852-3470. E-mail address: [email protected] (W.S. Kingery).
(VR1) which are selectively expressed by most of the small diameter primary afferents, including almost all the SP containing neurons [24]. Capsaicin activation of the VR1 causes an influx of calcium and sodium cations into the sensory neuron, triggering an excitotoxic effect. Injecting neonatal rats with capsaicin (50 mg/kg, s.c.) will selectively and permanently destroy virtually all of the SP containing neurons, thus inhibiting the evoked neurogenic extravasation response [9,19]. To test the hypothesis that capsaicin sensitive afferent neurons mediate the sequelae of nerve injury seen in this model of CRPS, the small diameter afferents were lesioned, and we determined the effects on neurogenic extravasation, acute heat and mechanical nociceptive thresholds, the development of hyperalgesia, autotomy behavior, and hindpaw edema. Male Sprague–Dawley rats (B&K Universal) were used in this study. Forty-eight hours after birth, rat pups were subcutaneously injected over the dorsal spine with either 50 mg/kg capsaicin (Sigma, St Louis, MO) or vehicle (10:10:80 v/v of Tween-80/ethanol/saline) in a volume of 5 ml/g. The 30 gauge needle was left in the skin for 60 s after the injection to ensure that the injectant did not reflux back out the needle tract. At the time of injection, each rat underwent either right
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 46 4- 8
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W.S. Kingery et al. / Neuroscience Letters 318 (2002) 39–43
or left ear punching to identify the treatment group. All further assays were performed in a blinded manner. Eight weeks after neonatal injection, the rats were deeply anesthetized with isoflurane and the saphenous nerve was exposed in the right thigh and a pair of platinum electrodes were applied. Evans blue (50 mg/kg; Sigma, St Louis, MO) was administered intravenously in a 50 mg/ml solution of 0.9% saline. After 10 min, the nerve was tightly ligated and then stimulated distal to the site of ligation (5 Hz, 0.5 ms pulse duration, 10 mA, for 10 min). The animals were then euthanized, and all the hindpaw skin was removed and placed in 4 ml of 99% formamide at room temperature for 72 h. The dye concentration in the formamide was then determined spectrophotometrically at a wavelength of 620 nm. Another type of neurogenic extravasation assay was performed using subcutaneous capsaicin injection. Capsaicin activates the VR1 on the SP containing sensory neurons [24], causing the release of SP with a subsequent increase in microvascular permeability. Eight weeks after neonatal injection, the rats were deeply anesthetized with isoflurane, and Evans blue (50 mg/kg; Sigma, St Louis, MO) was administered. Ten minutes later, the rats were subcutaneously injected over the dorsum of the hindpaw with a 50 ml solution of capsaicin (75 mg) or vehicle (7% ethanol). Ten minutes after subcutaneous injection, the animals were euthanized, and a 40 mg piece of dorsal hindpaw skin was excised from the center of the injection site and placed in formamide at room temperature for 72 h, after which the dye concentration was determined spectrophotometrically. Behavioral testing was performed in a dimly lit room, maintained between 22 and 248C. The rats were gently held during the nociceptive assay and only tested when they were quietly resting in the investigator’s hand. Heat nociceptive thresholds were determined as we have previously described [14], using the mean of three consecutive withdrawal thresholds to a Peltier device (4 £ 4 cm surface, CP1: 4-127-06L, Melcor, Trenton, NJ) applied to the medial dorsum of the hindpaw. A linear ramped temperature (18C/s, starting at 408C and with a cut-off of 528C) was used, and the examiner controlled the Peltier using a foot pedal switch. Mechanical nociceptive withdrawal responses were determined as we have previously described [14], using calibrated von Frey fibers (North Coast Medical, San Jose, CA). Four different fibers were used in graduating sequence (10, 23, 57 and 85 g), for a total of 12 consecutive fiber applications. The withdrawal threshold was the smallest fiber size that evoked at least two withdrawal responses during three consecutive applications with the same fiber. Autotomy behavior following sciatic transection was measured as described by Wall et al. [25]. A score of 1 was given for the removal of one or more toe-nails. An additional score of 1 was added for each distal half digit attacked. A further score of 1 was added for each proximal half digit autotomized. Thus, if all toe-nails and all parts of all toes innervated by the sciatic nerve (the three lateral digits) were removed in one hindpaw, a maximal score of
7 was achieved. During this investigation, autotomy behavior was only observed the insensate digits. Edema was measured as we have previously described [15], using a manual caliper to determine the maximum dorsal–ventral thickness of the hindpaw. Eight weeks after neonatal injection, the rats were trained for nociceptive testing for 2 consecutive days before baseline nociceptive thresholds and hindpaw thickness were determined on the third day. Thereafter, during isoflurane anesthesia, the right sciatic nerve was exposed at the midthigh level, a 1 cm segment of nerve excised, and the incision closed. Animals were tested at weekly intervals for 7 weeks post-operatively. The data were analyzed using two-way repeatedmeasures analysis of variance (ANOVA), comparing treatment groups (capsaicin vs. vehicle). Post-hoc differences at a given time were tested using the two-tailed Student’s ttest. A one-way repeated-measures ANOVA was used to determine change over time within one treatment group. Saphenous nerve stimulation caused extravasation of Evans blue dye in the hindpaw skin (19.6 ^ 2.8 mg vs. no measurable dye content after sham stimulation). Neonatal capsaicin treatment reduced saphenous nerve evoked extravasation by 68% (P , 0:01; Fig. 1a). In the neonatal vehicle treated rats, a subcutaneous capsaicin injection evoked significant cutaneous dye extravasation (P , 0:05; Fig. 1b). Neonatal capsaicin treatment had no effect on vehicle evoked extravasation, but completely blocked capsaicin
Fig. 1. Neonatal capsaicin treatment blocked neurogenic extravasation responses in adult rats to electrical stimulation (a), and subcutaneous capsaicin injection (b), indicating a loss of SP containing neurons. Capsaicin treated rats had a saphenous evoked hindpaw dye content of 6.3 ^ 3.1 vs. 19.6 ^ 2.8 mg for vehicle treated animals (n ¼ 7 per cohort). In the neonatal vehicle treated rats, a subcutaneous capsaicin injection evoked significantly more cutaneous dye extravasation (186 ^ 31 ng dye/mg skin) than an injection with vehicle alone (81 ^ 28 ng dye/mg skin). In rats that were neonatally treated with capsaicin, the extravasation response to a capsaicin injection (88 ^ 12 ng dye/mg skin) was completely blocked as compared with injection of vehicle (56 ^ 17 ng/mg; n ¼ 7 per cohort). *P , 0:05, **P , 0:01.
W.S. Kingery et al. / Neuroscience Letters 318 (2002) 39–43
evoked extravasation (compared with vehicle evoked extravasation; Fig. 1b). We examined the effects of neonatal capsaicin treatment on the development of hindpaw heat and mechanical hyperalgesia, autotomy behavior, and hindpaw edema after sciatic nerve transection (n ¼ 20 per cohort). Capsaicin treatment had no effect on baseline nociception when measured by: (1), medial dorsum heat withdrawal thresholds; (2), tail-flick latencies to radiant heat (3.18 ^ 0.06 vs. 3.29 ^ 0.05 s for vehicle treated, data not shown); or (3), medial dorsum mechanical withdrawal thresholds (Fig. 2). The vehicle treated rats developed significant hindpaw heat and mechanical hyperalgesia, autotomy behavior, and hindpaw edema within 2 weeks of sciatic section, which persisted for at least 7 weeks
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post-operatively. Neonatal capsaicin treatment prevented the development of hindpaw heat hyperalgesia and edema after sciatic section, but had no effect on the development of mechanical hyperalgesia or autotomy behavior. The results of this study indicate that capsaicin sensitive primary afferent neurons are required for neurogenic extravasation and the development of hindpaw edema after sciatic section. The contribution of neurogenic processes to the development and maintenance of chronic hindpaw edema has not been previously noted. The cutaneous injection of capsaicin, which acutely stimulates the release of SP from sensory C-fiber terminals, does evoke a brief edema response that has a large neurogenic extravasation component [2,7]. Some investigators have observed a neurogenic extravasa-
Fig. 2. This experiment evaluated the effect of neonatal capsaicin treatment on the development of hindpaw heat hyperalgesia (a) and mechanical hyperalgesia (b), autotomy behavior (c), and hindpaw edema (d) over 7 weeks after sciatic nerve transection (n ¼ 20 per cohort). Neonatal capsaicin treatment had no effect on baseline nociception for medial dorsum heat withdrawal thresholds (50.9 ^ 0.1 vs. 51.1 ^ 0.28C for vehicle treated) or medial dorsum mechanical withdrawal (67 ^ 3 vs. 73 ^ 4 g in vehicle treated). The vehicle treated rats developed significant heat and mechanical hyperalgesia within 2 weeks of sciatic section (one-way repeated-measures ANOVA, compared with the baseline threshold, P , 0:001), and this persisted for at least 7 weeks post-operatively. Neonatal capsaicin treatment prevented the development of hindpaw heat hyperalgesia, but not mechanical hyperalgesia after sciatic section (one-way repeatedmeasures ANOVA, compared with the baseline threshold). There was a significant difference between the heat thresholds of the neonatal capsaicin treatment group and the vehicle treated group (two-way repeated-measures ANOVA, P , 0:001), starting at 3 weeks after sciatic section and continuing for at least 7 weeks post-surgery. Neonatal capsaicin treatment had no effect on the development of autotomy behavior, but completely blocked the development of hindpaw edema after sciatic section. There was a significant difference in paw thickness between the treatment groups, starting at 14 days after sciatic section and continuing for at least 49 days post-surgery (two-way repeated-measures ANOVA, P , 0:001). **P , 0:01, ***P , 0:001.
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tion component in the transient (,24 h) hindpaw edema observed after formalin or carrageenan injection, cutaneous mustard oil application, and with heat injuries [5,7,17,21], but other investigators had contradictory results in these edema models [4,8]. The chronic edema elicited by injection of complete Freund’s adjuvant is not inhibited by neonatal capsaicin treatment, NK1 antagonists, or in mice with deletions of the genes encoding for SP or NK1 receptors [3,7,10]. Neonatal capsaicin treatment had no effect on acute heat and mechanical nociceptive thresholds, but did block the development of heat hyperalgesia after sciatic transection. The development of mechanical hyperalgesia and autotomy behavior after sciatic section was unaffected by neonatal capsaicin treatment. Other investigators have noted that neonatal capsaicin treatment inhibited the development of heat hyperalgesia in several different nerve injury models, including the spinal nerve ligation [16], sciatic loose ligation [18], and sciatic partial tight ligation models [23]. Furthermore, mice lacking the VR1 receptor had normal acute heat nociceptive thresholds in the hindpaw, but failed to develop heat hyperalgesia after an inflammatory injury [6]. In addition, neonatal capsaicin treatment did not prevent the development of mechanical allodynia [16,23] or autotomy behavior [20,22] in neuropathic models. Collectively, these data indicate that capsaicin sensitive primary afferent neurons are required for the development of heat hyperalgesia, but not mechanical hyperalgesia or autotomy behavior. Despite extensive investigation addressing the underlying mechanisms of CRPS, there is no consensus on either the pathophysiology or on the treatment for this condition [1,11,12]. Enhanced electrically evoked neurogenic extravasation responses have been observed in CRPS [26], leading us to postulate that facilitated SP signaling could mediate the development of hyperalgesia and limb edema in our CRPS model. This hypothesis is supported by the current evidence that capsaicin sensitive primary afferent neurons mediate neurogenic extravasation, heat hyperalgesia and edema in the sciatic section model. In this study, we found that neonatal capsaicin treatment dramatically inhibited cutaneous extravasation in adult rats when evoked by either electrical stimulation of the saphenous nerve or by subcutaneous capsaicin injection. Neonatal capsaicin treatment had no effect on acute nociception, but blocked the development of sciatic transection evoked hindpaw heat hyperalgesia and edema in the adult rats, evidence that small diameter afferents mediate these consequences of nerve injury. These results support the hypothesis that facilitated SP signaling underlies the development of hindpaw heat hyperalgesia and edema in this CRPS model.
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
This study was supported by National Institutes of Health Grant GM30232 (M.M.), a VA Merit Review (M.M.) and the Medical Research Council (M.M.). [1] Baron, R., Levine, J.D. and Fields, H.L., Causalgia and reflex sympathetic dystrophy: does the sympathetic nervous
[16]
[17]
system contribute to the generation of pain? Muscle Nerve, 22 (1999) 678–695. Bozic, C.R., Lu, B., Hopken, U.E., Gerard, C. and Gerard, N.P., Neurogenic amplification of immune complex inflammation, Science, 273 (1996) 1722–1725. Cao, Y.Q., Mantyh, P.W., Carlson, E.J., Gillespie, A.M., Epstein, C.J. and Basbaum, A.I., Primary afferent tachykinins are required to experience moderate to intense pain, Nature, 392 (1998) 390–394. Chapman, V., Buritova, J., Honore, P. and Besson, J.-M., Physiological contributions of neurokinin 1 receptor activation, and interaction with NMDA receptors, to inflammatory-evoked spinal c-fos expression, J. Neurophysiol., 76 (1996) 1817–1827. Damas, J. and Liegeois, J.-F., The inflammatory reaction induced by formalin in the rat paw, Naunyn-Schmiedeberg’s Arch. Pharmacol., 359 (1999) 220–227. Davis, J.B., Gray, J., Gunthorpe, M.J., Hatcher, J.P., Davey, P.T., Overend, P., Harries, M.H., Latcham, J., Clapham, C., Atkinson, K., Hughes, S.A., Rance, K., Grau, E., Harper, A.J., Pugh, P.L., Rogers, D.C., Bingham, S., Randall, A. and Sheardown, S.A., Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia, Nature, 405 (2000) 183–187. Felipe, C.D., Herrero, J.F., O’Brien, J.A., Palmer, J.A., Doyle, C.A., Smith, A.J.H., Laird, J.M.A., Belmonte, C., Cervero, F. and Hunt, S.P., Altered nociception, analgesia and aggression in mice lacking the receptor for substance P, Nature, 392 (1998) 394–397. Gamillscheg, A., Holzer, P., Donnerer, J. and Lembeck, F., Effect of neonatal treatment with capsaicin on carrageenaninduced paw edema in the rat, Naunyn-Schmiedeberg’s Arch. Pharmacol., 326 (1984) 340–342. Hammond, D.L. and Ruda, M.A., Developmental alterations in nociceptive threshold immunoreactive calcitonin generelated peptide and substance P, and fluoride-resistant acid phosphatase in neonatally capsaicin-treated rats, J. Comp. Neurol., 312 (1991) 436–450. Hylden, J.L.K., Noguchi, K. and Ruda, M.A., Neonatal capsaicin treatment attenuates spinal Fos activation and dynorphin gene expression following peripheral tissue inflammation and hyperalgesia, J. Neurosci., 12 (1992) 1716–1725. Kingery, W.S., A critical review of controlled clinical trials for peripheral neuropathic pain and complex regional pain syndromes, Pain, 73 (1997) 123–139. Kingery, W.S., Complex regional pain syndrome, In M. Grabois, S.J. Garrison, K.A. Hart and L.D. Lehmkuhl (Eds.), Physical Medicine and Rehabilitation: The Complete Approach, Blackwell Science, Malden, MA, 2000, pp. 1101– 1125. Kingery, W.S., Castellote, J.M. and Maze, M., Methylprednisolone prevents the development of autotomy and neuropathic edema in rats, but has no effect on nociceptive thresholds, Pain, 80 (1999) 555–566. Kingery, W.S., Agashe, G.S., Sawamura, S., Davies, M.F., Clark, J.D. and Maze, M., Glucocorticoid inhibition of neuropathic hyperalgesia and spinal Fos expression, Anesth. Analg., 92 (2001) 476–482. Kingery, W.S., Guo, T.Z., Agashe, G.S., Davies, M.F., Clark, J.D. and Maze, M., Glucocorticoid inhibition of neuropathic limb edema and cutaneous neurogenic extravasation, Brain Res., 913 (2001) 140–148. Kinnman, E. and Levine, J.D., Sensory and sympathetic contributions to nerve injury-induced sensory abnormalities in the rat, Neuroscience, 64 (1995) 751–767. Lofgren, O., Qi, Y. and Lundeberg, T., Inhibitory effects of
W.S. Kingery et al. / Neuroscience Letters 318 (2002) 39–43
[18]
[19]
[20]
[21]
tachykinin receptor antagonists on thermally induced inflammatory reactions in a rat model, Burns, 25 (1999) 125–129. Meller, S.T., Gebhart, G.F. and Maves, T.J., Neonatal capsaicin treatment prevents the development of the thermal hyperalgesia produced in a model of neuropathic pain in the rat, Pain, 51 (1992) 317–321. Nagy, J.I., Iversen, L.L., Goedert, M., Chapman, D. and Hunt, S.P., Dose-dependent effects of capsaicin on primary sensory neurons in the neonatal rat, J. Neurosci., 3 (1983) 399–406. Nagy, J.I., Buss, M. and Mallory, B., Autotomy in rats after peripheral nerve section: lack of effect of topical nerve or neonatal capsaicin treatment, Pain, 24 (1986) 75–86. Pinter, E., Brown, B., Hoult, J.R.S. and Brain, S.D., Lack of evidence for tachykinin NK1 receptor-mediated neutrophil accumulation in the rat cutaneous microvasculature by thermal injury, Eur. J. Pharmacol., 369 (1999) 91–98.
43
[22] Rodin, B.E. and Kruger, L., Deafferentation in animals as a model for the study of pain: an alternative hypothesis, Brain Res. Rev., 7 (1984) 213–228. [23] Shir, Y. and Seltzer, Z., A-fibers mediate mechanical hyperesthesia and allodynia and C-fibers mediate thermal hyperalgesia in a new model of causalgiform pain disorders in rats, Neurosci. Lett., 115 (1990) 62–67. [24] Tominaga, M., Caterina, M.J., Malmberg, A.B., Rosen, T.A., Gibert, H., Skinner, K., Raumann, B.E., Basbaum, A.I. and Julius, D., The cloned capsaicin receptor integrates multiple pain-producing stimuli, Neuron, 21 (1998) 531–543. [25] Wall, P.D., Scadding, J.W. and Tomkiewicz, M.M., The production and prevention of experimental anesthesia dolorosa, Pain, 6 (1979) 175–182. [26] Weber, M., Birklein, F., Neundorfer, B. and Schmelz, M., Facilitated neurogenic inflammation in complex regional pain syndrome, Pain, 91 (2001) 251–257.
Capsaicin sensitive afferents mediate the development of heat hyperalgesia and hindpaw edema after sciatic section in rats Wade S. Kingery a,b,*, Geeta S. Agashe c,d, Tian-Zhi Guo e, M. Frances Davies c,d, J. David Clark c,d, Mervyn Maze e a
Department of Functional Restoration, Stanford University School of Medicine, Stanford, CA, USA Physical Medicine and Rehabilitation Service, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA, USA c Department of Anesthesia, Stanford University School of Medicine, Stanford, CA, USA d Anesthesiology Service, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA, USA e Magill Department of Anaesthetics, Imperial College School of Medicine, London, UK
b
Received 2 October 2001; received in revised form 6 November 2001; accepted 6 November 2001
Abstract Sciatic section in rats evokes chronic hyperalgesia, autotomy pain behavior, and hindpaw edema, a constellation resembling complex regional pain syndrome (CRPS) in man. Glucocorticoid treatment inhibits these sequelae of sciatic section and also blocks neurogenic extravasation. Small diameter afferent neurons release substance P (SP), a mediator of both hyperalgesia and neurogenic extravasation. Now, we show that pre-emptive destruction of the small diameter fibers prevents neurogenic extravasation, and prevented the development of heat hyperalgesia and hindpaw edema after sciatic section. Thus, capsaicin sensitive primary afferent neurons which release SP are required for the development of heat hyperalgesia and hindpaw edema in this animal model of CRPS. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Edema; Neurogenic extravasation; Nerve injury; Complex regional pain syndrome
Nerve injuries can lead to the development of a complex regional pain syndrome (CRPS), including hyperalgesia (increased sensitivity to painful stimuli), spontaneous pain, distal limb edema, and increased spontaneous cutaneous protein extravasation [12]. We have shown that we can develop a model of this syndrome by sciatic nerve transection in rats [13–15]. In both the clinical condition of CRPS, as well as in the rat model, treatment with methylprednisolone can reverse edema and hyperalgesia; it also prevented the development of autotomy pain behavior in the rat model. We posited that the steroids inhibit neurogenic inflammation to produce these actions. Neurogenic inflammation is initiated by activation of small diameter primary afferent neurons through the release of substance P (SP), which binds to NK1 receptors on blood vessels; the resulting increase in vascular permeability causes fluid extravasation. Capsaicin, the pungent ingredient in hot chili peppers, activates the vanilloid receptors * Corresponding author. Physical Medicine and Rehabilitation Service (117), Veterans Affairs Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, CA 94304, USA. Tel.: 11-650493-5000 ext. 64768; fax: 11-650-852-3470. E-mail address: [email protected] (W.S. Kingery).
(VR1) which are selectively expressed by most of the small diameter primary afferents, including almost all the SP containing neurons [24]. Capsaicin activation of the VR1 causes an influx of calcium and sodium cations into the sensory neuron, triggering an excitotoxic effect. Injecting neonatal rats with capsaicin (50 mg/kg, s.c.) will selectively and permanently destroy virtually all of the SP containing neurons, thus inhibiting the evoked neurogenic extravasation response [9,19]. To test the hypothesis that capsaicin sensitive afferent neurons mediate the sequelae of nerve injury seen in this model of CRPS, the small diameter afferents were lesioned, and we determined the effects on neurogenic extravasation, acute heat and mechanical nociceptive thresholds, the development of hyperalgesia, autotomy behavior, and hindpaw edema. Male Sprague–Dawley rats (B&K Universal) were used in this study. Forty-eight hours after birth, rat pups were subcutaneously injected over the dorsal spine with either 50 mg/kg capsaicin (Sigma, St Louis, MO) or vehicle (10:10:80 v/v of Tween-80/ethanol/saline) in a volume of 5 ml/g. The 30 gauge needle was left in the skin for 60 s after the injection to ensure that the injectant did not reflux back out the needle tract. At the time of injection, each rat underwent either right
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 46 4- 8
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W.S. Kingery et al. / Neuroscience Letters 318 (2002) 39–43
or left ear punching to identify the treatment group. All further assays were performed in a blinded manner. Eight weeks after neonatal injection, the rats were deeply anesthetized with isoflurane and the saphenous nerve was exposed in the right thigh and a pair of platinum electrodes were applied. Evans blue (50 mg/kg; Sigma, St Louis, MO) was administered intravenously in a 50 mg/ml solution of 0.9% saline. After 10 min, the nerve was tightly ligated and then stimulated distal to the site of ligation (5 Hz, 0.5 ms pulse duration, 10 mA, for 10 min). The animals were then euthanized, and all the hindpaw skin was removed and placed in 4 ml of 99% formamide at room temperature for 72 h. The dye concentration in the formamide was then determined spectrophotometrically at a wavelength of 620 nm. Another type of neurogenic extravasation assay was performed using subcutaneous capsaicin injection. Capsaicin activates the VR1 on the SP containing sensory neurons [24], causing the release of SP with a subsequent increase in microvascular permeability. Eight weeks after neonatal injection, the rats were deeply anesthetized with isoflurane, and Evans blue (50 mg/kg; Sigma, St Louis, MO) was administered. Ten minutes later, the rats were subcutaneously injected over the dorsum of the hindpaw with a 50 ml solution of capsaicin (75 mg) or vehicle (7% ethanol). Ten minutes after subcutaneous injection, the animals were euthanized, and a 40 mg piece of dorsal hindpaw skin was excised from the center of the injection site and placed in formamide at room temperature for 72 h, after which the dye concentration was determined spectrophotometrically. Behavioral testing was performed in a dimly lit room, maintained between 22 and 248C. The rats were gently held during the nociceptive assay and only tested when they were quietly resting in the investigator’s hand. Heat nociceptive thresholds were determined as we have previously described [14], using the mean of three consecutive withdrawal thresholds to a Peltier device (4 £ 4 cm surface, CP1: 4-127-06L, Melcor, Trenton, NJ) applied to the medial dorsum of the hindpaw. A linear ramped temperature (18C/s, starting at 408C and with a cut-off of 528C) was used, and the examiner controlled the Peltier using a foot pedal switch. Mechanical nociceptive withdrawal responses were determined as we have previously described [14], using calibrated von Frey fibers (North Coast Medical, San Jose, CA). Four different fibers were used in graduating sequence (10, 23, 57 and 85 g), for a total of 12 consecutive fiber applications. The withdrawal threshold was the smallest fiber size that evoked at least two withdrawal responses during three consecutive applications with the same fiber. Autotomy behavior following sciatic transection was measured as described by Wall et al. [25]. A score of 1 was given for the removal of one or more toe-nails. An additional score of 1 was added for each distal half digit attacked. A further score of 1 was added for each proximal half digit autotomized. Thus, if all toe-nails and all parts of all toes innervated by the sciatic nerve (the three lateral digits) were removed in one hindpaw, a maximal score of
7 was achieved. During this investigation, autotomy behavior was only observed the insensate digits. Edema was measured as we have previously described [15], using a manual caliper to determine the maximum dorsal–ventral thickness of the hindpaw. Eight weeks after neonatal injection, the rats were trained for nociceptive testing for 2 consecutive days before baseline nociceptive thresholds and hindpaw thickness were determined on the third day. Thereafter, during isoflurane anesthesia, the right sciatic nerve was exposed at the midthigh level, a 1 cm segment of nerve excised, and the incision closed. Animals were tested at weekly intervals for 7 weeks post-operatively. The data were analyzed using two-way repeatedmeasures analysis of variance (ANOVA), comparing treatment groups (capsaicin vs. vehicle). Post-hoc differences at a given time were tested using the two-tailed Student’s ttest. A one-way repeated-measures ANOVA was used to determine change over time within one treatment group. Saphenous nerve stimulation caused extravasation of Evans blue dye in the hindpaw skin (19.6 ^ 2.8 mg vs. no measurable dye content after sham stimulation). Neonatal capsaicin treatment reduced saphenous nerve evoked extravasation by 68% (P , 0:01; Fig. 1a). In the neonatal vehicle treated rats, a subcutaneous capsaicin injection evoked significant cutaneous dye extravasation (P , 0:05; Fig. 1b). Neonatal capsaicin treatment had no effect on vehicle evoked extravasation, but completely blocked capsaicin
Fig. 1. Neonatal capsaicin treatment blocked neurogenic extravasation responses in adult rats to electrical stimulation (a), and subcutaneous capsaicin injection (b), indicating a loss of SP containing neurons. Capsaicin treated rats had a saphenous evoked hindpaw dye content of 6.3 ^ 3.1 vs. 19.6 ^ 2.8 mg for vehicle treated animals (n ¼ 7 per cohort). In the neonatal vehicle treated rats, a subcutaneous capsaicin injection evoked significantly more cutaneous dye extravasation (186 ^ 31 ng dye/mg skin) than an injection with vehicle alone (81 ^ 28 ng dye/mg skin). In rats that were neonatally treated with capsaicin, the extravasation response to a capsaicin injection (88 ^ 12 ng dye/mg skin) was completely blocked as compared with injection of vehicle (56 ^ 17 ng/mg; n ¼ 7 per cohort). *P , 0:05, **P , 0:01.
W.S. Kingery et al. / Neuroscience Letters 318 (2002) 39–43
evoked extravasation (compared with vehicle evoked extravasation; Fig. 1b). We examined the effects of neonatal capsaicin treatment on the development of hindpaw heat and mechanical hyperalgesia, autotomy behavior, and hindpaw edema after sciatic nerve transection (n ¼ 20 per cohort). Capsaicin treatment had no effect on baseline nociception when measured by: (1), medial dorsum heat withdrawal thresholds; (2), tail-flick latencies to radiant heat (3.18 ^ 0.06 vs. 3.29 ^ 0.05 s for vehicle treated, data not shown); or (3), medial dorsum mechanical withdrawal thresholds (Fig. 2). The vehicle treated rats developed significant hindpaw heat and mechanical hyperalgesia, autotomy behavior, and hindpaw edema within 2 weeks of sciatic section, which persisted for at least 7 weeks
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post-operatively. Neonatal capsaicin treatment prevented the development of hindpaw heat hyperalgesia and edema after sciatic section, but had no effect on the development of mechanical hyperalgesia or autotomy behavior. The results of this study indicate that capsaicin sensitive primary afferent neurons are required for neurogenic extravasation and the development of hindpaw edema after sciatic section. The contribution of neurogenic processes to the development and maintenance of chronic hindpaw edema has not been previously noted. The cutaneous injection of capsaicin, which acutely stimulates the release of SP from sensory C-fiber terminals, does evoke a brief edema response that has a large neurogenic extravasation component [2,7]. Some investigators have observed a neurogenic extravasa-
Fig. 2. This experiment evaluated the effect of neonatal capsaicin treatment on the development of hindpaw heat hyperalgesia (a) and mechanical hyperalgesia (b), autotomy behavior (c), and hindpaw edema (d) over 7 weeks after sciatic nerve transection (n ¼ 20 per cohort). Neonatal capsaicin treatment had no effect on baseline nociception for medial dorsum heat withdrawal thresholds (50.9 ^ 0.1 vs. 51.1 ^ 0.28C for vehicle treated) or medial dorsum mechanical withdrawal (67 ^ 3 vs. 73 ^ 4 g in vehicle treated). The vehicle treated rats developed significant heat and mechanical hyperalgesia within 2 weeks of sciatic section (one-way repeated-measures ANOVA, compared with the baseline threshold, P , 0:001), and this persisted for at least 7 weeks post-operatively. Neonatal capsaicin treatment prevented the development of hindpaw heat hyperalgesia, but not mechanical hyperalgesia after sciatic section (one-way repeatedmeasures ANOVA, compared with the baseline threshold). There was a significant difference between the heat thresholds of the neonatal capsaicin treatment group and the vehicle treated group (two-way repeated-measures ANOVA, P , 0:001), starting at 3 weeks after sciatic section and continuing for at least 7 weeks post-surgery. Neonatal capsaicin treatment had no effect on the development of autotomy behavior, but completely blocked the development of hindpaw edema after sciatic section. There was a significant difference in paw thickness between the treatment groups, starting at 14 days after sciatic section and continuing for at least 49 days post-surgery (two-way repeated-measures ANOVA, P , 0:001). **P , 0:01, ***P , 0:001.
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tion component in the transient (,24 h) hindpaw edema observed after formalin or carrageenan injection, cutaneous mustard oil application, and with heat injuries [5,7,17,21], but other investigators had contradictory results in these edema models [4,8]. The chronic edema elicited by injection of complete Freund’s adjuvant is not inhibited by neonatal capsaicin treatment, NK1 antagonists, or in mice with deletions of the genes encoding for SP or NK1 receptors [3,7,10]. Neonatal capsaicin treatment had no effect on acute heat and mechanical nociceptive thresholds, but did block the development of heat hyperalgesia after sciatic transection. The development of mechanical hyperalgesia and autotomy behavior after sciatic section was unaffected by neonatal capsaicin treatment. Other investigators have noted that neonatal capsaicin treatment inhibited the development of heat hyperalgesia in several different nerve injury models, including the spinal nerve ligation [16], sciatic loose ligation [18], and sciatic partial tight ligation models [23]. Furthermore, mice lacking the VR1 receptor had normal acute heat nociceptive thresholds in the hindpaw, but failed to develop heat hyperalgesia after an inflammatory injury [6]. In addition, neonatal capsaicin treatment did not prevent the development of mechanical allodynia [16,23] or autotomy behavior [20,22] in neuropathic models. Collectively, these data indicate that capsaicin sensitive primary afferent neurons are required for the development of heat hyperalgesia, but not mechanical hyperalgesia or autotomy behavior. Despite extensive investigation addressing the underlying mechanisms of CRPS, there is no consensus on either the pathophysiology or on the treatment for this condition [1,11,12]. Enhanced electrically evoked neurogenic extravasation responses have been observed in CRPS [26], leading us to postulate that facilitated SP signaling could mediate the development of hyperalgesia and limb edema in our CRPS model. This hypothesis is supported by the current evidence that capsaicin sensitive primary afferent neurons mediate neurogenic extravasation, heat hyperalgesia and edema in the sciatic section model. In this study, we found that neonatal capsaicin treatment dramatically inhibited cutaneous extravasation in adult rats when evoked by either electrical stimulation of the saphenous nerve or by subcutaneous capsaicin injection. Neonatal capsaicin treatment had no effect on acute nociception, but blocked the development of sciatic transection evoked hindpaw heat hyperalgesia and edema in the adult rats, evidence that small diameter afferents mediate these consequences of nerve injury. These results support the hypothesis that facilitated SP signaling underlies the development of hindpaw heat hyperalgesia and edema in this CRPS model.
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
This study was supported by National Institutes of Health Grant GM30232 (M.M.), a VA Merit Review (M.M.) and the Medical Research Council (M.M.). [1] Baron, R., Levine, J.D. and Fields, H.L., Causalgia and reflex sympathetic dystrophy: does the sympathetic nervous
[16]
[17]
system contribute to the generation of pain? Muscle Nerve, 22 (1999) 678–695. Bozic, C.R., Lu, B., Hopken, U.E., Gerard, C. and Gerard, N.P., Neurogenic amplification of immune complex inflammation, Science, 273 (1996) 1722–1725. Cao, Y.Q., Mantyh, P.W., Carlson, E.J., Gillespie, A.M., Epstein, C.J. and Basbaum, A.I., Primary afferent tachykinins are required to experience moderate to intense pain, Nature, 392 (1998) 390–394. Chapman, V., Buritova, J., Honore, P. and Besson, J.-M., Physiological contributions of neurokinin 1 receptor activation, and interaction with NMDA receptors, to inflammatory-evoked spinal c-fos expression, J. Neurophysiol., 76 (1996) 1817–1827. Damas, J. and Liegeois, J.-F., The inflammatory reaction induced by formalin in the rat paw, Naunyn-Schmiedeberg’s Arch. Pharmacol., 359 (1999) 220–227. Davis, J.B., Gray, J., Gunthorpe, M.J., Hatcher, J.P., Davey, P.T., Overend, P., Harries, M.H., Latcham, J., Clapham, C., Atkinson, K., Hughes, S.A., Rance, K., Grau, E., Harper, A.J., Pugh, P.L., Rogers, D.C., Bingham, S., Randall, A. and Sheardown, S.A., Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia, Nature, 405 (2000) 183–187. Felipe, C.D., Herrero, J.F., O’Brien, J.A., Palmer, J.A., Doyle, C.A., Smith, A.J.H., Laird, J.M.A., Belmonte, C., Cervero, F. and Hunt, S.P., Altered nociception, analgesia and aggression in mice lacking the receptor for substance P, Nature, 392 (1998) 394–397. Gamillscheg, A., Holzer, P., Donnerer, J. and Lembeck, F., Effect of neonatal treatment with capsaicin on carrageenaninduced paw edema in the rat, Naunyn-Schmiedeberg’s Arch. Pharmacol., 326 (1984) 340–342. Hammond, D.L. and Ruda, M.A., Developmental alterations in nociceptive threshold immunoreactive calcitonin generelated peptide and substance P, and fluoride-resistant acid phosphatase in neonatally capsaicin-treated rats, J. Comp. Neurol., 312 (1991) 436–450. Hylden, J.L.K., Noguchi, K. and Ruda, M.A., Neonatal capsaicin treatment attenuates spinal Fos activation and dynorphin gene expression following peripheral tissue inflammation and hyperalgesia, J. Neurosci., 12 (1992) 1716–1725. Kingery, W.S., A critical review of controlled clinical trials for peripheral neuropathic pain and complex regional pain syndromes, Pain, 73 (1997) 123–139. Kingery, W.S., Complex regional pain syndrome, In M. Grabois, S.J. Garrison, K.A. Hart and L.D. Lehmkuhl (Eds.), Physical Medicine and Rehabilitation: The Complete Approach, Blackwell Science, Malden, MA, 2000, pp. 1101– 1125. Kingery, W.S., Castellote, J.M. and Maze, M., Methylprednisolone prevents the development of autotomy and neuropathic edema in rats, but has no effect on nociceptive thresholds, Pain, 80 (1999) 555–566. Kingery, W.S., Agashe, G.S., Sawamura, S., Davies, M.F., Clark, J.D. and Maze, M., Glucocorticoid inhibition of neuropathic hyperalgesia and spinal Fos expression, Anesth. Analg., 92 (2001) 476–482. Kingery, W.S., Guo, T.Z., Agashe, G.S., Davies, M.F., Clark, J.D. and Maze, M., Glucocorticoid inhibition of neuropathic limb edema and cutaneous neurogenic extravasation, Brain Res., 913 (2001) 140–148. Kinnman, E. and Levine, J.D., Sensory and sympathetic contributions to nerve injury-induced sensory abnormalities in the rat, Neuroscience, 64 (1995) 751–767. Lofgren, O., Qi, Y. and Lundeberg, T., Inhibitory effects of
W.S. Kingery et al. / Neuroscience Letters 318 (2002) 39–43
[18]
[19]
[20]
[21]
tachykinin receptor antagonists on thermally induced inflammatory reactions in a rat model, Burns, 25 (1999) 125–129. Meller, S.T., Gebhart, G.F. and Maves, T.J., Neonatal capsaicin treatment prevents the development of the thermal hyperalgesia produced in a model of neuropathic pain in the rat, Pain, 51 (1992) 317–321. Nagy, J.I., Iversen, L.L., Goedert, M., Chapman, D. and Hunt, S.P., Dose-dependent effects of capsaicin on primary sensory neurons in the neonatal rat, J. Neurosci., 3 (1983) 399–406. Nagy, J.I., Buss, M. and Mallory, B., Autotomy in rats after peripheral nerve section: lack of effect of topical nerve or neonatal capsaicin treatment, Pain, 24 (1986) 75–86. Pinter, E., Brown, B., Hoult, J.R.S. and Brain, S.D., Lack of evidence for tachykinin NK1 receptor-mediated neutrophil accumulation in the rat cutaneous microvasculature by thermal injury, Eur. J. Pharmacol., 369 (1999) 91–98.
43
[22] Rodin, B.E. and Kruger, L., Deafferentation in animals as a model for the study of pain: an alternative hypothesis, Brain Res. Rev., 7 (1984) 213–228. [23] Shir, Y. and Seltzer, Z., A-fibers mediate mechanical hyperesthesia and allodynia and C-fibers mediate thermal hyperalgesia in a new model of causalgiform pain disorders in rats, Neurosci. Lett., 115 (1990) 62–67. [24] Tominaga, M., Caterina, M.J., Malmberg, A.B., Rosen, T.A., Gibert, H., Skinner, K., Raumann, B.E., Basbaum, A.I. and Julius, D., The cloned capsaicin receptor integrates multiple pain-producing stimuli, Neuron, 21 (1998) 531–543. [25] Wall, P.D., Scadding, J.W. and Tomkiewicz, M.M., The production and prevention of experimental anesthesia dolorosa, Pain, 6 (1979) 175–182. [26] Weber, M., Birklein, F., Neundorfer, B. and Schmelz, M., Facilitated neurogenic inflammation in complex regional pain syndrome, Pain, 91 (2001) 251–257.