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Role of spinal cholecystokinin in neuropathic pain after spinal cord hemisection in rats
Neuroscience Letters 462 (2009) 303–307
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Role of spinal cholecystokinin in neuropathic pain after spinal cord hemisection in rats Junesun Kim a , Jung Hoon Kim b , Youngkyung Kim b , Hwi-young Cho b , Seung Kil Hong b , Young Wook Yoon b,∗ a b
Department of Physical Therapy, Korea University College of Health Science, Seoul, 136-703, Republic of Korea Department of Physiology, Korea University College of Medicine, 126-1, Anam-dong, Sungbuk-gu, Seoul, 136-705, Republic of Korea
a r t i c l e
i n f o
Article history: Received 6 June 2009 Received in revised form 13 July 2009 Accepted 13 July 2009 Keywords: Cholecystokinin CI-988 Allodynia Central pain Spinal cord injury Neuropathic pain
a b s t r a c t In the present study we determined whether spinal cholecystokinin (CCK) or the cholecystokinin receptor is involved in below-level neuropathic pain of spinal cord injury (SCI). The effect of the CCKB receptor antagonist, CI-988 on mechanical allodynia and the expression level of CCK and CCKB receptor were investigated. Spinal hemisection was done at the T13 level in rats under enflurane anesthesia. CI-988 was administered intraperitoneally and intrathecally and behavioral tests were conducted. After systemic injection, mechanical allodynia was reduced by higher doses of CI-988 (10 and 20 mg/kg). Intrathecal CI-988 (100, 200 and 500 g) dose-dependently increased the paw withdrawal threshold in both paws. Following spinal hemisection, CCK mRNA expression increased on the ipsilateral side at the spinal segments caudal to the injury and both sides of the spinal L4-5 segments without any significant changes in CCKB receptor mRNA levels. These results suggest that up-regulation of spinal CCK may contribute to maintenance of mechanical allodynia following SCI and that clinical application of CI-988 or similar drugs may be useful therapeutic agents for management of central neuropathic pain. © 2009 Elsevier Ireland Ltd. All rights reserved.
Neuropathic pain following spinal cord injury (SCI) includes spontaneous pain syndromes, hyperalgesia and allodynia, which occur above, at or below the level of the lesion [17] in about half of patients with SCI [18]. These pain syndromes are usually chronic and may cause persistent problems for SCI patients in rehabilitation. The underlying mechanism is not fully understood. Injury to the spinal cord leads to neuroplastic changes including anatomical, physiological and molecular changes within the spinal cord and brain. Previous studies suggest that alterations in neuropeptides such as substance P, calcitonin gene-related peptide and cholecystokinin are involved in generation of neuropathic pain [10,25]. Of these peptides, cholecystokinin (CCK) is well known as an antagonist of endogenous opioids in the central nervous system [3,22]. Experimental evidence has shown that CCK mRNA increases after injury to the nervous system [2,25]. Xu et al. [25] reported up-regulation of CCK and CCK receptor mRNA in dorsal root ganglia after sciatic nerve injury in rats. Brewer et al. [2] reported an increase in CCK mRNA in the cortex of painful rats compared to painless rats in an excitotoxic SCI model. Previous reports clearly show that neuropathic pain can be controlled by blocking the actions of CCK in rats [28]. Xu et al. [24] reported, using an ischemic SCI model, that systemic opioids did not effectively
∗ Corresponding author. Tel.: +82 2 920 6278; fax: +82 2 925 5492. E-mail address: [email protected] (Y.W. Yoon). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.07.042
reduce mechanical allodynia but systemic CI-988, an antagonist of the CCKB receptor alleviated mechanical allodynia. Kovelowski et al. [12] demonstrated blockade of the action of CCK by injection of L365,260, a CCKB receptor antagonist, into the rostroventromedial medulla (RVM) reversed tactile allodynia and thermal hyperalgesia after spinal nerve injury, suggesting that endogenous CCK may contribute to maintenance of neuropathic pain following nerve injury. However, most studies are limited to peripheral nerve injury or certain types of SCI, especially ischemic SCI [23,24]. Thus, in the present study, we investigated the role of CCK in the maintenance of pain below the level of thoracic spinal hemisection. All experiment procedures were done in accordance with guidelines set by the Korea University College of Medicine Animal Research Policies Committee. Male Sprague–Dawley rats (n = 68, 150–200 g, at the time of operation) were used for this experiment. The animals were kept in a 12-h light/12-h dark cycle with light on 7:00 A.M. Under enflurane anesthesia (by mixture of 4% enflurane and 95% O2 ), a longitudinal incision was made exposing several segments, a laminectomy was performed, and the spinal cord was hemisected at T13 on the left side with a no. 11 scalpel blade. The wound was closed in anatomical layers, the skin with stainless steel wound clips. Behavioral tests for motor function and mechanical allodynia were performed preoperatively and postoperatively for the hindpaws. The tests were performed on each rat 1 day prior to surgery
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J. Kim et al. / Neuroscience Letters 462 (2009) 303–307
and 1, 4, 7 and 14 days postoperatively (PO), before the study of drug effects. Rats that showed a little mechanical allodynia or displayed contralateral hindlimb motor deficits were excluded. Mechanical allodynia was assessed by measuring the threshold of brisk paw withdrawal response to graded mechanical stimulus with a series of von Frey filaments (0.41, 0.70, 1.20, 2.00, 3.63, 5.50, 8.50, and 15.10 g, Stoelting, Wood Dale, IL, USA). To do this, the rat was placed under a transparent plastic dome (28 cm × 28 cm × 10 cm) on a metal mesh floor and a von Frey filament was applied to the plantar surface of the foot. A von Frey filament was applied for 3–4 s to each hind paw while the filament was bent. The 50% withdrawal threshold was determined using the up–down method [4], starting with a 2.0 g (4.31 mN) strength of filament. A withdrawal response was cause to present the next weaker stimulus, and lack of withdrawal led to presentation of the next stronger stimulus. Stimuli were presented at intervals of several seconds. A brisk foot withdrawal to application of a von Frey filament was regarded as a positive response. Interpolation of the 50% threshold was carried out according to the method of Dixon [6]. The investigator conducting the behavioral tests was blinded about the injected drug. Tests were carried out 3 weeks after spinal hemisection. During this period, motor function in hemisected rats fully recovered and signs of mechanical allodynia were well established as described in our previous report [11]. More than one test was performed on some rats. Intervals between repeated tests were at least 3 days. CI-988 (5, 10 and 20 mg/kg) or vehicle (saline) was injected intraperitoneally. Rats were randomly assigned to different treatment groups. Behavioral signs of mechanical allodynia were measured 30 min before and 15, 30, 45, 60, 90 and 120 min after injection of a drug. In order to determine the effect of intrathecally (IT) administered drugs on mechanical allodynia, rats for IT drug administration were implanted with catheters 2 weeks after hemisection. Under enflurane anesthesia (by mixture of 4% enflurane and 95% O2 ), the occipital muscles were separated from their attachment point and retracted caudally to expose the cisternal membrane at the base of the skull. Sterilized PE-10 tubing was threaded through an incision in the atlanto-occipital membrane to the 1 or 2 segments rostral to the hemisection site (5.5 cm). Animals showing evidence of neuromuscular dysfunction were excluded from further tests. After the experiment, the position of the IT catheter was confirmed by injection of Evans Blue dye following laminectomy. Drug was injected IT in a volume of 10 l followed by 10 l saline to flush the catheter. Behavioral signs of mechanical allodynia were measured 30 min before and 15, 30, 60, 90, 120 and 180 min after the injection. CI-988 was a generous gift from Pfizer Inc. (Groton, CT, USA). To exclude the possibility that motor impairment might contribute to changes in withdrawal threshold during testing, a modification of the combined behavioral score (CBS) of Gale et al. [8] was performed at the time of the behavioral test. The CBS assigns a weight to each of the tests and combines them into one total score that represents the degree of motor impairment. Tests were as follows: motor scores, toe spread, righting reflex, extension withdrawal reflex, placing reflex and inclined plane. Neurological function was evaluated by a scoring system that ranged from 0 for a normal rat to 90 for a completely paralyzed rat. To determine whether there were changes in expression levels of CCK and CCKB receptor mRNA, segments of the spinal cord at T11-12, L1-2 and L4-5 levels were removed and dissected into ipsilateral and contralateral dorsal quadrants from SCI rats (n = 8) and sham rats (n = 6). T11-12 and L1-2 levels of the spinal cord were segments just rostral and caudal, respectively, to the level of injury. The L4-5 spinal cord segment was also examined because it receives mainly sensory inputs from the foot, pain responses from
which were measured in the present study. The tissues were immediately frozen in liquid nitrogen and stored at −70 ◦ C. Total RNA extraction was performed using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. First-strand cDNA synthesis was performed using 50 pM oligo (dT)20 primer with Moloney-Mouse Leukemia Virus reverse transcriptase (Invitrogen, #28025-013, Carlsbad, CA, USA) at 37 ◦ C for 50 min. Total RNA and cDNA concentrations were determined at 260 nm by UV spectrophotometer (GeneQuant, Pharmacia, USA). We amplified 500 ng of cDNA for cholecystokinin (CCK), CCKB receptor (CCKBR) and 375 ng of cDNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with i-star taq DNA polymerase (Intron, #25162, Sungnam, Korea). PCR primer and conditions are presented in Table 1. PCR products were detected on a 1% agarose gel. A picture was taken using a digital camera (Canon A620) and we measured the density of the PCR bands using a computer-assisted image analysis system (NIH image software). All values are expressed as mean ± SEM. The Friedman repeated measures of analysis of variance followed by multiple comparison tests were used to compare behavioral test results before and after drug administration with the same animal. The absorbencies of the PCR bands were measured using Scion image software. Normal and injured group comparisons were performed using a one-way ANOVA with Scheffe’s post hoc test. Rats with SCI showed significant decrease in withdrawal threshold to von Frey stimulation with maximal motor recovery 3 weeks after spinal hemisection, as in our previous report [11]. The effects of systemic CI-988 on the hindlimb withdrawal threshold to mechanical stimulation applied to the plantar surface of the right foot and the left foot are shown in Fig. 1A. CI-988 at 5 mg/kg had no significant effects on paw withdrawal threshold, whereas doses of 10 and 20 mg/kg significantly increased paw withdrawal threshold without change of motor function tested using a CBS scale (data not shown). This anti-allodynic effect was significant at 15 min and persisted up to 60 min after injection in both feet compared to pretreatment values. There were no significant differences in threshold changes induced by CI-988 between ipsilateral and contralateral hind paws. Saline had no effect on the responsiveness to mechanical stimuli with von Frey stimulation. Intrathecally injected CI-988 (100, 200 and 500 g) significantly increased paw withdrawal threshold to mechanical stimulation applied to the plantar surface of the foot in a dose-dependent manner (Fig. 1B). Even a lower dose of CI-988 (100 g, n = 12) significantly increased withdrawal threshold from 15 to 60 min. The effect was peak at 15–30 min after injection, gradually diminished, and persisted for 90 min after higher doses of CI-988 (200 and 500 g). Saline (n = 9) did not change the withdrawal threshold throughout the test period. No changes in the CBS scale were observed after injection of any of the doses that were tested in this study (data not shown). We attempted to determine whether there were changes in expression level of CCK and CCKB receptor mRNA in the spinal cord after hemisection. The expression of CCK mRNA was assessed for both ipsilateral and contralateral sides at T11-12, L1-2 and L4-5 spinal cord segments and was compared to levels in normal rats (Fig. 2). CCK mRNA expression was significantly increased in both ipsilateral and contralateral sides at the L4-5 level compared to control (p < 0.05). At the L1-2 spinal cord level, CCK mRNA expression was significantly increased on the ipsilateral side (p < 0.05). On the contralateral side, CCK mRNA expression increased slightly, but there was no significant difference. At the T11-12 spinal cord level, CCK mRNA expression was similar to that of control on both ipsilateral and contralateral sides. In contrast to expression of CCK mRNA, CCKB receptor mRNA expression in the spinal cord did not show significant differences between hemisected rats and normal rats at all of the sites tested in this study.
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Table 1 PCR primer and condition.
Cholecystokinin (CCK) Cholecystokinin receptor (CCKBR) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
Length
Forward
Reverse
413 bp 361 bp 276 bp
GAA GTG GAC CCT ATG GA CAA GGC CAT TTC CTA CCT CA GAG TCT ACT GGC GTC TTC AC
TGC ATT GCACAC TCT GAA CA TAG GTA GAG TTC GCG GGA GA CCA TCC ACA GTC TTC TGA GT
The present data demonstrate that the mechanical allodynia induced by spinal cord hemisection was reduced by systemic and IT CI-988. CCK mRNA expression was significantly increased on both ipsilateral and contralateral sides at L4-5 and at the ipsilateral L1-2 spinal segment, with no change in the expression of CCKB receptor mRNA after spinal left hemisection. Systemically injected CI-988 significantly increased the paw withdrawal threshold, although the prominent anti-allodynic effects were achieved at the higher doses (10 and 20 mg/kg) in this study. The doses (5, 10 and 20 mg/kg) of systemic CI-988 tested were chosen because a previous report showed that 10 mg/kg of systemic CI-988 did not produce significant motor changes [16]. Our results are similar to those of a previous report [24], in which systemic CI-988 was effective in reducing vocalization threshold to mechanical stimulation applied to the back skin, and those analgesic effects were reversed by naloxone in an ischemic SCI model. However, higher doses of systemic CI-988 were needed to produce
PCR condition Temperature and time
Cycle
94 ◦ C:40 s, 62 ◦ C:30 s, 72 ◦ C:40 s 95 ◦ C:1 min, 59 ◦ C:1 min, 72 ◦ C:1 min 95 ◦ C:1 min, 61 ◦ C:1 min, 72 ◦ C:1 min
25 26 23
significant inhibition of mechanical allodynia, suggesting that the mechanical allodynia resulting from spinal hemisection is relatively resistant to systemic CI-988. IT CI-988 applied to the injured site increased the withdrawal threshold to von Frey stimulation applied to the plantar surface of the foot in a dose-dependent manner without any motor depression. Results that application of CI-988 at lesion sites reduced below-level neuropathic pain suggest that the lesion site could be a useful target for managing below-level pain. The relative ineffectiveness of systemic CI-988 and the relative effectiveness of IT administration suggest that a relatively high concentration of CI-988 in the spinal cord is required to produce the effects. Considering that CCK mRNA was increased in the spinal cord caudal to the injury, and that IT injection was reported to spread a few centimeters rostrocaudally from the tip of the catheter [27], analgesic effects of IT CI-988 may come from its action on caudal as well as rostral segments. A previous study [5] reported that a sig-
Fig. 1. Effects of systemic (A) and intrathecal (B) CI-988 on withdrawal threshold to mechanical stimulation applied to the plantar surface of the contralateral (left column) and ipsilateral foot (right column). Asterisks indicate the values significantly different from the pre-injection control value (PRE) (p < 0.05).
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Fig. 2. (A). A representative blot showing CCK mRNA and CCKB receptor mRNA from T11-12, L1-2 and L4-5 spinal segments. Also shown are relative gene expression levels of CCK mRNA (B) and CCKB receptor mRNA (C) for T11-12, L1-2 and L4-5 spinal cord segments. T11-12I: ipsilateral T11-12 spinal cord segments, T11-12C: contralateral T11-12 spinal cord segments, T11-12N; T11-12 spinal cord segments from normal rats, L1-2I: ipsilateral L1-2 spinal cord segments, L1-2C: contralateral L1-2 spinal cord segments, L1-2N; L1-2 spinal cord segments from normal rats, L45I; ipsilateral L4-5 spinal cord segments, L4-5C: contralateral L4-5 cord segments, L4-5N: L4-5 spinal cord segments from normal rats. Asterisks indicate that the values were significantly different from the corresponding control value (p < 0.05).
nificant anti-allodynic effect was achieved with a dose of 100 g IT of CI-988 after peripheral nerve injury. Based on our present results, the analgesic effect of CI-988 on allodynia may reflect an involvement of the endogenous CCKergic system in maintenance of chronic neuropathic pain after SCI. Blockade of CCKB receptors in the spinal cord attenuated thermal hyperalgesia after constriction injury to the sciatic nerve [28]. Further, CCKB receptor deficient mice showed significant increases in withdrawal threshold for mechanical sensitivity and they also did not display mechanical hyperalgesia after sciatic nerve ligation [13]. Thus, blocking spinal CCK receptors seems to be effective in reducing mechanical allodynia in experimental neuropathic pain models, including our current results. Previous studies suggest a more complicated picture of the nature of neuropathic pain such as a reduction in opioid receptors [14] and an increase in anti-opioid peptides such as CCK [9]. Recent studies have concentrated on the activation of descending pain facilitation in chronic pain states [20,21]. It is well known that enhanced descending facilitation under pathological conditions can be driven by CCK in the RVM [12,15,20]. Kovelowski et al. [12] demonstrated that microinjection of a CCKB receptor antagonist, L365,260, into the RVM reversed tactile allodynia and thermal
hyperalgesia after spinal nerve injury. Although the mechanisms for the effects of descending facilitation on chronic pain have been studied, the role of the spinal CCKergic system in central pain is still unclear. It is important to know where changes in the endogenous CCKergic system take place in the CNS, whether changes occur after spinal hemisection, and what types of changes occur. According to previous research, CCK is released into the spinal cord by intensive electrical stimulation of peripheral nerves [26] and electrical stimulation reduces the analgesic effects of IT morphine, but not of intracerebroventricular morphine [7], suggesting that the spinal cord is a major site of action of endogenous CCKergic systems. It suggests that changes in the spinal CCKergic system are associated with the development of neuropathic pain following SCI. To determine changes in the spinal CCKergic system following injury, we assessed expression levels of CCK and CCKB receptor mRNA in spinal segments rostral and caudal to the injured site. A recent report described an up-regulation of CCK mRNA in the cortex of rats with pain-like symptoms following SCI [2]. Another study demonstrated that CCK-like immunoreactivity is elevated 3fold in the cerebrospinal fluid (CSF) in painful rats compared to painless rats after spinal ischemia [23]. Our results demonstrate that CCK mRNA expression is increased on both sides at the L4-5 spinal cord level and on the ipsilateral side at the L1-2 spinal cord level, suggesting that up-regulation of spinal CCK may contribute to maintenance of central pain after SCI. The relationship between severity of pain and expression level of CCK mRNA was not assessed here because most of spinal hemisected rats showed a fairly high degree of mechanical allodynia. It should be noted that we found a difference in expression levels of CCK mRNA among the three sites tested in this study. CCK mRNA level was elevated in the spinal segments below the injured site, whereas there were no significant changes in the level of CCK mRNA expression in rostral segments. The present data may reflect the effects of cutting the lateral half of the spinal cord on descending facilitatory influences. Previous literature showed that descending facilitation projects bilaterally from the RVM [1] and nociceptive stimulation produces increased facilitation bilaterally [19]. Earlier studies showed that CCK receptor mRNA as well as CCK mRNA expression levels are up-regulated in dorsal root ganglion cells after unilateral section of the sciatic nerve in rats [25]. Interestingly, however, no significant difference was observed in CCKB receptor mRNA expression in any spinal segments measured after spinal hemisection. Together, these results suggest that enhanced CCK synthesis and release in spinal segments caudal to the level of injury rather than up-regulation of CCKB receptors is associated with spinal hemisection. A possible explanation for the role of the spinal CCKergic system in central pain following SCI is that an increase in CCK mRNA level results in enhancement of endogenous CCK levels in the spinal dorsal horn and the excess CCK acts on CCKB receptors that are maintained at near normal levels. Increased CCK facilitates pain, and therefore blocking CCK receptors reduce pain. The number of CCK receptors did not change in the present study, but there may have been functional changes which further studies will elucidate. In summary, the CCKB receptor antagonist, CI-988 effectively reduced below-level mechanical allodynia after SCI and upregulation of CCK may contribute to development or maintenance of neuropathic pain after spinal hemisection. Thus, CCK receptor antagonism may represent a strategy for development of new therapeutic agents to manage mechanical allodynia in this type of central neuropathic pain.
Acknowledgment This work was supported by a grant of the Korea University College of Health Science (K0718991).
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References [1] M. Antal, M. Petko, E. Polgar, C.W. Heizmann, J. Storm-Mathisen, Direct evidence of an extensive GABAergic innervation of the spinal dorsal horn by fibres descending from the rostral ventromedial medulla, Neuroscience 73 (1996) 509–518. [2] K.L. Brewer, D. McMillan, T. Nolan, K. Shum, Cortical changes in cholecystokinin mRNA are related to spontaneous pain behaviors following excitotoxic spinal cord injury in the rat, Brain Res. Mol. Brain Res. 118 (2003) 171–174. [3] F. Cesselin, Opioid and anti-opioid peptides, Fundam. Clin. Pharmacol. 9 (1995) 409–433. [4] S.R. Chaplan, F.W. Bach, J.W. Pogrel, J.M. Chung, T.L. Yaksh, Quantitative assessment of tactile allodynia in the rat paw, J. Neurosci. Methods 53 (1994) 55–63. [5] M.A. Coudore-Civiale, C. Courteix, J. Fialip, M. Boucher, A. Eschalier, Spinal effect of the cholecystokinin-B receptor antagonist CI-988 on hyperalgesia, allodynia and morphine-induced analgesia in diabetic and mononeuropathic rats, Pain 88 (2000) 15–22. [6] W.J. Dixon, Efficient analysis of experimental observations, Annu. Rev. Pharmacol. Toxicol. 20 (1980) 441–462. [7] Y. Fukazawa, T. Maeda, W. Hamabe, K. Kumamoto, Y. Gao, C. Yamamoto, M. Ozaki, S. Kishioka, Activation of spinal anti-analgesic system following electroacupuncture stimulation in rats, J. Pharmacol. Sci. 99 (2005) 408–414. [8] K. Gale, H. Kerasidis, J.R. Wrathall, Spinal cord contusion in the rat: behavioral analysis of functional neurologic impairment, Exp. Neurol. 88 (1985) 123–134. [9] M.M. Heinricher, M.J. Neubert, Neural basis for the hyperalgesic action of cholecystokinin in the rostral ventromedial medulla, J. Neurophysiol. 92 (2004) 1982–1989. [10] T. Hokfelt, X. Zhang, Z. Wiesenfeld-Hallin, Messenger plasticity in primary sensory neurons following axotomy and its functional implications, Trends Neurosci. 17 (1994) 22–30. [11] J. Kim, Y.W. Yoon, S.K. Hong, H.S. Na, Cold and mechanical allodynia in both hindpaws and tail following thoracic spinal cord hemisection in rats: time courses and their correlates, Neurosci. Lett. 343 (2003) 200–204. [12] C.J. Kovelowski, M.H. Ossipov, H. Sun, J. Lai, T.P. Malan, F. Porreca, Supraspinal cholecystokinin may drive tonic descending facilitation mechanisms to maintain neuropathic pain in the rat, Pain 87 (2000) 265–273. [13] K. Kurrikoff, S. Koks, T. Matsui, M. Bourin, A. Arend, M. Aunapuu, E. Vasar, Deletion of the CCK2 receptor gene reduces mechanical sensitivity and abolishes the development of hyperalgesia in mononeuropathic mice, Eur. J. Neurosci. 20 (2004) 1577–1586. [14] M.H. Ossipov, Y. Lopez, M.L. Nichols, D. Bian, F. Porreca, Inhibition by spinal morphine of the tail-flick response is attenuated in rats with nerve ligation injury, Neurosci. Lett. 199 (1995) 83–86.
307
[15] F. Porreca, M.H. Ossipov, G.F. Gebhart, Chronic pain and medullary descending facilitation, Trends Neurosci. 25 (2002) 319–325. [16] M. Qian, A.E. Johnson, L. Kallstrom, H. Carrer, P. Sodersten, Cholecystokinin, dopamine D2 and N-methyl-d-aspartate binding sites in the nucleus of the solitary tract of the rat: possible relationship to ingestive behavior, Neuroscience 77 (1997) 1077–1089. [17] P.J. Siddall, R.P. Yezierski, J.D. Loeser, K.J. Burchiel, Taxonomy and Epidemiology of Spinal Cord Injury Pain. Spinal Cord Injury Pain: Assessment, Mechanisms, Management, IASP Press, Seattle, 2002, pp. 9–24. [18] R.R. Tasker, Central pain states, in: J.D. Loeser (Ed.), Bonica’s Management of Pain, Lippincott Williams Wilkins, Philadelphia, 2001, pp. 433–457. [19] R. Terayama, R. Dubner, K. Ren, The roles of NMDA receptor activation and nucleus reticularis gigantocellularis in the time-dependent changes in descending inhibition after inflammation, Pain 97 (2002) 171–181. [20] D.V. Tillu, G.F. Gebhart, K.A. Sluka, Descending facilitatory pathways from the RVM initiate and maintain bilateral hyperalgesia after muscle insult, Pain 136 (2008) 331–339. [21] L.P. Vera-Portocarrero, E.T. Zhang, M.H. Ossipov, J.Y. Xie, T. King, J. Lai, F. Porreca, Descending facilitation from the rostral ventromedial medulla maintains nerve injury-induced central sensitization, Neuroscience 140 (2006) 1311– 1320. [22] Z. Wiesenfeld-Hallin, G. de Arauja Lucas, P. Alster, X.J. Xu, T. Hokfelt, Cholecystokinin/opioid interactions, Brain Res. 848 (1999) 78–89. [23] X.J. Xu, P. Alster, W.P. Wu, J.X. Hao, Z. Wiesenfeld-Hallin, Increased level of cholecystokinin in cerebrospinal fluid is associated with chronic pain-like behavior in spinally injured rats, Peptides 22 (2001) 1305–1308. [24] X.J. Xu, J.X. Hao, A. Seiger, J. Hughes, T. Hokfelt, Z. Wiesenfeld-Hallin, Chronic pain-related behaviors in spinally injured rats: evidence for functional alterations of the endogenous cholecystokinin and opioid systems, Pain 56 (1994) 271–277. [25] X.J. Xu, M.J. Puke, V.M. Verge, Z. Wiesenfeld-Hallin, J. Hughes, T. Hokfelt, Upregulation of cholecystokinin in primary sensory neurons is associated with morphine insensitivity in experimental neuropathic pain in the rat, Neurosci. Lett. 152 (1993) 129–132. [26] T.L. Yaksh, E.O. Abay II, V.L. Go, Studies on the location and release of cholecystokinin and vasoactive intestinal peptide in rat and cat spinal cord, Brain Res. 242 (1982) 279–290. [27] T.L. Yaksh, T.A. Rudy, Chronic catheterization of the spinal subarachnoid space, Physiol. Behav. 17 (1976) 1031–1036. [28] T. Yamamoto, N. Nozaki-Taguchi, Role of cholecystokinin-B receptor in the maintenance of thermal hyperalgesia induced by unilateral constriction injury to the sciatic nerve in the rat, Neurosci. Lett. 202 (1995) 89–92.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Role of spinal cholecystokinin in neuropathic pain after spinal cord hemisection in rats Junesun Kim a , Jung Hoon Kim b , Youngkyung Kim b , Hwi-young Cho b , Seung Kil Hong b , Young Wook Yoon b,∗ a b
Department of Physical Therapy, Korea University College of Health Science, Seoul, 136-703, Republic of Korea Department of Physiology, Korea University College of Medicine, 126-1, Anam-dong, Sungbuk-gu, Seoul, 136-705, Republic of Korea
a r t i c l e
i n f o
Article history: Received 6 June 2009 Received in revised form 13 July 2009 Accepted 13 July 2009 Keywords: Cholecystokinin CI-988 Allodynia Central pain Spinal cord injury Neuropathic pain
a b s t r a c t In the present study we determined whether spinal cholecystokinin (CCK) or the cholecystokinin receptor is involved in below-level neuropathic pain of spinal cord injury (SCI). The effect of the CCKB receptor antagonist, CI-988 on mechanical allodynia and the expression level of CCK and CCKB receptor were investigated. Spinal hemisection was done at the T13 level in rats under enflurane anesthesia. CI-988 was administered intraperitoneally and intrathecally and behavioral tests were conducted. After systemic injection, mechanical allodynia was reduced by higher doses of CI-988 (10 and 20 mg/kg). Intrathecal CI-988 (100, 200 and 500 g) dose-dependently increased the paw withdrawal threshold in both paws. Following spinal hemisection, CCK mRNA expression increased on the ipsilateral side at the spinal segments caudal to the injury and both sides of the spinal L4-5 segments without any significant changes in CCKB receptor mRNA levels. These results suggest that up-regulation of spinal CCK may contribute to maintenance of mechanical allodynia following SCI and that clinical application of CI-988 or similar drugs may be useful therapeutic agents for management of central neuropathic pain. © 2009 Elsevier Ireland Ltd. All rights reserved.
Neuropathic pain following spinal cord injury (SCI) includes spontaneous pain syndromes, hyperalgesia and allodynia, which occur above, at or below the level of the lesion [17] in about half of patients with SCI [18]. These pain syndromes are usually chronic and may cause persistent problems for SCI patients in rehabilitation. The underlying mechanism is not fully understood. Injury to the spinal cord leads to neuroplastic changes including anatomical, physiological and molecular changes within the spinal cord and brain. Previous studies suggest that alterations in neuropeptides such as substance P, calcitonin gene-related peptide and cholecystokinin are involved in generation of neuropathic pain [10,25]. Of these peptides, cholecystokinin (CCK) is well known as an antagonist of endogenous opioids in the central nervous system [3,22]. Experimental evidence has shown that CCK mRNA increases after injury to the nervous system [2,25]. Xu et al. [25] reported up-regulation of CCK and CCK receptor mRNA in dorsal root ganglia after sciatic nerve injury in rats. Brewer et al. [2] reported an increase in CCK mRNA in the cortex of painful rats compared to painless rats in an excitotoxic SCI model. Previous reports clearly show that neuropathic pain can be controlled by blocking the actions of CCK in rats [28]. Xu et al. [24] reported, using an ischemic SCI model, that systemic opioids did not effectively
∗ Corresponding author. Tel.: +82 2 920 6278; fax: +82 2 925 5492. E-mail address: [email protected] (Y.W. Yoon). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.07.042
reduce mechanical allodynia but systemic CI-988, an antagonist of the CCKB receptor alleviated mechanical allodynia. Kovelowski et al. [12] demonstrated blockade of the action of CCK by injection of L365,260, a CCKB receptor antagonist, into the rostroventromedial medulla (RVM) reversed tactile allodynia and thermal hyperalgesia after spinal nerve injury, suggesting that endogenous CCK may contribute to maintenance of neuropathic pain following nerve injury. However, most studies are limited to peripheral nerve injury or certain types of SCI, especially ischemic SCI [23,24]. Thus, in the present study, we investigated the role of CCK in the maintenance of pain below the level of thoracic spinal hemisection. All experiment procedures were done in accordance with guidelines set by the Korea University College of Medicine Animal Research Policies Committee. Male Sprague–Dawley rats (n = 68, 150–200 g, at the time of operation) were used for this experiment. The animals were kept in a 12-h light/12-h dark cycle with light on 7:00 A.M. Under enflurane anesthesia (by mixture of 4% enflurane and 95% O2 ), a longitudinal incision was made exposing several segments, a laminectomy was performed, and the spinal cord was hemisected at T13 on the left side with a no. 11 scalpel blade. The wound was closed in anatomical layers, the skin with stainless steel wound clips. Behavioral tests for motor function and mechanical allodynia were performed preoperatively and postoperatively for the hindpaws. The tests were performed on each rat 1 day prior to surgery
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and 1, 4, 7 and 14 days postoperatively (PO), before the study of drug effects. Rats that showed a little mechanical allodynia or displayed contralateral hindlimb motor deficits were excluded. Mechanical allodynia was assessed by measuring the threshold of brisk paw withdrawal response to graded mechanical stimulus with a series of von Frey filaments (0.41, 0.70, 1.20, 2.00, 3.63, 5.50, 8.50, and 15.10 g, Stoelting, Wood Dale, IL, USA). To do this, the rat was placed under a transparent plastic dome (28 cm × 28 cm × 10 cm) on a metal mesh floor and a von Frey filament was applied to the plantar surface of the foot. A von Frey filament was applied for 3–4 s to each hind paw while the filament was bent. The 50% withdrawal threshold was determined using the up–down method [4], starting with a 2.0 g (4.31 mN) strength of filament. A withdrawal response was cause to present the next weaker stimulus, and lack of withdrawal led to presentation of the next stronger stimulus. Stimuli were presented at intervals of several seconds. A brisk foot withdrawal to application of a von Frey filament was regarded as a positive response. Interpolation of the 50% threshold was carried out according to the method of Dixon [6]. The investigator conducting the behavioral tests was blinded about the injected drug. Tests were carried out 3 weeks after spinal hemisection. During this period, motor function in hemisected rats fully recovered and signs of mechanical allodynia were well established as described in our previous report [11]. More than one test was performed on some rats. Intervals between repeated tests were at least 3 days. CI-988 (5, 10 and 20 mg/kg) or vehicle (saline) was injected intraperitoneally. Rats were randomly assigned to different treatment groups. Behavioral signs of mechanical allodynia were measured 30 min before and 15, 30, 45, 60, 90 and 120 min after injection of a drug. In order to determine the effect of intrathecally (IT) administered drugs on mechanical allodynia, rats for IT drug administration were implanted with catheters 2 weeks after hemisection. Under enflurane anesthesia (by mixture of 4% enflurane and 95% O2 ), the occipital muscles were separated from their attachment point and retracted caudally to expose the cisternal membrane at the base of the skull. Sterilized PE-10 tubing was threaded through an incision in the atlanto-occipital membrane to the 1 or 2 segments rostral to the hemisection site (5.5 cm). Animals showing evidence of neuromuscular dysfunction were excluded from further tests. After the experiment, the position of the IT catheter was confirmed by injection of Evans Blue dye following laminectomy. Drug was injected IT in a volume of 10 l followed by 10 l saline to flush the catheter. Behavioral signs of mechanical allodynia were measured 30 min before and 15, 30, 60, 90, 120 and 180 min after the injection. CI-988 was a generous gift from Pfizer Inc. (Groton, CT, USA). To exclude the possibility that motor impairment might contribute to changes in withdrawal threshold during testing, a modification of the combined behavioral score (CBS) of Gale et al. [8] was performed at the time of the behavioral test. The CBS assigns a weight to each of the tests and combines them into one total score that represents the degree of motor impairment. Tests were as follows: motor scores, toe spread, righting reflex, extension withdrawal reflex, placing reflex and inclined plane. Neurological function was evaluated by a scoring system that ranged from 0 for a normal rat to 90 for a completely paralyzed rat. To determine whether there were changes in expression levels of CCK and CCKB receptor mRNA, segments of the spinal cord at T11-12, L1-2 and L4-5 levels were removed and dissected into ipsilateral and contralateral dorsal quadrants from SCI rats (n = 8) and sham rats (n = 6). T11-12 and L1-2 levels of the spinal cord were segments just rostral and caudal, respectively, to the level of injury. The L4-5 spinal cord segment was also examined because it receives mainly sensory inputs from the foot, pain responses from
which were measured in the present study. The tissues were immediately frozen in liquid nitrogen and stored at −70 ◦ C. Total RNA extraction was performed using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. First-strand cDNA synthesis was performed using 50 pM oligo (dT)20 primer with Moloney-Mouse Leukemia Virus reverse transcriptase (Invitrogen, #28025-013, Carlsbad, CA, USA) at 37 ◦ C for 50 min. Total RNA and cDNA concentrations were determined at 260 nm by UV spectrophotometer (GeneQuant, Pharmacia, USA). We amplified 500 ng of cDNA for cholecystokinin (CCK), CCKB receptor (CCKBR) and 375 ng of cDNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with i-star taq DNA polymerase (Intron, #25162, Sungnam, Korea). PCR primer and conditions are presented in Table 1. PCR products were detected on a 1% agarose gel. A picture was taken using a digital camera (Canon A620) and we measured the density of the PCR bands using a computer-assisted image analysis system (NIH image software). All values are expressed as mean ± SEM. The Friedman repeated measures of analysis of variance followed by multiple comparison tests were used to compare behavioral test results before and after drug administration with the same animal. The absorbencies of the PCR bands were measured using Scion image software. Normal and injured group comparisons were performed using a one-way ANOVA with Scheffe’s post hoc test. Rats with SCI showed significant decrease in withdrawal threshold to von Frey stimulation with maximal motor recovery 3 weeks after spinal hemisection, as in our previous report [11]. The effects of systemic CI-988 on the hindlimb withdrawal threshold to mechanical stimulation applied to the plantar surface of the right foot and the left foot are shown in Fig. 1A. CI-988 at 5 mg/kg had no significant effects on paw withdrawal threshold, whereas doses of 10 and 20 mg/kg significantly increased paw withdrawal threshold without change of motor function tested using a CBS scale (data not shown). This anti-allodynic effect was significant at 15 min and persisted up to 60 min after injection in both feet compared to pretreatment values. There were no significant differences in threshold changes induced by CI-988 between ipsilateral and contralateral hind paws. Saline had no effect on the responsiveness to mechanical stimuli with von Frey stimulation. Intrathecally injected CI-988 (100, 200 and 500 g) significantly increased paw withdrawal threshold to mechanical stimulation applied to the plantar surface of the foot in a dose-dependent manner (Fig. 1B). Even a lower dose of CI-988 (100 g, n = 12) significantly increased withdrawal threshold from 15 to 60 min. The effect was peak at 15–30 min after injection, gradually diminished, and persisted for 90 min after higher doses of CI-988 (200 and 500 g). Saline (n = 9) did not change the withdrawal threshold throughout the test period. No changes in the CBS scale were observed after injection of any of the doses that were tested in this study (data not shown). We attempted to determine whether there were changes in expression level of CCK and CCKB receptor mRNA in the spinal cord after hemisection. The expression of CCK mRNA was assessed for both ipsilateral and contralateral sides at T11-12, L1-2 and L4-5 spinal cord segments and was compared to levels in normal rats (Fig. 2). CCK mRNA expression was significantly increased in both ipsilateral and contralateral sides at the L4-5 level compared to control (p < 0.05). At the L1-2 spinal cord level, CCK mRNA expression was significantly increased on the ipsilateral side (p < 0.05). On the contralateral side, CCK mRNA expression increased slightly, but there was no significant difference. At the T11-12 spinal cord level, CCK mRNA expression was similar to that of control on both ipsilateral and contralateral sides. In contrast to expression of CCK mRNA, CCKB receptor mRNA expression in the spinal cord did not show significant differences between hemisected rats and normal rats at all of the sites tested in this study.
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Table 1 PCR primer and condition.
Cholecystokinin (CCK) Cholecystokinin receptor (CCKBR) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
Length
Forward
Reverse
413 bp 361 bp 276 bp
GAA GTG GAC CCT ATG GA CAA GGC CAT TTC CTA CCT CA GAG TCT ACT GGC GTC TTC AC
TGC ATT GCACAC TCT GAA CA TAG GTA GAG TTC GCG GGA GA CCA TCC ACA GTC TTC TGA GT
The present data demonstrate that the mechanical allodynia induced by spinal cord hemisection was reduced by systemic and IT CI-988. CCK mRNA expression was significantly increased on both ipsilateral and contralateral sides at L4-5 and at the ipsilateral L1-2 spinal segment, with no change in the expression of CCKB receptor mRNA after spinal left hemisection. Systemically injected CI-988 significantly increased the paw withdrawal threshold, although the prominent anti-allodynic effects were achieved at the higher doses (10 and 20 mg/kg) in this study. The doses (5, 10 and 20 mg/kg) of systemic CI-988 tested were chosen because a previous report showed that 10 mg/kg of systemic CI-988 did not produce significant motor changes [16]. Our results are similar to those of a previous report [24], in which systemic CI-988 was effective in reducing vocalization threshold to mechanical stimulation applied to the back skin, and those analgesic effects were reversed by naloxone in an ischemic SCI model. However, higher doses of systemic CI-988 were needed to produce
PCR condition Temperature and time
Cycle
94 ◦ C:40 s, 62 ◦ C:30 s, 72 ◦ C:40 s 95 ◦ C:1 min, 59 ◦ C:1 min, 72 ◦ C:1 min 95 ◦ C:1 min, 61 ◦ C:1 min, 72 ◦ C:1 min
25 26 23
significant inhibition of mechanical allodynia, suggesting that the mechanical allodynia resulting from spinal hemisection is relatively resistant to systemic CI-988. IT CI-988 applied to the injured site increased the withdrawal threshold to von Frey stimulation applied to the plantar surface of the foot in a dose-dependent manner without any motor depression. Results that application of CI-988 at lesion sites reduced below-level neuropathic pain suggest that the lesion site could be a useful target for managing below-level pain. The relative ineffectiveness of systemic CI-988 and the relative effectiveness of IT administration suggest that a relatively high concentration of CI-988 in the spinal cord is required to produce the effects. Considering that CCK mRNA was increased in the spinal cord caudal to the injury, and that IT injection was reported to spread a few centimeters rostrocaudally from the tip of the catheter [27], analgesic effects of IT CI-988 may come from its action on caudal as well as rostral segments. A previous study [5] reported that a sig-
Fig. 1. Effects of systemic (A) and intrathecal (B) CI-988 on withdrawal threshold to mechanical stimulation applied to the plantar surface of the contralateral (left column) and ipsilateral foot (right column). Asterisks indicate the values significantly different from the pre-injection control value (PRE) (p < 0.05).
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Fig. 2. (A). A representative blot showing CCK mRNA and CCKB receptor mRNA from T11-12, L1-2 and L4-5 spinal segments. Also shown are relative gene expression levels of CCK mRNA (B) and CCKB receptor mRNA (C) for T11-12, L1-2 and L4-5 spinal cord segments. T11-12I: ipsilateral T11-12 spinal cord segments, T11-12C: contralateral T11-12 spinal cord segments, T11-12N; T11-12 spinal cord segments from normal rats, L1-2I: ipsilateral L1-2 spinal cord segments, L1-2C: contralateral L1-2 spinal cord segments, L1-2N; L1-2 spinal cord segments from normal rats, L45I; ipsilateral L4-5 spinal cord segments, L4-5C: contralateral L4-5 cord segments, L4-5N: L4-5 spinal cord segments from normal rats. Asterisks indicate that the values were significantly different from the corresponding control value (p < 0.05).
nificant anti-allodynic effect was achieved with a dose of 100 g IT of CI-988 after peripheral nerve injury. Based on our present results, the analgesic effect of CI-988 on allodynia may reflect an involvement of the endogenous CCKergic system in maintenance of chronic neuropathic pain after SCI. Blockade of CCKB receptors in the spinal cord attenuated thermal hyperalgesia after constriction injury to the sciatic nerve [28]. Further, CCKB receptor deficient mice showed significant increases in withdrawal threshold for mechanical sensitivity and they also did not display mechanical hyperalgesia after sciatic nerve ligation [13]. Thus, blocking spinal CCK receptors seems to be effective in reducing mechanical allodynia in experimental neuropathic pain models, including our current results. Previous studies suggest a more complicated picture of the nature of neuropathic pain such as a reduction in opioid receptors [14] and an increase in anti-opioid peptides such as CCK [9]. Recent studies have concentrated on the activation of descending pain facilitation in chronic pain states [20,21]. It is well known that enhanced descending facilitation under pathological conditions can be driven by CCK in the RVM [12,15,20]. Kovelowski et al. [12] demonstrated that microinjection of a CCKB receptor antagonist, L365,260, into the RVM reversed tactile allodynia and thermal
hyperalgesia after spinal nerve injury. Although the mechanisms for the effects of descending facilitation on chronic pain have been studied, the role of the spinal CCKergic system in central pain is still unclear. It is important to know where changes in the endogenous CCKergic system take place in the CNS, whether changes occur after spinal hemisection, and what types of changes occur. According to previous research, CCK is released into the spinal cord by intensive electrical stimulation of peripheral nerves [26] and electrical stimulation reduces the analgesic effects of IT morphine, but not of intracerebroventricular morphine [7], suggesting that the spinal cord is a major site of action of endogenous CCKergic systems. It suggests that changes in the spinal CCKergic system are associated with the development of neuropathic pain following SCI. To determine changes in the spinal CCKergic system following injury, we assessed expression levels of CCK and CCKB receptor mRNA in spinal segments rostral and caudal to the injured site. A recent report described an up-regulation of CCK mRNA in the cortex of rats with pain-like symptoms following SCI [2]. Another study demonstrated that CCK-like immunoreactivity is elevated 3fold in the cerebrospinal fluid (CSF) in painful rats compared to painless rats after spinal ischemia [23]. Our results demonstrate that CCK mRNA expression is increased on both sides at the L4-5 spinal cord level and on the ipsilateral side at the L1-2 spinal cord level, suggesting that up-regulation of spinal CCK may contribute to maintenance of central pain after SCI. The relationship between severity of pain and expression level of CCK mRNA was not assessed here because most of spinal hemisected rats showed a fairly high degree of mechanical allodynia. It should be noted that we found a difference in expression levels of CCK mRNA among the three sites tested in this study. CCK mRNA level was elevated in the spinal segments below the injured site, whereas there were no significant changes in the level of CCK mRNA expression in rostral segments. The present data may reflect the effects of cutting the lateral half of the spinal cord on descending facilitatory influences. Previous literature showed that descending facilitation projects bilaterally from the RVM [1] and nociceptive stimulation produces increased facilitation bilaterally [19]. Earlier studies showed that CCK receptor mRNA as well as CCK mRNA expression levels are up-regulated in dorsal root ganglion cells after unilateral section of the sciatic nerve in rats [25]. Interestingly, however, no significant difference was observed in CCKB receptor mRNA expression in any spinal segments measured after spinal hemisection. Together, these results suggest that enhanced CCK synthesis and release in spinal segments caudal to the level of injury rather than up-regulation of CCKB receptors is associated with spinal hemisection. A possible explanation for the role of the spinal CCKergic system in central pain following SCI is that an increase in CCK mRNA level results in enhancement of endogenous CCK levels in the spinal dorsal horn and the excess CCK acts on CCKB receptors that are maintained at near normal levels. Increased CCK facilitates pain, and therefore blocking CCK receptors reduce pain. The number of CCK receptors did not change in the present study, but there may have been functional changes which further studies will elucidate. In summary, the CCKB receptor antagonist, CI-988 effectively reduced below-level mechanical allodynia after SCI and upregulation of CCK may contribute to development or maintenance of neuropathic pain after spinal hemisection. Thus, CCK receptor antagonism may represent a strategy for development of new therapeutic agents to manage mechanical allodynia in this type of central neuropathic pain.
Acknowledgment This work was supported by a grant of the Korea University College of Health Science (K0718991).
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References [1] M. Antal, M. Petko, E. Polgar, C.W. Heizmann, J. Storm-Mathisen, Direct evidence of an extensive GABAergic innervation of the spinal dorsal horn by fibres descending from the rostral ventromedial medulla, Neuroscience 73 (1996) 509–518. [2] K.L. Brewer, D. McMillan, T. Nolan, K. Shum, Cortical changes in cholecystokinin mRNA are related to spontaneous pain behaviors following excitotoxic spinal cord injury in the rat, Brain Res. Mol. Brain Res. 118 (2003) 171–174. [3] F. Cesselin, Opioid and anti-opioid peptides, Fundam. Clin. Pharmacol. 9 (1995) 409–433. [4] S.R. Chaplan, F.W. Bach, J.W. Pogrel, J.M. Chung, T.L. Yaksh, Quantitative assessment of tactile allodynia in the rat paw, J. Neurosci. Methods 53 (1994) 55–63. [5] M.A. Coudore-Civiale, C. Courteix, J. Fialip, M. Boucher, A. Eschalier, Spinal effect of the cholecystokinin-B receptor antagonist CI-988 on hyperalgesia, allodynia and morphine-induced analgesia in diabetic and mononeuropathic rats, Pain 88 (2000) 15–22. [6] W.J. Dixon, Efficient analysis of experimental observations, Annu. Rev. Pharmacol. Toxicol. 20 (1980) 441–462. [7] Y. Fukazawa, T. Maeda, W. Hamabe, K. Kumamoto, Y. Gao, C. Yamamoto, M. Ozaki, S. Kishioka, Activation of spinal anti-analgesic system following electroacupuncture stimulation in rats, J. Pharmacol. Sci. 99 (2005) 408–414. [8] K. Gale, H. Kerasidis, J.R. Wrathall, Spinal cord contusion in the rat: behavioral analysis of functional neurologic impairment, Exp. Neurol. 88 (1985) 123–134. [9] M.M. Heinricher, M.J. Neubert, Neural basis for the hyperalgesic action of cholecystokinin in the rostral ventromedial medulla, J. Neurophysiol. 92 (2004) 1982–1989. [10] T. Hokfelt, X. Zhang, Z. Wiesenfeld-Hallin, Messenger plasticity in primary sensory neurons following axotomy and its functional implications, Trends Neurosci. 17 (1994) 22–30. [11] J. Kim, Y.W. Yoon, S.K. Hong, H.S. Na, Cold and mechanical allodynia in both hindpaws and tail following thoracic spinal cord hemisection in rats: time courses and their correlates, Neurosci. Lett. 343 (2003) 200–204. [12] C.J. Kovelowski, M.H. Ossipov, H. Sun, J. Lai, T.P. Malan, F. Porreca, Supraspinal cholecystokinin may drive tonic descending facilitation mechanisms to maintain neuropathic pain in the rat, Pain 87 (2000) 265–273. [13] K. Kurrikoff, S. Koks, T. Matsui, M. Bourin, A. Arend, M. Aunapuu, E. Vasar, Deletion of the CCK2 receptor gene reduces mechanical sensitivity and abolishes the development of hyperalgesia in mononeuropathic mice, Eur. J. Neurosci. 20 (2004) 1577–1586. [14] M.H. Ossipov, Y. Lopez, M.L. Nichols, D. Bian, F. Porreca, Inhibition by spinal morphine of the tail-flick response is attenuated in rats with nerve ligation injury, Neurosci. Lett. 199 (1995) 83–86.
307
[15] F. Porreca, M.H. Ossipov, G.F. Gebhart, Chronic pain and medullary descending facilitation, Trends Neurosci. 25 (2002) 319–325. [16] M. Qian, A.E. Johnson, L. Kallstrom, H. Carrer, P. Sodersten, Cholecystokinin, dopamine D2 and N-methyl-d-aspartate binding sites in the nucleus of the solitary tract of the rat: possible relationship to ingestive behavior, Neuroscience 77 (1997) 1077–1089. [17] P.J. Siddall, R.P. Yezierski, J.D. Loeser, K.J. Burchiel, Taxonomy and Epidemiology of Spinal Cord Injury Pain. Spinal Cord Injury Pain: Assessment, Mechanisms, Management, IASP Press, Seattle, 2002, pp. 9–24. [18] R.R. Tasker, Central pain states, in: J.D. Loeser (Ed.), Bonica’s Management of Pain, Lippincott Williams Wilkins, Philadelphia, 2001, pp. 433–457. [19] R. Terayama, R. Dubner, K. Ren, The roles of NMDA receptor activation and nucleus reticularis gigantocellularis in the time-dependent changes in descending inhibition after inflammation, Pain 97 (2002) 171–181. [20] D.V. Tillu, G.F. Gebhart, K.A. Sluka, Descending facilitatory pathways from the RVM initiate and maintain bilateral hyperalgesia after muscle insult, Pain 136 (2008) 331–339. [21] L.P. Vera-Portocarrero, E.T. Zhang, M.H. Ossipov, J.Y. Xie, T. King, J. Lai, F. Porreca, Descending facilitation from the rostral ventromedial medulla maintains nerve injury-induced central sensitization, Neuroscience 140 (2006) 1311– 1320. [22] Z. Wiesenfeld-Hallin, G. de Arauja Lucas, P. Alster, X.J. Xu, T. Hokfelt, Cholecystokinin/opioid interactions, Brain Res. 848 (1999) 78–89. [23] X.J. Xu, P. Alster, W.P. Wu, J.X. Hao, Z. Wiesenfeld-Hallin, Increased level of cholecystokinin in cerebrospinal fluid is associated with chronic pain-like behavior in spinally injured rats, Peptides 22 (2001) 1305–1308. [24] X.J. Xu, J.X. Hao, A. Seiger, J. Hughes, T. Hokfelt, Z. Wiesenfeld-Hallin, Chronic pain-related behaviors in spinally injured rats: evidence for functional alterations of the endogenous cholecystokinin and opioid systems, Pain 56 (1994) 271–277. [25] X.J. Xu, M.J. Puke, V.M. Verge, Z. Wiesenfeld-Hallin, J. Hughes, T. Hokfelt, Upregulation of cholecystokinin in primary sensory neurons is associated with morphine insensitivity in experimental neuropathic pain in the rat, Neurosci. Lett. 152 (1993) 129–132. [26] T.L. Yaksh, E.O. Abay II, V.L. Go, Studies on the location and release of cholecystokinin and vasoactive intestinal peptide in rat and cat spinal cord, Brain Res. 242 (1982) 279–290. [27] T.L. Yaksh, T.A. Rudy, Chronic catheterization of the spinal subarachnoid space, Physiol. Behav. 17 (1976) 1031–1036. [28] T. Yamamoto, N. Nozaki-Taguchi, Role of cholecystokinin-B receptor in the maintenance of thermal hyperalgesia induced by unilateral constriction injury to the sciatic nerve in the rat, Neurosci. Lett. 202 (1995) 89–92.