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Antiallodynic effect through spinal endothelin-B receptor antagonism in rat models of complex regional pain syndrome
Neuroscience Letters 584 (2015) 45–49
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Antiallodynic effect through spinal endothelin-B receptor antagonism in rat models of complex regional pain syndrome Yeo Ok Kim a,1 , In Ji Kim a,1 , Myung Ha Yoon a,b,∗ a b
Department of Anesthesiology and Pain Medicine, Chonnam National University, Medical School, Gwangju, South Korea Center for Creative Biomedical Scientists at Chonnam National University, Gwangju, South Korea
h i g h l i g h t s • • • • •
Each CRPS-I and -II rat model was made by O-ring application or by spinal nerve ligation. The level of ET-1 in the spinal cord was increased in both CRPS models. Intrathecal ET-B receptor antagonist increased the withdrawal threshold in both CRPS types. Intrathecal ET-A receptor antagonist did not affect the withdrawal threshold in either CRPS type. Intrathecal ET-B receptor antagonist decreased the spinal ET-1 level in both CRPS rats.
a r t i c l e
i n f o
Article history: Received 17 August 2014 Received in revised form 21 September 2014 Accepted 6 October 2014 Available online 22 October 2014 Keywords: Allodynia CRPS Endothelin(ET)-1 ET-B receptor Spinal cord
a b s t r a c t Complex regional pain syndrome (CRPS) is a very complicated chronic pain disorder that has been classified into two types (I and II). Endothelin (ET) receptors are involved in pain conditions at the spinal level. We investigated the role of spinal ET receptors in CRPS. Chronic post-ischemia pain (CPIP) was induced in male Sprague–Dawley rats as a model for CRPS-I by placing a tourniquet (O-ring) at the ankle joint for 3 h, and removing it to allow reperfusion. Ligation of L5 and L6 spinal nerves to induce neuropathic pain was performed as a model for CRPS-II. After O-ring application and spinal nerve ligation, the paw withdrawal threshold was significantly decreased at injured sites. Intrathecal administration of the selective ET-B receptor antagonist BQ 788 dose-dependently increased the withdrawal threshold in both CRPS-I and CRPS-II. In contrast, ET-A receptor antagonist BQ 123 did not affect the withdrawal threshold in either CRPS type. The ET-1 levels of plasma and spinal cord increased in both CRPS types. Intrathecal BQ 788 decreased the spinal ET-1 level. These results suggest that ET-1 is involved in the development of mechanical allodynia in CRPS. Furthermore, the ET-B receptor appears to be involved in spinal cord-related CRPS. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Complex regional pain syndrome (CRPS) is a debilitating chronic pain disorder that is difficult to treat satisfactorily [12]. Therefore, clinicians often experience emotional stress while treating such patients, while the patients often feel frustration and suffer a loss of quality of life. CRPS is classified into type I (previously called reflex sympathetic dystrophy) and type II (previously termed causalgia) [13]. CRPS-I occurs after fracture, soft tissue injury, or
∗ Corresponding author at: 42 Jebongro, Donggu, Gwangju 501-757, South Korea. Tel.: +82 62 220 6893; fax: +82 62 232 6294. E-mail address: [email protected] (M.H. Yoon). 1 Both Yeo Ok Kim and In Ji Kim contributed equally as a first author. http://dx.doi.org/10.1016/j.neulet.2014.10.005 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
crush injury [5]. CRPS-II is similar, but also exhibits clinically verified nerve injury [25]. Typical symptoms of CRPS are allodynia and hyperalgesia [14]. Although the underlying mechanisms of CRPS have been studied increasingly over the past decade, knowledge of the detailed mechanisms is lacking because of the complex pathophysiology of this syndrome. Therefore, CRPS treatment remains a problematic issue and effective studies that shed more light on this disorder are clearly needed. Recently, the chronic post-ischemia pain (CPIP) model was proposed as a CRPS-I animal model [4]. A classical neuropathic pain model induced by spinal nerve ligation (SNL) may be considered an animal model of CRPS-II. Endothelins (ET) are one of the many signaling systems involved in various chronic pain conditions. Behaviorally, ET-1 induces pain,
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Y.O. Kim et al. / Neuroscience Letters 584 (2015) 45–49
whereas local or systemic ET-receptor antagonists attenuate a variety of nociceptive states [1,3,7,11,15,18–20,26]. However, little is known about the role of ET receptors found in the spinal cord in nociception [23]. Therefore, we investigated the modulatory role of spinal ET receptors using rat models of CRPS-I and CRPS-II, as well as the possible modification of the ET-1 levels in plasma and spinal cord.
2. Materials and methods 2.1. Animal preparation This study was approved by The Institutional Animal Care and Use Committee of Chonnam National University. Male Sprague–Dawley rats weighing 100–120 and 320–340 g were used in all experiments. While in the home–cage environment, the animals had free access to a standard rat diet and tap water. Room temperature was maintained at 20–23 ◦ C with a 12:12-hour light/dark cycle.
2.2. Animal model of CRPS-I (CPIP) and CRPS-II (SNL) Both CRPS-I model and CRPS-II model were produced by O-ring application and by spinal nerve ligation as described previously [4,17]. In CRPS-I, a Nitrile 70 Durometer O-ring (Orings West, Seattle, WA, USA) with 7/32 in. internal diameter was placed around the rat’s left hind limb just proximal to the ankle joint for 3 h under sevoflurane anesthesia, and then removed. In CRPS-II, the left L5 and L6 spinal nerves of rats were isolated adjacent to the vertebral column during sevoflurane anesthesia and tightly ligated with a 6-0 silk suture. Sham rats received the same procedure, except that the O-ring was cut so that it fit loosely around the ankle without occluding the blood flow to the hind paw or without ligation of the spinal nerves. Animals were considered to have CRPS-I and CRPS-II when a paw-flinching response occurred upon applying a bending force of <5 and <4 g, respectively. Following procedure, the mechanical sensitivity of the injured paw was daily evaluated for 21 days.
2.3. Implantation of intrathecal catheter At 2 and 5 days after hind paw ischemia and reperfusion or spinal nerve ligation, a polyethylene-10 tube was inserted into the subarachnoid space through a slit made in the atlantooccipital membrane under sevoflurane anesthesia [27]. Rats with neurological deficit after catheterization were excluded and euthanized immediately with an overdose of volatile anesthetics. There was a recovery period of 5 days after catheterization before commencing the behavioral study.
2.4. Drugs Cyclo[d-Trp-d-Asp-Pro-d-Val-Leu] (BQ 123, Tocris Cookson Ltd., Bristol, UK) was used as an ET-A receptor antagonist while N-[(cis-2,6-dimethyl-1-piperidinyl)carbonyl]-4-methyl-l-leucyl1-(methoxycarbonyl)-d-tryptophyl-d-norleucine sodium salt (BQ 788, Tocris) was administered as an ET-B receptor antagonist. BQ 123 and BQ 788 were dissolved in 0.9% saline or dimethylsulfoxide (DMSO), respectively. Intrathecal administration of these agents was performed using a hand-driven, gear-operated syringe pump. The drugs were delivered as a 10 L solution, followed by an additional 10 L normal saline to flush the catheter.
2.5. Assessment of mechanical allodynia To determine withdrawal threshold, rats were placed individually in plastic cages with a plastic mesh floor. The animals were tested after acclimation to the environment, typically 20–30 min after placement in the cage. The paw withdrawal threshold in response to mechanical stimulation was measured using the up and down method [2] by applying calibrated von Frey filaments (Stoelting, Wood Dale, IL, USA) to the hind paw from underneath the cage through openings in the mesh floor. A series of eight von Frey filaments (0.4, 0.7, 1.2, 2.0, 3.6, 5.5, 8.5, and 15 g) were applied vertically to the plantar surface of the hind paw for 5 s while the hair was bent. Brisk withdrawal or paw flinching was considered a positive response. The absence of a response in the animals at a pressure of 15 g was considered the cutoff value. 2.6. Experimental paradigm The rats were allocated to receive BQ 123 or BQ 788 on the day of the experiment, while the control animals received solvent alone (saline or DMSO, respectively). Animals were tested only once. All experiments were carried out by an observer blinded to the drug treatments. 2.7. Effects of intrathecal BQ 123 and BQ 788 The effects of the ET-A receptor antagonist BQ 123 (20 and 50 g) and the ET-B receptor antagonist BQ 788 (10, 20, and 40 g) were investigated in CPIP and neuropathic painstate rats. Measurement of the mechanical threshold prior to ischemia–reperfusion induction or spinal nerve ligation was taken as the baseline threshold. The withdrawal threshold was determined at 15, 30, 60, 90, 120, 150, and 180 min after intrathecal administration of the drugs. The withdrawal threshold measured immediately before intrathecal delivery of drugs was taken as the control. The highest drug doses were selected based on their lack of neurologic impairment with maximal solubility from pilot experiments. Hence, the highest drug doses administered were considered the maximum doses. 2.8. Measurement of ET-1 levels The levels of ET-1 in plasma and spinal cord were measured in sham, CRPS and ET-B receptor antagonist BQ 788-delivered rats. In CRPS models, ET-1 levels were determined 7 and 10 days after ischemia–reperfusion or spinal nerve ligation, and 60–90 min after BQ 788 administration. The ET-1 level was quantified using ELISA kits obtained from Assay Designs (Ann Arbor, Michigan, USA). 2.9. General behavior Behavioral changes in response to BQ 123 and BQ 788 treatment were evaluated in additional rats 5, 10, 20, 30, 40, 50, and 60 min after intrathecal administration of the highest drug doses. Motor functions were determined by examining the righting and placing-stepping reflexes. Righting was evaluated by placing the rat horizontally with its back on a table, which normally gives rise to an immediate coordinated twisting of the body to an upright position. Placing-stepping reflexes were evoked by drawing the dorsum of either hind paw across the edge of the table. Rats normally attempt to place their paws forward into a walking position. Pinna and corneal reflexes was also evaluated and considered present or absent. Abnormal behavior, including serpentine movement or tremors, was also monitored.
Y.O. Kim et al. / Neuroscience Letters 584 (2015) 45–49
Fig. 1. Time course of hind paw withdrawal response to von Frey filaments after ischemic reperfusion (A) and spinal nerve ligation (B). Data are presented as the withdrawal threshold. Each line represents mean ± SEM (n = 5–6). BL: baseline withdrawal threshold measured before ischemic reperfusion or spinal nerve ligation. Significant difference between the injured and non-injured site is indicated; * P < 0.05, † P < 0.01, ‡ P < 0.001.
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Fig. 2. Effect of intrathecal ET-A receptor antagonist BQ 123 on hind paw withdrawal response to von Frey filaments after ischemic reperfusion and spinal nerve ligation. Data are presented as the withdrawal threshold. Each line represents mean ± SEM (n = 5–6). BL: baseline withdrawal threshold measured before ischemic reperfusion or spinal nerve ligation. Control data were measured immediately before intrathecal drug delivery. Intrathecal BQ 123 did not affect the withdrawal threshold in the injured paw.
2.10. Statistical analysis Data are expressed as mean ± SEM. Time–response data are presented as the withdrawal threshold in grams. Dose–response data are presented as the percentage of the maximum possible effect (%MPE). Withdrawal threshold data from von Frey filament testing were converted into %MPE according to the formula: %MPE = [(postdrug threshold − post-injured baseline threshold)/(cutoff threshold − post-injured baseline threshold)] × 100. Dose–response data were analyzed using one-way analysis of variance followed by Scheffe’s post hoc test. The differences in the withdrawal threshold or the ET-1 level between injured and non-injured sites were analyzed using an unpaired t-test. P < 0.05 was considered statistically significant.
No differences in the post-injured baseline withdrawal threshold (control) among the drug treatment groups were found in CPIP and neuropathic pain rats (Figs. 2 and 3). Intrathecal BQ 123 did not alter the withdrawal threshold in the injured paw of CPIP and neuropathic pain rats (Fig. 2). In contrast, intrathecal BQ 788 dose-dependently increased the withdrawal threshold in the injured paw of CPIP and neuropathic pain rats (Fig. 3). The ET-1 levels of plasma and spinal cord increased in CPIP and neuropathic pain rats compared to sham rats. In addition, intrathecal BQ 788 decreased the ET-1 level in the spinal cord of CRPS rats (Tables 1 and 2).
3. Results Righting and placing-stepping reflexes were normal after intrathecal delivery of BQ 123 and BQ 788, and pinna and corneal reflexes were present at the dosage used. No overt abnormal behavioral changes were observed. The withdrawal threshold was significantly decreased and persisted for almost 21 days at the O-ring application site and at the ligation site compared to the sham group (Fig. 1). The withdrawal thresholds in the sham groups did not change during the observational period.
Table 1 The level of ET-1 (pg/ml) in the plasma. CRPS-I
ET-1
CRPS-II
Sham
CPIP
Sham
SNL
0.2 ± 0.2
1.8 ± 1.3*
0.8 ± 0.9
2.3 ± 1.0†
ET-1 = endothelin-1; CRPS = complex regional pain syndrome; CPIP = chronic postischemia pain; SNL = spinal nerve ligation. Values are mean ± SEM (n = 4–5). * P < 0.05 compared to sham. † P < 0.01 compared to sham.
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Fig. 3. Effects of intrathecal ET-B receptor antagonist BQ 788 on hind paw withdrawal response to von Frey filaments after ischemic reperfusion and spinal nerve ligation. Data are presented as the withdrawal threshold or the percent of maximal possible effect (%MPE). Each line represents mean ± SEM (n = 6–7). BL: baseline withdrawal threshold measured before ischemic reperfusion or spinal nerve ligation. Control data were measured immediately before intrathecal drug delivery. Intrathecal BQ 788 produced a dose-dependent increase in the withdrawal threshold in the injured paw; * P < 0.05, † P < 0.01.
4. Discussion The withdrawal threshold in CPIP and neuropathic rats was increased by a selective ET-B receptor antagonist but not by a selective ET-A receptor antagonist. The ET-1 levels of the plasma and the spinal cord increased in CPIP and neuropathic pain rats. These findings suggest that ET-1 may contribute to the development of CRPS, potentially through the spinal ET-B receptor. In contrast, the spinal ET-A receptor does not appear to be associated with CRPS. CRPS is currently generally considered a systemic condition that involves both central and peripheral components of the neuraxis [14] and interactions between the immune and nervous systems [25]. Clinical characteristics of CRPS include pain, abnormal regulation of blood flow and sweating, edema of the skin and subcutaneous tissues, trophic changes to the skin, skin appendages and subcutaneous tissues, and active and passive movement disorders [14]. Although CRPS is classified into types I and II [13], the underlying mechanisms of both remain largely unknown. Several studies
on laboratory animals and humans have indicated that ET participates in the pathogenesis of the pain-related process. ET is an endogenous 21-amino-acid peptide family composed of ET-1, ET2, and ET-3 that regulates blood flow, cell proliferation, secretion, ion transport, and pain perception [23]. ET-1–3 are synthesized as propeptides, which are transformed into the active form by sequential endopeptidase and ER-converting enzyme-mediated cleavage. ET-1 is the predominant and most potent isoform, and its biological effects are transduced by two pharmacologically distinguishable receptor subtypes, the ET-A and ET-B receptors [23]. ET may play a role in nociceptive processing, independently of its cardiovascular properties [10,11,16]. Local injection of ET-1 produces pain-like behavior through activation of the ET-A receptor in rats [7]. Local administration of ET-A and ET-B receptor antagonists inhibit thermal and inflammatory hyperalgesia in mice [1]. Intraplantar ET-A and ET-B receptor antagonists inhibit ET-1-induced nociception in rats [19,20]. Intraperitoneal ET-A receptor antagonist improves thermal and mechanical hyperalgesia induced by chronic constriction injury of the rat sciatic nerve and attenuates tactile allodynia
Table 2 The level of ET-1 (pg/ml) in the spinal cord. CRPS-I
ET-1
CRPS-II
Sham
CPIP
CPIP: BQ 788
Sham
SNL
SNL: BQ 788
3.5 ± 0.3
5.0 ± 0.3*
3.4 ± 0.4#
3.4 ± 0.2
8.7 ± 0.9†
4.4 ± 0.1‡
ET-1 = endothelin-1; CRPS = complex regional pain syndrome; CPIP = chronic post-ischemia pain; SNL = spinal nerve ligation. Values are mean ± SEM (n = 5–6). * P < 0.05 compared to sham. # P < 0.05 compared to CPIP. † P < 0.01 compared to sham. ‡ P < 0.01 compared to SNL.
Y.O. Kim et al. / Neuroscience Letters 584 (2015) 45–49
in a diabetic rat model of neuropathic pain [15,18]. Intravenous ET-B, but not ET-A, receptor antagonist reduces mechanical allodynia in rats with trigeminal neuropathic pain [3]. Intraplantar ET-A and ET-B receptor antagonists diminish cold allodynia induced by spinal nerve ligation, whereas only the ET-A receptor antagonist is effective against thermal hyperalgesia [26]. An oral ET-A receptor antagonist relieves sciatica in pulmonary hypertension patients [22]. In contrast, the present study demonstrated that intrathecal ET-B, but not ET-A, receptor antagonist reduced mechanical allodynia induced in animal models of CPIP and spinal nerve ligation-induced neuropathic pain. Furthermore, ET-A and ET-B receptors have been reported to exist in the spinal cord [23]. Other studies have reported ET-1 levels that are different than what we found in the present study. In CRPS-I patients, the ET-1 level increases in blister fluid and tissue [8,9]. However, plasma and cerebrospinal fluid ET-1 levels are not different between CRPSI patients and normal volunteers [6,21]. A significant increase in spinal ET concentration has been reported after spinal cord injury in rats [24]. In the present study, the plasma ET-1 level increased after ischemia–reperfusion and spinal nerve ligation. Furthermore, the increased ET-1 level in the spina cord of CRPS rats was attenuated by ET-B receptor anatgonist, but not by ET-A receptor anatgonist. Therefore, the current results together with previous findings suggest that an increased ET-1 after ischemia–reperfusion or nerve injury may contribute to mechanical allodynia through the activation of the ET-B receptor in the spinal cord. This is the first study to demonstrate the relevance of spinal ET-B receptor to allodynia in CRPS. In addition, the plasma ET-1 level may be a biomarker for CRPS. However, further research needs to apply this tool to human. Furthermore, CPIP and SNL modeling used in this study did not cause the spinal cord damage. Thus, those procedures little affected the plasma or spinal ET-1 level. There are some limitations to this study. First, there were insufficient observational parameters. CRPS is a complex disorder manifesting as several symptoms, including allodynia, vasomotor and sudomotor function, trophic skin changes, and movement changes. However, we only evaluated allodynia. Thus, other parameters mentioned above should also be assessed. Second, there was a lack of data on the ET-A receptor antagonist. BQ 123 was only used at a maximum dose of 50 g, above which it was insoluble. In addition, the effect of other ET-A receptor antagonists was not evaluated. Thus, further studies are required to consider these issues. Spinal ET-B receptor antagonists are not yet available in clinics. However, they may be clinically applied in the treatment of CRPS in the future. In conclusion, the ET-1 levels of plasma and spinal cord increased after ischemic reperfusion and spinal nerve ligation. In addition, intrathecal ET-B receptor, but not ET-A receptor, antagonist alleviated mechanical allodynia induced by ischemic reperfusion and spinal nerve ligation. These findings suggest that ET-1 may be involved in the spinal signaling mechanism operated by the ET-B receptor in mechanical allodynia in CRPS. Accordingly, the spinal ET-B receptor antagonist may be useful for managing mechanical allodynia in CRPS.
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Antiallodynic effect through spinal endothelin-B receptor antagonism in rat models of complex regional pain syndrome Yeo Ok Kim a,1 , In Ji Kim a,1 , Myung Ha Yoon a,b,∗ a b
Department of Anesthesiology and Pain Medicine, Chonnam National University, Medical School, Gwangju, South Korea Center for Creative Biomedical Scientists at Chonnam National University, Gwangju, South Korea
h i g h l i g h t s • • • • •
Each CRPS-I and -II rat model was made by O-ring application or by spinal nerve ligation. The level of ET-1 in the spinal cord was increased in both CRPS models. Intrathecal ET-B receptor antagonist increased the withdrawal threshold in both CRPS types. Intrathecal ET-A receptor antagonist did not affect the withdrawal threshold in either CRPS type. Intrathecal ET-B receptor antagonist decreased the spinal ET-1 level in both CRPS rats.
a r t i c l e
i n f o
Article history: Received 17 August 2014 Received in revised form 21 September 2014 Accepted 6 October 2014 Available online 22 October 2014 Keywords: Allodynia CRPS Endothelin(ET)-1 ET-B receptor Spinal cord
a b s t r a c t Complex regional pain syndrome (CRPS) is a very complicated chronic pain disorder that has been classified into two types (I and II). Endothelin (ET) receptors are involved in pain conditions at the spinal level. We investigated the role of spinal ET receptors in CRPS. Chronic post-ischemia pain (CPIP) was induced in male Sprague–Dawley rats as a model for CRPS-I by placing a tourniquet (O-ring) at the ankle joint for 3 h, and removing it to allow reperfusion. Ligation of L5 and L6 spinal nerves to induce neuropathic pain was performed as a model for CRPS-II. After O-ring application and spinal nerve ligation, the paw withdrawal threshold was significantly decreased at injured sites. Intrathecal administration of the selective ET-B receptor antagonist BQ 788 dose-dependently increased the withdrawal threshold in both CRPS-I and CRPS-II. In contrast, ET-A receptor antagonist BQ 123 did not affect the withdrawal threshold in either CRPS type. The ET-1 levels of plasma and spinal cord increased in both CRPS types. Intrathecal BQ 788 decreased the spinal ET-1 level. These results suggest that ET-1 is involved in the development of mechanical allodynia in CRPS. Furthermore, the ET-B receptor appears to be involved in spinal cord-related CRPS. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Complex regional pain syndrome (CRPS) is a debilitating chronic pain disorder that is difficult to treat satisfactorily [12]. Therefore, clinicians often experience emotional stress while treating such patients, while the patients often feel frustration and suffer a loss of quality of life. CRPS is classified into type I (previously called reflex sympathetic dystrophy) and type II (previously termed causalgia) [13]. CRPS-I occurs after fracture, soft tissue injury, or
∗ Corresponding author at: 42 Jebongro, Donggu, Gwangju 501-757, South Korea. Tel.: +82 62 220 6893; fax: +82 62 232 6294. E-mail address: [email protected] (M.H. Yoon). 1 Both Yeo Ok Kim and In Ji Kim contributed equally as a first author. http://dx.doi.org/10.1016/j.neulet.2014.10.005 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
crush injury [5]. CRPS-II is similar, but also exhibits clinically verified nerve injury [25]. Typical symptoms of CRPS are allodynia and hyperalgesia [14]. Although the underlying mechanisms of CRPS have been studied increasingly over the past decade, knowledge of the detailed mechanisms is lacking because of the complex pathophysiology of this syndrome. Therefore, CRPS treatment remains a problematic issue and effective studies that shed more light on this disorder are clearly needed. Recently, the chronic post-ischemia pain (CPIP) model was proposed as a CRPS-I animal model [4]. A classical neuropathic pain model induced by spinal nerve ligation (SNL) may be considered an animal model of CRPS-II. Endothelins (ET) are one of the many signaling systems involved in various chronic pain conditions. Behaviorally, ET-1 induces pain,
46
Y.O. Kim et al. / Neuroscience Letters 584 (2015) 45–49
whereas local or systemic ET-receptor antagonists attenuate a variety of nociceptive states [1,3,7,11,15,18–20,26]. However, little is known about the role of ET receptors found in the spinal cord in nociception [23]. Therefore, we investigated the modulatory role of spinal ET receptors using rat models of CRPS-I and CRPS-II, as well as the possible modification of the ET-1 levels in plasma and spinal cord.
2. Materials and methods 2.1. Animal preparation This study was approved by The Institutional Animal Care and Use Committee of Chonnam National University. Male Sprague–Dawley rats weighing 100–120 and 320–340 g were used in all experiments. While in the home–cage environment, the animals had free access to a standard rat diet and tap water. Room temperature was maintained at 20–23 ◦ C with a 12:12-hour light/dark cycle.
2.2. Animal model of CRPS-I (CPIP) and CRPS-II (SNL) Both CRPS-I model and CRPS-II model were produced by O-ring application and by spinal nerve ligation as described previously [4,17]. In CRPS-I, a Nitrile 70 Durometer O-ring (Orings West, Seattle, WA, USA) with 7/32 in. internal diameter was placed around the rat’s left hind limb just proximal to the ankle joint for 3 h under sevoflurane anesthesia, and then removed. In CRPS-II, the left L5 and L6 spinal nerves of rats were isolated adjacent to the vertebral column during sevoflurane anesthesia and tightly ligated with a 6-0 silk suture. Sham rats received the same procedure, except that the O-ring was cut so that it fit loosely around the ankle without occluding the blood flow to the hind paw or without ligation of the spinal nerves. Animals were considered to have CRPS-I and CRPS-II when a paw-flinching response occurred upon applying a bending force of <5 and <4 g, respectively. Following procedure, the mechanical sensitivity of the injured paw was daily evaluated for 21 days.
2.3. Implantation of intrathecal catheter At 2 and 5 days after hind paw ischemia and reperfusion or spinal nerve ligation, a polyethylene-10 tube was inserted into the subarachnoid space through a slit made in the atlantooccipital membrane under sevoflurane anesthesia [27]. Rats with neurological deficit after catheterization were excluded and euthanized immediately with an overdose of volatile anesthetics. There was a recovery period of 5 days after catheterization before commencing the behavioral study.
2.4. Drugs Cyclo[d-Trp-d-Asp-Pro-d-Val-Leu] (BQ 123, Tocris Cookson Ltd., Bristol, UK) was used as an ET-A receptor antagonist while N-[(cis-2,6-dimethyl-1-piperidinyl)carbonyl]-4-methyl-l-leucyl1-(methoxycarbonyl)-d-tryptophyl-d-norleucine sodium salt (BQ 788, Tocris) was administered as an ET-B receptor antagonist. BQ 123 and BQ 788 were dissolved in 0.9% saline or dimethylsulfoxide (DMSO), respectively. Intrathecal administration of these agents was performed using a hand-driven, gear-operated syringe pump. The drugs were delivered as a 10 L solution, followed by an additional 10 L normal saline to flush the catheter.
2.5. Assessment of mechanical allodynia To determine withdrawal threshold, rats were placed individually in plastic cages with a plastic mesh floor. The animals were tested after acclimation to the environment, typically 20–30 min after placement in the cage. The paw withdrawal threshold in response to mechanical stimulation was measured using the up and down method [2] by applying calibrated von Frey filaments (Stoelting, Wood Dale, IL, USA) to the hind paw from underneath the cage through openings in the mesh floor. A series of eight von Frey filaments (0.4, 0.7, 1.2, 2.0, 3.6, 5.5, 8.5, and 15 g) were applied vertically to the plantar surface of the hind paw for 5 s while the hair was bent. Brisk withdrawal or paw flinching was considered a positive response. The absence of a response in the animals at a pressure of 15 g was considered the cutoff value. 2.6. Experimental paradigm The rats were allocated to receive BQ 123 or BQ 788 on the day of the experiment, while the control animals received solvent alone (saline or DMSO, respectively). Animals were tested only once. All experiments were carried out by an observer blinded to the drug treatments. 2.7. Effects of intrathecal BQ 123 and BQ 788 The effects of the ET-A receptor antagonist BQ 123 (20 and 50 g) and the ET-B receptor antagonist BQ 788 (10, 20, and 40 g) were investigated in CPIP and neuropathic painstate rats. Measurement of the mechanical threshold prior to ischemia–reperfusion induction or spinal nerve ligation was taken as the baseline threshold. The withdrawal threshold was determined at 15, 30, 60, 90, 120, 150, and 180 min after intrathecal administration of the drugs. The withdrawal threshold measured immediately before intrathecal delivery of drugs was taken as the control. The highest drug doses were selected based on their lack of neurologic impairment with maximal solubility from pilot experiments. Hence, the highest drug doses administered were considered the maximum doses. 2.8. Measurement of ET-1 levels The levels of ET-1 in plasma and spinal cord were measured in sham, CRPS and ET-B receptor antagonist BQ 788-delivered rats. In CRPS models, ET-1 levels were determined 7 and 10 days after ischemia–reperfusion or spinal nerve ligation, and 60–90 min after BQ 788 administration. The ET-1 level was quantified using ELISA kits obtained from Assay Designs (Ann Arbor, Michigan, USA). 2.9. General behavior Behavioral changes in response to BQ 123 and BQ 788 treatment were evaluated in additional rats 5, 10, 20, 30, 40, 50, and 60 min after intrathecal administration of the highest drug doses. Motor functions were determined by examining the righting and placing-stepping reflexes. Righting was evaluated by placing the rat horizontally with its back on a table, which normally gives rise to an immediate coordinated twisting of the body to an upright position. Placing-stepping reflexes were evoked by drawing the dorsum of either hind paw across the edge of the table. Rats normally attempt to place their paws forward into a walking position. Pinna and corneal reflexes was also evaluated and considered present or absent. Abnormal behavior, including serpentine movement or tremors, was also monitored.
Y.O. Kim et al. / Neuroscience Letters 584 (2015) 45–49
Fig. 1. Time course of hind paw withdrawal response to von Frey filaments after ischemic reperfusion (A) and spinal nerve ligation (B). Data are presented as the withdrawal threshold. Each line represents mean ± SEM (n = 5–6). BL: baseline withdrawal threshold measured before ischemic reperfusion or spinal nerve ligation. Significant difference between the injured and non-injured site is indicated; * P < 0.05, † P < 0.01, ‡ P < 0.001.
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Fig. 2. Effect of intrathecal ET-A receptor antagonist BQ 123 on hind paw withdrawal response to von Frey filaments after ischemic reperfusion and spinal nerve ligation. Data are presented as the withdrawal threshold. Each line represents mean ± SEM (n = 5–6). BL: baseline withdrawal threshold measured before ischemic reperfusion or spinal nerve ligation. Control data were measured immediately before intrathecal drug delivery. Intrathecal BQ 123 did not affect the withdrawal threshold in the injured paw.
2.10. Statistical analysis Data are expressed as mean ± SEM. Time–response data are presented as the withdrawal threshold in grams. Dose–response data are presented as the percentage of the maximum possible effect (%MPE). Withdrawal threshold data from von Frey filament testing were converted into %MPE according to the formula: %MPE = [(postdrug threshold − post-injured baseline threshold)/(cutoff threshold − post-injured baseline threshold)] × 100. Dose–response data were analyzed using one-way analysis of variance followed by Scheffe’s post hoc test. The differences in the withdrawal threshold or the ET-1 level between injured and non-injured sites were analyzed using an unpaired t-test. P < 0.05 was considered statistically significant.
No differences in the post-injured baseline withdrawal threshold (control) among the drug treatment groups were found in CPIP and neuropathic pain rats (Figs. 2 and 3). Intrathecal BQ 123 did not alter the withdrawal threshold in the injured paw of CPIP and neuropathic pain rats (Fig. 2). In contrast, intrathecal BQ 788 dose-dependently increased the withdrawal threshold in the injured paw of CPIP and neuropathic pain rats (Fig. 3). The ET-1 levels of plasma and spinal cord increased in CPIP and neuropathic pain rats compared to sham rats. In addition, intrathecal BQ 788 decreased the ET-1 level in the spinal cord of CRPS rats (Tables 1 and 2).
3. Results Righting and placing-stepping reflexes were normal after intrathecal delivery of BQ 123 and BQ 788, and pinna and corneal reflexes were present at the dosage used. No overt abnormal behavioral changes were observed. The withdrawal threshold was significantly decreased and persisted for almost 21 days at the O-ring application site and at the ligation site compared to the sham group (Fig. 1). The withdrawal thresholds in the sham groups did not change during the observational period.
Table 1 The level of ET-1 (pg/ml) in the plasma. CRPS-I
ET-1
CRPS-II
Sham
CPIP
Sham
SNL
0.2 ± 0.2
1.8 ± 1.3*
0.8 ± 0.9
2.3 ± 1.0†
ET-1 = endothelin-1; CRPS = complex regional pain syndrome; CPIP = chronic postischemia pain; SNL = spinal nerve ligation. Values are mean ± SEM (n = 4–5). * P < 0.05 compared to sham. † P < 0.01 compared to sham.
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Fig. 3. Effects of intrathecal ET-B receptor antagonist BQ 788 on hind paw withdrawal response to von Frey filaments after ischemic reperfusion and spinal nerve ligation. Data are presented as the withdrawal threshold or the percent of maximal possible effect (%MPE). Each line represents mean ± SEM (n = 6–7). BL: baseline withdrawal threshold measured before ischemic reperfusion or spinal nerve ligation. Control data were measured immediately before intrathecal drug delivery. Intrathecal BQ 788 produced a dose-dependent increase in the withdrawal threshold in the injured paw; * P < 0.05, † P < 0.01.
4. Discussion The withdrawal threshold in CPIP and neuropathic rats was increased by a selective ET-B receptor antagonist but not by a selective ET-A receptor antagonist. The ET-1 levels of the plasma and the spinal cord increased in CPIP and neuropathic pain rats. These findings suggest that ET-1 may contribute to the development of CRPS, potentially through the spinal ET-B receptor. In contrast, the spinal ET-A receptor does not appear to be associated with CRPS. CRPS is currently generally considered a systemic condition that involves both central and peripheral components of the neuraxis [14] and interactions between the immune and nervous systems [25]. Clinical characteristics of CRPS include pain, abnormal regulation of blood flow and sweating, edema of the skin and subcutaneous tissues, trophic changes to the skin, skin appendages and subcutaneous tissues, and active and passive movement disorders [14]. Although CRPS is classified into types I and II [13], the underlying mechanisms of both remain largely unknown. Several studies
on laboratory animals and humans have indicated that ET participates in the pathogenesis of the pain-related process. ET is an endogenous 21-amino-acid peptide family composed of ET-1, ET2, and ET-3 that regulates blood flow, cell proliferation, secretion, ion transport, and pain perception [23]. ET-1–3 are synthesized as propeptides, which are transformed into the active form by sequential endopeptidase and ER-converting enzyme-mediated cleavage. ET-1 is the predominant and most potent isoform, and its biological effects are transduced by two pharmacologically distinguishable receptor subtypes, the ET-A and ET-B receptors [23]. ET may play a role in nociceptive processing, independently of its cardiovascular properties [10,11,16]. Local injection of ET-1 produces pain-like behavior through activation of the ET-A receptor in rats [7]. Local administration of ET-A and ET-B receptor antagonists inhibit thermal and inflammatory hyperalgesia in mice [1]. Intraplantar ET-A and ET-B receptor antagonists inhibit ET-1-induced nociception in rats [19,20]. Intraperitoneal ET-A receptor antagonist improves thermal and mechanical hyperalgesia induced by chronic constriction injury of the rat sciatic nerve and attenuates tactile allodynia
Table 2 The level of ET-1 (pg/ml) in the spinal cord. CRPS-I
ET-1
CRPS-II
Sham
CPIP
CPIP: BQ 788
Sham
SNL
SNL: BQ 788
3.5 ± 0.3
5.0 ± 0.3*
3.4 ± 0.4#
3.4 ± 0.2
8.7 ± 0.9†
4.4 ± 0.1‡
ET-1 = endothelin-1; CRPS = complex regional pain syndrome; CPIP = chronic post-ischemia pain; SNL = spinal nerve ligation. Values are mean ± SEM (n = 5–6). * P < 0.05 compared to sham. # P < 0.05 compared to CPIP. † P < 0.01 compared to sham. ‡ P < 0.01 compared to SNL.
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in a diabetic rat model of neuropathic pain [15,18]. Intravenous ET-B, but not ET-A, receptor antagonist reduces mechanical allodynia in rats with trigeminal neuropathic pain [3]. Intraplantar ET-A and ET-B receptor antagonists diminish cold allodynia induced by spinal nerve ligation, whereas only the ET-A receptor antagonist is effective against thermal hyperalgesia [26]. An oral ET-A receptor antagonist relieves sciatica in pulmonary hypertension patients [22]. In contrast, the present study demonstrated that intrathecal ET-B, but not ET-A, receptor antagonist reduced mechanical allodynia induced in animal models of CPIP and spinal nerve ligation-induced neuropathic pain. Furthermore, ET-A and ET-B receptors have been reported to exist in the spinal cord [23]. Other studies have reported ET-1 levels that are different than what we found in the present study. In CRPS-I patients, the ET-1 level increases in blister fluid and tissue [8,9]. However, plasma and cerebrospinal fluid ET-1 levels are not different between CRPSI patients and normal volunteers [6,21]. A significant increase in spinal ET concentration has been reported after spinal cord injury in rats [24]. In the present study, the plasma ET-1 level increased after ischemia–reperfusion and spinal nerve ligation. Furthermore, the increased ET-1 level in the spina cord of CRPS rats was attenuated by ET-B receptor anatgonist, but not by ET-A receptor anatgonist. Therefore, the current results together with previous findings suggest that an increased ET-1 after ischemia–reperfusion or nerve injury may contribute to mechanical allodynia through the activation of the ET-B receptor in the spinal cord. This is the first study to demonstrate the relevance of spinal ET-B receptor to allodynia in CRPS. In addition, the plasma ET-1 level may be a biomarker for CRPS. However, further research needs to apply this tool to human. Furthermore, CPIP and SNL modeling used in this study did not cause the spinal cord damage. Thus, those procedures little affected the plasma or spinal ET-1 level. There are some limitations to this study. First, there were insufficient observational parameters. CRPS is a complex disorder manifesting as several symptoms, including allodynia, vasomotor and sudomotor function, trophic skin changes, and movement changes. However, we only evaluated allodynia. Thus, other parameters mentioned above should also be assessed. Second, there was a lack of data on the ET-A receptor antagonist. BQ 123 was only used at a maximum dose of 50 g, above which it was insoluble. In addition, the effect of other ET-A receptor antagonists was not evaluated. Thus, further studies are required to consider these issues. Spinal ET-B receptor antagonists are not yet available in clinics. However, they may be clinically applied in the treatment of CRPS in the future. In conclusion, the ET-1 levels of plasma and spinal cord increased after ischemic reperfusion and spinal nerve ligation. In addition, intrathecal ET-B receptor, but not ET-A receptor, antagonist alleviated mechanical allodynia induced by ischemic reperfusion and spinal nerve ligation. These findings suggest that ET-1 may be involved in the spinal signaling mechanism operated by the ET-B receptor in mechanical allodynia in CRPS. Accordingly, the spinal ET-B receptor antagonist may be useful for managing mechanical allodynia in CRPS.
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