| JOURNAL HOME | CME HOME | THIS MONTH | PAST ISSUES | ETOC | COLLECTIONS |
| AUTHORS | REVIEWERS | EDITORIAL BOARD | FEEDBACK | RSS | HELP |
| ||||||||||||||
| ||||||||||||||
|
|
|
|
||||||||||||
|
||||||||||||||
Departments of Anesthesiology and Cell Biology, Emory University School of Medicine, Atlanta, Georgia
Address correspondence and reprint requests to Marie Csete MD, PhD, Emory Anesthesiology Laboratories, 1462 Clifton Rd NE, Room 420, Atlanta GA 30322. Address e-mail to marie.csete{at}emoryhealthcare.org.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Injection of ET-1 causes acute pain (9,10), as does its direct application to the sciatic nerve (11) but the pain is not simply the result of vasoconstriction. Both ETA and mixed ETA-ETB receptor antagonists can ameliorate pain in animal models depending on the type of pain, the dose of ET-1 used in the model, and the study design (8,10). Significantly, activation of ETB receptors has been shown to have analgesic effects in some models via peripheral release of endorphins from keratinocytes (12) ultimately negatively modulating the initial pain signal. Thus ET-1 mediated peripheral pain signaling is a complex integration of ETA-mediated activation of nociceptors on primary sensory neurons and ETB–mediated signals. Dorsal root ganglion small diameter neurons contain abundant ETA receptors (13) and peripheral ETA receptors have also been identified in keratinocytes (12).
Thus an impressive literature shows that exogenously administered ET-1 causes pain and that ETA antagonists decrease ET-1 mediated pain. ETA antagonism also reduces tactile allodynia in diabetic rats (14). Endothelin secretion from tumors has also been associated with pain, and this pain can be ameliorated by ETA antagonism (15). In this study we explored the possibility that ET may mediate neuropathic pain in the rat sciatic nerve chronic constriction injury (CCI) model. We demonstrate upregulation of ET-1 and modulation of expression of its receptors in the sciatic nerve at the site of injury and relief of pain behavior by the ETA antagonist atrasentan (16). This study suggests that ET-1 may be a mediator of pain that develops as a consequence of nonspecific injuries and that pharmacologic antagonism of the ETA receptor deserves investigation as a therapy for neuropathic pain.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
300 g) were given free access to food and water. Baseline paw withdrawal latency (PWL) to thermal stimulus was used to assess thermal and mechanical sensitivity. PWL assays were conducted after the rats were acclimated for 10 min (e.g., made postural adjustments). A high-intensity radiant heat source projected through a round aperture in the cage floor was used for the tests. A photoelectric cell detected light reflected off the paw, and paw withdrawal turned off the lamp and an electronic timer (17). Five sets of tests were done on each hindpaw at each time point.
Mechanical allodynia was evaluated by paw withdrawal response (PWR) to progressive application of force with von Frey monofilaments applied from beneath the cage. Each filament was applied once to an area approximately 1 cm from the heel, starting with 4.08 g and continuing until a withdrawal response occurred or the force of 6.65 g was reached. Three PWR testing sessions were performed before surgery. The least force (bending force) from the three tests producing a response was considered the withdrawal threshold. Five sets of tests were done on each hindpaw at each session. All of the postoperative tests were pooled and compared with the grand mean of the three baseline preoperative tests. The observer performing behavioral testing was blinded as to the treatment group.
The surgical technique for left sciatic nerve CCI was adapted from Bennett and Xie (18). Rats were anesthetized with pentobarbital 40 mg/kg IP and then given 2–4 mg/kg as necessary to maintain adequate anesthetic depth. Strict aseptic technique was used. After adequate depth of anesthesia was verified by lack of response to tail or toe pinch, skin incision was made from the left sciatic notch to the distal thigh. Subcutaneous tissue was bluntly dissected to expose the biceps femoris. The muscle was incised at the sciatic notch and its fibers spread. Mayo scissors were placed through the muscle and a 2-cm cut in the muscle made toward the knee. With blunt dissection, the muscle was freed from below, and the sciatic nerve was freed from investing fascia. Four ligatures (4-0 Chromic), 1 mm apart, were placed around the sciatic nerve and tightened until the suture just indented the nerve. The muscle was closed with 4-0 Vicryl and the skin with 3-0 Vicryl (Ethicon, Piscataway NJ). Identical surgery was then performed on the opposite (right) side except that ligatures were not placed (sham surgery). This sham surgery control creates equivalent amounts of muscle injury as the CCI but purposefully leaves the nerve uninjured, allowing comparison of behavioral tests as a function of surgery/muscle injury versus CCI.
Five days after surgery mechanical and thermal sensitivity were again assayed. Only animals that fulfilled published (19) predetermined criteria for the presence of neuropathic pain (PWL on the left side at least 20% shorter than on the right or PWLleft/PWLright < 0.8) were studied further. These rats were randomly assigned to receive 5 mL/kg (IP) of the selective ETA antagonist atrasentan (Abbott Laboratories, Abbott Park, IL) or saline. Atrasentan is a highly potent and specific ETA antagonist with selectivity >1862-fold at the ETA receptor versus the ETB receptor in the rat (20). Thirty minutes later another set of behavioral tests was performed to evaluate the acute effect of ETA blockade on PWL and PWR. The rats were then killed with pentobarbital overdose and tissue samples were harvested for analyses.
PWL values for the CCI limb are expressed as a percentage of the corresponding value for the sham limb ([PWLL/PWLR] x 100). Differences between preoperative and postoperative PWL were assessed by using a one-way analysis of variance for repeated measures followed by a post hoc Scheffé test for multiple comparisons. Because they are noncontinuous medians, differences between preoperative and postoperative PWRs were analyzed using a Wilcoxon’s signed rank test or nonparametric repeated-measures analysis of variance with a Dunnett’s multiple comparison test, as appropriate. P values of <0.05 were considered to indicate significant differences among groups.
The number of rats per group was 12: One group of rats had CCI plus atrasentan and one group of rats had CCI plus saline. Because sham surgery itself might have an effect on subsequent ET-1 expression, a third group of rats (n = 6) received an intraperitoneal injection of saline to induce a nonspecific stress response but no surgery.
After euthanasia, sciatic nerves were dissected out then divided into three specimens: center of nerve (site of CCI), proximal to injury, and distal to injury. The nerves were homogenized in Trizol® (Invitrogen, Carlsbad CA), and total RNA was isolated using acid-guanidium-phenol-chloroform extraction. RNA concentration was measured spectrophotometrically. Quality of RNA was confirmed by measuring the ratio of products at 260 and 280 
(ratio was 1.6–1.8). Aliquots of total RNA (1 µg) were reverse transcribed to cDNA using Moloney murine leukemia virus RT, 100 mM DTT, RNAse inhibitor, 10 mM dNTPs and buffer at 37°C for 60 min (all reagents from Promega, Madison WI), and cDNA stored at –80°C.
Semiquantitative polymerase chain reaction (PCR) (for ET-1, ETA, ETB messages) was performed using 10 mM dNTP, appropriate primers, and Taq polymerase (Promega). To confirm linear range of PCR, reactions were run over a number of amplification cycles. For ET-1: 26, 31, and 36 cycles were run after 2 min at 94°C as follows: 94°C for 1 min (denaturation), 62°C for 1 min (annealing), and 72°C for 2 min (elongation) followed by a final extension for 10 min at 72°C. PCR for ETA and ETB was performed using the following conditions: 30, 35, and 40 cycles of 94°C for 30 s (denaturation), 58°C for 1 min (annealing), and 72°C for 2 min (extension) followed by a final extension for 10 min at 72°C. The amplification products for ET-1 were predicted to contain 382 base pairs (bp), 398 bp for ETA receptor, and 421 bp for ETB. Amplicon size was determined on 1.2% agarose gels.
All primer sets crossed at least one intron/exon boundary of genomic sequence. Primers used to amplify ET-1 were forward 5'- TTG CTC CTG CTC CTC CTT GAT G -3' and reverse 5'- GGT CTT GAT GCT GTT GCT GAT G -3'. Primers for amplification of ETA receptor were forward 5'- CAG TGC TAA TCT AAG CAG CC -3' and reverse 5'- TGC AGA GAA ACA CTC CAA AAT C -3'. Primers for ETB receptor were forward 5'- AGG CCA CAC CAT CTC TTC TC -3' and reverse 5'- AGC TTG CAC ATC TCA GCT C -3'. For western analyses, nerve sections were collected in lysis buffer and stored at –80°C until assayed. Thawed samples were homogenized on ice and sonicated, and protein concentration was determined. The western protocol for detecting ETA and ETB protein was directly adapted from published protocols (21). Thirty micrograms protein was loaded per well on 12% agarose gels (Bio-Rad, Hercules CA), proteins were transferred to nitrocellulose membranes (Amersham, Piscataway NJ), then blocked with Tris-buffered saline (TBS) + 5% nonfat milk and 0.1% Tween-20 for 2 h at room temperature. Anti-ETA and anti-ETB rabbit polyclonal antibodies (Chemicon, Temecula CA) were incubated overnight at 4°C (final concentrations of 12 µg/mL and 1.5 µg/mL, respectively). After TBS-T washes, membranes were incubated with goat anti-rabbit immunoglobulin (Ig) G conjugated to horseradish peroxidase (1:5000) in 5% nonfat milk for 1 h at room temperature then washed again. Detection of bands was performed using chemiluminescence (ECL, Amersham, Piscataway NJ).
To ensure exact identification of the band of interest, control competition assays were performed as follows: primary antibody was incubated with blocking peptide (24 µg antibody against both receptors, 24 µg ETA antigen and 6 µg ETB antigen from Chemicon) for 1 h at room temperature in 100 µL buffer. The entire reaction was then added to 5% milk buffer and the western protocol was repeated as above. For these competition assays three samples were used (left mid, right proximal, and positive internal control).
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
|
Western blot analysis confirmed upregulation of ETA receptor in the CCI-treated nerve relative to the sham side; anti-ET-1 antibody suitable for western analysis is not available. On the other hand, there was not clear concordance between message level and protein level expression of the ETB receptor. At the protein level, ETB was slightly more abundant on the sham surgery side than in the CCI-lesioned nerve. The ETA band ran slightly above the 37-kD ladder marker, and the ETB receptor band ran slightly below the same marker. Details of the protein-level expression pattern are shown in Figure 2. Specificity of the bands on the Western blots was confirmed by competition using purified receptor peptides incubated with antibody before the membranes were exposed to the primary antibody. In both cases, the bands shown in Figure 2 were competed away completely (not shown).
|
The selective ETA receptor antagonist atrasentan reduces thermal hyperalgesia acutely in the CCI-induced pain model. The presence of thermal hyperalgesia after CCI was demonstrated by a highly significant decrease in PWL postoperatively when compared with preoperative control values (67.5% ± 6.09% versus 108.85% ± 13.20%, P < 0.01) (Fig. 3). A single intraperitoneal injection of the selective ETA receptor antagonist atrasentan resulted in significant improvement of CCI-induced thermal hyperalgesia 5 days after CCI, mediating a significant increase in PWL, though not a complete reversal to baseline (Fig. 3).
|
Atrasentan completely reverses CCI-induced mechanical hyperalgesia acutely. CCI resulted in the development of significant mechanical allodynia, as demonstrated by a significant decrease in PWR compared with preoperative values (5.07 ± 0.46 versus 5.88 ± 0.22, P < 0.05) (Fig. 4). After a single intraperitoneal injection of the selective ETA receptor antagonist atrasentan, CCI-induced mechanical allodynia was completely reversed (i.e., not different from preoperative values, Fig. 4). For both PWR and PWL measurements, saline administration had no apparent effect. PWR measurements on the sham surgery side demonstrated no change after surgery compared to preoperative measurements and no change as a function of atrasentan or saline injection after surgery (not shown).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Atrasentan improves deficits in peripheral nerve conduction and blood flow in rats with streptozotocin-induced diabetes (23). In this same model, atrasentan decreases tactile allodynia (14), supporting a role for ETA receptor antagonism in the treatment of neuropathic pain. Furthermore, the authors of this study cite evidence that improved blood flow alone is insufficient to explain the decreased tactile allodynia (11). More similar to our model, the placement of two tight ligatures around rat sciatic nerve resulted in increased binding sites for radioactively-labeled ET-1 in the nerve itself, particularly in areas rich in peripheral supporting cells of the nerve (13). Future studies will determine whether pretreatment of animals with ETA antagonists prevents establishment of pain after injury and whether chronic ETA antagonism is useful in treating clinical neuropathic pain.
The ETA antagonist atrasentan is likely to be seen by anesthesiologists in clinical contexts besides pain management. Large clinical trials suggest that atrasentan slows the progression of hormone-refractory prostate cancer (24). Prostate tumors secrete ET, and, in addition to the chemotherapeutic value of atrasentan, clinical trials have suggested improved pain scores in atrasentan-treated prostate cancer patients (15). Preclinical studies suggest that atrasentan may also be a useful chemotherapeutic drug for ovarian carcinoma (5). In primate models, blockade of ETB receptors causes hypertension, which can be alleviated by atrasentan (4). In clinical studies to determine largest (oral) doses tolerated by cancer patients, the common side effects were headache, rhinitis, and peripheral edema (up to 60 mg/day). At the largest doses given (75 mg/day), some patients experienced dose-limiting hypotension or hyponatremia (25). Pain was not measured as an outcome in this pharmacokinetic study.
Despite the expanding number of drugs that are reported to be useful for treatment of neuropathic pain, considerable patient variability in response to available therapies makes pain management difficult to predict. In addition, many patients with chronic neuropathic pain are not afforded complete pain relief. Data presented here suggest that ETA receptor antagonism deserves further study as a novel therapy for neuropathic pain.
|
Footnotes |
|---|
Accepted for publication July 13, 2005.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  D. T. Hamamoto, S. G. Khasabov, D. M. Cain, and D. A. Simone Tumor-Evoked Sensitization of C Nociceptors: A Role for Endothelin J Neurophysiol, October 1, 2008; 100(4): 2300 - 2311. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Klass, V. Gavrikov, D. Drury, B. Stewart, S. Hunter, D. D. Denson, A. Hord, and M. Csete Intravenous Mononuclear Marrow Cells Reverse Neuropathic Pain from Experimental Mononeuropathy Anesth. Analg., April 1, 2007; 104(4): 944 - 948. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 U. C. Kopp, M. Z. Cicha, and L. A. Smith Differential effects of endothelin on activation of renal mechanosensory nerves: stimulatory in high-sodium diet and inhibitory in low-sodium diet Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2006; 291(5): R1545 - R1556. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
= 500 bp; 
= 1000 bp).
