| JOURNAL HOME | CME HOME | THIS MONTH | PAST ISSUES | ETOC | COLLECTIONS |
| AUTHORS | REVIEWERS | EDITORIAL BOARD | FEEDBACK | RSS | HELP |
 
|
|
|
||
|
|
Anesth Analg 2006;102:1781-1788 © 2006 International Anesthesia Research Society doi: 10.1213/01.ane.0000205755.43562.2b
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Polyamines are thought to be involved in the regulation of numerous metabolic and electrophysiological processes in the nervous system. In this study we evaluated the effect of a synthetic polyamine-deficient diet on pain in a carrageenan (Car)-induced inflammatory rat model. Inflammation was induced with a unilateral subcutaneous injection of Car in a plantar hindpaw in rats fed without (control group) or with (deficiency group) a polyamine-deficient diet. Ipsilateral and contralateral hyperalgesia was evaluated using the Randall-Sellito pressure test. Heart rate changes were also recorded under general anesthesia. Then, the effects of a bupivacaine sciatic nerve block and subcutaneous injection of naloxone or ketamine were evaluated for Car-induced hyperalgesia. Data were analyzed using analysis of variance followed by unpaired Student's t-test (significance P < 0.05). Before Car injection, no significant difference was observed in response to mechanical stimuli between the control and the deficiency groups (n = 114 in pooled data). Car injection induced significant ipsilateral and contralateral hyperalgesia in the control groups, whereas a significant analgesic effect appeared in the deficient groups on both the ipsilateral and contralateral hindpaws. This analgesic effect was confirmed by the electrocardiogram recording that showed a significant increase in heart rate in the control group after Car injection compared with the deficiency group that showed a decrease in heart rate under general anesthesia. Bupivacaine sciatic nerve block had no significant effect on hypoalgesia phenomena induced by polyamine deficiency. Naloxone administration had no effect in the control group but reversed the analgesic effect in the deficiency group. Ketamine administration induced a significant analgesic effect in the control group and partly reversed the analgesic effect in the deficiency group. In conclusion, a synthetic polyamine-deficient diet had a significant general analgesic effect on Car-induced mechanical hyperalgesia. The mechanism of analgesic action remains to be elucidated.
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Polyamines are ubiquitous aliphatic amines that regulate cell division and proliferation and have been considered a promising target for cancer therapy (1,2). Polyamines are involved in regulation of numerous metabolic and electrophysiological processes in the nervous system (3). Polyamine metabolism has been suggested to play a role in pain pathways, in that complete polyamine deprivation modifies the latency of the response to mechanical stimuli reducing pain-related behaviors in a mechanical or thermic injury animal model (4). However, these results were obtained with complete polyamine deprivation achieved through an efficient therapy including synthetic diet preparations (i.e., exogenous source), decontamination of gastrointestinal tract (neomycin and metronidazole), and inhibition of ornithine decarboxylase (
-difluoromethylornithine) (i.e., endogenous source). The aim of this preclinical study was to assess the effects of polyamine deprivation obtained through only a synthetic diet in a carrageenan (Car)-induced inflammatory hyperalgesia model. Further, bupivacaine (sciatic nerve block), naloxone and ketamine given by subcutaneous injection were evaluated to try to understand the possible mechanism of action of the polyamine deprivation. |
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Experiments were performed in 114 adult male Sprague-Dawley rats weighing 270–320 g. The animals were housed 2 by 2 with a 12-h light–dark cycle and with unrestricted water. The study was conducted in accordance with the ethical guidelines of the International Association for Study of Pain and was approved by the Local Animal Research Committee.
Rats were randomly assigned into 2 groups receiving 2 different diets for 4 days before the tests. Control groups received normal synthetic diets (containing putrescine 54 mg/kg, spermidine 27 mg/kg, spermine 7 mg/kg, and cadaverine 37 mg/kg), and deficient (DEF) groups received a synthetic polyamine-deficient diet (containing <10 µg polyamines) without antibiotic (neomycin) or -difluoromethylornithine.
The experimental design is summarized in Table 1. In the first step, the effect of Car injection alone was recorded (group Car and group Def; n = 15). Then, an electrocardiogram (ECG) was performed under general anesthesia (group ECG and ECG-Def; n = 7) to compare heart rate (HR) without stress.
|
In a second step, to understand the mechanisms of the possible action of polyamine deficiency on pain, the effect of a sciatic nerve block with bupivacaine (B) (CarB and CarB-Def groups; n = 15) was evaluated 1 h after Car injection. The effects of a subcutaneous injection 3 h after Car injection (4 h after the beginning of study) of 1 mg/kg of naloxone (N) (Narcan®; Dupont Pharma SA, Paris, France) (CarN and CarN-Def groups; n = 10) and 10 mg/kg of ketamine (K) (Ketalar®; Parke Davis, Courbevoie, France) (CarK and CarK-Def groups; n = 10) were also evaluated.
Inflammation was induced 1 h after the beginning of the study by the injection of 0.2 mL of 2% Car (Sigma Chemical, Saint-Quentin, France) injected with a 25-gauge needle subcutaneously in the right plantar hindpaw; the animals were anesthetized with 2%–3% halothane. To estimate the local inflammatory effect, edema was measured with a thread and caliper as previously described (5). The circumference of the paw was measured with a thread, to the nearest mm, at the metatarsal level.
After inflammation, pain was evaluated for 24 h in response to a mechanical injury (i.e., Randall-Sellito test). The withdrawal threshold of the paw in response to increasing pressure using an Analgesy–Meter (Ugo Basile, Milan, Italy) was used (6). Ipsilateral hyperalgesia was determined in the inflamed paw by positioning the paw under a pressure pad, the probe (tip diameter of the pusher: 1 mm) being applied to the dorsal, lateral, and external parts of the paw, avoiding its saphenous nerve innervation. Contralateral paw hyperalgesia was determined by the change in withdrawal threshold on the left contralateral paw (7). The choice of a cutoff value of 400 g was necessary to limit injury to the paw (8). Every test was performed in triplicate and mean measures were recorded. Seven days before the beginning of the study, animals were trained on the pressure test.
Concerning the ECG groups, ECG probes were implanted under general anesthesia with halothane 2% and baseline HR was then recorded at steady-state for 5 min under slight anesthesia with halothane 1% (baseline). Three hours after this recording, Car injection was performed under a new slight general anesthesia at steady-state (halothane 1%) and HR was then recorded for 5 min. Then sciatic nerve block was performed and HR was recorded for 6 min and anesthesia was stopped.
For the sciatic nerve block, the landmarks for sciatic nerve stimulation under short general anesthesia (halothane: 2%) have been previously described (9). They were easily found by means of palpation (i.e., greater trochanter and sciatic notch of the pelvis). The sciatic nerve was identified using a nerve stimulator under aseptic conditions. Nerve block with an injection of 0.5 mL of 0.25% of B was performed using electrical stimulation (HNS 111; Braun Melsungen; Germany) via a 25-mm insulated needle (0.7-mm inner diameter; Stimuplex A; Braun). When muscle twitches of the hindpaw were elicited at 0.5 mA impulses, and after a negative blood aspiration test, B was injected. Nerve twitches disappeared immediately after the beginning of the injecting of solution and reappeared when the intensity of nerve stimulation was increased. At the end of injection the insulated needle was removed. Clinical evaluation was performed when rats were completely awake. The baseline was defined by the data obtained before the Car injection and the sciatic nerve block. Measurements were recorded hourly up to 3 h after the recovery of motor block and then extended to 24 h. To confirm the onset of the block, motor tests were performed by an evaluation of the rat's ability to hop and to place weight on the hindleg. The nerve block was then evaluated with a mechanical stimulus.
Sample-size calculation was performed based on a previous study using the same model (n = 10) (10). Raw data were expressed in grams for motor reaction threshold to pressure and millimeters for paw circumference. Data (mean ± sd) were analyzed using analysis of variance followed by the unpaired Student's t-test with Bonferroni correction. Statistical significance was defined as P < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
With 4 days of synthetic diet, there was a slight but significant difference in the weight of rats between control (303 ± 24 g) and Def groups (296 ± 22 g) (P = 0.01).
Polyamine deprivation obtained through a synthetic diet reduced Car-induced hyperalgesia. Before Car injection, there was no difference between the control group (Car group) and the polyamine-deficient group (Car-Def). After Car injection alone, no significant difference in edema was observed for 24 h between the 2 groups (P = 0.7). In the control group, Car injection induced ipsilateral hyperalgesia (P < 0.001) and contralateral hyperalgesia from 2 h to 25 h (P < 0.001). At the same time, in the polyamine-deficient group, a significant hypoalgesic effect appeared compared to the baseline defined by the first 2 measurements before Car injection (P < 0.001). Moreover a significant bilateral hypoalgesic effect appeared in the deficient group as compared with the control group from 2 h to 25 h (P < 0.01) (Fig. 1).
|
and discontinuous line) were evaluated. For the group receiving a synthetic polyamine-deficient diet (group Car-Def): right paw (ipsilateral to Car injection; 
and continuous line) and left paw (contralateral to Car injection; 
and discontinuous line) were evaluated. n = 15 in each group. *P < 0.01 control vs deficient for ipsilateral paw; +P < 0.01 control vs deficient for contralateral paw.Concerning HR, there was no difference between the control (ECG group) and deficient group (ECG-Def group) before Car injection. After Car injection, HR increased significantly in the control group whereas HR was stable in the polyamine-deficient group. After sciatic nerve block, no effect on HR was observed in the control group whereas a significant decrease of HR was noted compared with the baseline before Car injection in the deficient group (P < 0.05) The HR difference between the two groups remained significant (P < 0.05) (Fig. 2).
|
Before and after carrageenan injection in control group: P < 0.05; 
before and after carrageenan injection in deficient group: P < 0.05; *control group vs deficient group: P < 0.05.Sciatic nerve blockade with bupivacaine does not alter the hypoalgesic effect of polyamine deprivation (Fig. 3).
|
Concerning the sciatic nerve block, there was no significant difference between the polyamine deficient (CarB-Def group) and the control groups (CarB group) in the onset or the duration (4 ± 0.5 h) of motor block (i.e., withdrawal thresholds evaluation). After recovery from the block, in the control group ipsilateral and contralateral hyperalgesia was significant from 6 to 25 h (P < 0.01), whereas in the deficient group a significant bilateral analgesic effect appeared compared with the baseline before Car injection (P = 0.009). This hypoalgesic effect was similar to the one observed in the deficient group with Car injection alone (Car-Def group). Moreover, a significant hypoalgesic effect (P < 00.1) was observed between the deficient group and control group from 4 to 25 h on the ipsilateral paw and from 6 to 25 h in the contralateral paw. Sciatic nerve block had no significant effect on the edema.
Naloxone more than ketamine reverses the analgesic effect of polyamine deprivation (Figs. 4 and 5).
|
|
When naloxone was subcutaneously administered 3 h after Car injection, there was no effect on ipsilateral and contralateral hyperalgesia in the control group (group CarN group). When N was subcutaneously administrated 3 h after Car infiltration in the polyamine-deficient group (Group CarN-Def), there was a disappearance of the hypoalgesic effect observed after inflammation (P < 0.001). In this group, an ipsilateral hyperalgesia state (under baseline) was significant from 6 to 8 h (P = 0.01) on the ipsilateral paw and from 5 to 12 h on the contralateral paw (P < 0.05). Finally, at the end of the experience a significant hypoalgesia (P < 0.01) appeared again in the deficient group compared with the control group from 7 to 25 h on the ipsilateral paw and from 13 to 25 h on the contralateral paw.
Subcutaneous K administration 3 h after Car injection induced a significant analgesia (compared with baseline before Car injection) from 5 to 9 h in the control group (Fig. 5; P < 0.001). Inversely, in the deficient group, K injection caused a decrease in the hypoalgesic state from 5 to 9 h (compared with baseline; P = 0.04) on the ipsilateral paw. However, a significant residual hypoalgesic effect was again observed (compared with the baseline before Car injection) at the end of the experience. There was no significant difference in paw pressure test between the deficient and the control group within 6 to 8 h. Then, a significant hypoalgesic effect was observed again in the CarK-Def group compared with the CarK group from 9 to 25 h (P = 0.01). No significant difference was observed during edema evaluation.
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
This is the first study reporting an analgesic effect in an inflammatory pain model after a partial polyamine deficiency obtained with only a polyamine-deficient synthetic diet.
When we pooled all our data (n = 57 in each group), no difference in withdrawal thresholds was recorded before Car-induced injury between polyamine-deficient and control animals. For all the control groups our hyperalgesia results observed after inflammation are in agreement with previous reports (10,11). However, after inflammation, in the deficient groups a hypoalgesic effect appeared, suggesting that polyamine deprivation can prevent painful behavior. Only one study with rats has shown a hypoalgesic effect with a complete polyamine deficiency obtained with a tritherapy including synthetic diet preparations (i.e., exogenous source), decontamination of the gastrointestinal tract (neomycin and metronidazole), and inhibition of ornithine decarboxylase (-difluoromethylornithine) (i.e., endogenous source) (4). In this study, animals only treated with the deficient diet (i.e., without decontamination of the gastrointestinal tract or inhibition of ornithine decarboxylase) did not react differently from the control group. However, the stimulus was mechanic or thermic and no inflammatory injury was performed before the stimulus as in our study. Yet, in our study, we noted a hypoalgesic effect only after inflammation, suggesting surprisingly that Car injection produced analgesia with a polyamine-deficient diet.
In rats, as in humans, a painful stimulus generally leads to an adrenergic response. In clinical practice, HR changes are usually used as an indicator of pain. It is interesting to note that the HR evaluation (that can be considered a nonbehavioral index of pain) was in agreement with the results obtained with the Analgesy–Meter methods (i.e., difference appeared only after inflammation). The absence of analgesia after sciatic nerve block in the control group (ECG-Def group) was probably attributable to the inflammation pain from the saphenous nerve innervation.
Hypoalgesic or analgesic effects observed with polyamine deficiency do not seem to be mediated by a peripheral action. Indeed, no difference was observed concerning the B sciatic nerve block between the control group and the deficiency group (CarB and CarB-Def groups). Duration of block and apparition of ipsilateral and contralateral hyperalgesia are in agreements with previous studies (7,8,10,11). HR evolution also seems to suggest a central effect because the sciatic block was not sufficient to avoid an increase of HR (i.e., saphenous nerve innervation), whereas there was a decrease of HR in the deficient group. We must note that edema (an inflammation marker) was not modified by the diet. The effect of a polyamine-deficient diet seems to be attributable to a central action, as a hypoalgesic effect was observed in the contralateral paw (hypoalgesia in Groups Car-Def, CarB-def, or "normal"-algesia in groups CarK-Def and CarN-Def; but never hyperalgesia).
Naloxone, as antagonist of opiates, did not affect Car-induced hyperalgesia in the control group but, surprisingly, did induce a profound decrease of the polyamine deprivation analgesic effect in the deficiency group. This effect of naxolone suggests that polyamine deprivation may act by maximizing the potential of opioids' effects. These results seem to be in agreement with the result of a preliminary clinical study. In fact, this study concerning patients with metastatic hormone-refractory prostate cancer treated with a polyamine-reduced diet showed a decrease of morphine consumption (12).
Furthermore, it has been shown that NMDA receptors, which have subunit NR2B, have a recognition site of polyamines (13,14). We also know that NR2B subunit-containing NMDA receptors are localized in areas that play a key role in pain transmission (dorsal horn and front brain). It has been shown that the phosphorylation of NR2B subunit containing the NMDA receptor is associated with inflammatory hyperalgesia (15) and that block of this binding site provokes analgesia (16). All these results are in agreement and seem to show NMDA receptor activation by polyamines in algic phenomenon, in painful phenomenon, and analgesia provoked by their inhibition. However, surprisingly, in our study hypoalgesia provoked by polyamine deprivation was partly reversed by K (a NMDA receptor antagonist). In another study, Coderre and Van Empel (17) showed that spermine could enhance hypoalgesia action of MK-801 (an anesthetic binding site of NMDA receptor antagonist). All these results highlight the complex interaction between polyamines and NMDA receptors. It seems that these receptors may be both activated or inhibited by polyamines.
The hypoalgesic effect of polyamine deprivation seems to act by the opiate system and NMDA receptors. Two studies show an interaction between these two systems. The first showed, in inflammatory pain, that sensitization of dorsal horn neurons increased by morphine can be prevented by K administration (18). The second showed that blocking the polyamine binding site NMDA receptor increases morphine efficacy in mice (16).
We conclude that a synthetic polyamine-deficient diet has a significant general analgesic effect on inflammatory pain. These results are in accordance with previous results obtained with complete polyamine deprivation (synthetic diet, decontamination of the gastrointestinal tract, and inhibition of ornithine decarboylase). A simple synthetic polyamine-deficient diet could have clinical benefit for reducing hypersensitivity to pain in the postoperative period. However, further studies using other analgesic drugs (i.e., nonsteroidal antiinflammatory drugs, clonidine, neostigmine with a systemic or neuroaxial administration) are needed to better understand the mechanism of polyamine action on pain. These promising results must be confirmed in patients to understand the potential of this diet for treatment of acute and chronic pain.
The authors are indebted to Veronica I. Shubayev, MD, and Anthony Rubin, MD, for their critical review of this manuscript.
|
Footnotes |
|---|
Accepted for publication January 12, 2006.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
- Le Roch N, Douaud F, Havouis R, et al. Dimethylsilane polyamines: cytostatic compounds with potentials as anticancer drugs. II. Uptake and potential cytotoxic mechanisms. Anticancer Res 2002;22:3765–76.[Medline]
- Devens BH, Weeks RS, Burns MR, et al. Polyamine depletion therapy in prostate cancer. Prostate Cancer Prostatic Dis 2000; 3:275–9.[Web of Science][Medline]
- Williams K. Interactions of polyamines with ion channels. Biochem J 1997;15:325:289–97.
- Kergozien S, Bansard JY, Delcros JG, et al. Polyamine deprivation provokes an antalgic effect. Life Sci 1996;58:2209–15.[Medline]
- Fletcher D, Kayser V, Guilbaud G. Influence of timing of administration on the analgesic effect of bupivacaine infiltration in carrageenan-injected rats. Anesthesiology 1996;84:1129–37.[Web of Science][Medline]
- Randall LO, Sellito JJ. A method for measurement of analgesic activity on inflamed tissue. Arch Int Pharmacodynam 1957; 111:409–19.
- Kissin I, Lee SS, Bradley EL. Effect of prolonged nerve block on inflammatory hyperalgesia in rats. Anesthesiology 1998;88:224–32.[Web of Science][Medline]
- Gentili ME, Mazoit JX, Samii K, Fletcher D. The effect of a sciatic nerve block on the development of inflammation in carrageenan injected rats. Anesth Analg 1999;89:979–84.
[Abstract/Free Full Text] - Grant GJ, Vermeulen K, Zakowski MI, et al. A rat sciatic nerve model for independant assessment of sensory and motor block induced by local anesthetics. Anesth Analg 1992;75:889–94.
[Abstract/Free Full Text] - Estebe JP, Gentili M, Le Corre P, et al. Sciatic nerve block with bupivacaine-loaded microspheres prevents hyperalgesia in an inflammatory animal model. Can J Anaesth 2002;49:690–3.[Web of Science][Medline]
- Estebe JP, Gentili ME, Le Corre P, et al. Controlateral effect of amitriptyline and bupivacaine for sciatic nerve block in an animal model of inflammation. Br J Anaesth 2004;93:705–9.
[Abstract/Free Full Text] - Cipolla B, Guilli F, Moulinoux JP. Polyamine-reduced diet in metastatic hormone-refractory prostate cancer (HRPC) patients. Biochem Soc Trans 2003;31:384–7.[Medline]
- Parsons CG. NMDA receptors as targets for drug action in neuropathic pain. Eur J Pharmacol 2001;429:71–8.[Web of Science][Medline]
- Banke TG, Traynelis SF. Activation of NR1/NR2B NMDA receptors. Nat Neurosci 2003;6:144–52.[Web of Science][Medline]
- Guo W, Zou S, Guan Y, et al. Tyrosine phosphorylation of the NR2B subunit of the NMDA receptor in the spinal cord during the development and maintenance of inflammatory hyperalgesia. J Neurosci 2002;22:6208–17.
[Abstract/Free Full Text] - Bernardi M, Bertolini A, Szczawinska K, Genedani S. Blockade of the polyamine site of NMDA receptors produces antinociception and enhances the effect of morphine in mice. Eur J Pharmacol 1996;298:51–5.[Web of Science][Medline]
- Coderre TJ, Van Empel I. The utility of excitatory amino acid (EAA) antagonists as analgesic agents. II. Assessment of the antinociceptive activity of combination and non-competitive NMDA antagonists with agents acting at allosteric-glycine and polyamine receptor sites. Pain 1994;59:353–9.[Web of Science][Medline]
- Rivat C, Laulin JP, Corcuff JB, et al. Fentanyl enhancement of carrageenan-induced long-lasting hyperalgesia in rats. Anesthesiology 2002;96:381–91.[Web of Science][Medline]
This article has been cited by other articles:
|
|
 |
|
  J. Sandkuhler Models and Mechanisms of Hyperalgesia and Allodynia Physiol Rev, April 1, 2009; 89(2): 707 - 758. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
