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Combined systemic administration of morphine and magnesium sulfate attenuates pain-related behavior in mononeuropathic rats
Brain Research 943 (2002) 101–104 www.elsevier.com / locate / bres
Short communication
Combined systemic administration of morphine and magnesium sulfate attenuates pain-related behavior in mononeuropathic rats Ahmet Ulugol*, Aysegul Aslantas, Yesim Ipci, Alev Tuncer, Cetin Hakan Karadag, Ismet Dokmeci Department of Pharmacology, Faculty of Medicine, Trakya University, 22030 Edirne, Turkey Accepted 22 November 2001
Abstract The response to opioids is reduced in neuropathic pain states. We examined the effect of the combination of morphine (0.1 mg / kg) and magnesium sulfate (125 mg / kg) on behavioral signs of neuropathic pain in spinal nerve ligated rats. Administered alone, neither drug produced any effect, but the combination exerted a significant anti-allodynic effect, which was partially reversed by naloxone. These results suggest that combining low doses of magnesium sulfate with opiates might be an alternative in treating neuropathic pain, with reduced risk of side effects. 2002 Elsevier Science B.V. All rights reserved. Theme: Sensory systems Topic: Pain modulation: pharmacology Keywords: Neuropathic pain; Allodynia; Morphine; Magnesium
Peripheral nerve injury may lead to neuropathic pain, which is characterized by spontaneous pain, hyperalgesia (augmented pain response to normally painful stimuli) and allodynia (nociceptive responses to normally innocuous stimuli), analogous to clinical conditions of neuropathic pain. Several animal models, including spinal nerve ligation (SNL), have recently been developed to study the mechanisms of neuropathic pain [17]. Although the effectiveness of opiates in the treatment of neuropathic pain states is controversial, this condition is generally accepted to respond poorly to opioids [3,19,24]. Dose escalations for treatment are not devoid of side effects [3]. N-methyl-D-aspartate (NMDA) receptors are known to be involved in the etiology of chronic neuropathic pain [5,8,9,11]. NMDA receptor antagonists have been shown to be effective in various experimental models of neuropathic pain; however, their adverse effects also preclude their use in this condition [20,22]. Therapeutic benefit may also be improved by combining low doses of opioids with *Corresponding author. Tel.: 190-532-431-6449; fax: 190-284-2352476. E-mail addresses: [email protected] (A. Ulugol), [email protected] (A. Ulugol).
NMDA antagonists [1,6]. Mg 21 ions have been shown to block inward current flow through ion channels linked to NMDA receptors, and prevent extracellular Ca 21 to enter the cell [21]. Accordingly, magnesium ions have been effective in different pain models [2], and potentiated morphine antinociception after surgical incision [18]. The aim of this study was to determine the influence of systemically injected magnesium sulfate on the anti-allodynic effect of morphine in spinal nerve ligated rats. This study was conducted according to the guidelines of the Ethical Committee of the International Association for the Study of Pain, and had been approved by the ‘Animal Care Ethics Committee’ of our faculty. Wistar rats of either sex (DETAM, Istanbul, Turkey), weighing 300–350 g at time of operation, were used for the experiments. SNL model of neuropathic pain, previously described by Kim and Chung [17], was used. Briefly, under sodium pentobarbital anesthesia (40 mg / kg, i.p.), the left L5 and L6 spinal nerves were isolated and tightly ligated with 6-0 silk thread. The rats were placed under a glass cover on a metal mesh floor and mechanical allodynia thresholds were assessed, as described previously [4]. Briefly, eight von Frey filaments, with approximately equal logarithmic incremental bending forces, were chosen (von Frey num-
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )02618-5
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A. Ulugol et al. / Brain Research 943 (2002) 101 – 104
bers: 3.22, 3.61, 4.08, 4.31, 4.56, 4.93, 5.18, 5.46; equivalent to 0.16, 0.4, 1, 2, 4, 8, 15, 26 g). Each hair was pressed perpendicularly against the plantar surface of the hindpaw until slight buckling was observed, and lifting of the paw was recorded as a positive response. Starting with the 2.0 g hair, 50% withdrawal thresholds were determined using the up-down method of Dixon [12]. If the rat responded, the next weaker hair was applied. In case of a negative response, the next stronger hair was applied. The test was continued until four measurements after the first change in response had been obtained. The response to cold stimulation was tested by spraying ethyl chloride (Walter Ritter-Pharmaceutica, Germany) to the plantar surface of the paw (2–3 s) from an approximate distance of 2 cm and classified as: 0, no response; 1, startle response without paw withdrawal; 2, brief withdrawal of the paw; 3, prolonged withdrawal (5–30 s); 4, prolonged and repetitive withdrawal (.30 s) combined with flinching and / or licking [16]. A significant increase in cold scores in response to ethyl chloride application was interpreted as cold allodynia. Tests took place 2–4 weeks after spinal nerve ligation. Mechanical allodynia thresholds and cold allodynia scores were assessed immediately before, and at 0.5, 1, and 2 h after drug injections. After testing two doses of magnesium sulfate (125, 250 mg / kg, i.p.), the effect of morphine (0.1 mg / kg, i.p.) and magnesium sulfate (125 mg / kg, i.p.) combination on mechanical and cold allodynia was investigated. The subthreshold dose of morphine was defined from results, both from the literature and from earlier observations in our laboratory. Magnesium sulfate was administered 5 min prior to morphine. Morphine hydrchloride (Haver, Istanbul, Turkey) was diluted from commercial preparations. Magnesium sulfate was obtained from Merck, and naloxone was purchased from Research Biochemicals Inc. All chemicals were dissolved in isotonic NaCl and administered in a volume of 0.1 ml / 100 g body weight. Analysis of variance (ANOVA) followed by Newman-Keuls test was used for analyzing the data from mechanical allodynia, and Mann–Whitney U-test for analyzing the data from cold allodynia. Values of P,0.05 were considered to be significant. All data are expressed as mean6S.E.M. As described previously [17], rats subjected to L5 and L6 SNL developed pain-related behavior, marked as mechanical and cold allodynia. Prior to operation, 50% paw withdrawal threshold for mechanical allodynia and cold scores for ethyl chloride applications were 14.361.3 and 1.160.1, respectively. Two to 4 weeks after nerve injury, significant reduction in paw withdrawal thresholds to von Frey filaments (2.460.3, P,0.05) and significant increase in cold scores (3.160.2, P,0.05) were observed. With its higher dose, magnesium sulfate (250 mg / kg) increased paw withdrawal thresholds to von Frey hairs (P,0.05, Fig. 1A), and decreased cold scores of ethyl chloride applications (P,0.05, Fig. 2A), compared to
Fig. 1. Effects of i.p. injections of saline and two doses of magnesium sulfate (125–250 mg / kg) on mechanical allodynia (A). Antiallodynic effect of the combination of subthreshold doses of morphine (M, 0.1 mg / kg) and magnesium sulfate (Mg, 125 mg / kg), and prevention of this effect on mechanical allodynia by naloxone (N, 1 mg / kg) (B). Preoperative (Pre) values were determined before the operation (SNL). n56–7 per group. (*P,0.05, compared to corresponding values in the saline control group; 1 P,0.05, compared to corresponding values in the combination group).
corresponding values in the saline control group (0.1 ml / 100 g). When subthreshold doses of magnesium sulfate (125 mg / kg) and morphine (0.1 mg / kg) was combined, an anti-allodynic effect was observed (P,0.05, Figs. 1B and 2B). This effect was partially reversed with 1 mg / kg of naloxone, which may show that this anti-allodynic effect of the combination is mainly mediated via opioid receptors. The doses of 1 mg / kg of naloxone and 0.1 mg / kg of morphine were chosen since it had no effect by their own in previous studies. The response of chronic pain states to opioid treatment is controversial, and neuropathic pain is generally accepted to be particularly insensitive to opioid analgesics [3,19,24]. The loss of opioid receptors expressed on C-fibre afferents,
A. Ulugol et al. / Brain Research 943 (2002) 101 – 104
Fig. 2. Effects of i.p. injections of saline and two doses of magnesium sulfate (125–250 mg / kg) on cold allodynia (A). Antiallodynic effect of the combination of subthreshold doses of morphine (M, 0.1 mg / kg) and magnesium sulfate (Mg, 125 mg / kg), and prevention of this effect on cold allodynia by naloxone (N, 1 mg / kg) (B). Preoperative (Pre) values were determined before the operation (SNL). n56–7 per group. (*P, 0.05, compared to corresponding values in the saline control group; 1 P,0.05, compared to corresponding values in the combination group).
the increase in the levels of cholecystokinin, the accumulation of morphine-3-glucuronide, and the activation of NMDA receptors may lead to reduced sensitivity to morphine in neuropathic pain states [10,11,23,26]. NMDA receptor antagonists, either alone, or in combination with opioids, have been indicated to be effective both in experimental pain models and in clinical studies; however, their side effects generally limit their use as effective agents [1,6,20,22]. At resting membrane potentials, Mg 21 blocks the NMDA receptor’s ion channel and prevents ligand gating of channel conductance. Release of substance P, its actions on the neurokinin-1 receptor, and probably the contribution of other peptides, removes the Mg 21 block of the NMDA
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receptor [11]. Removal of this block may underlie the participation of NMDA receptor in the ‘wind-up’ phenomenon [8,9]. It can be hypothesized that magnesium could have similar antinociceptive effects as NMDA antagonists; however, results obtained from experimental studies are controversial. Magnesium sulfate has been shown to prevent autotomy in peripherally deafferented rats [14], and suppressed neuropathic pain responses via a spinal site of action [25]. Recently, magnesium has also been shown to reverse hyperalgesia both in nerve-injured and diabetic rats [2]. On the other hand, magnesium sulfate did not exert remarkable antinociceptive effects in acute pain models [15]. Clinical use of magnesium is also controversial; Felsby et al. indicated that Mg 21 infusion failed to reduce pain significantly in chronic states [13]; however, Crosby et al. suggested magnesium sulfate as a safe, well-tolerated and effective agent in patients with neuropathic pain due to cancer [7]. We found that magnesium sulfate exerts a significant anti-allodynic effect with its higher dose (250 mg / kg); however, this is a fairly low dose which dose not induce any side effects [2,25]. We also showed that the combination of subthreshold doses of morphine and magnesium sulfate attenuated mechanical and cold allodynia in spinal nerve ligated rats, similar to the observation of Kroin et al., where the same combination have been effective against nociception after surgical incision [18]. When Mg 21 blockade of NMDA receptor’s ion channel is taken into consideration, this observation may reflect the importance of NMDA receptor-mediated sustained afferent drive in the activity of morphine in neuropathic pain states. These results suggest that magnesium sulfate could be used to lower dosages of morphine, and this combination therapy might be useful in the management of human neuropathic pain.
References [1] C. Advokat, F.Q. Rhein, Potentiation of morphine-induced antinociception in acute spinal rats by the NMDA antagonist dextrorphan, Brain Res. 699 (1995) 157–160. http: / / journals.bmn.com / medline / search / record?uid5MDLN.96188365&rendertype5full. [2] S. Begon, G. Pickering, A. Eschalier, C. Dubray, Magnesium and MK-801 have a similar effect in two experimental models of neuropathic pain, Brain Res. 887 (2000) 436–439. http: / / www.neuroscion.com / abstract / JOURNAL.BRES.nol0145a 00068993 v0887i02 00030286. ] ] ] [3] F. Benedetti, S. Vighetti, M. Amanzio, C. Casadio, A. Oliaro, B. Bergamasco, G. Maggi, Dose-response relationship of opioids in nociceptive and neuropathic postoperative pain, Pain 74 (1998) 205–211. http: / / www.elsevier.nl / gej-ng / 10 / 35 / 50 / 35 / 24 / 34 / abstract.html. [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. http: / / journals.bmn.com / medline / search / record?uid5MDLN.95082408&rendertype5full. [5] V. Chapman, J.E. Haley, A.H. Dickenson, Electrophysiological analysis of preemptive effects of spinal opioids on N-methyl-D-
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Short communication
Combined systemic administration of morphine and magnesium sulfate attenuates pain-related behavior in mononeuropathic rats Ahmet Ulugol*, Aysegul Aslantas, Yesim Ipci, Alev Tuncer, Cetin Hakan Karadag, Ismet Dokmeci Department of Pharmacology, Faculty of Medicine, Trakya University, 22030 Edirne, Turkey Accepted 22 November 2001
Abstract The response to opioids is reduced in neuropathic pain states. We examined the effect of the combination of morphine (0.1 mg / kg) and magnesium sulfate (125 mg / kg) on behavioral signs of neuropathic pain in spinal nerve ligated rats. Administered alone, neither drug produced any effect, but the combination exerted a significant anti-allodynic effect, which was partially reversed by naloxone. These results suggest that combining low doses of magnesium sulfate with opiates might be an alternative in treating neuropathic pain, with reduced risk of side effects. 2002 Elsevier Science B.V. All rights reserved. Theme: Sensory systems Topic: Pain modulation: pharmacology Keywords: Neuropathic pain; Allodynia; Morphine; Magnesium
Peripheral nerve injury may lead to neuropathic pain, which is characterized by spontaneous pain, hyperalgesia (augmented pain response to normally painful stimuli) and allodynia (nociceptive responses to normally innocuous stimuli), analogous to clinical conditions of neuropathic pain. Several animal models, including spinal nerve ligation (SNL), have recently been developed to study the mechanisms of neuropathic pain [17]. Although the effectiveness of opiates in the treatment of neuropathic pain states is controversial, this condition is generally accepted to respond poorly to opioids [3,19,24]. Dose escalations for treatment are not devoid of side effects [3]. N-methyl-D-aspartate (NMDA) receptors are known to be involved in the etiology of chronic neuropathic pain [5,8,9,11]. NMDA receptor antagonists have been shown to be effective in various experimental models of neuropathic pain; however, their adverse effects also preclude their use in this condition [20,22]. Therapeutic benefit may also be improved by combining low doses of opioids with *Corresponding author. Tel.: 190-532-431-6449; fax: 190-284-2352476. E-mail addresses: [email protected] (A. Ulugol), [email protected] (A. Ulugol).
NMDA antagonists [1,6]. Mg 21 ions have been shown to block inward current flow through ion channels linked to NMDA receptors, and prevent extracellular Ca 21 to enter the cell [21]. Accordingly, magnesium ions have been effective in different pain models [2], and potentiated morphine antinociception after surgical incision [18]. The aim of this study was to determine the influence of systemically injected magnesium sulfate on the anti-allodynic effect of morphine in spinal nerve ligated rats. This study was conducted according to the guidelines of the Ethical Committee of the International Association for the Study of Pain, and had been approved by the ‘Animal Care Ethics Committee’ of our faculty. Wistar rats of either sex (DETAM, Istanbul, Turkey), weighing 300–350 g at time of operation, were used for the experiments. SNL model of neuropathic pain, previously described by Kim and Chung [17], was used. Briefly, under sodium pentobarbital anesthesia (40 mg / kg, i.p.), the left L5 and L6 spinal nerves were isolated and tightly ligated with 6-0 silk thread. The rats were placed under a glass cover on a metal mesh floor and mechanical allodynia thresholds were assessed, as described previously [4]. Briefly, eight von Frey filaments, with approximately equal logarithmic incremental bending forces, were chosen (von Frey num-
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )02618-5
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A. Ulugol et al. / Brain Research 943 (2002) 101 – 104
bers: 3.22, 3.61, 4.08, 4.31, 4.56, 4.93, 5.18, 5.46; equivalent to 0.16, 0.4, 1, 2, 4, 8, 15, 26 g). Each hair was pressed perpendicularly against the plantar surface of the hindpaw until slight buckling was observed, and lifting of the paw was recorded as a positive response. Starting with the 2.0 g hair, 50% withdrawal thresholds were determined using the up-down method of Dixon [12]. If the rat responded, the next weaker hair was applied. In case of a negative response, the next stronger hair was applied. The test was continued until four measurements after the first change in response had been obtained. The response to cold stimulation was tested by spraying ethyl chloride (Walter Ritter-Pharmaceutica, Germany) to the plantar surface of the paw (2–3 s) from an approximate distance of 2 cm and classified as: 0, no response; 1, startle response without paw withdrawal; 2, brief withdrawal of the paw; 3, prolonged withdrawal (5–30 s); 4, prolonged and repetitive withdrawal (.30 s) combined with flinching and / or licking [16]. A significant increase in cold scores in response to ethyl chloride application was interpreted as cold allodynia. Tests took place 2–4 weeks after spinal nerve ligation. Mechanical allodynia thresholds and cold allodynia scores were assessed immediately before, and at 0.5, 1, and 2 h after drug injections. After testing two doses of magnesium sulfate (125, 250 mg / kg, i.p.), the effect of morphine (0.1 mg / kg, i.p.) and magnesium sulfate (125 mg / kg, i.p.) combination on mechanical and cold allodynia was investigated. The subthreshold dose of morphine was defined from results, both from the literature and from earlier observations in our laboratory. Magnesium sulfate was administered 5 min prior to morphine. Morphine hydrchloride (Haver, Istanbul, Turkey) was diluted from commercial preparations. Magnesium sulfate was obtained from Merck, and naloxone was purchased from Research Biochemicals Inc. All chemicals were dissolved in isotonic NaCl and administered in a volume of 0.1 ml / 100 g body weight. Analysis of variance (ANOVA) followed by Newman-Keuls test was used for analyzing the data from mechanical allodynia, and Mann–Whitney U-test for analyzing the data from cold allodynia. Values of P,0.05 were considered to be significant. All data are expressed as mean6S.E.M. As described previously [17], rats subjected to L5 and L6 SNL developed pain-related behavior, marked as mechanical and cold allodynia. Prior to operation, 50% paw withdrawal threshold for mechanical allodynia and cold scores for ethyl chloride applications were 14.361.3 and 1.160.1, respectively. Two to 4 weeks after nerve injury, significant reduction in paw withdrawal thresholds to von Frey filaments (2.460.3, P,0.05) and significant increase in cold scores (3.160.2, P,0.05) were observed. With its higher dose, magnesium sulfate (250 mg / kg) increased paw withdrawal thresholds to von Frey hairs (P,0.05, Fig. 1A), and decreased cold scores of ethyl chloride applications (P,0.05, Fig. 2A), compared to
Fig. 1. Effects of i.p. injections of saline and two doses of magnesium sulfate (125–250 mg / kg) on mechanical allodynia (A). Antiallodynic effect of the combination of subthreshold doses of morphine (M, 0.1 mg / kg) and magnesium sulfate (Mg, 125 mg / kg), and prevention of this effect on mechanical allodynia by naloxone (N, 1 mg / kg) (B). Preoperative (Pre) values were determined before the operation (SNL). n56–7 per group. (*P,0.05, compared to corresponding values in the saline control group; 1 P,0.05, compared to corresponding values in the combination group).
corresponding values in the saline control group (0.1 ml / 100 g). When subthreshold doses of magnesium sulfate (125 mg / kg) and morphine (0.1 mg / kg) was combined, an anti-allodynic effect was observed (P,0.05, Figs. 1B and 2B). This effect was partially reversed with 1 mg / kg of naloxone, which may show that this anti-allodynic effect of the combination is mainly mediated via opioid receptors. The doses of 1 mg / kg of naloxone and 0.1 mg / kg of morphine were chosen since it had no effect by their own in previous studies. The response of chronic pain states to opioid treatment is controversial, and neuropathic pain is generally accepted to be particularly insensitive to opioid analgesics [3,19,24]. The loss of opioid receptors expressed on C-fibre afferents,
A. Ulugol et al. / Brain Research 943 (2002) 101 – 104
Fig. 2. Effects of i.p. injections of saline and two doses of magnesium sulfate (125–250 mg / kg) on cold allodynia (A). Antiallodynic effect of the combination of subthreshold doses of morphine (M, 0.1 mg / kg) and magnesium sulfate (Mg, 125 mg / kg), and prevention of this effect on cold allodynia by naloxone (N, 1 mg / kg) (B). Preoperative (Pre) values were determined before the operation (SNL). n56–7 per group. (*P, 0.05, compared to corresponding values in the saline control group; 1 P,0.05, compared to corresponding values in the combination group).
the increase in the levels of cholecystokinin, the accumulation of morphine-3-glucuronide, and the activation of NMDA receptors may lead to reduced sensitivity to morphine in neuropathic pain states [10,11,23,26]. NMDA receptor antagonists, either alone, or in combination with opioids, have been indicated to be effective both in experimental pain models and in clinical studies; however, their side effects generally limit their use as effective agents [1,6,20,22]. At resting membrane potentials, Mg 21 blocks the NMDA receptor’s ion channel and prevents ligand gating of channel conductance. Release of substance P, its actions on the neurokinin-1 receptor, and probably the contribution of other peptides, removes the Mg 21 block of the NMDA
103
receptor [11]. Removal of this block may underlie the participation of NMDA receptor in the ‘wind-up’ phenomenon [8,9]. It can be hypothesized that magnesium could have similar antinociceptive effects as NMDA antagonists; however, results obtained from experimental studies are controversial. Magnesium sulfate has been shown to prevent autotomy in peripherally deafferented rats [14], and suppressed neuropathic pain responses via a spinal site of action [25]. Recently, magnesium has also been shown to reverse hyperalgesia both in nerve-injured and diabetic rats [2]. On the other hand, magnesium sulfate did not exert remarkable antinociceptive effects in acute pain models [15]. Clinical use of magnesium is also controversial; Felsby et al. indicated that Mg 21 infusion failed to reduce pain significantly in chronic states [13]; however, Crosby et al. suggested magnesium sulfate as a safe, well-tolerated and effective agent in patients with neuropathic pain due to cancer [7]. We found that magnesium sulfate exerts a significant anti-allodynic effect with its higher dose (250 mg / kg); however, this is a fairly low dose which dose not induce any side effects [2,25]. We also showed that the combination of subthreshold doses of morphine and magnesium sulfate attenuated mechanical and cold allodynia in spinal nerve ligated rats, similar to the observation of Kroin et al., where the same combination have been effective against nociception after surgical incision [18]. When Mg 21 blockade of NMDA receptor’s ion channel is taken into consideration, this observation may reflect the importance of NMDA receptor-mediated sustained afferent drive in the activity of morphine in neuropathic pain states. These results suggest that magnesium sulfate could be used to lower dosages of morphine, and this combination therapy might be useful in the management of human neuropathic pain.
References [1] C. Advokat, F.Q. Rhein, Potentiation of morphine-induced antinociception in acute spinal rats by the NMDA antagonist dextrorphan, Brain Res. 699 (1995) 157–160. http: / / journals.bmn.com / medline / search / record?uid5MDLN.96188365&rendertype5full. [2] S. Begon, G. Pickering, A. Eschalier, C. Dubray, Magnesium and MK-801 have a similar effect in two experimental models of neuropathic pain, Brain Res. 887 (2000) 436–439. http: / / www.neuroscion.com / abstract / JOURNAL.BRES.nol0145a 00068993 v0887i02 00030286. ] ] ] [3] F. Benedetti, S. Vighetti, M. Amanzio, C. Casadio, A. Oliaro, B. Bergamasco, G. Maggi, Dose-response relationship of opioids in nociceptive and neuropathic postoperative pain, Pain 74 (1998) 205–211. http: / / www.elsevier.nl / gej-ng / 10 / 35 / 50 / 35 / 24 / 34 / abstract.html. [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. http: / / journals.bmn.com / medline / search / record?uid5MDLN.95082408&rendertype5full. [5] V. Chapman, J.E. Haley, A.H. Dickenson, Electrophysiological analysis of preemptive effects of spinal opioids on N-methyl-D-
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