Research report 315
Postinjury treatment with magnesium sulfate attenuates neuropathic pains following spinal cord injury in male rats Leila Farsia,b, Khashayar Afsharib, Mansoor Keshavarzb,c, Maryam NaghibZadehb, Fereidoon Memarid and Abbas Norouzi-Javidana Spinal cord injury (SCI) has a number of severe and disabling consequences including chronic pain. Approximately 40% of patients experience neuropathic pain, which appears to be persistent. Previous studies have demonstrated the neuroprotective effects of magnesium sulfate (MgSO4). We aimed to investigate the effect of MgSO4 on neuropathic pains following SCI in male rats. Thirty-two adult male rats (weight 300–350 g) were used. After laminectomy, a complete SCI was induced by compression of the spinal cord for 1 min with an aneurysm clip. A single dose of 300 or 600 mg/kg MgSO4 was injected intraperitoneally. Tail-flick latency and acetone drop test scores were evaluated before surgery and once a week for 4 weeks after surgery. Rats in groups SCI + Mg300 and SCI + Mg600 showed significantly higher mean tail-flick latencies and lower mean scores in the acetone test compared with those in the SCI + veh group 4 weeks after
surgery (P < 0.05). These findings revealed that systemic single-dose administration of MgSO4 can attenuate thermal hyperalgesia and cold allodynia induced by SCI in rats. Behavioural Pharmacology 26:315–320 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Introduction
whether administered before, shortly after, or hours after the injury (Bareyre et al., 2000; Saatman et al., 2001; Temkin et al., 2007).
Spinal cord injury (SCI) has severe and disabling consequences including chronic pain and neuropathic pain, which occur in around 40% of patients (Baastrup and Finnerup, 2008) and appear to be persistent despite various treatment methods. In recent years, more information on SCI pain mechanisms has been derived from experimental models and clinical studies, but treatment is still difficult and insufficient (Finnerup and Baastrup, 2012). Many agents have been found to be neuroprotective, one of which is magnesium. Magnesium has long been documented as an essential cation necessary for the proper functioning of more than 300 key enzymes involved in energy renovation, lipid and nucleic acid metabolism, and protein synthesis (Vink and Cernak, 2000). Furthermore, magnesium is a compulsory requirement for any reaction that either produces or consumes ATP (Ebel and Gunther, 1980), including glycolysis and oxidative phosphorylation. Consequently, any decrease in the magnesium concentration following neurotrauma will decrease a cells ability to preserve its membrane potential and to repair itself (Vink and Cernak, 2000). It has been shown that after head injuries in humans, total serum and ionized magnesium concentrations decline (Memon et al., 1995), which is a critical factor leading to irreversible tissue damage following direct or indirect neurotrauma (Vink and Cernak, 2000). Magnesium supplementation improves treatment results 0955-8810 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Behavioural Pharmacology 2015, 26:315–320 Keywords: hyperalgesia, magnesium sulfate, neuropathic pain, rat, spinal cord injury, tail flick a Brain and Spinal Cord Injury Research Center, bElectrophysiology Research Center, Neuroscience Institute, cDepartment of Physiology, School of Medicine and dCancer Institute, Imam Khomeini Hospital, Tehran University of Medical Sciences, Tehran, Iran
Correspondence to Mansoor Keshavarz, MD, PhD, Department of Physiology, School of Medicine, Tehran University of Medical Sciences, Poursina Street, 1456833581 Tehran, Iran E-mail: [email protected] Received 3 September 2013 Accepted as revised 20 September 2014
Moreover, magnesium deficiency is found in association with acute medical/surgical conditions in which pain or stress is present (Dubray et al., 1997), and it causes hyperalgesia that can be ameliorated by NMDA antagonists (Weissberg et al., 1991). Magnesium has been used extensively in clinical approaches for the treatment of pre-eclampsia (Sibai, 2005), stroke (Muir et al., 2004), arrhythmia (Piotrowski and Kalus, 2004), and myocardial infarction (Gowda and Khan, 2004). Magnesium is also cheap and readily accessible, and its clear pharmacological profile confers substantial advantages over other neuroprotective agents (Kohno et al., 2007). Its role in the prevention and treatment of diabetic neuropathy has also been demonstrated (Hasanein et al., 2006; Rondon et al., 2010). In light of the neuroprotective effect of magnesium in SCI (Kohno et al., 2007) and its established role in the prevention and treatment of neuropathic pain (Hasanein et al., 2006; Rondon et al., 2010), we aimed to investigate the effect of magnesium sulfate (MgSO4) on neuropathic pain following SCI in male rats.
Methods Subjects
This study was performed on 32 adult male rats (weight 300–350 g at the start of the experiment) that were DOI: 10.1097/FBP.0000000000000103
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
316 Behavioural Pharmacology 2015, Vol 26 No 3
purchased from Pasteur Institute, Tehran, Iran. The animals were kept at room temperature (23 ± 2°C) and a humidity of 50 ± 5%, in a 12-h light/dark cycle (light: 08.00–20.00 h), with free access to tap water and a standard pellet chow. They were handled in accordance with the criteria of the ‘Guide for the Care and Use of Laboratory Animals’ (NIH US publication 23-86 revised 1985). This study also followed the International Association for the Study of Pain guidelines for animal experiments (Rondon et al., 2010). Body weight, SCI complications such as autophagia, and mortality were measured and recorded. The animals were randomly divided into four groups (n = 6–8 per group): Sham + veh group: only laminectomy, without SCI, was performed. Animals received a 1 ml intraperitoneal injection of normal saline within 30 min after the surgery. SCI + veh group: SCI was induced and animals received a 1 ml intraperitoneal injection of normal saline within 30 min after the surgery. SCI + Mg300 group: animals received a 300 mg/kg intraperitoneal injection of MgSO4 in 1 ml of saline vehicle within 30 min after SCI. SCI + Mg600 group: animals received a 600 mg/kg intraperitoneal injection of MgSO4 in 1 ml of saline vehicle within 30 min after SCI. According to the literature, magnesium has no effect in sham-operated groups (Xiao-Jun et al., 1994); hence, we did not include sham + magnesium groups.Rats from each group were randomly selected and handled by a blind observer (blinded to the doses of magnesium and vehicle in the SCI groups).
Surgery and drug administration
Rats were anesthetized with an intraperitoneal injection of 50 mg/kg ketamine (Trittau, Germany). Before surgery, the operation site was shaved and disinfected with a 10% solution of polyvidon iodine. The surgery was performed using a standard sterile technique. An incision was made over the thoracic spine at the T7–T12 level. After the incision of the dermal and subdermal tissues at the midline, paravertebral muscles were bluntly dissected to expose the lamina bilaterally. Complete laminectomies were performed to expose the spinal cord at T7–T12. SCI was induced by 1-min compression of the spinal cord at the T9 level horizontally and extradurally with an aneurysm clip. The wounds were then closed with a 3/0 silk suture. Within 30 min after the surgery, 1 ml of 300 or 600 mg/kg MgSO4 (suspended in sterile distilled water), provided by Sigma (St Louis, Missouri, USA), was injected intraperitoneally in the magnesiumtreated groups.
In a pilot study, we used four doses of MgSO4 (100, 300, 600, and 900 mg/kg). We observed that 100 mg/kg MgSO4 had no significant effect and 900 mg/kg MgSO4 had a similar effect to 600 mg/kg MgSO4. On the basis of this, we decided to use 300 and 600 mg/kg MgSO4 for this study. Similar doses were used in several previous studies: for example, Xiao-Jun et al. (1994) reported that 150 and 200 mg/kg magnesium had no hyperalgesic effects, but 300 mg/kg was clearly active. Postoperative care included controlling the body temperature and prophylactic antibiotic administration to prevent infection (70 mg/kg cephazolin for 7 days). SCI rats underwent manual bladder expression twice daily for 10–14 days until their bladder functions fully recovered. Following the method used by Wiseman et al. (2009), we killed the rats after 4 weeks for histological studies to confirm that the SCI site was correct. Measurement of thermal hyperalgesia
Tail-flick latency (TFL) was measured using a Tail-Flick Analgesia Meter (IITC life science model 33t; Los Angeles, California, USA) before the surgery and at days 0, 7, 14, 21, and 28. After a 45-min acclimatization period, TFL was measured by exposing the dorsal surface of the animal’s tail to a radiant heat source, and the time taken for the conscious rats to move their tails from the noxious thermal stimulus was recorded. To reach proper baseline intensity, each animal was subjected to five test trials and the strength of the stimulus was adjusted so that TFLs would be between 7 and 8 s. A cutoff time of 8 s was used to prevent tail injury. The mean intensity level was then calculated and used in the following tail-flick tests. Cold allodynia (acetone drop test)
After a 45-min acclimatization period, the response to cold stimulation was tested by spraying acetone onto the plantar surface of the paw (2–3 s) from an estimated distance of 2 cm. The result was 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) along with flinching and/or licking (Kauppila, 2000). A significant increase in the response to acetone application was interpreted as cold allodynia (Ulugol et al., 2002). Statistical analysis
For data analysis, two-way repeated measure analysis of variance was used, followed by Tukey’s post-hoc test. P less than 0.05 was considered significant. All data are expressed as mean ± SEM. SPSS18 was used for analysis (SPSS Inc., Chicago, Illinois, USA).
Results There were no significant differences between groups in any variable on day 0 of the study. SCI caused a significant decrease in the thermal nociceptive threshold
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
Magnesium attenuates neuropathic pain Farsi et al. 317
Tail-flick latency
In general, the SCI groups showed a reduction in the mean TFL after surgery in response to thermal stimuli (tail-flick test). Intraperitoneal administration of MgSO4 inhibited the nociceptive phenomena induced by SCI over time (Fig. 1). Two-way analysis of variance of TFL showed significant main effects of dose [F(3, 20) = 40.38, P < 0.001] and time [F(4,80) = 23.20, P < 0.001], and a significant dose × time interaction [F(12,80) = 8.66, P < 0.001]. Cold allodynia
SCI rats showed significantly increased sensitivity to cold stimuli (acetone drop on the paw) compared with that in presurgical tests (Fig. 2). Statistical analysis showed significant main effect of time [F(4,80) = 37.97, P < 0.001] and dose [F(3,20) = 13.02, P < 0.001], and a significant dose × time interaction [F(12,80) = 5.30, P < 0.001].
Fig. 2
3 Acetone test (score)
(measured by the Tail-Flick Analgesia Meter) and an increase in the cold allodynia score (measured by the acetone drop test), as compared with the baseline values obtained before surgery.
SCI+Mg300
SCI+Mg600
SCI+vehicle
Sham
∗
2.5 2 1.5 1 0.5 0 0
1
2 Weeks after surgery
3
4
Influence of post-SCI administration of MgSO4 on the acetone test scores of the rats over the next 4 weeks. SCI + Mg300 and SCI + Mg600 indicate SCI rats treated with 300 mg/kg magnesium (intraperitoneally) and 600 mg/kg magnesium (intraperitoneally), respectively. The SCI + veh group includes SCI rats that received normal saline. The data are presented as mean ± SEM (six to eight rats per groups). *P < 0.05 indicates a significant difference in the mean acetone score of the SCI + veh group compared with other groups. MgSO4, magnesium sulphate; SCI, spinal cord injury; veh, vehicle.
Fig. 3
Body weight
SCI+vehicle
SCI+Mg300
SCI+Mg600
Sham
420 370 Weight (g)
After SCI surgery, there was weight loss in all SCI groups. This was more severe in the SCI + veh group (Fig. 3). Statistical analysis showed significant main effects of time [F(4,80) = 15.67, P < 0.001] and dose [F(3,20) = 14.63, P < 0.001], and a significant dose × time interaction [F(12,80) = 11.20, P < 0.001].
320 270
*
*
*
220 Fig. 1 SCI+vehicle Sham
9
∗
Tail-flick test (s)
8
170
SCI+Mg300 SCI+Mg600
∗
7 6 5
∗
∗
3
4
0
1
2 Weeks after surgery
3
4
Influence of postinjury administration of MgSO4 on the body weight of the rats over the next 4 weeks. SCI + Mg300 and SCI + Mg600 indicate SCI rats treated with 300 mg/kg magnesium (intraperitoneally) and 600 mg/kg magnesium (intraperitoneally), respectively. The SCI + veh group includes SCI rats that received normal saline. The data are presented as mean ± SEM (six to eight rats per groups). *P < 0.05 indicates a significant difference between the SCI + veh group and other groups: sham, SCI + Mg300. MgSO4, magnesium sulphate; SCI, spinal cord injury; veh, vehicle.
4 3 0
1
2 Weeks after surgery
Influence of postinjury administration of MgSO4 on the mean tail-flick latencies of the rats over the next 4 weeks. SCI + Mg300 and SCI + Mg600 indicate SCI rats treated with 300 mg/kg magnesium (intraperitoneally) and 600 mg/kg magnesium (intraperitoneally), respectively. The SCI + veh group includes SCI rats that received normal saline. The data are presented as mean ± SEM (six to eight rats per groups). *P < 0.05 indicates a significant difference compared with other groups. MgSO4, magnesium sulphate; SCI, spinal cord injury; veh, vehicle.
Discussion In the present study, we report that a single-dose intraperitoneal injection of MgSO4 within 30 min after SCI resulted in an improvement in hyperalgesia and allodynia over a 4-week period after SCI surgery. Several studies have shown that SCI causes chronic neuropathic pain (Baastrup and Finnerup, 2008; Finnerup and Baastrup, 2012; Yezierski et al., 2013). The present study also found symptoms of neuropathic pain after SCI, including lower
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
318 Behavioural Pharmacology 2015, Vol 26 No 3
thermal pain thresholds, indicating thermal hyperalgesia, and a higher score in the acetone drop test, indicating cold allodynia. Magnesium has long been recognized as an essential cation necessary for the appropriate functioning of over 300 key enzymes (Vink and Cernak, 2000). Moreover, any reaction that either produces or consumes ATP demands magnesium (Ebel and Gunther, 1980). Previous studies have reported therapeutic effects of magnesium for diabetes and its complications (Soltani et al., 2005a, 2005b, 2007; Hasanein et al., 2006). MgSO4 is a prominent neuroprotective agent against experimental neurodegeneration and central nervous system damage (Wolf et al., 1991; Simpson et al., 1994; Lang-Lazdunski et al., 2000; Gok et al., 2007; Temkin et al., 2007), and it has been demonstrated to be effective in several types of CNS injuries such as SCI, traumatic brain injury, and ischemia (Temkin et al., 2007; Wiseman et al., 2009). In three carefully randomized placebocontrolled trials, antenatal MgSO4 was found to decrease the risk for cerebral palsy in children who survived from early preterm birth (Crowther et al., 2004; Marret et al., 2008; Rouse et al., 2008). It was concluded that MgSO4 decreased the risk for cerebral palsy by 32% of children, but it had no effect on fetal or infant mortality (Doyle et al., 2009). According to Wiseman et al. (2009), magnesium improved the neurological function of SCI rats compared with controls. Lampl et al. (2001), in a randomized, placebocontrolled, double-blind study, examined the protective effect of MgSO4 administered intravenously to patients within 24 h after a stroke and reported a significant positive effect on the outcome. It has been demonstrated that the decline in the tissue magnesium level is an essential pathophysiological factor in secondary SCI (Lemke et al., 1990), and the use of MgSO4 reduced apoptotic cell death after SCI (Suzer et al., 1999). The present study found moderate weight loss in the SCI groups for 2 weeks, which was significantly lower in the magnesium-treated groups compared with the SCI + veh group. This is assumed to be due to the pain and disability after SCI, and the difficulty in food and water consumption. Two weeks after SCI, the mean weight of the SCI rats remained constant until the third week, and then it started to increase. Better neurological and functional conditions (Ditor et al., 2007; Wiseman et al., 2009) in rats treated with magnesium may lead to higher weight gain in this group. [However, Wiseman et al. (2009) did not report weight loss in SCI rats after surgery in either control or magnesium-treated groups.] Analgesic and antihyperalgesic effects of magnesium have been reported in several studies (Crosby et al., 2000; Hasanein et al., 2006; Song et al., 2011; Arai et al., 2013), but its effect on neuropathic pain after SCI has not previously been reported. However, although magnesium attenuates hyperalgesia, it has been reported to have no
effect on sham-operated rats without hyperalgesia. Xiao-Jun et al. (1994) administered magnesium in chronic constriction injury (CCI) rats and compared withdrawal latencies (heat hyperalgesia) bilaterally (on the CCI side with hyperalgesia and on the contralateral side without CCI and hyperalgesia). Magnesium had no significant effect on withdrawal latencies (heat hyperalgesia) in the sham-operated side. It has been shown that SCI rats have significantly lower TFLs compared with normal rats (Xiao-Jun et al., 1994). In the present study, SCI groups treated with 300 or 600 mg/kg MgSO4 had significantly higher mean TFLs compared with the SCI + veh group after 4 weeks, indicating lower heat hyperalgesia relative to the SCI + veh group. Rondon et al. (2010) showed that magnesium eliminates thermal hyperalgesia in a rat model of diabetic neuropathic pain. Hasanein et al. (2006) also showed that oral magnesium administration at the time of diabetes induction may abolish thermal hyperalgesia in diabetic rats. Mert et al. (2009) reported that intraplantar coadministration of fentanyl and magnesium can prevent delayed thermal hyperalgesia in rats. These observations are consistent with our findings. However, in a clinical trial on patients suffering from neuropathic pain, Pickering et al. (2011) could not demonstrate any significant difference in pain alleviation between patients under 1 month of oral treatment with magnesium and those who were administered placebo. This inconsistency may be due to different forms of drug administration: Pickering et al. (2011) administered oral MgCl2 capsules, whereas in the present study we administered MgSO4 intraperitoneally in rats. Similar to the previous studies showing that SCI can cause cold allodynia (Lindsey et al., 2000), the present results show that after SCI there was cold allodynia in the SCI groups, which was attenuated in the fourth week. In a study on oxaliplatin-induced peripheral neuropathy in rats, Sakurai et al. (2009) showed that administration of calcium or magnesium (0.5 mmol/kg, intravenously) before oxaliplatin or oxalate administration prevented cold hyperalgesia. In another study of spinal nerveligated rats that suffered from neuropathic pain, Ulugol et al. (2002) showed that MgSO4 exerts a significant antiallodynic effect, attenuating cold allodynia at a dose of 250 mg/kg. One hypothesis of the pathophysiology of neuropathic pain following SCI took into consideration the role of spinal cord NMDA receptor channels in central sensitization (Hasanein et al., 2006). As magnesium is an antagonist of these channels (Mayer et al., 1984), this mechanism may also be implicated in the elevation of the thermal pain threshold in magnesium-treated SCI rats (Hasanein et al., 2006). Magnesium has a neuroprotective action through a number of mechanisms, such as dilatation of cerebrovascular arteries, blockage of NMDA
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
Magnesium attenuates neuropathic pain Farsi et al. 319
receptors, and blockage of voltage-gated calcium channels. Furthermore, by directly inhibiting lipid peroxidation and preventing the depletion of glutathione, this element may reduce the severity of endothelial and neuronal reperfusion injury (Dickens et al., 1992; Regan et al., 1998; Peker et al., 2004). In this regard, Gok et al. (2007) showed that treatment with a single 600 mg/kg dose of MgSO4 instantly after SCI prevents neutrophil infiltration into the spinal cord after a contusion injury, by attenuating chemotaxis. Neutrophil adhesion and diapedesis leads to blood–spinal cord barrier breakdown, and the phagocytic property of neutrophils plays a fundamental role in tissue damage. Activated neutrophils are also known to produce a group of inflammatory mediators including proteases, cytokines, chemokines, and free radicals (Harlan, 1987; Xu et al., 1990; Cuzzocrea et al., 2005; Genovese et al., 2005). According to a study by Miles et al. (2001), MgSO4 significantly reduces neuronal death when administered up to 24 h after transient global ischemia, offering a neuroprotective mechanism of action for magnesium that broadens beyond excitotoxicity. After ischemia, there is a spread interruption in many cellular functions, including enzymatic reactions related to ATP, mitochondrial and plasma membrane integrity, protein synthesis, calcium transport, and oxidative phosphorylation (Harding, 1992; McIntosh, 1993; McLean, 1994; Muir and Lees, 1995). Restoration of any of these processes in postischemic neurons may be an extra or alternate mechanism of action of magnesium in preventing neuronal death (Miles et al., 2001). Moreover, magnesium may cause vasodilatation of spinal cord vessels by stimulating endothelial prostacyclin release (Kaptanoglu et al., 2003b), inhibiting lipid peroxidation (Regan et al., 1998), or preventing thrombosis of critical segmental vessels by inhibiting platelet reactivity (Kaptanoglu et al., 2003a). Oxidative stress is suggested to be an essential etiological factor in neuropathy (Yagihashi, 1995), and magnesium may indirectly exert an antioxidant action in reducing oxidative stress (Bonnefont-Rousselot, 2004).
Conclusion
Regardless of the mechanism, our findings indicate that magnesium can attenuate thermal hyperalgesia and cold allodynia following SCI. Further studies are required to explain the mechanisms for the neuroprotective properties of MgSO4 and to elucidate its potential use in human SCI.
Acknowledgements The authors thank Dr Dehpour and Dr Javadi-paydar for their collaborations. This study was financially supported by Tehran University of Medical Sciences (proposal code of 91-03-139-18681), in collaboration with the Brain and Spinal Cord Injury Research Center.
Conflicts of interest
There are no conflicts of interest.
References Arai Y-C P., Hatakeyama N, Nishihara M, Ikeuchi M, Kurisuno M, Ikemoto T (2013). Intravenous lidocaine and magnesium for management of intractable trigeminal neuralgia: a case series of nine patients. J Anesth 27:960–962. Baastrup C, Finnerup NB (2008). Pharmacological management of neuropathic pain following spinal cord injury. CNS Drugs 22:455–475. Bareyre FM, Saatman KE, Raghupathi R, McIntosh TK (2000). Postinjury treatment with magnesium chloride attenuates cortical damage after traumatic brain injury in rats. J Neurotrauma 17:1029–1039. Bonnefont-Rousselot D (2004). The role of antioxidant micronutrients in the prevention of diabetic complications. Treat Endocrinol 3:41–52. Crosby V, Wilcock A, Corcoran R (2000). The safety and efficacy of a single dose (500 mg or 1 g) of intravenous magnesium sulfate in neuropathic pain poorly responsive to strong opioid analgesics in patients with cancer. J Pain Symptom Manage 19:35–39. Crowther CA, Hiller JE, Doyle LW, Haslam RR (2004). Effect of magnesium sulfate given for neuroprotection before preterm birth: a randomized, controlled trial. Obstet Gynecol Surv 59:330–332. Cuzzocrea S, Rossi A, Mazzon E, Di Paola R, Genovese T, Muià C, et al. (2005). 5-Lipoxygenase modulates colitis through the regulation of adhesion molecule expression and neutrophil migration. Lab Invest 85:808–822. Dickens BF, Weglicki WB, Li YS, Mak IT (1992). Magnesium deficiency in vitro enhances free radical-induced intracellular oxidation and cytotoxicity in endothelial cells. FEBS Lett 311:187–191. Ditor DS, John SM, Roy J, Marx JC, Kittmer C, Weaver LC (2007). Effects of polyethylene glycol and magnesium sulfate administration on clinically relevant neurological outcomes after spinal cord injury in the rat. J Neurosci Res 85:1458–1467. Doyle LW, Crowther CA, Middleton P, Marret S, Rouse D (2009). Magnesium sulphate for women at risk of preterm birth for neuroprotection of the fetus. Cochrane Database Syst Rev 21:CD004661. Dubray C, Alloui A, Bardin L, Rock E, Mazur A, Rayssiguier Y, et al. (1997). Magnesium deficiency induces an hyperalgesia reversed by the NMDA receptor antagonist MK801. Neuroreport 8:1383–1386. Ebel H, Günther T (1980). Magnesium metabolism: a review. J Clin Chem Clin Biochem 18:257–270. Finnerup NB, Baastrup C (2012). Spinal cord injury pain: mechanisms and management. Curr Pain Headache Rep 16:207–216. Genovese T, Mazzon E, Rossi A, Di Paola R, Cannavò G, Muià C, et al. (2005). Involvement of 5-lipoxygenase in spinal cord injury. J Neuroimmunol 166:55–64. Gok B, Okutan O, Beskonakli E, Kilinc K (2007). Effects of magnesium sulphate following spinal cord injury in rats. Chin J Physiol 50:93–97. Gowda RM, Khan IA (2004). Magnesium in treatment of acute myocardial infarction. Int J Cardiol 96:467–469. Harding JJ (1992). Pharmacological treatment strategies in age-related cataracts. Drugs Aging 2:287–300. Harlan JM (1987). Consequences of leukocyte-vessel wall interactions in inflammatory and immune reactions. Semin Thromb Hemost 13:434–444. Hasanein P, Parviz M, Keshavarz M, Javanmardi K, Mansoori M, Soltani N (2006). Oral magnesium administration prevents thermal hyperalgesia induced by diabetes in rats. Diabetes Res Clin Pract 73:17–22. Kaptanoglu E, Beskonakli E, Okutan O, Selcuk Surucu H, Taskin Y (2003a). Effect of magnesium sulphate in experimental spinal cord injury: evaluation with ultrastructural findings and early clinical results. J Clin Neurosci 10:329–334. Kaptanoglu E, Beskonakli E, Solaroglu I, Kilinc A, Taskin Y (2003b). Magnesium sulfate treatment in experimental spinal cord injury: emphasis on vascular changes and early clinical results. Neurosurg Rev 26:283–287. Kauppila T (2000). Cold exposure enhances tactile allodynia transiently in mononeuropathic rats. Exp Neurol 161:740–744. Kohno H, Ishida A, Imamaki M, Shimura H, Miyazaki M (2007). Efficacy and vasodilatory benefit of magnesium prophylaxis for protection against spinal cord ischemia. Ann Vasc Surg 21:352–359. Lampl Y, Gilad R, Geva D, Eshel Y, Sadeh M (2001). Intravenous administration of magnesium sulfate in acute stroke: a randomized double-blind study. Clin Neuropharmacol 24:11–15. Lang-Lazdunski L, Heurteaux C, Dupont H, Widmann C, Lazdunski M (2000). Prevention of ischemic spinal cord injury: comparative effects of magnesium sulfate and riluzole. J Vasc Surg 32:179–189. Lemke M, Yum SW, Faden AI (1990). Lipid alterations correlate with tissue magnesium decrease following impact trauma in rabbit spinal cord. Mol Chem Neuropathol 12:147–165.
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
320 Behavioural Pharmacology 2015, Vol 26 No 3
Lindsey AE, Loverso RL, Tovar CA, Hill CE, Beattie MS, Bresnahan JC (2000). An analysis of changes in sensory thresholds to mild tactile and cold stimuli after experimental spinal cord injury in the rat. Neurorehabil Neural Repair 14:287–300. Marret S, Marpeau L, Follet-Bouhamed C, Cambonie G, Astruc D, Delaporte B, et al. (2008). Effect of magnesium sulphate on mortality and neurologic morbidity of the very-preterm newborn (of less than 33 weeks) with two-year neurological outcome: results of the prospective PREMAG trial. Gynecol Obstet Fertil 36:278–288. Mayer ML, Westbrook GL, Guthrie PB (1984). Voltage-dependent block by Mg2 + of NMDA responses in spinal cord neurones. Nature 309:261–263. McIntosh TK (1993). Novel pharmacologic therapies in the treatment of experimental traumatic brain injury: a review. J Neurotrauma 10:215–261. McLean RM (1994). Magnesium and its therapeutic uses: a review. Am J Med 96:63–76. Memon Z, Altura B, Benjamin J, Cracco R, Altura B (1995). Predictive value of serum ionized but not total magnesium levels in head injuries. Scand J Clin Lab Invest 55:671–677. Mert T, Gunes Y, Ozcengiz D, Gunay I (2009). Magnesium modifies fentanylinduced local antinociception and hyperalgesia. Naunyn Schmiedebergs Arch Pharmacol 380:415–420. Miles AN, Majda BT, Meloni BP, Knuckey NW (2001). Postischemic intravenous administration of magnesium sulfate inhibits hippocampal CA1 neuronal death after transient global ischemia in rats. Neurosurgery 49:1443–1450, Discussion 1450-1. Muir KW, Lees KR (1995). A randomized, double-blind, placebo-controlled pilot trial of intravenous magnesium sulfate in acute stroke. Stroke 26:1183–1188. Muir K, Lees K, Ford I, Davis S. Intravenous Magnesium Efficacy in Stroke (IMAGES) Study Investigators (2004). Magnesium for acute stroke (Intravenous Magnesium Efficacy in Stroke trial): randomised controlled trial. Lancet 363:439–445. Peker S, Abacioglu U, Sun I, Konya D, Yuksel M, Pamir NM (2004). Prophylactic effects of magnesium and vitamin E in rat spinal cord radiation damage: evaluation based on lipid peroxidation levels. Life Sci 75:1523–1530. Pickering G, Morel V, Simen E, Cardot JM, Moustafa F, Delage N, et al. (2011). Oral magnesium treatment in patients with neuropathic pain: a randomized clinical trial. Magnes Res 24:28–35. Piotrowski AA, Kalus JS (2004). Magnesium for the treatment and prevention of atrial tachyarrhythmias. Pharmacotherapy 24:879–895. Regan RF, Jasper E, Guo Y, Panter SS (1998). The effect of magnesium on oxidative neuronal injury in vitro. J Neurochem 70:77–85. Rondón LJ, Privat AM, Daulhac L, Davin N, Mazur A, Fialip J, et al. (2010). Magnesium attenuates chronic hypersensitivity and spinal cord NMDA receptor phosphorylation in a rat model of diabetic neuropathic pain. J Physiol 588:4205–4215. Rouse DJ, Hirtz DG, Thom E, Varner MW, Spong CY, Mercer BM, et al. Eunice Kennedy Shriver NICHD Maternal-Fetal Medicine Units Network (2008). A randomized, controlled trial of magnesium sulfate for the prevention of cerebral palsy. N Engl J Med 359:895–905. Saatman KE, Bareyre FM, Grady MS, McIntosh TK (2001). Acute cytoskeletal alterations and cell death induced by experimental brain injury are attenuated by magnesium treatment and exacerbated by magnesium deficiency. J Neuropathol Exp Neurol 60:183–194. Sakurai M, Egashira N, Kawashiri T, Yano T, Ikesue H, Oishi R (2009). Oxaliplatininduced neuropathy in the rat: involvement of oxalate in cold hyperalgesia but not mechanical allodynia. Pain 147:165–174.
Sibai BM (2005). Magnesium sulfate prophylaxis in preeclampsia: evidence from randomized trials. Clin Obstet Gynecol 48:478–488. Simpson JI, Eide TR, Schiff GA, Clagnaz JF, Hossain I, Tverskoy A, Koski G (1994). Intrathecal magnesium sulfate protects the spinal cord from ischemic injury during thoracic aortic cross-clamping. Anesthesiology 81:1493–1499, Discussion 26A-27A. Soltani N, Keshavarz M, Minaii B, Mirershadi F, Asl SZ, Dehpour AR (2005a). Effects of administration of oral magnesium on plasma glucose and pathological changes in the aorta and pancreas of diabetic rats. Clin Exp Pharmacol Physiol 32:604–610. Soltani N, Keshavarz M, Sohanaki H, Dehpour AR, Zahedi Asl SZ (2005b). Oral magnesium administration prevents vascular complications in STZdiabetic rats. Life Sci 76:1455–1464. Soltani N, Keshavarz M, Dehpour AR (2007). Effect of oral magnesium sulfate administration on blood pressure and lipid profile in streptozocin diabetic rat. Eur J Pharmacol 560:201–205. Song JW, Lee YW, Yoon KB, Park SJ, Shim YH (2011). Magnesium sulfate prevents remifentanil-induced postoperative hyperalgesia in patients undergoing thyroidectomy. Anesth Analg 113:390–397. Süzer T, Coskun E, Islekel H, Tahta K (1999). Neuroprotective effect of magnesium on lipid peroxidation and axonal function after experimental spinal cord injury. Spinal Cord 37:480–484. Temkin NR, Anderson GD, Winn HR, Ellenbogen RG, Britz GW, Schuster J, et al. (2007). Magnesium sulfate for neuroprotection after traumatic brain injury: a randomised controlled trial. Lancet Neurol 6:29–38. Ulugol A, Aslantas A, Ipci Y, Tuncer A, Hakan Karadag C, Dokmeci I (2002). Combined systemic administration of morphine and magnesium sulfate attenuates pain-related behavior in mononeuropathic rats. Brain Res 943:101–104. Vink R, Cernak I (2000). Regulation of intracellular free magnesium in central nervous system injury. Front Biosci 5:D656–D665. Weissberg N, Schwartz G, Shemesh O, Brooks B, Algur N, Eylath U, Abraham A (1991). Serum and intracellular electrolytes in patients with and without pain. Magnes Res 4:49. Wiseman DB, Dailey AT, Lundin D, Zhou J, Lipson A, Falicov A, Shaffrey CI (2009). Magnesium efficacy in a rat spinal cord injury model. J Neurosurg Spine 10:308–314. Wolf G, Fischer S, Hass P, Abicht K, Keilhoff G (1991). Magnesium sulphate subcutaneously injected protects against kainate-induced convulsions and neurodegeneration: in vivo study on the rat hippocampus. Neuroscience 43:31–34. Xu XJ, Hao JX, Seiger Å, Hughes J, Hökfelt T, Wiesenfeld-Hallin Z (1994). Chronic pain-related behaviors in spinally injured rats: evidence for functional alterations of the endogenous cholecystokinin and opioid systems. Pain 56:271–277. Xu J, Hsu CY, Liu TH, Hogan EL, Perot PL, Tai HH (1990). Leukotriene B4 release and polymorphonuclear cell infiltration in spinal cord injury. J Neurochem 55:907–912. Yagihashi S (1995). Pathology and pathogenetic mechanisms of diabetic neuropathy. Diabetes Metab Rev 11:193–225. Yezierski RP, Green M, Murphy K, Vierck CJ (2013). Effects of gabapentin on thermal sensitivity following spinal nerve ligation or spinal cord compression. Behav Pharmacol 24:598–609.
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
Postinjury treatment with magnesium sulfate attenuates neuropathic pains following spinal cord injury in male rats Leila Farsia,b, Khashayar Afsharib, Mansoor Keshavarzb,c, Maryam NaghibZadehb, Fereidoon Memarid and Abbas Norouzi-Javidana Spinal cord injury (SCI) has a number of severe and disabling consequences including chronic pain. Approximately 40% of patients experience neuropathic pain, which appears to be persistent. Previous studies have demonstrated the neuroprotective effects of magnesium sulfate (MgSO4). We aimed to investigate the effect of MgSO4 on neuropathic pains following SCI in male rats. Thirty-two adult male rats (weight 300–350 g) were used. After laminectomy, a complete SCI was induced by compression of the spinal cord for 1 min with an aneurysm clip. A single dose of 300 or 600 mg/kg MgSO4 was injected intraperitoneally. Tail-flick latency and acetone drop test scores were evaluated before surgery and once a week for 4 weeks after surgery. Rats in groups SCI + Mg300 and SCI + Mg600 showed significantly higher mean tail-flick latencies and lower mean scores in the acetone test compared with those in the SCI + veh group 4 weeks after
surgery (P < 0.05). These findings revealed that systemic single-dose administration of MgSO4 can attenuate thermal hyperalgesia and cold allodynia induced by SCI in rats. Behavioural Pharmacology 26:315–320 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Introduction
whether administered before, shortly after, or hours after the injury (Bareyre et al., 2000; Saatman et al., 2001; Temkin et al., 2007).
Spinal cord injury (SCI) has severe and disabling consequences including chronic pain and neuropathic pain, which occur in around 40% of patients (Baastrup and Finnerup, 2008) and appear to be persistent despite various treatment methods. In recent years, more information on SCI pain mechanisms has been derived from experimental models and clinical studies, but treatment is still difficult and insufficient (Finnerup and Baastrup, 2012). Many agents have been found to be neuroprotective, one of which is magnesium. Magnesium has long been documented as an essential cation necessary for the proper functioning of more than 300 key enzymes involved in energy renovation, lipid and nucleic acid metabolism, and protein synthesis (Vink and Cernak, 2000). Furthermore, magnesium is a compulsory requirement for any reaction that either produces or consumes ATP (Ebel and Gunther, 1980), including glycolysis and oxidative phosphorylation. Consequently, any decrease in the magnesium concentration following neurotrauma will decrease a cells ability to preserve its membrane potential and to repair itself (Vink and Cernak, 2000). It has been shown that after head injuries in humans, total serum and ionized magnesium concentrations decline (Memon et al., 1995), which is a critical factor leading to irreversible tissue damage following direct or indirect neurotrauma (Vink and Cernak, 2000). Magnesium supplementation improves treatment results 0955-8810 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Behavioural Pharmacology 2015, 26:315–320 Keywords: hyperalgesia, magnesium sulfate, neuropathic pain, rat, spinal cord injury, tail flick a Brain and Spinal Cord Injury Research Center, bElectrophysiology Research Center, Neuroscience Institute, cDepartment of Physiology, School of Medicine and dCancer Institute, Imam Khomeini Hospital, Tehran University of Medical Sciences, Tehran, Iran
Correspondence to Mansoor Keshavarz, MD, PhD, Department of Physiology, School of Medicine, Tehran University of Medical Sciences, Poursina Street, 1456833581 Tehran, Iran E-mail: [email protected] Received 3 September 2013 Accepted as revised 20 September 2014
Moreover, magnesium deficiency is found in association with acute medical/surgical conditions in which pain or stress is present (Dubray et al., 1997), and it causes hyperalgesia that can be ameliorated by NMDA antagonists (Weissberg et al., 1991). Magnesium has been used extensively in clinical approaches for the treatment of pre-eclampsia (Sibai, 2005), stroke (Muir et al., 2004), arrhythmia (Piotrowski and Kalus, 2004), and myocardial infarction (Gowda and Khan, 2004). Magnesium is also cheap and readily accessible, and its clear pharmacological profile confers substantial advantages over other neuroprotective agents (Kohno et al., 2007). Its role in the prevention and treatment of diabetic neuropathy has also been demonstrated (Hasanein et al., 2006; Rondon et al., 2010). In light of the neuroprotective effect of magnesium in SCI (Kohno et al., 2007) and its established role in the prevention and treatment of neuropathic pain (Hasanein et al., 2006; Rondon et al., 2010), we aimed to investigate the effect of magnesium sulfate (MgSO4) on neuropathic pain following SCI in male rats.
Methods Subjects
This study was performed on 32 adult male rats (weight 300–350 g at the start of the experiment) that were DOI: 10.1097/FBP.0000000000000103
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
316 Behavioural Pharmacology 2015, Vol 26 No 3
purchased from Pasteur Institute, Tehran, Iran. The animals were kept at room temperature (23 ± 2°C) and a humidity of 50 ± 5%, in a 12-h light/dark cycle (light: 08.00–20.00 h), with free access to tap water and a standard pellet chow. They were handled in accordance with the criteria of the ‘Guide for the Care and Use of Laboratory Animals’ (NIH US publication 23-86 revised 1985). This study also followed the International Association for the Study of Pain guidelines for animal experiments (Rondon et al., 2010). Body weight, SCI complications such as autophagia, and mortality were measured and recorded. The animals were randomly divided into four groups (n = 6–8 per group): Sham + veh group: only laminectomy, without SCI, was performed. Animals received a 1 ml intraperitoneal injection of normal saline within 30 min after the surgery. SCI + veh group: SCI was induced and animals received a 1 ml intraperitoneal injection of normal saline within 30 min after the surgery. SCI + Mg300 group: animals received a 300 mg/kg intraperitoneal injection of MgSO4 in 1 ml of saline vehicle within 30 min after SCI. SCI + Mg600 group: animals received a 600 mg/kg intraperitoneal injection of MgSO4 in 1 ml of saline vehicle within 30 min after SCI. According to the literature, magnesium has no effect in sham-operated groups (Xiao-Jun et al., 1994); hence, we did not include sham + magnesium groups.Rats from each group were randomly selected and handled by a blind observer (blinded to the doses of magnesium and vehicle in the SCI groups).
Surgery and drug administration
Rats were anesthetized with an intraperitoneal injection of 50 mg/kg ketamine (Trittau, Germany). Before surgery, the operation site was shaved and disinfected with a 10% solution of polyvidon iodine. The surgery was performed using a standard sterile technique. An incision was made over the thoracic spine at the T7–T12 level. After the incision of the dermal and subdermal tissues at the midline, paravertebral muscles were bluntly dissected to expose the lamina bilaterally. Complete laminectomies were performed to expose the spinal cord at T7–T12. SCI was induced by 1-min compression of the spinal cord at the T9 level horizontally and extradurally with an aneurysm clip. The wounds were then closed with a 3/0 silk suture. Within 30 min after the surgery, 1 ml of 300 or 600 mg/kg MgSO4 (suspended in sterile distilled water), provided by Sigma (St Louis, Missouri, USA), was injected intraperitoneally in the magnesiumtreated groups.
In a pilot study, we used four doses of MgSO4 (100, 300, 600, and 900 mg/kg). We observed that 100 mg/kg MgSO4 had no significant effect and 900 mg/kg MgSO4 had a similar effect to 600 mg/kg MgSO4. On the basis of this, we decided to use 300 and 600 mg/kg MgSO4 for this study. Similar doses were used in several previous studies: for example, Xiao-Jun et al. (1994) reported that 150 and 200 mg/kg magnesium had no hyperalgesic effects, but 300 mg/kg was clearly active. Postoperative care included controlling the body temperature and prophylactic antibiotic administration to prevent infection (70 mg/kg cephazolin for 7 days). SCI rats underwent manual bladder expression twice daily for 10–14 days until their bladder functions fully recovered. Following the method used by Wiseman et al. (2009), we killed the rats after 4 weeks for histological studies to confirm that the SCI site was correct. Measurement of thermal hyperalgesia
Tail-flick latency (TFL) was measured using a Tail-Flick Analgesia Meter (IITC life science model 33t; Los Angeles, California, USA) before the surgery and at days 0, 7, 14, 21, and 28. After a 45-min acclimatization period, TFL was measured by exposing the dorsal surface of the animal’s tail to a radiant heat source, and the time taken for the conscious rats to move their tails from the noxious thermal stimulus was recorded. To reach proper baseline intensity, each animal was subjected to five test trials and the strength of the stimulus was adjusted so that TFLs would be between 7 and 8 s. A cutoff time of 8 s was used to prevent tail injury. The mean intensity level was then calculated and used in the following tail-flick tests. Cold allodynia (acetone drop test)
After a 45-min acclimatization period, the response to cold stimulation was tested by spraying acetone onto the plantar surface of the paw (2–3 s) from an estimated distance of 2 cm. The result was 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) along with flinching and/or licking (Kauppila, 2000). A significant increase in the response to acetone application was interpreted as cold allodynia (Ulugol et al., 2002). Statistical analysis
For data analysis, two-way repeated measure analysis of variance was used, followed by Tukey’s post-hoc test. P less than 0.05 was considered significant. All data are expressed as mean ± SEM. SPSS18 was used for analysis (SPSS Inc., Chicago, Illinois, USA).
Results There were no significant differences between groups in any variable on day 0 of the study. SCI caused a significant decrease in the thermal nociceptive threshold
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
Magnesium attenuates neuropathic pain Farsi et al. 317
Tail-flick latency
In general, the SCI groups showed a reduction in the mean TFL after surgery in response to thermal stimuli (tail-flick test). Intraperitoneal administration of MgSO4 inhibited the nociceptive phenomena induced by SCI over time (Fig. 1). Two-way analysis of variance of TFL showed significant main effects of dose [F(3, 20) = 40.38, P < 0.001] and time [F(4,80) = 23.20, P < 0.001], and a significant dose × time interaction [F(12,80) = 8.66, P < 0.001]. Cold allodynia
SCI rats showed significantly increased sensitivity to cold stimuli (acetone drop on the paw) compared with that in presurgical tests (Fig. 2). Statistical analysis showed significant main effect of time [F(4,80) = 37.97, P < 0.001] and dose [F(3,20) = 13.02, P < 0.001], and a significant dose × time interaction [F(12,80) = 5.30, P < 0.001].
Fig. 2
3 Acetone test (score)
(measured by the Tail-Flick Analgesia Meter) and an increase in the cold allodynia score (measured by the acetone drop test), as compared with the baseline values obtained before surgery.
SCI+Mg300
SCI+Mg600
SCI+vehicle
Sham
∗
2.5 2 1.5 1 0.5 0 0
1
2 Weeks after surgery
3
4
Influence of post-SCI administration of MgSO4 on the acetone test scores of the rats over the next 4 weeks. SCI + Mg300 and SCI + Mg600 indicate SCI rats treated with 300 mg/kg magnesium (intraperitoneally) and 600 mg/kg magnesium (intraperitoneally), respectively. The SCI + veh group includes SCI rats that received normal saline. The data are presented as mean ± SEM (six to eight rats per groups). *P < 0.05 indicates a significant difference in the mean acetone score of the SCI + veh group compared with other groups. MgSO4, magnesium sulphate; SCI, spinal cord injury; veh, vehicle.
Fig. 3
Body weight
SCI+vehicle
SCI+Mg300
SCI+Mg600
Sham
420 370 Weight (g)
After SCI surgery, there was weight loss in all SCI groups. This was more severe in the SCI + veh group (Fig. 3). Statistical analysis showed significant main effects of time [F(4,80) = 15.67, P < 0.001] and dose [F(3,20) = 14.63, P < 0.001], and a significant dose × time interaction [F(12,80) = 11.20, P < 0.001].
320 270
*
*
*
220 Fig. 1 SCI+vehicle Sham
9
∗
Tail-flick test (s)
8
170
SCI+Mg300 SCI+Mg600
∗
7 6 5
∗
∗
3
4
0
1
2 Weeks after surgery
3
4
Influence of postinjury administration of MgSO4 on the body weight of the rats over the next 4 weeks. SCI + Mg300 and SCI + Mg600 indicate SCI rats treated with 300 mg/kg magnesium (intraperitoneally) and 600 mg/kg magnesium (intraperitoneally), respectively. The SCI + veh group includes SCI rats that received normal saline. The data are presented as mean ± SEM (six to eight rats per groups). *P < 0.05 indicates a significant difference between the SCI + veh group and other groups: sham, SCI + Mg300. MgSO4, magnesium sulphate; SCI, spinal cord injury; veh, vehicle.
4 3 0
1
2 Weeks after surgery
Influence of postinjury administration of MgSO4 on the mean tail-flick latencies of the rats over the next 4 weeks. SCI + Mg300 and SCI + Mg600 indicate SCI rats treated with 300 mg/kg magnesium (intraperitoneally) and 600 mg/kg magnesium (intraperitoneally), respectively. The SCI + veh group includes SCI rats that received normal saline. The data are presented as mean ± SEM (six to eight rats per groups). *P < 0.05 indicates a significant difference compared with other groups. MgSO4, magnesium sulphate; SCI, spinal cord injury; veh, vehicle.
Discussion In the present study, we report that a single-dose intraperitoneal injection of MgSO4 within 30 min after SCI resulted in an improvement in hyperalgesia and allodynia over a 4-week period after SCI surgery. Several studies have shown that SCI causes chronic neuropathic pain (Baastrup and Finnerup, 2008; Finnerup and Baastrup, 2012; Yezierski et al., 2013). The present study also found symptoms of neuropathic pain after SCI, including lower
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
318 Behavioural Pharmacology 2015, Vol 26 No 3
thermal pain thresholds, indicating thermal hyperalgesia, and a higher score in the acetone drop test, indicating cold allodynia. Magnesium has long been recognized as an essential cation necessary for the appropriate functioning of over 300 key enzymes (Vink and Cernak, 2000). Moreover, any reaction that either produces or consumes ATP demands magnesium (Ebel and Gunther, 1980). Previous studies have reported therapeutic effects of magnesium for diabetes and its complications (Soltani et al., 2005a, 2005b, 2007; Hasanein et al., 2006). MgSO4 is a prominent neuroprotective agent against experimental neurodegeneration and central nervous system damage (Wolf et al., 1991; Simpson et al., 1994; Lang-Lazdunski et al., 2000; Gok et al., 2007; Temkin et al., 2007), and it has been demonstrated to be effective in several types of CNS injuries such as SCI, traumatic brain injury, and ischemia (Temkin et al., 2007; Wiseman et al., 2009). In three carefully randomized placebocontrolled trials, antenatal MgSO4 was found to decrease the risk for cerebral palsy in children who survived from early preterm birth (Crowther et al., 2004; Marret et al., 2008; Rouse et al., 2008). It was concluded that MgSO4 decreased the risk for cerebral palsy by 32% of children, but it had no effect on fetal or infant mortality (Doyle et al., 2009). According to Wiseman et al. (2009), magnesium improved the neurological function of SCI rats compared with controls. Lampl et al. (2001), in a randomized, placebocontrolled, double-blind study, examined the protective effect of MgSO4 administered intravenously to patients within 24 h after a stroke and reported a significant positive effect on the outcome. It has been demonstrated that the decline in the tissue magnesium level is an essential pathophysiological factor in secondary SCI (Lemke et al., 1990), and the use of MgSO4 reduced apoptotic cell death after SCI (Suzer et al., 1999). The present study found moderate weight loss in the SCI groups for 2 weeks, which was significantly lower in the magnesium-treated groups compared with the SCI + veh group. This is assumed to be due to the pain and disability after SCI, and the difficulty in food and water consumption. Two weeks after SCI, the mean weight of the SCI rats remained constant until the third week, and then it started to increase. Better neurological and functional conditions (Ditor et al., 2007; Wiseman et al., 2009) in rats treated with magnesium may lead to higher weight gain in this group. [However, Wiseman et al. (2009) did not report weight loss in SCI rats after surgery in either control or magnesium-treated groups.] Analgesic and antihyperalgesic effects of magnesium have been reported in several studies (Crosby et al., 2000; Hasanein et al., 2006; Song et al., 2011; Arai et al., 2013), but its effect on neuropathic pain after SCI has not previously been reported. However, although magnesium attenuates hyperalgesia, it has been reported to have no
effect on sham-operated rats without hyperalgesia. Xiao-Jun et al. (1994) administered magnesium in chronic constriction injury (CCI) rats and compared withdrawal latencies (heat hyperalgesia) bilaterally (on the CCI side with hyperalgesia and on the contralateral side without CCI and hyperalgesia). Magnesium had no significant effect on withdrawal latencies (heat hyperalgesia) in the sham-operated side. It has been shown that SCI rats have significantly lower TFLs compared with normal rats (Xiao-Jun et al., 1994). In the present study, SCI groups treated with 300 or 600 mg/kg MgSO4 had significantly higher mean TFLs compared with the SCI + veh group after 4 weeks, indicating lower heat hyperalgesia relative to the SCI + veh group. Rondon et al. (2010) showed that magnesium eliminates thermal hyperalgesia in a rat model of diabetic neuropathic pain. Hasanein et al. (2006) also showed that oral magnesium administration at the time of diabetes induction may abolish thermal hyperalgesia in diabetic rats. Mert et al. (2009) reported that intraplantar coadministration of fentanyl and magnesium can prevent delayed thermal hyperalgesia in rats. These observations are consistent with our findings. However, in a clinical trial on patients suffering from neuropathic pain, Pickering et al. (2011) could not demonstrate any significant difference in pain alleviation between patients under 1 month of oral treatment with magnesium and those who were administered placebo. This inconsistency may be due to different forms of drug administration: Pickering et al. (2011) administered oral MgCl2 capsules, whereas in the present study we administered MgSO4 intraperitoneally in rats. Similar to the previous studies showing that SCI can cause cold allodynia (Lindsey et al., 2000), the present results show that after SCI there was cold allodynia in the SCI groups, which was attenuated in the fourth week. In a study on oxaliplatin-induced peripheral neuropathy in rats, Sakurai et al. (2009) showed that administration of calcium or magnesium (0.5 mmol/kg, intravenously) before oxaliplatin or oxalate administration prevented cold hyperalgesia. In another study of spinal nerveligated rats that suffered from neuropathic pain, Ulugol et al. (2002) showed that MgSO4 exerts a significant antiallodynic effect, attenuating cold allodynia at a dose of 250 mg/kg. One hypothesis of the pathophysiology of neuropathic pain following SCI took into consideration the role of spinal cord NMDA receptor channels in central sensitization (Hasanein et al., 2006). As magnesium is an antagonist of these channels (Mayer et al., 1984), this mechanism may also be implicated in the elevation of the thermal pain threshold in magnesium-treated SCI rats (Hasanein et al., 2006). Magnesium has a neuroprotective action through a number of mechanisms, such as dilatation of cerebrovascular arteries, blockage of NMDA
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
Magnesium attenuates neuropathic pain Farsi et al. 319
receptors, and blockage of voltage-gated calcium channels. Furthermore, by directly inhibiting lipid peroxidation and preventing the depletion of glutathione, this element may reduce the severity of endothelial and neuronal reperfusion injury (Dickens et al., 1992; Regan et al., 1998; Peker et al., 2004). In this regard, Gok et al. (2007) showed that treatment with a single 600 mg/kg dose of MgSO4 instantly after SCI prevents neutrophil infiltration into the spinal cord after a contusion injury, by attenuating chemotaxis. Neutrophil adhesion and diapedesis leads to blood–spinal cord barrier breakdown, and the phagocytic property of neutrophils plays a fundamental role in tissue damage. Activated neutrophils are also known to produce a group of inflammatory mediators including proteases, cytokines, chemokines, and free radicals (Harlan, 1987; Xu et al., 1990; Cuzzocrea et al., 2005; Genovese et al., 2005). According to a study by Miles et al. (2001), MgSO4 significantly reduces neuronal death when administered up to 24 h after transient global ischemia, offering a neuroprotective mechanism of action for magnesium that broadens beyond excitotoxicity. After ischemia, there is a spread interruption in many cellular functions, including enzymatic reactions related to ATP, mitochondrial and plasma membrane integrity, protein synthesis, calcium transport, and oxidative phosphorylation (Harding, 1992; McIntosh, 1993; McLean, 1994; Muir and Lees, 1995). Restoration of any of these processes in postischemic neurons may be an extra or alternate mechanism of action of magnesium in preventing neuronal death (Miles et al., 2001). Moreover, magnesium may cause vasodilatation of spinal cord vessels by stimulating endothelial prostacyclin release (Kaptanoglu et al., 2003b), inhibiting lipid peroxidation (Regan et al., 1998), or preventing thrombosis of critical segmental vessels by inhibiting platelet reactivity (Kaptanoglu et al., 2003a). Oxidative stress is suggested to be an essential etiological factor in neuropathy (Yagihashi, 1995), and magnesium may indirectly exert an antioxidant action in reducing oxidative stress (Bonnefont-Rousselot, 2004).
Conclusion
Regardless of the mechanism, our findings indicate that magnesium can attenuate thermal hyperalgesia and cold allodynia following SCI. Further studies are required to explain the mechanisms for the neuroprotective properties of MgSO4 and to elucidate its potential use in human SCI.
Acknowledgements The authors thank Dr Dehpour and Dr Javadi-paydar for their collaborations. This study was financially supported by Tehran University of Medical Sciences (proposal code of 91-03-139-18681), in collaboration with the Brain and Spinal Cord Injury Research Center.
Conflicts of interest
There are no conflicts of interest.
References Arai Y-C P., Hatakeyama N, Nishihara M, Ikeuchi M, Kurisuno M, Ikemoto T (2013). Intravenous lidocaine and magnesium for management of intractable trigeminal neuralgia: a case series of nine patients. J Anesth 27:960–962. Baastrup C, Finnerup NB (2008). Pharmacological management of neuropathic pain following spinal cord injury. CNS Drugs 22:455–475. Bareyre FM, Saatman KE, Raghupathi R, McIntosh TK (2000). Postinjury treatment with magnesium chloride attenuates cortical damage after traumatic brain injury in rats. J Neurotrauma 17:1029–1039. Bonnefont-Rousselot D (2004). The role of antioxidant micronutrients in the prevention of diabetic complications. Treat Endocrinol 3:41–52. Crosby V, Wilcock A, Corcoran R (2000). The safety and efficacy of a single dose (500 mg or 1 g) of intravenous magnesium sulfate in neuropathic pain poorly responsive to strong opioid analgesics in patients with cancer. J Pain Symptom Manage 19:35–39. Crowther CA, Hiller JE, Doyle LW, Haslam RR (2004). Effect of magnesium sulfate given for neuroprotection before preterm birth: a randomized, controlled trial. Obstet Gynecol Surv 59:330–332. Cuzzocrea S, Rossi A, Mazzon E, Di Paola R, Genovese T, Muià C, et al. (2005). 5-Lipoxygenase modulates colitis through the regulation of adhesion molecule expression and neutrophil migration. Lab Invest 85:808–822. Dickens BF, Weglicki WB, Li YS, Mak IT (1992). Magnesium deficiency in vitro enhances free radical-induced intracellular oxidation and cytotoxicity in endothelial cells. FEBS Lett 311:187–191. Ditor DS, John SM, Roy J, Marx JC, Kittmer C, Weaver LC (2007). Effects of polyethylene glycol and magnesium sulfate administration on clinically relevant neurological outcomes after spinal cord injury in the rat. J Neurosci Res 85:1458–1467. Doyle LW, Crowther CA, Middleton P, Marret S, Rouse D (2009). Magnesium sulphate for women at risk of preterm birth for neuroprotection of the fetus. Cochrane Database Syst Rev 21:CD004661. Dubray C, Alloui A, Bardin L, Rock E, Mazur A, Rayssiguier Y, et al. (1997). Magnesium deficiency induces an hyperalgesia reversed by the NMDA receptor antagonist MK801. Neuroreport 8:1383–1386. Ebel H, Günther T (1980). Magnesium metabolism: a review. J Clin Chem Clin Biochem 18:257–270. Finnerup NB, Baastrup C (2012). Spinal cord injury pain: mechanisms and management. Curr Pain Headache Rep 16:207–216. Genovese T, Mazzon E, Rossi A, Di Paola R, Cannavò G, Muià C, et al. (2005). Involvement of 5-lipoxygenase in spinal cord injury. J Neuroimmunol 166:55–64. Gok B, Okutan O, Beskonakli E, Kilinc K (2007). Effects of magnesium sulphate following spinal cord injury in rats. Chin J Physiol 50:93–97. Gowda RM, Khan IA (2004). Magnesium in treatment of acute myocardial infarction. Int J Cardiol 96:467–469. Harding JJ (1992). Pharmacological treatment strategies in age-related cataracts. Drugs Aging 2:287–300. Harlan JM (1987). Consequences of leukocyte-vessel wall interactions in inflammatory and immune reactions. Semin Thromb Hemost 13:434–444. Hasanein P, Parviz M, Keshavarz M, Javanmardi K, Mansoori M, Soltani N (2006). Oral magnesium administration prevents thermal hyperalgesia induced by diabetes in rats. Diabetes Res Clin Pract 73:17–22. Kaptanoglu E, Beskonakli E, Okutan O, Selcuk Surucu H, Taskin Y (2003a). Effect of magnesium sulphate in experimental spinal cord injury: evaluation with ultrastructural findings and early clinical results. J Clin Neurosci 10:329–334. Kaptanoglu E, Beskonakli E, Solaroglu I, Kilinc A, Taskin Y (2003b). Magnesium sulfate treatment in experimental spinal cord injury: emphasis on vascular changes and early clinical results. Neurosurg Rev 26:283–287. Kauppila T (2000). Cold exposure enhances tactile allodynia transiently in mononeuropathic rats. Exp Neurol 161:740–744. Kohno H, Ishida A, Imamaki M, Shimura H, Miyazaki M (2007). Efficacy and vasodilatory benefit of magnesium prophylaxis for protection against spinal cord ischemia. Ann Vasc Surg 21:352–359. Lampl Y, Gilad R, Geva D, Eshel Y, Sadeh M (2001). Intravenous administration of magnesium sulfate in acute stroke: a randomized double-blind study. Clin Neuropharmacol 24:11–15. Lang-Lazdunski L, Heurteaux C, Dupont H, Widmann C, Lazdunski M (2000). Prevention of ischemic spinal cord injury: comparative effects of magnesium sulfate and riluzole. J Vasc Surg 32:179–189. Lemke M, Yum SW, Faden AI (1990). Lipid alterations correlate with tissue magnesium decrease following impact trauma in rabbit spinal cord. Mol Chem Neuropathol 12:147–165.
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
320 Behavioural Pharmacology 2015, Vol 26 No 3
Lindsey AE, Loverso RL, Tovar CA, Hill CE, Beattie MS, Bresnahan JC (2000). An analysis of changes in sensory thresholds to mild tactile and cold stimuli after experimental spinal cord injury in the rat. Neurorehabil Neural Repair 14:287–300. Marret S, Marpeau L, Follet-Bouhamed C, Cambonie G, Astruc D, Delaporte B, et al. (2008). Effect of magnesium sulphate on mortality and neurologic morbidity of the very-preterm newborn (of less than 33 weeks) with two-year neurological outcome: results of the prospective PREMAG trial. Gynecol Obstet Fertil 36:278–288. Mayer ML, Westbrook GL, Guthrie PB (1984). Voltage-dependent block by Mg2 + of NMDA responses in spinal cord neurones. Nature 309:261–263. McIntosh TK (1993). Novel pharmacologic therapies in the treatment of experimental traumatic brain injury: a review. J Neurotrauma 10:215–261. McLean RM (1994). Magnesium and its therapeutic uses: a review. Am J Med 96:63–76. Memon Z, Altura B, Benjamin J, Cracco R, Altura B (1995). Predictive value of serum ionized but not total magnesium levels in head injuries. Scand J Clin Lab Invest 55:671–677. Mert T, Gunes Y, Ozcengiz D, Gunay I (2009). Magnesium modifies fentanylinduced local antinociception and hyperalgesia. Naunyn Schmiedebergs Arch Pharmacol 380:415–420. Miles AN, Majda BT, Meloni BP, Knuckey NW (2001). Postischemic intravenous administration of magnesium sulfate inhibits hippocampal CA1 neuronal death after transient global ischemia in rats. Neurosurgery 49:1443–1450, Discussion 1450-1. Muir KW, Lees KR (1995). A randomized, double-blind, placebo-controlled pilot trial of intravenous magnesium sulfate in acute stroke. Stroke 26:1183–1188. Muir K, Lees K, Ford I, Davis S. Intravenous Magnesium Efficacy in Stroke (IMAGES) Study Investigators (2004). Magnesium for acute stroke (Intravenous Magnesium Efficacy in Stroke trial): randomised controlled trial. Lancet 363:439–445. Peker S, Abacioglu U, Sun I, Konya D, Yuksel M, Pamir NM (2004). Prophylactic effects of magnesium and vitamin E in rat spinal cord radiation damage: evaluation based on lipid peroxidation levels. Life Sci 75:1523–1530. Pickering G, Morel V, Simen E, Cardot JM, Moustafa F, Delage N, et al. (2011). Oral magnesium treatment in patients with neuropathic pain: a randomized clinical trial. Magnes Res 24:28–35. Piotrowski AA, Kalus JS (2004). Magnesium for the treatment and prevention of atrial tachyarrhythmias. Pharmacotherapy 24:879–895. Regan RF, Jasper E, Guo Y, Panter SS (1998). The effect of magnesium on oxidative neuronal injury in vitro. J Neurochem 70:77–85. Rondón LJ, Privat AM, Daulhac L, Davin N, Mazur A, Fialip J, et al. (2010). Magnesium attenuates chronic hypersensitivity and spinal cord NMDA receptor phosphorylation in a rat model of diabetic neuropathic pain. J Physiol 588:4205–4215. Rouse DJ, Hirtz DG, Thom E, Varner MW, Spong CY, Mercer BM, et al. Eunice Kennedy Shriver NICHD Maternal-Fetal Medicine Units Network (2008). A randomized, controlled trial of magnesium sulfate for the prevention of cerebral palsy. N Engl J Med 359:895–905. Saatman KE, Bareyre FM, Grady MS, McIntosh TK (2001). Acute cytoskeletal alterations and cell death induced by experimental brain injury are attenuated by magnesium treatment and exacerbated by magnesium deficiency. J Neuropathol Exp Neurol 60:183–194. Sakurai M, Egashira N, Kawashiri T, Yano T, Ikesue H, Oishi R (2009). Oxaliplatininduced neuropathy in the rat: involvement of oxalate in cold hyperalgesia but not mechanical allodynia. Pain 147:165–174.
Sibai BM (2005). Magnesium sulfate prophylaxis in preeclampsia: evidence from randomized trials. Clin Obstet Gynecol 48:478–488. Simpson JI, Eide TR, Schiff GA, Clagnaz JF, Hossain I, Tverskoy A, Koski G (1994). Intrathecal magnesium sulfate protects the spinal cord from ischemic injury during thoracic aortic cross-clamping. Anesthesiology 81:1493–1499, Discussion 26A-27A. Soltani N, Keshavarz M, Minaii B, Mirershadi F, Asl SZ, Dehpour AR (2005a). Effects of administration of oral magnesium on plasma glucose and pathological changes in the aorta and pancreas of diabetic rats. Clin Exp Pharmacol Physiol 32:604–610. Soltani N, Keshavarz M, Sohanaki H, Dehpour AR, Zahedi Asl SZ (2005b). Oral magnesium administration prevents vascular complications in STZdiabetic rats. Life Sci 76:1455–1464. Soltani N, Keshavarz M, Dehpour AR (2007). Effect of oral magnesium sulfate administration on blood pressure and lipid profile in streptozocin diabetic rat. Eur J Pharmacol 560:201–205. Song JW, Lee YW, Yoon KB, Park SJ, Shim YH (2011). Magnesium sulfate prevents remifentanil-induced postoperative hyperalgesia in patients undergoing thyroidectomy. Anesth Analg 113:390–397. Süzer T, Coskun E, Islekel H, Tahta K (1999). Neuroprotective effect of magnesium on lipid peroxidation and axonal function after experimental spinal cord injury. Spinal Cord 37:480–484. Temkin NR, Anderson GD, Winn HR, Ellenbogen RG, Britz GW, Schuster J, et al. (2007). Magnesium sulfate for neuroprotection after traumatic brain injury: a randomised controlled trial. Lancet Neurol 6:29–38. Ulugol A, Aslantas A, Ipci Y, Tuncer A, Hakan Karadag C, Dokmeci I (2002). Combined systemic administration of morphine and magnesium sulfate attenuates pain-related behavior in mononeuropathic rats. Brain Res 943:101–104. Vink R, Cernak I (2000). Regulation of intracellular free magnesium in central nervous system injury. Front Biosci 5:D656–D665. Weissberg N, Schwartz G, Shemesh O, Brooks B, Algur N, Eylath U, Abraham A (1991). Serum and intracellular electrolytes in patients with and without pain. Magnes Res 4:49. Wiseman DB, Dailey AT, Lundin D, Zhou J, Lipson A, Falicov A, Shaffrey CI (2009). Magnesium efficacy in a rat spinal cord injury model. J Neurosurg Spine 10:308–314. Wolf G, Fischer S, Hass P, Abicht K, Keilhoff G (1991). Magnesium sulphate subcutaneously injected protects against kainate-induced convulsions and neurodegeneration: in vivo study on the rat hippocampus. Neuroscience 43:31–34. Xu XJ, Hao JX, Seiger Å, Hughes J, Hökfelt T, Wiesenfeld-Hallin Z (1994). Chronic pain-related behaviors in spinally injured rats: evidence for functional alterations of the endogenous cholecystokinin and opioid systems. Pain 56:271–277. Xu J, Hsu CY, Liu TH, Hogan EL, Perot PL, Tai HH (1990). Leukotriene B4 release and polymorphonuclear cell infiltration in spinal cord injury. J Neurochem 55:907–912. Yagihashi S (1995). Pathology and pathogenetic mechanisms of diabetic neuropathy. Diabetes Metab Rev 11:193–225. Yezierski RP, Green M, Murphy K, Vierck CJ (2013). Effects of gabapentin on thermal sensitivity following spinal nerve ligation or spinal cord compression. Behav Pharmacol 24:598–609.
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited.
