opper is one of the essential micronutrients of the body which plays a key role as a cofactor in the function of metabolic enzymes such as cytochrome oxidase C, superoxide dismutase, metallothionein, dopamine-beta-hydroxylase, lysyl oxidase as well as coagulation factor V and VIII. It is also involved in cellular processes like energy production in mitochondria, detoxification of free radicals, melanin structure, synthesis of neurotransmitters, and stability of connective tissue (1). Previous studies have revealed that disruption in copper homeostasis will cause diseases such as Wilson disease and Menkes syndrome (2).
Seizure is one of the neurological complications in Menkes and Wilson's diseases. Previous studies have shown that oral administration of copper inhibits long-term potentiation in the CA1 region of rat hippocampal slices (3,4). Since long-term potentiation is believed to involve in learning and also seizures, it seems that the effect
Mohammad Reza Palizvan
Department of Physiology, Faculty of Medicine, Arak University of Medical Sciences.
of copper on long-term potentiation could be associated with effects of this element on epilepsy and seizures (5-7). The aim of this study was to assess the influence of intraperitoneal injection of on seizures induced by pentylenetetrazole in mice, and comparing it with sodium valproate.
2. Materials and Methods
In this experimental study, male NMRI mice, weighing 25 to 30 g, were housed five per cage with free access to food and water. Mice were kept in a vivarium under controlled laboratory conditions (temperature, 22-26°C) with an artificial 12-h light/dark cycle. All animals were allowed to acclimate for ≥5 days before testing with food and water "ad libitum". All experimental procedures were approved by the Ethics Committee of the Arak University of Medical Sciences. Animals were randomly divided into seven groups with eight mice per group. Animals received saline, copper sulfate (10, 50 and 100 mg/kg, i.p.) and sodium valproate (50, 150 and 300 mg/kg, i.p.). Twenty minutes after injection, all mice received 100 mg/kg of pentylenetetrazole (PTZ) intraperitoneally (8). Mice behaviors were monitored for seizures immediately after PTZ injection for a period of 30 min and seizure responses were assessed. Seizure stages were classified into four phases; stage 1: hypoactivity, stage 2: partial clonus (clonic seizure activity affecting face, head, and/or forelimb or forelimbs), stage 3: generalized clonus (sudden loss of upright posture, whole-body clonus involving all four limbs and tail, rearing, and autonomic signs), and stage 4: tonic-clonic (maximal) seizure (generalized seizure characterized by tonic hindlimb extension) (9). Tonic-clonic maximal seizures were associated with death. Some mice recovered spontaneously. It was not unusual for mice to exhibit multiple episodes of tonic hind limb extension within the 30 min observation period. In this research, latency to stage 2 seizure, stage 3 seizure and from the onset of stage 3 to death were recorded.
2.1. Data analysis
Data of seizure stages are expressed as means± standard error of the mean (S.E.M.). Data were analyzed by one-way analysis of variance (ANOVA) and followed by Tukey's post hoc test. In all experiments, a P-value
Figure 1 shows the effect of intraperitoneal administration of different doses of CuSO4 (10, 50, 100 mg/kg) on PTZ-induced seizure. One-way ANOVA (F3, 31=37.184, p
Figure 1. Comparison of the effect of copper sulfate and sodium valproate on stage 2 latency. Statistical analysis (analysis of variance) revealed that both copper sulfate and sodium valprote dose-dependently increase stage 2 latency.
in latency to stage two seizures (S2L) for CuSO4 at doses of 50 and 100 mg compared with saline-treated control animals. Intraperitoneal injections of sodium valproate (50, 150 and 300 mg/kg) also increased S2L in valproate-treated animals as compared to controls (Figure 1). Statistical analysis with ANOVA revealed no significant difference between two groups (F6, 58=41.667, p
All doses of sodium valproate and copper sulfate significantly decrease the reverse of time that need to reach to stage 3 seizure multiplied to 100 as compared with the control group (F6,58 = 153.736, p
Figure 2. Comparison of effect of copper sulfate and sodium valproate on stage 3 latency. Statistical analysis (analysis of variance) revealed that both copper sulfate and sodium valprote dose-dependently increase stage 3 latency.
Figure 3. Comparison of the effect of copper sulfate and sodium valproate on revers of time from stage 3 to death. Statistical analysis (analysis of variance) revealed that both copper sulfate and sodium valprote dose-dependently decrease this parameter.
These results showed that intraperitoneal injection of copper sulfate has an inhibitory effect on seizure parameters in mice. Many studies have been conducted on the relationship between copper and epilepsy, and the identification of toxic effects of copper on the body systems (10). In line with results obtained in this study, it has been reported that seizures and epilepsy are neurological symptoms in patients with Menkes disease. In this x-linked disease, copper transport is impaired and serum levels of copper and ceruloplasmin protein decrease (11). Also, in animal models of Menkes disease, there is increased risk of seizures and neuronal damage that can be removed with copper-containing supplements. Many articles attribute the seizures in this disease to a reduction of serum copper concentration (12-14). Routine injection of copper to mice also eliminates the seizure potentials created by the combination of microwaves and chlorpromazine and convulsive seizures by adding penicillin to sensorimotor cortex of the brain (15). Brunia et al in 1972 reported that plasma copper concentrations increase in patients with different types of epilepsy (16).
On the other hand, Kuzuya and colleagues in 1993 acclaimed that although serum copper increases in epileptic patients who use anticonvulsant drugs, this increase is not significantly different from control group (17). Verroti and his colleagues in 2002 also showed that before and after treatment with anticonvulsant drugs, there is no relationship between plasma copper and epilepsy (18). However, Doretto et al in 2002 reported that serum copper concentrations in mice with audiogenic seizures is more than normal mice (19). The results of our study showed inhibitory effects of copper sulfate injection on seizures induced by PTZ.
The results of this study showed that intraperitoneal injection of copper sulfate has inhibitory effect on seizures induced by intraperitoneal injection of PTZ in NMRI strain mice. These results can help to investigate the chronic effects of copper sulfate on the seizure and anticonvulsant effects of copper and other metals used in enzymes building.
1. Makinoda S, Mikuni M, Sogame M, Kobamatsu Y, Furuta I, Yamada H, et al. Erythropoietin, granulocyte-colony stimulating factor, interleukin-1[beta] and interleukin-6 during the normal menstrual cycle. International Journal of Gynecology and Obstetrics 1996; 55: 265-71.
2. Fujiwara N, Iso H, Kitanaka N, Kitanaka J, Eguchi H, Ookawara T, et al. Effects of copper metabolism on neurological functions in Wistar and Wilson's disease model rats. Biochemical and Biophysical Research Communications 2006; 349:1079-86.
3. Goldschmith A, Infante C, Leiva J, Motles E, Palestini M. Interference of chronically ingested copper in long-term potentiation (LTP) of rat hippocampus. Brain Research 2005; 1056:176-82.
4. Leiva J, Gaete P, Palestini M. Copper interaction on the long-term potentiation. Archives Italiennes de Biologie 2003; 141:149-55.
5. Ebert U, Rundfeldt C, Löscher W. Development and pharmacological suppression of secondary afterdischarges in the hippocampus of amygdala-kindled rats.The European Journal of Neuroscience 1995; 7:732-41.
6. Kamphuis W, Gorter JA, da Silva FL. A long-lasting decrease in the inhibitory effect of GABA on glutamateresponses of hippocampal pyramidal neurons induced by kindling epileptogenesis. Neuroscience 1991;41:425-31.
7. Mattson RH, Cramer JA, Collins JF. A Comparison of Valproate with Carbamazepine for the Treatment of Complex Partial Seizures and Secondarily Generalized Tonic–Clonic Seizures in Adults. New England Journal of Medicine 1992; 327:765-71.
8. Luszczki JJ, Czernecki R, Dudra-Jastrzebska M, Borowicz KK, Czuczwar SJ. Influence of agmatine on the protective action of numerous antiepileptic drugs against pentetrazole-induced seizures in mice. Pharmacological Reports 2009; 61:252-60.
9. Ferraro TN, Golden GT, Smith GG, St Jean P, Schork NJ, Mulholland N, et al. Mapping loci for pentylenetetrazol-induced seizure susceptibility in mice. The Journal of Neuroscience 1999; 19:6733-9.
10. Armstrong C, Leong W, Lees GJ. Comparative effects of metal chelating agents on the neuronal cytotoxicity induced by copper (Cu+2), iron (Fe+3) and zinc in the hippocampus. Brain Research 2001; 892:51-62.
11. Tümer Z, Møller LB. Menkes disease. European Journal of Human Genetics 2010; 18:511-8.
12. Luszczki JJ, Czernecki R, Dudra-Jastrzebska M, Borowicz KK, Czuczwar SJ. Influence of agmatine on the protective action of numerous antiepileptic drugs against pentetrazole-induced seizures in mice. Pharmacological Reports 2009; 61:252-60.
13. Kaler SG, Liew CJ, Donsante A, Hicks JD, Sato S, Greenfield JC. Molecular correlates of epilepsy in early diagnosed and treated Menkes disease. Journal of Inherited Metabolic Disease 2010; 33:583-9.
14. Kreuder J, Otten A, Fuder H, Tümer Z, Tønnesen T, Horn N, et al. Clinical and biochemical consequences of copper-histidine therapy in Menkes disease. European Journal of Pediatrics 1993; 152:828-32.
15. Tsaryuk VV, Potapovich AI, Kostyuk VA. Protective effect of copper-rutin complex in animals with experimental epilepsy. Bulletin of Experimental Biology and Medicine 2002; 133:334-5.
16. Brunia C, Buyze G. Serum copper levels and epilepsy. Epilepsia 1972; 13:621-5.
17. Kuzuya T, Hasegawa T, Shimizu K, Nabeshima T. Effect of anti-epileptic drugs on serum zinc and copper concentrations in epileptic patients. International Journal of Clinical Pharmacology, Therapy and Toxicology 1993; 31: 61-5.
18. Verrotti A, Basciani F, Trotta D, Pomilio MP, Morgese G, Chiarelli F. Serum copper, zinc, selenium, glutathione peroxidase and superoxide dismutase levels in epileptic children before and after 1 year of sodium valproate and carbamazepine therapy. Epilepsy Resarch 2002; 48:71-5.
19. Doretto MC, Simões S, Paiva AM, Osório-Neto E. Zinc, magnesium and copper profiles in three experimental models of epilepsy. Brain Research 2002; 956:166-72.