Yue W et al / Acta Pharmacol Sin 2002 Sep; 23 (9): 847-850

Inhibitory effects of succinic acid on chemical kindling and amygdala electrical kindling in rats¹

YUE Wang², LIU Yan-Xia, ZANG Dong-Lian, ZHOU Ming, ZHANG Fang, WANG Lei

Department of Pharmacology, Medical College, Qingdao University, Qingdao 266021, China

¹ Project supported by the Educational Committee of China, ¡í [1995]806.

² Correspondence to Prof YUE Wang. Phn 86-532-383-8480, ext 3756. Fax 86-532-380-1449. E-mail faoqmc@public.qd.sd.cn

Received 2001-06-26 Accepted 2002-05-30

KEY WORDS kindling; epilepsy; succinic acid; picrotoxin; GABA-A receptors

ABSTRACT

AIM: To investigate the effects and mechanism of succinic acid on pentylenetetrazol (PTZ) chemical kindling and amygdala electrical kindling in rats. METHODS: PTZ chemical kindling and amygdala electrical kindling models were established in rats. The effects of succinic acid on the behavior and afterdischarge of kindled rats were observed. The mice were pretreated with succinic acid, 30 min later, picrotoxin, a GABA_A receptor antagonist was given by ip, then the effects of succinic acid on mice were observed. RESULTS: Succinic acid (100-400 mg/kg, ip) dose-dependently inhibited PTZ chemical and amygdala kindled seizure (P<0.05, P<0.01), elevated the afterdischarge threshold, and reduced the Racine's stage of amygdala kindling rats (P<0.05, P<0.01); succinic acid (200-400 mg/kg, ip) inhibited picrotoxin-convulsion in mice (P<0.05, P<0.01). CONCLUSION: Succinc acid inhibits PTZ chemical and amygdala electrical kindling in rats, and the inhibition mechanism may be related to the enhancement of GABAergic system action in the brain, especially through GABA_A receptors.

INTRODUCTION

Succinic acid was so named because it is found mainly in amber, a famous mineral pharmacy^[1], which is also found in organisms such as aquatic alga. It has been demonstrated that there is a specific glucose metabolic by-pass in the brain, glutamic acid-GABA-succinic acid by-pass. Its inhibitory effects on central nervous system and anti-convulsion property have been reported^[2]. However, antiepileptic effect of succinic acid has not been reported. The present study was designed to investigate the antiepileptic effects of succinic acid and mechanism on PTZ chemical and amygdala electrical kindling in rats.

MATERIALS AND METHODS

Drugs Succinic acid was produced by Beijing Chemical Plant(China); pentylenetetrazol (PTZ), picrotoxin, and pentobarbital were purchased from Sigma (St Louis, USA).

Animals Wistar rats (¡â,weighing 180 g±10 g) were provided by the Animal Center, Qingdao Institute of Drug Control (certificate ¡í 000697, grade II). Four rats per cage were under controlled temperature (23 ¡æ±2 ¡æ) and on a 12 h-light/12 h-dark lighting cycle with free access to food and water. Kunming mice (¡â,weighing 20 g±2 g) were from Animal Center, Qingdao Institute of Drug Control (certificate ¡í 000209, grade II).

Chemical kindling PTZ (1.75 % solution) were administered to rats, 35 mg/kg^[3] (subconvulsive dosage) ip, and the behavioral seizure severity was recorded within 1 h after administration. Seizures induced were classified into 7 behavioral categories (grade 0-6) according to Ono^[4]: grade 0, no behavioral seizures; grade 1, head nodding or twitching; grade 2, myoclonic jerks; grade 3, head twisting, forelimb clonic convulsions; grade 4, kangaroo position; grade 5, falling down; grade 6, tonic convulsions. Rats which displayed 5 consecutive grade 2 or higher seizures were defined as kindled rats.

The kindled rats (n=9) were given succinic acid of various doses (50, 100, 200, and 400 mg/kg, ip) at a volume of 2 mL/kg. Succinic acid was diluted in normal saline (NS). The day before that, the rats were given equal volume of NS as self-control. Behavioral seizure severity was recorded within 1 h and seizure percentage on grade 6 was calculated.

Amygdala kindling The rats were anesthetized with sodium pentobarbital (40 mg/kg, ip) and given stereotaxic implantation of a bipolar electrode into each basolateral nucleus of the amygdala. The electrode consisted of two twisted stainless-steel wires (0.25 mm in diameter) which were separated at the tip by 0.25 mm. according to the brain atlas of König et al^[5], the following stereotaxic coordinates were used: AP 3.0 mm, L 4.8 mm, DV 8.8 mm. All coordinates were measured from bregma. The electrode pair was anchored to the skull with miniature screws and dental cement. Following the electrode implantation, the animals were treated with sodium benzylpenicillin for 3 d to prevent infection.

Two weeks after the operation, constant current stimulations (400 mA, 60 Hz, 1 ms, monophasic square wave for 1 s) were delivered to the right amygdala once a day. EEG and seizure stage were recorded and analyzed. Behavioral seizure severity was classified according to the classification described by Racine^[6]: stage 1, facial clonus; stage 2, head nodding; stage 3, bilateral forelimb clonus; stage 4, rearing; stage 5, rearing and falling. Animals which displayed 5 consecutive seizures on stage 5 were defined as kindled.

The kindled rats (n=9) were given succinic acid of various doses (50, 100, 200, and 400 mg/kg, ip)at a volume of 2 mL/kg. After 30 min of injection, the afterdischarge threshold (ADT) was determined with the ramp method. A constant-voltage stimulus (1 V) was delivered to determine ADT. And then the voltage was increased by 0.2 V every 2 min till an afterdischarge of at least 3 s duration was evoked. Afterdischarge, seizure percentage, and seizure severity were monitered at the ADT. The rats were given the same volume of NS as self-control before they were given succinic acid. The interval of experiments was at least 4 d.

Effect of succinic acid on mouse picrotoxin-convulsion^[7] Kunming mice (n=30) were randomly divided into three groups (n=10). Succinic acid-treated groups were treated with succinic acid (200 and 400 mg/kg), while the control group were given the same volume of NS. Then 30 min later, the mice were administered picrotoxin^[8], a GABA_A receptor antagonist, 7.5 mg/kg ip^[9] . Picrotoxin was diluted in Me₂SO.

Statistical analysis Data were expressed as mean±SD. Intergroup differences were analyzed using unpaired and paired t-test.

RESULTS

Inhibition of succinic acid on PTZ chemical kindling Succinic acid (100-400 mg/kg, ip) decreased Ono grade 6 percentage (P<0.05, P<0.01) and dose-dependently inhibited PTZ chemical kindling seizure (P<0.05, P<0.01). The dose of 50 mg/kg ip, had no effect on kindling seizure compared with the controls (Tab 1).

Tab 1. Effect of succinic acid on behavioral seizure in PTZ kindled rats. n=9 rats. Mean±SD. ^aP>0.05, ^bP<0.05, ^cP<0.01 vs individual vehicle control.

	Ono grade		Ono grade 6 seizure
Dose/			percentage/%
mg¡¤kg-1	Vehicle	Succinic	Vehicle	Succinic
		acid-treated		acid- treated
50	6.0	5.3¡À1.0a	100.0	66.7a
100	6.0	4.6¡À1.1b	100.0	25.0b
200	6.0	3.3¡À1.4c	100.0	16.7c
400	6.0	3.0¡À1.3c	100.0	12.5c

Effect of succinic acid on behavioral seizures in amygdala kindled rats Succinic acid (50 mg/kg, ip) had no effect on kindled rats. The dose of 100-400 mg/kg ip increased ADT (P<0.05, P<0.01), reduced Racine stage (P<0.05, P<0.01), and dose-dependently decreased seizure severity (P<0.05, P<0.01) (Tab 2).

Tab 2. Effects of succinic acid on ADT and behavior seizure in amygdala kindled rats. n=9 rats. Mean±SD. ^aP>0.05, ^bP<0.05, ^cP<0.01 vs individual vehicle control.

Effect of succinic acid on picrotoxin induced convulsion Succinic acid (200 and 400 mg/kg, ip) prolonged the latency of picrotoxin-induced convulsion in mice (P<0.05, P<0.01, Tab 3) .

Tab 3. Effect of succinic acid on mice picrotoxin-convulsion. n=10 mice. Mean±SD. ^bP<0.05, ^cP<0.01vs individual vehicle control.

DISCUSSION

PTZ chemical kindling and amygdala electrical kindling models are two commonly used kindling models. The former is an ideal model in the study of the grand mal of human primary epilepsy^[10], while the latter is similar to human epilepsy in seizure activity , EEG, and the epileptiform discharge^[11]. The kindling model in rats is considered to be a good model of human chronic epilepsy.

The present study showed that succinic acid (100-400 mg/kg, ip) dose-dependently inhibited PTZ chemical kindling and amygdala electrical kindling , decreasing Ono seizure severity and the percentage of Ono grade 6 in PTZ chemical kindling; elevating the ADT and reducing seizure severity and Racine stage in amygdala electrical kindling. Succinic acid (200-400 mg/kg, ip) prolonged the latency of convulsant mice caused by picrotoxin, which suggests succinic acid has inhibitory effect on GABA_A receptor, according with the enhancement effect on GABAergic system of semicarbazide convulsant model reported in the preamble^[1].

Succinic acid is similar in molecular structure to inhibitory amino acids neurotransmitter GABA and excitatory amino acid neurotransmitter glutamic acid. It is interesting that in structure it is also similar to sodium oxybate, a clinical local anesthetic^[12]. Excitatory and inhibitory neurotransmitters, such as glutamic acid and GABA, take part in maintaining the balance of excitation and inhibition in the brain, where there is a specific by-pass of succinic acid. This suggests that succinic acid is an endogenous neuro-active substance. Succinic acid may act through GABA receptor or through glutamic acid receptor. To investigate the inhibitory effect of succinic acid on kindling, we studied its effect on picrotoxin-induced convulsion according to Zhang et al^[7] . The results indicated that the inhibitory effect of succinic acid on kindling probably involved the enhancement of GABA_A subtype receptor, while the inhibitory effect on glutamic acid receptor could not be excluded.

In conclusion, succinic acid inhibits the PTZ chemical kindling and amygdala electrical kindling , and the mechanism is related to the enhancement of the GABAergic system action in the brain . Since succinic acid has low toxicity^[13] , it is of pharmacological value to further study the anti-epileptic effect of succinic acid.

ACKNOWLEDGMENT The authors wish to thank Prof ZHANG Shi-Shan for his kind assistance.

REFERENCES

• 1 Jin Y, Zhang SS. Inhibitory effect of succinic acid on central nervous system. Acta Pharm Sin 1980; 15: 761-63.
• 2 Jin Y, Yue W. Pharmacological advancement of succinic acid. Chin Pharm Bull 1983; 18: 36-8.
• 3 Yue W, Wang L, Zhang F, Zhong WZ, Liu ZT. Inhibition of nicardipine on amygdala kindling in rats. Acta Pharmacol Sin 2001; 22: 365-8.
• 4 Ono J, Vieth RF, Walson PD. Electrocorticographical observation of seizures induced by pentylenetetrazol (PTZ) injection in rats. Functional Neurol 1990; 5: 345-52.
• 5 König JFR, Klippel RA, editors. The rat brain. Hamburg: Krieger RE Publishing Co; 1967. p 71-2.
• 6 Racine RJ. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroenceph Clin Neurophysiol 1972; 32: 281-94.
• 7 Zhang JT, Le F, Liu Y. GABA receptor-ligand binding experiment. In: Zhang JT, editor. Modern experimental methods in pharmacology. 1st ed. Beijing: Union Medical College Press; 1998. p 561-2.
• 8 Tatsuo MA, Yokoro CML, Salgado JV, Pesquero SM, Santana MA,Francischi JN. Hyperalgetic effect induced by barbiturates, midazolam and ethanol: pharmacological evidence for GABA_A receptor involvement. Braz J Med Biol Res 1997; 30: 251-6.
• 9 Zhang SS. Anticonvulsion experimental methods in medicine.In: Xu SY, Bian RL, Chen X, editors. Experimental methods pharmacology. 2nd ed. Beijing: People's Medical Publishing House ; 1994. p 675-9.
• 10 Löscher W, Schimidt D. Which animal models should be used in the search for new antiepileptic drug? A proposal based on experimental and clinical considerations. Epilepsy Res 1988; 2: 145-56.
• 11 Albertson TE, Joy RM, Stark LG. Cargamazepine: a pharmacological study in the kindling model of epilepsy. Neuropharmacology 1984; 23: 1117-23.
• 12 Duan SM. General anesthetics in veins. In: Duan SM, editor. Anesthetical pharmacology. Beijing: People's Medical Publishing House; 2000. p 96-106.
• 13 Jiangsu New Medical College. Encyclopedia of Chinese materia medica. v II. Beijing: People's Medical Publishing House; 2001. p 2278-9.