Neuroscience Letters 314 (2001) 65–68 www.elsevier.com/locate/neulet
Ether fraction of methanol extracts of Gastrodia elata, a traditional medicinal herb, protects against kainic acid-induced neuronal damage in the mouse hippocampus Hyeon-Ju Kim a, Kwang-Deog Moon a, Sang-Young Oh b, Sang-Pyo Kim c, Seong-Ryong Lee d,* a
Department of Food Science and Technology, Kyungpook National University, Taegu, 702-701 South Korea b Department of Food Engineering, Sangju National University, Sangju, 742-170 South Korea c Department of Pathology, School of Medicine, Keimyung University, Taegu, 700-712 South Korea d Department of Pharmacology and Brain Research Institute, School of Medicine, Keimyung University, 194 Dongsan dong, Taegu, 700-712 South Korea Received 16 May 2001; received in revised form 29 August 2001; accepted 12 September 2001
Abstract Gastrodia elata (GE) has been used traditionally for the treatment of convulsive diseases such as epilepsy in oriental countries including South Korea and still occupies an important place in traditional medicine in Asia. We studied the anticonvulsive effect and protective effect of the ether fraction of methanol extracts (EFME) of GE against hippocampal neuronal damage after kainic acid administration in mice. Mice were treated with the EFME of GE (200 or 500 mg/kg per day, p.o.) for 14 days before kainic acid injection (45 mg/kg, i.p.). The EFME of GE (at the dose of 500 mg/kg) delayed the onset time of neurobehavioral change (P , 0:01) and reduced the severity of convulsions (P , 0:05) and hippocampal neuronal damage in the CA1 (P , 0:01) and CA3 (P , 0:05) regions. Our results show that The EFME of GE has anticonvulsive effect and putative neuroprotective effect against excitotoxicity induced by kainic acid. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Gastrodia elata; Kainic acid; Excitotoxicity; Seizure; Hippocampus; Neuroprotection
Excessive release of glutamate and related excitatory amino acids may play an important role in the pathogenesis of neuronal injury [2]. Kainic acid is a powerful excitatory amino acid agonist with potent excitotoxic and convulsant properties [16]. Kainic acid has been used to study a variety of central nervous system disorders involving excitation, excitotoxicity, and acute cell loss. Administration of kainic acid can trigger characteristic limbic seizures and selective neuronal cell death in the hippocampal CA1 and CA3 regions in addition to other regions [19]. Gastrodia elata (GE) is a traditional herbal agent that has been used as an anti-convulsant in oriental countries for centuries. In addition, GE is also used as an analgesic and a sedative against vertigo, general paralysis, and tetanus. Vanilly alcohol and gastrodin from GE are known to have anticonvulsive action [20]. Recently Hsieh and colleagues * Corresponding author. Tel.: 182-53-250-7478; fax: 182-53252-1605. E-mail address:
[email protected] (S.-R. Lee).
[5] reported anticonvulsive effect of GE in kainic acid-treated rats. The constituents of GE inhibited glutamate-induced apoptosis in neuronal cells [12]. In previous studies, the ether fraction of methanol extracts (EFME) of GE attenuates the decrease of the GABA and the increase of glutamate content and shows anticonvulsant effect in the pentylenetetrazole-induced seizures [3,6]. In the present study, we investigated whether the EFME of GE has the neuroprotective action against kainic acid-induced excitotoxicity in mice. Male ICR mice (25–35 g) were used for this study. These animals were kept in cages under a light-dark cycle with the light on from 06:00–18:00 h. Food and water was available ad libitum. The experimental procedure was approved by our institutional animal care and use committee. Kainic acid (Sigma Chemical Co., St Louis, MO) was dissolved in distilled water, the pH was adjusted to 7.4, and the solution (45 mg/kg) was injected intraperitoneally into the mice. GE was obtained from Suk-Bo Gastrodia farm in Kyoungbuk province of South Korea. Three-years-old GE was used in
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 29 6- 0
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the mice showed myoclonic twitches of the forelimbs (piano-playing) without falling; (4) grade 3, generalized seizures or aggressive seizure jump. Behavioral changes were examined for 3 h after kainic acid administration. In addition, the onset time (min) of first behavioral change and the mortality rate were examined. Four days after administration of kainic acid, the animals were deeply anesthetized with chloral hydrate solution and perfused transcardially with heparinized phosphate-buffered saline (PBS, pH 7.2), followed by perfusion with 10% formalin in PBS. The brains were immediately removed from the skull and fixed in the same fixative for 24–48 h. Thereafter, the brains were embedded in paraffin and representative coronal sections (6mm thick), which included the dorsal hippocampus, were obtained using a rotary microtome. Tissue sections were
Fig. 1. Effect of the ether fraction of methanol extracts (EFME) of GE on kainic acid-induced seizures in mice. (A) Onset time of the first neurobehavioral change after kainic acid treatment. (B) Grade of kainic acid-induced seizures. Data are presented as the mean ^ SEM. VEH, kainic acid and vehicle-treated (n ¼ 14); GE, kainic acid and the EFME of GE-treated mice (n ¼ 14). *P , 0:05, **P , 0:01 in comparison with kainic acid and vehicle-treated mice.
this study. Washed and chopped fresh GE was frozen in deep freezer (at 2708C) for 24 h. GE was freeze-dried and powdered. The dried powder of GE was added three times weight of methanol and reflux extracted three times at 608C. Methanol extracted material was filtered, concentrated, and suspended in distilled water. Thereafter, the suspension was extracted three times with an equal volume of ether, which was evaporated at low temperature under reduced pressure. Ether fraction was suspended in 1% aqueous carboxymethyl cellulose solution as suspending solution [6]. Mice were administered orally with the suspended material of EFME of GE (200 or 500 mg/kg per day) using esophagus needle for 14 days before and 4 days after kainic acid injection. Control mice were administered with same volume of 1% aqueous carboxymethyl cellulose solution without GE. Neurobehavioral changes after kainic acid administration were assessed using the method for mice described by Kondo et al. [8] with some modification. Behavioral findings were graded according to seizure severity as follows. Grade 0, in which the mice showed no seizure; grade 1, in which mice showed tailarch, staring posture or automatism; (3) grade 2, in which
Fig. 2. Effect of the ether fraction of methanol extracts (EFME) of GE on the morphology of the CA1 and CA3 regions of hippocampus 4 days after kainic acid treatment in mice (hematoxylin-eosin staining). CA1 (A) and CA3 (B) regions in control mice treated with same volume of saline and vehicle. CA1 (C) and CA3 (D) regions in mice with kainic acid and vehicle. CA1 (E) and CA3 (F) regions in mice with kainic acid and the EFME of GE (200 mg/kg). CA1 (G) and CA3 (H) regions in mice with kainic acid and the EFME of GE (500 mg/kg). After kainic acid treatment, only a few normal cells are seen with round cell bodies and clear nuclei and nucleoli. Damaged cells are shrunken and distorted, with small dense nuclear remnants. Arrows indicate examples of damaged cells in CA1 and CA3 regions. Bar ¼ 50 mm.
H.-J. Kim et al. / Neuroscience Letters 314 (2001) 65–68
stained with hematoxylin and eosin. A blinded investigator performed the histological examination. All normal-appearing CA1 pyramidal neurons (per 500 mm) and CA3 pyramidal neurons under a high power field ( £ 200) of light microscopy were counted bilaterally and averaged. All data are presented as mean ^ SEM. Statistical assessments were performed using ANOVA followed by post-hoc Scheffe’s test (for histology) or unpaired Student’s t-test (for seizure grade and onset time of first neurobehavioral change). Statistical significance refers to results where P , 0:05 was obtained. The onset time of neurobehavioral change was significantly delayed in the kainic acid and ether fraction-treated mice (at the dose of 500 mg/kg) as compared to that of kainic acid and vehicle-treated mice (P , 0:01, Fig. 1A). Furthermore, the grade of neurobehavioral changes was reduced (P , 0:05, Fig. 1B). However, the EFME of GE at the dose of 200 mg/kg failed to reduce the neurobehavioral changes
Fig. 3. (A) Effect of the ether fraction of methanol extract (EFME) of GE on the number of surviving cells in the hippocampal CA1 region in mice 4 days after kainic acid administration. CTL: saline and vehicle-treated control (n ¼ 5); VEH, kainic acid and vehicletreated (n ¼ 6); GE, kainic acid and the EFME of GE-treated mice (n ¼ 6). (B) Effect of the EFME of GE on the number of surviving cells in the hippocampal CA3 region in mice 4 days after kainic acid administration. CTL, saline and vehicle-treated control (n ¼ 5); VEH, kainic acid and vehicle-treated (n ¼ 6); GE, kainic acid and the EFME of GE-treated mice (n ¼ 6). HPF: high power field ( £ 200). Data are presented as the mean ^ SEM. *P , 0:05, **P , 0:01 in comparison with kainic acid and vehicle-treated mice.
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induced by kainic acid (data not shown). Histological examination demonstrated a marked decrease of viable neuronal cells in the CA1 and CA3 regions of the hippocampus in kainic acid-treated mice compared to controls (Figs. 2 and 3). These decreases in the number of viable pyramidal neuronal cells were significantly attenuated by the EFME of GE administrations (at the dose of 500 mg/kg) in the hippocampal CA1 (P , 0:01) and CA3 regions (P , 0:05) (Figs. 2 and 3). The EFME of GE administered at the dose of 200 mg/kg didn’t reduce hippocampal neuronal damage. In the present study the EFME of GE showed an anticonvulsive effect and attenuated kainic acid-induced excitotoxic hippocampal neuronal damages in mice. Previously, it has been demonstrated that GE has an anticonvulsive effect in rats treated with kainic acid, and Lee et al. [11] reported attenuation of pentylenetetrazole-induced seizures by the EFME of GE. Although there was no complete inhibition of convulsions, in our study, the EFME of GE delayed the onset time of neurobehavioral changes and reduced kainic acid-induced convulsion severity. Supporting these findings, Huh et al. [6] found that the EFME of GE can inhibit the increase of glutamate content and decrease of GABA content in rat brain after pentylenetetrazole-induced convulsion. In addition, extracts of GE inhibit kainic acid binding to glutamate receptors [1]. GE is reported to have free radical scavenging and antioxidant effects [13] and has protective effect against apoptotic neuronal cell death related to excitotoxicity [12]. It has been suggested that antioxidant effects and anti-apoptosis action of constituents of GE may account at least in part for the basis of the their anti-epileptic activities [12]. Several antioxidants and free radical scavengers are known to inhibit the kainic acid-induced neuronal injury and convulsions [7,10,11,15]. Kainic acid binds a subtype of ionotropic receptor of glutamate and leads to intracellular calcium overload, which in turn generates reactive oxygen species including hydrogen peroxide, superoxide anion, and hydroxyl radicals [17]. There are several reports about the evidences of apoptotic neuronal injury induced by kainic acid [14,18] and the protective effect of anti-apoptotic strategies against kainic acid-induced neuronal injury [4,9]. In the present study, the EFME of GE administration attenuated kainic acid-induced neuronal injuries in the hippocampal CA1 and CA3 regions. Although further investigation is necessary to elucidate the mechanisms of action, our data suggests that the EFME of GE has anticonvulsant and neuroprotective effects. We would thank to Eun-Im Pyo for her excellent technical assistance. We also thank to Lee Brinegar and Dr Tom L. Nguyen for their English corrections in preparing the manuscript.
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