Effects of ethanol extracts of scorpion on hippocampal apoptosis and caspase-3 expression in lithium chloride-pilocarpine-induced status
epilepticus rats**★
Liang Yu, Hongbin Sun, Yi Liang, Yan Xie, Baoming He, Fei Xu

Department of Neurology, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Chengdu  610072, Sichuan Province, China

Liang Yu★, Master, Associate chief physician, Department of Neurology, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Chengdu  610072, Sichuan Province, China

Corresponding author: Hongbin Sun, Master of Medical Science, Chief physician, Department of Neurology, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Chengdu  610072, Sichuan Province, China
Shb1369@yahoo.com.cn

Supported by: the National Natural Science Foundation of China, No. 30740035*; the Tackle Key Program of Sichuan Province, No. 05SG1672*

Abstract
BACKGROUND: Previous studies have demonstrated that scorpion venom in the scorpion can inhibit epilepsy and apoptosis. However, it remains unclear whether ethanol extracts of scorpion (EES) exhibit similar effects.
OBJECTIVE: To investigate the effects of EES on hippocampal apoptosis and caspase-3 expression, and to compare the effects on sodium valproate (positive control drug) in a rat model of status epilepticus induced by lithium chloride-pilocarpine. 
DESIGN, TIME AND SETTING: This randomized, controlled study was conducted at the Drug Research and Development Center, Kanghong Pharmaceuticals Group, and the Department of Pathology, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, China from May 2007 to April 2008.
MATERIALS: EES were prepared by Huashen Pharmaceutical, China. Sodium valproate (Hunan Xiangzhong Pharmaceutical, China) and lithium chloride-pilocarpine (Sigma, USA) were also used in the present study.
METHODS: From a total of 156 rats, six served as normal controls. The remaining rats were intraperitoneally injected with lithium chloride-pilocarpine to establish status epilepticus models, and then assigned to five groups (n = 30, respectively). Animals in each group were administered drugs at 15 minutes after epileptic seizure by gavage. i.e. in the normal control and model groups, rats were treated with 1 mL/0.1 kg saline. The sodium valproate group was administered 120 mg/kg/d sodium valproate. The low-, moderate-, and high-dose EES groups received treatments of 290, 580, and 1 160 mg/kg/d EES. The dispensed concentration was 1 mL/0.1 kg. Rat seizure behavior was observed. If status epilepticus did not terminated after 1 hour, the rats were intraperitoneally administered atropine (1 mg/kg) and diazepam (10 mg/kg) to terminate seizure. These rats were continuously observed for 6 hours to ensure seizure termination. Then rats were treated with the above-mentioned drugs at 8: 00 am each day until sacrifice, which took place 4 hours after drug administration.
MAIN OUTCOME MEASURES: Terminal dUTP nick end labeling (TUNEL)-positive cells and caspase-3 expression were, respectively, determined by TUNEL and immunohistochemistry at 6, 24, 48, and 72 hours, as well as 7 days, after status epilepticus. Behavioral changes were also measured.
RESULTS: A few caspase-3-positive cells were observed. TUNEL- and caspase-3-positive cells were mainly visible in the hippocampal CA1 and CA3 regions 6 hours following status epilepticus in the model and drug intervention groups. The number of TUNEL-positive cells reached a peak at 48 hours following status epilepticus in the sodium valproate group, as well as the moderate- and high-dose EES groups, and number of TUNEL-positive cells reached a peak at 72 hours in the model and low-dose EES groups. The number of caspase-3-positive cells reached a peak at 48 hours in each group. Following treatment of sodium valproate and EES, the number of TUNEL- and caspase-3-positive cells significantly decreased compared with the model group at various time points (P < 0.05). The number of TUNEL- and caspase-3-positive cells was greatest in the low-dose EES group, followed by the moderate- and high-dose EES groups. The number of TUNEL- and caspase-3-positive cells was similar between the sodium valproate and high-dose EES groups. Epileptic seizure was significantly improved in the sodium valproate group, as well as the moderate- and high-dose EES groups, compared with the model group (P < 0.05 or P < 0.01). Treatment with sodium valproate and high-dose EES resulted in the best outcome, although the results were similar (P > 0.05).
CONCLUSION: A dose of 1 160 mg/kg/d EES significantly inhibited status epilepticus. This outcome corresponded to a decreased number of apoptotic cells and caspase-3-positive cells, which was similar to sodium valproate. These results suggest that it is not necessary to extract a component from the scorpion for the treatment of epilepsy. The high dose of EES significantly inhibited epilepsy, which correlated with decreased hippocampal caspase-3 expression.
Key Words: ethanol extracts of scorpion; apoptosis; terminal dUTP nick-end labeling; caspase-3; model of status epilepticus; lithium chloride-pilocarpine; brain injury; neural regeneration

INTRODUCTION
  
Various apoptosis-related proteins and enzymes are abnormally expressed following status epilepticus, which ultimately results in hippocampal apoptosis[1-5]. These factors are also major factors in the recurrence of convulsion[4-7]. The currently used anti-epileptic drugs are successful in controlling seizure, but it remains unclear whether these drugs also resist apoptosis[8-14].
Many Chinese medicines have been shown to inhibit non-epilepsy-induced apoptosis, such as cerebral ischemia, spinal cord injury, and retinal light damage, and also exhibit neuroprotective effects[15]. However, it remains to be shown whether anti-epileptic Chinese medicines resist apoptosis following seizure. Among the various scorpion species, Buthus martensii Karsch, a widely distributed scorpion species in Asia, has received great attention, because it has been used in Chinese traditional medicine for a long time to treat many nervous system diseases, including epilepsy. Because of the various components of the scorpion, the relationship between scorpion ingredients remains unclear, and it is difficult to determine the precise action mechanism, in particular with regard to apoptosis following anti-status epilepticus. Previous studies have extracted scorpion venom or a component from the scorpion[16-17], such as anti-epilepsy peptide, B. martensii Karsch I, B. martensii Karsch IM, and B. martensii Karsch αIV[18-21], which do not completely represent the pharmacological action of the scorpion. Ethanol and aqueous extracts of scorpion exist[22], and although the main components are comprised of proteins and amino acids, the ethanol extract contains the effective ingredients of the scorpion[22]. Therefore, the present study selected ethanol extracts of scorpion (EES) and utilized a rat model of status epilepticus induced by lithium chloride-pilocarpine[23] to observe the effects of EES against status epilepticus and hippocampal apoptosis.
The present study analyzed the anti-epileptic and apoptotic effects of EES, as well as the optimal dose, following status epilepticus. In addition, the mechanisms were related to caspase-3 expression.

MATERIALS AND METHODS

Design
A randomized, controlled study.
Time and setting
Experiments were conducted at the Drug Research and Development Center, Kanghong Pharmaceuticals Group, and the Department of Pathology, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, China from May 2007 to April 2008.
Materials
A total of 156 clean, healthy, adult, male, Sprague Dawley rats, weighing (200 ± 20) g, were supplied by the Experimental Animal Center, Zhengzhou University, China (license No. SCXK(yu)2005-0001). The rats were individually housed at (21 ± 2) °C, with 30%–35% relative humidity, 12-hour light/dark cycles, and free access to food and water. No difference in exposure factors was determined between the groups. The study was performed after the rats were allowed to acclimate for 1 week. Protocols were performed in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of the People’s Republic of China[24].
Drugs and reagents are as follows:

Methods
EES preparation
EES was prepared by Huashen Pharmaceutical, China according to previously published methods[22]. Briefly, Asian scorpion B. martensii Karsch, 6 cm long, was crushed. A total of 2 kg powder was placed in a 5 L round-bottom flask and was supplemented with 4 L 60% ethanol. The mixture was heated to reflux for 1 hour and then filtered. The filtrate was collected for further use, and the filter residue was mixed with 3 L 60% ethanol, heated to reflux for 0.5 hour, and then filtered. The two filtrates were then mixed. Pressure was reduced to collect the ethanol (–0.08 MPa, 70 °C). A total of 2 L extract was obtained and stored in the cold for further use.
The equivalent dose reduction factor (W) was determined between Sprague Dawley rat and human. The drug dose for rats (mg/kg) was calculated by W × human dose (mg/kg). In accordance with Pharmacopoeia of the People’s Republic of China[25], the scorpion dose was 5–12 g/d, and 5 g/d was used in the present study. An adult human weight of 60 kg served as the standard weight, which was substituted into the above-mentioned formula. The result was considered a moderate dose for rats, half the moderate dose was the low dose, and twice the moderate dose was the high dose. Finally, the low-, moderate-, and high-dose EES were 290, 580, 1 160 mg/kg/d, respectively.
Model induction, animal grouping, and management
Of 156 rats, six rats served as normal controls. The remaining rats were selected to establish status epilepticus models[26]. First, the rats were intraperitoneally injected with lithium chloride (127 mg/kg) for 18–24 hours, intraperitoneally administered atropine (1 mg/kg) for    30 minutes, and subsequently intraperitoneally treated with pilocarpine (initially 30 mg/kg, but if no seizure was determined after 30 minutes, 10 mg/kg was added every 15 minutes until grade IV or V seizures were presented, without significant intermittent seizures).
Behavioral seizures were scored according to the Racine scale[27]: grade 0, normal behavior; grade I, facial twitches; grade II, chewing, nodding; grade III, forelimb clonus; grade IV, rearing with forelimb clonus; grade V, rearing and falling on the back. Rats with IV–V grades and with greater than 15 minutes of successive seizure were considered to be successful epilepsy models.
The successful rat models were randomly assigned to model and sodium valproate groups, as well as low-, moderate-, and high-dose EES groups (n = 30). Each group was randomly assigned to post-status epilepticus 6-, 24-, 48-, and 72-hour, as well as 7-day, subgroups (n = 6). 
Animals in each group were administered drugs at 15 minutes after epileptic seizure by gavage. i.e. in the normal control and model groups, rats were treated with  1 mL/0.1 kg saline. The sodium valproate group was administered 120 mg/kg/d sodium valproate. The low-, moderate-, and high-dose EES groups received treatments of 290, 580, and 1 160 mg/kg/d EES. The dispensed concentration was 1 mL/0.1 kg. Rat seizure behavior was observed. If status epilepticus did not terminate after 1 hour, the rats were intraperitoneally administered atropine (1 mg/kg) and diazepam (10 mg/kg) to terminate seizure. These rats were continuously observed for 6 hours to ensure seizure termination. Then rats were treated with the above-mentioned drugs at 8: 00 am each day until sacrifice, which took place 4 hours after drug administration.
Terminal dUTP nick end labeling (TUNEL) staining
The rats were intraperitoneally anesthetized with 1% sodium pentobarbital (40 mg/kg) at post-status epilepticus corresponding time points (normal controls were sacrificed at 72 hours following status epilepticus), and immediately fixed by intracardiac perfusion. Hippocampal sections were washed overnight in water, dehydrated, permeabilized, embedded in paraffin, and cut into 10-μm thick sections. Three consecutive sections from each sample underwent TUNEL staining in accordance with TUNEL staining kit instructions and previously published methods[28]. The sections were deparaffinized, rehydrated, and incubated in protease K (20 μg/mL dissolved in Tris/HCl, pH 7.4–8.0) for 30 minutes at room temperature. The sections were then incubated with 50 μL TUNEL reaction mixture at 37 °C in a humidified chamber for 60 minutes. Converter-POD solution (50 μL) was added to the slides and incubated at 37 °C for 30 minutes. 3, 3’-diaminobenzidine substrate solution (50–100 μL) was applied and incubated at room temperature for 10 minutes. A phosphate-buffered saline (PBS) wash was performed between each step. Subsequently, the sections were counterstained with hematoxylin for 2 minutes, dehydrated, permeabilized, and mounted on microscope slides. Positive and negative controls were also set up. Following TUNEL staining, apoptotic cell nuclei were stained brown or yellow, whereas negative cell nuclei were stained blue. TUNEL-positive cells from three consecutive sections were quantified in the CA3 and CA1 regions from each animal under optical microscopy at a magnification of 200. The mean value was used for statistical analysis.
Immunohistochemical staining for caspase-3
Sample preparation was identical to the TUNEL staining method. In accordance with immunohistochemical staining kit instructions and previously published methods[29], the sections were baked at 60 °C for 1 hour, deparaffinized with graded concentrations of xylene and ethanol, blocked in 3% H2O2 (one sample) + distilled water (10 samples) at room temperature for 10 minutes to inactivate endogenous enzyme, and then washed three times in distilled water (5 minutes each). Antigen retrieval was performed with 0.01 mol/L sodium citrate buffer (pH 6.0) and boiling in a microwave oven, with an interval of 5–10 minutes, which was repeated twice. Following cooling, the sections were washed twice with PBS (pH 7.2–7.6) for 5 minutes each. The sections were then blocked in 5% bovine serum albumin for 30 minutes at room temperature. Excess liquid was removed prior to incubation with rabbit anti-caspase-3 polyclonal antibody (1: 100) overnight at 4 °C, followed by three wash steps in PBS washes for 2 minutes each. The samples were subsequently incubated in biotin-labeled goat anti-rabbit IgG (1: 100) at 37 °C for 30 minutes, rinsed three times in PBS for 2 minutes each, horseradish peroxidase/A-V for 30 minutes, and 4 × 5-minute PBS washes. Sections were developed with 3, 3’-diaminobenzidine, counterstained twice with hematoxylin, dehydrated, permeabilized, and mounted on microscope slides. Caspase-3-positive cell nuclei were brown or yellow, whereas negative cell nuclei were blue. Caspase-3-positive cells were quantified using the same method as TUNEL-positive cells.
Main outcome measures
Rat hippocampal apoptosis and caspase-3 expression in the CA1 and CA3 regions were respectively determined by TUNEL and immunohistochemistry at 6, 24, 48, and 72 hours, as well as 7 days, following status epilepticus.
Statistical analysis
Data were analyzed using SPSS 11.5 software (SPSS, IL, Chicago, USA). Measurement data were expressed as Mean ± SD, and grouped data were expressed as a relative number. Measurement data among groups were compared by completely randomized design analysis of variance. Pairwise comparison among groups was conducted using q-test and partitioning Chi-square. Grouped data from multiple samples were compared using the chi-square test for contingency table. A value of P < 0.05 was considered statistically significant.

RESULTS

Quantitative analysis and behavioral changes
All rats appeared normal at 18–24 hours following lithium chloride injection. However, at 1-5 minutes following pilocarpine injection, the rats developed peripheral cholinergic nervous excitation (muscarinic action), such as contracted pupils, piloerection, bloody tears, conjunctival congestion, profuse sweating, salivation, and diarrhea. At 5–15 minutes later, the rats exhibited grade I–II seizures. At 17–37 minutes later, they exhibited grade IV–V seizures, which represented successful models of status epilepticus. Dead or unsuccessful rat models were replaced by new rats. In total, 186 rats were included in the final analysis. Of these, 177 rats developed status epilepticus following intraperitoneal injection of pilocarpine. The remaining nine rats exhibited only affected grade I-II seizures, and their activities recovered to normal 2–3 hours later. The success rate of status epilepticus was 95.2% (177/186).
Following 15 minutes of seizures, the rats were treated with EES and sodium valproate, resulting in significantly reduced seizure grade, as well as significantly decreased seizure duration, compared with the model group (P < 0.05 or P < 0.01). Results from high-dose EES and sodium valproate were optimal, and no significant difference was determined between them (P > 0.05). Results from low-dose EES were insignificant (P > 0.05) (Table 1). The rats, whose seizures were not terminated, received diazepam and atropine to terminate seizures 1 hour following status epilepticus. Rats in the normal control group did not develop epileptic seizures or status epilepticus. In total, 27 rats died during model induction, resulting in a death rate of 14.5% (27/186).

Rat hippocampal cell apoptosis
TUNEL-positive cells were not visible in the normal control group. A few TUNEL-positive cells were apparent in the hippocampal CA1 and CA3 regions at 6 hours following status epilepticus in the model and drug intervention groups. The number of TUNEL-positive cells reached a peak at 48 hours following status epilepticus in the sodium valproate group, as well as the moderate- and high-dose EES groups, and the number of TUNEL-positive cells reached a peak at 72 hours in the model and low-dose EES groups, decreasing gradually thereafter. The number of TUNEL-positive cells was significantly reduced in the moderate- and high-dose EES groups, as well as the sodium valproate group, compared with the model group at various time points (P < 0.05). Low-, moderate-, and high-dose EES reduced hippocampal apoptosis in a dose-dependent manner. The number of TUNEL-positive cells was similar between the sodium valproate and high-dose EES groups (Table 2, Figure 1).
 

Caspase 3-positive cells in the rat hippocampus
A few caspase 3-positive cells were observed in the normal control group. Caspase 3-positive cells were mainly visible in the hippocampal CA1 and CA3 regions at 6 hours following status epilepticus in the model and drug intervention groups.
The number of caspase 3-positive cells reached a peak at 48 hours in each group, and then gradually diminished. In the moderate- and high-dose EES groups, as well as the sodium valproate group, the number of caspase 3-positive cells was significantly less compared with the model group (P < 0.05). The difference in caspase 3-positive cells was similar to TUNEL-positive cells in the low-, moderate-, and high-dose EES groups in a dose-dependent manner (Table 3, Figure 2).
 

DISCUSSION

Lithium chloride preconditioning reduces the required pilocarpine dose and decreases the death rate of epileptic animals in lithium chloride-pilocarpine-induced status epilepticus models[30]. The above-described models were characterized by (1) clear presentation of epileptic seizure; (2) a large gap between effective and lethal dose, suggesting good safety; (3) long duration of spontaneous recurrent seizure; and (4) characteristic molecular biology similar to human epilepsy. Moreover, lithium chloride-pilocarpine-induced status epilepticus models are commonly used to simulate human temporal epilepsy[23]. In addition, lithium chloride-pilocarpine has been commonly used to induce models of status epilepticus and complex partial seizures in studies of temporal epilepsy and anti-epileptic drug selection. During model induction, the application of low-dose pilocarpine (15 mg/kg) has been shown to result in a low death rate. However, repeated injections are required, with a low success rate for model establishment. High-dose pilocarpine (50 mg/kg) results in high death and incidence rates, and A moderate-dose pilocarpine (30 mg/kg) results in high incidence and low death rates[31]. Therefore, the present study initially used 30 mg/kg pilocarpine. However, if seizure was not observed 30 minutes later, 10 mg/kg was added every 15 minutes until grade IV or V seizures presented, without significant intermittent seizures. The success rate of model establishment in the present study was higher than previously results[32]. This could be due to a short interval of pilocarpine addition, additional frequency, and increased dose. Moreover, the death rate in the present study did not increase, but rather decreased, which suggested that increased additional doses and shortened additional intervals resulted in better outcomes during model induction. Nevertheless, a previous study[33] demonstrated that rats, which did not respond to several low-dose pilocarpine treatments by exhibiting epilepsy, presented genetic variations to successful models. Therefore, additional frequency was not increased, and 6 drug additions were used for the present study. When the total amount reached 90 mg/kg, the rats were replaced with new rats if epileptic seizure did not occur. Following EES intervention, results demonstrated significantly reduced seizure grade, as well as significantly reduced seizure duration, in status epilepticus rats, which was dose-dependent. Results were similar between sodium valproate and high-dose EES. In addition, high-dose EES was the optimal EES dose in the present study. These results demonstrate that it is not necessary to extract a scorpion component for the treatment of epilepsy. The anti-epilepsy mechanisms of EES have been shown to include (1) regulation of a neurotransmitter and its receptors; EES also reduces damage to gamma-aminobutyric acid (GABA)-positive interneurons[34-35], increases GABA release[34], counteracts proenkephalin mRNA expression[36], induces hippocampal neuron neuropeptide Y-positive reaction and neuropeptide Y mRNA expression[17], prevents decreased κ opioid receptor and NR2B expression in cells from rats of kainic acid-induced seizures[37], and selectively increases prodynorphin mRNA and cholecystokinin mRNA expression in the hippocampus of seizure-sensitive rats[38-39]. The components of EES (with exception to scorpion venom) increase cerebral cortex GABA A receptor binding activity and reduce N-methyl-D-aspartate receptor binding activity[40]. Regulation of ion channel: scorpion venom thermostable protein from EES inhibits sodium channel activation and promotes its inactivation[41]. B. martensii Karsch IM elevates Na+ current threshold and diminishes glutamic acid release by blocking sodium channels[23]. A series of short-chain peptides in EES block voltage-gated Ca2+-activated potassium current[42]. (3) Inhibition of glial cell proliferation and glial scar formation: scorpion venom downregulates transcription factor activity in glial fibrillary acidic protein gene[43], prevents astrocyte proliferation, and counteracts glial scar formation following epilepsy onset[35, 43]. (4) Inhibition of apoptosis: scorpion venom upregulates bcl-2[44-45] and downregulates bax[45] gene expression. Unknown compounds from EES have been shown to decrease caspase-8 expression[46].
The pathological results from the present study demonstrated TUNEL-positive cells mainly in the hippocampal CA1 and CA3 regions 6 hours following status epilepticus. The number of TUNEL-positive cells reached a peak at 72 hours following status epilepticus, and then gradually decreased. The number of TUNEL-positive cells remained increased after 7 days, which is consistent with previously published results[47]. Caspase-3-positive cells were also visible in the CA1 and CA3 regions at 6 hours following status epilepticus. The number of caspase-3-positive cells reached a peak at 48 hours in each group. It is well known that increased caspase-3 expression occurs earlier than DNA fragmentation and the peak of apoptotic cell number. Apoptosis participates in damage to hippocampal neurons following status epilepticus, and is closely associated with caspase-3 activation[47]. In the present study, when status epilepticus was controlled by sodium valproate and EES, the number of apoptotic cells and caspase-3-positive cells significantly decreased in the CA1 and CA3 regions at various time points, which indicated that anti-epilepsy treatment effectively reduced and resisted the development of these pathological changes. Simultaneously, the anti-epilepsy effectiveness of various-doses of EES was consistent with decreased numbers of apoptotic and caspase-3-positive cells. EES exhibited a positive-dose, anti-epileptic effect, as well as a neuroprotective effects, and prevented apoptosis of brain cells. The number of apoptotic and caspase-3-positive cells was significantly reduced in the low-dose EES group compared with the model group, but status epilepticus was not improved. These results suggested that EES inhibited apoptosis by blocking hippocampal caspase-3 expression.
A previous study demonstrated the presence of taurine, tyrosine, leucine, isoleucine, and phenylalanine in EES[48]. However, amino acids are not truly active, and so these results did not determine the active components of EES. Scorpion venom is the main active component in EES. Further studies are needed to determine whether scorpion venom, or its interaction with other components, exerts anti-epileptic and anti-apoptotic effects, as well as the identification of the other components and the correlation between scorpion venom and other components. Moreover, because scorpion venom is a mixture of various components, additional studies are needed to determine the effects of these remaining components and the use of these components in the treatment of epilepsy.
In conclusion, EES was shown to contain the effective ingredients of scorpion, and drug intervention was performed following model induction, which was consistent with the clinical practice of status epilepticus. In addition, this was the first study to address the effects of EES on hippocampal apoptosis and caspase-3 expression in the rat at various time points following anti-status epilepticus treatment. Results from the present study demonstrated that EES resists status epilepticus, inhibits rat hippocampal neural cell apoptosis and caspase-3 expression following status epilepticus, and exhibits neuroprotective effects.

REFERENCES

[1] Greene ND, Bamidele A, Choy M, et al. Proteome changes associated with hippocampal MRI abnormalities in the lithium pilocarpine-induced model of convulsive status epilepticus. Proteomics. 2007;7(8):1336-1344.
[2] Tang SH, Zhang L, Sun JY, et al. Mitochondrial and nuclear changes in hippocampal neurons in a lithium-pilocarpine-induced status epilepticus rat model. Neural Regen Res. 2009;4(3): 200-204.
[3] Mikati MA, Zeinieh M, Habib RA, et al. Changes in sphingomyelinases, ceramide, Bax, Bcl(2), and caspase-3 during and after experimental status epilepticus. Epilepsy Res. 2008; 81(2-3):161-166.
[4] Kamida T, Fujiki M, Ooba H, et al. Neuroprotective effects of edaravone, a free radical scavenger, on the rat hippocampus after pilocarpine-induced status epilepticus. Seizure. 2009;18(1):71-75.
[5] Chuang YC, Lin JW, Chen SD, et al. Preservation of mitochondrial integrity and energy metabolism during experimental status epilepticus leads to neuronal apoptotic cell death in the hippocampus of the rat. Seizure. 2009;18(6):420-428.
[6] Borges K, Gearing M, Mcdermott DL, et al. Neuronal and glial pathological changes during epileptogenesis in the mouse pilocarpine model? Exp Neurol. 2003;182(1):21-34.
[7] Brandt C, Glien M, Potschka H, et al. Epileptogenesis and neuropatheology after different types of status epilepticus induced by prolonged electrical stimulation of the basolateral amygdala in rats. Epilepsy Res. 2003;55(1-2):83-103.
[8] Rigoulot MA, Koning E, Ferrandon A, et al. Neuroprotective properties of topiramate in the lithium-pilocarpine model of epilepsy. J Pharmacol Exp Ther. 2004;308(2):787-795.
[9] Pitk?nen A, Kubova H. Antiepileptic drugs in neuroprotection. Expert Opin Pharmacother. 2004;5(4):777-798.
[10] Kaindl AM, Asimiadou S, Manthey D, et al. Antiepileptic drugs and the developing brain. Cell Mol Life Sci. 2006; 63(4): 399-413.
[11] Kim JS, Kondratyev A, Tomita Y, et al. Neurodevelopmental impact of antiepileptic drugs and seizures in the immature brain. Epilepsia. 2007;48 Suppl 5:19-26.
[12] Stefovska VG, Uckermann O, Czuczwar M, et al. Sedative and anticonvulsant drugs suppress postnatal neurogenesis. Ann Neurol. 2008;64(4):434-445.
[13] Katz I, Kim J, Gale K, et al. Effects of lamotrigine alone and in combination with MK-801, phenobarbital, or phenytoin on cell death in the neonatal rat brain. J Pharmacol Exp Ther. 2007; 322(2):494-500.
[14] Parinejad N, Keshavarzi S, Movahedin M, et al. Behavioral and histological assessment of the effect of intermittent feeding in the pilocarpine model of temporal lobe epilepsy. Epilepsy Res. 2009;86(1):54-65.
[15] Chen YJ, Deng JX, Lin YL. Advance in research of Chinese medicine affecting cell apoptosis. Fujian Xumu Shouyi. 2008;30(1):3-5.
[16] Zhang HS, Wang JJ. Advance in pharmacological research of Chinese medicine Scorpio. Zhongguo Zhongyi Jizheng. 2007;16(2):224-226.
[17] Feng YH, Yu DQ, Peng Y, et al. Effects of scorpion venom heat-resistant protein on kainic acid induced-damage of cultured primitive rat hippocampal neuropeptide Y-nergic neurons. Zhongguo Yingyong Shenglixue Zazhi. 2007;23(3):315-318.
[18] Wang CG, He XL, Shao F, et al. Molecular characterization of an anti-epilepsy peptide from the scorpion Buthus martensii Karsch. Eur J Biochem. 2001;268(8):2480-2485.
[19] Chai ZF, Zhu MM, Bai ZT, et al. Chinese-scorpion (Buthus martensii Karsch) toxin BmK αIV, a novel modulator of sodium channels: from genomic organization to functional analysis. Biochem J. 2006;399(3):445-453.
[20] He X, Peng F, Zhang J, et al. Inhibitory effects of recombinant neurotoxin BmK IM on seizures induced by pentylenetetrazol in rats. Chin Med J (Engl). 2003;116(12):1898-1903.
[21] Bai ZT, Zhao R, Zhang XY, et al. The epileptic seizures induced by BmK I, a modulator of sodium channels. Exp Neurol. 2006;197(1): 167-176.
[22] Li WZ, Yao JC, He Q. Study on the ethanol extracting technology for scorpion and centipede. Weifang Yixueyuan Xuebao. 2006; 28(5):345-346.
[23] Murdoch D. Mechanisms of status epilepticus: an evidence-based review. Curr Opin Neurol. 2007;20(2):213-216.
[24] The Ministry of Science and Technology of the People’s Republic of China. Guidance Suggestions for the Care and Use of Laboratory Animals. 2006-09-30.
[25] China Pharmacopoeia Committee. Pharmacopoeia of the People's Republic of China. 2005 ed. Beijing: Chemical Industry Press. 2005.
[26] Chu K, Kim M, Jung KH, et al. Human neural stem cell transplantation reduces spontaneous recurrent seizures following pilocarpine-induced status epilepticus in adult rats. Brain Res. 2004;1023(2):213-221.
[27] Racine RJ. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysiol. 1972;32(3):281-294.
[28] Ansari B, Coates PJ, Greenstein BD, et al. In situ end-labeling detects DNA strand breaks in apoptosis and other physiological and pathological states. J Pathol. 1993;170(1):1-8.
[29] Niquet J, Auvin S, Archie M, et al. Status epilepticus triggers caspase-3 activation and necrosis in the immature rat brain. Epilepsia. 2007;48(6):1203-1206.
[30] Voutsinos-Porche B, Koning E, Kaplan H, et al. Temporal patterns of the cerebral inflammatory response in the rat lithium- pilocarpine model of temporal lobe epilepsy. Neurobiol Dis. 2004; 17(3):385-402.
[31] Zhang JQ, Zhao HN, Wang XM. Establishment and appraisement of lithium-pilocarpine induced rat model of acute epilepsy. Chuanbei Yixueyuan Xuebao. 2007;22(6):537-539.
[32] Wu YL, Liang JC, Zhan XL, et al. Temporal lobe epilepsy model induced by lithium-pilocarpin and progression of mossy fiber sprouting. Zhongguo Bijiao Yixue Zazhi. 2009;19(6):43-46.
[33] Glien M, Brandt C, Potschka H, et al. Repeated low-dose treatment of rats with pilocarpine: low mortality but high proportion of rats developing epilepsy. Epilepsy Res. 2001;46(2):111-119.
[34] Jiang CL, Zhang WQ. Effect of Scorpion venom on the release of GABA in hippocampus of epileptic rats induced by kainic acid. Shengli Xuebao. 1999;51(6):609-614.
[35] Cheng WG, Zhang JP, Wang B, et al. The effects of Scorpion venom on GFAP and GABA in entorhinal cortex of epileptic rats induced by kainic acid. Zhonghua Zhongxiyi Zazhi. 2003;4(8): 1126-1128.
[36] Li DD, Gong J, Li XF, et al. The opioid mechanism on anti-seizure susceptibility by Scorpion. Zhongguo Weishengtai Xue Zazhi. 1999;11(2):75-77.
[37] Du XL, Gao MH, Zhao J, et al. The change of expression of kappa opioid receptors and NR2B immunoreactivity in area tempestas of seizure sensitive rats. Shenjing Jipouxue Zazhi. 2004;20(5): 463-467
[38] Jiang CL, Zhang WQ. Effect of Scorpion venom on the expression of PDYN mRNA in hippocampus of epileptic rats. Zhongguo Yingyong Shenglixue Zazhi. 2000;16(1):72-74.
[39] Jiang CL, Peng Y, Zhang WQ. Effect of Scorpion venom on the expression of PCCK mRNA in hippocampus of epileptic rats induced by kainic acid. Zhongguo Yingyong Shenglixue Zazhi. 2000;16(2):108-110.
[40] Huang YC, Zuo PP. The modulation of Buthus martensiic Karch extract(BmKE)on Cerebral Cortex NMDA receptor and GABAA receptor in epileptic mice. Shizhen Guoyi Guoyao. 2007;18(1): 71-73.
[41] Zhang XY, Wang Y, Zhang J, et al. Inhibition of sodium channels in acutely isolated hippocampal neurons by Scorpion venom heat resistant protein. Shengli Xuebao. 2007;59 (3):278-284.
[42] Xu CQ, Br?ne B, Wicher D, et al. BmBKTx1, a novel Ca2+-activated K+ channel blocker purified from the Asian scorpion Buthus martensii Karsch. J Biol Chem. 2004;279(33): 34562-34569.
[43] Jiang CL, Zhang WQ. The GFAP gene regulation mechanism on anti-epileptic seizures by Scorpion venom. Zhongguo Yingyong Shenlixue Zazhi. 2002;18(4):406-407.
[44] Du XL, Gao MH, Zhao J, et al. The change of expression of bcl-2 in area tempestas of seizure sensitive rats. Shenjing Jipouxue Zazhi. 2005;21(2):185-189.
[45] Liu CQ, Gao X, Peng Y. Effect of scorpion venom on the expression of Bcl-2/Bax in dopaminergic neurons of PD mice. Jichu Yixue yu Linchuang. 2004;24(6):623-628.
[46] Fattorusso R, Frutos S, Sun X, et al. Traditional Chinese medicines with caspase-inhibitory activity. Phytomedicine. 2006;13(1-2):16-22.
[47] Zhang YQ, Liao WH, Chi LX, et al. Dynamic changes of apoptotic neuron in hippocampus after pilocarpine induced status epilepticus in rats. Disan Junyi Daxue Xuebao. 2007;29(1): 71-73.
[48] Jin ZX, Liu JJ, Jin LY, et al. Chemical components of Scorpio ethanol extract. Heilongjiang Yiyao. 1995;8(2):69-70.
 (Edited by Zhou JG/Qiu Y/Song LP)