Scorpion ethanol extract and valproic acid effects on hippocampal glial fibrillary acidic protein ex

NEURAL REGENERATION RESEARCHVolume 7, Issue 6, February 2012www.nrronline.orgCite this article as: Neural Regen Res 2012; 7 (6): 426-433Scorpion ethanol extract and valproic acid effectson hippocampal glial fibrillary acidic proteinexpression in a rat model of chronic-kindlingepilepsy induced by lithium chloride-pilocarpineYi Liang, Hongbin Sun, Liang Yu, Baoming He, Yan Xieuan Academy of Medical Sciences Sichuan Provincial People's Hospital, Chengdu 610072, SichuanAbstractThe present study analyzed the effects of ethanol extracts of scorpion on epilepsy prevention andhippocampal expression of glial fibrillary acidic protein in a lithium chloride-pilocarpine epilepticrat model. Results were subsequently compared with valproic acid. Results showed graduallyNeurology, Sichuanincreased hippocampal glial fibrillary acidic protein expression followng model establishmentglial fibrillary acidic protein mRNA expression was significantly increased at 3 days, reached aHospital, Chengdu 610072,peak at 7 days, and then gradually decreased thereafter. Ethanol extracts of scorpion doses of580 and 1 160 mg/kg, as well as 120 mg/kg valproic acid, led to a decreased number of glialfibrillary acidic protein-positive cells and glial fibrillary acidic protein mRNA expression, as well asdecreased seizure grades and frequency of spontaneously recurrent seizures. The effects of160 mg/kg ethanol extracts of scorpion were equal to those of 120 mg/kg valproic acid. Theseresults suggested that the anti-epileptic effect of ethanol extracts of scorpion were associatedwith decreased hippocampal glial fibrillary acidic protein expression in a rat model of lithiumchloride-pilocarpine induced epilepsyKey Words: Chinese herbs; epilepsy; glial fibrillary acidic protein; lithium chloride-pilooshb1369@yahoo.comscorpion ethanol extraction; valproic acidReceived: 2011-09-01distributed scorpion species in Asia, it haINTRODUCTIONbeen used in Chinese traditional medicine BM, Xie Y scorpion ethanolfor a long time to treat epilepsy However, extract and v alproic acidThe brain undergoes a variety ofthe antiepileptic mechanisms remains poorlypathological changes in response to seizure, understood and the scorpion componentssuch as neuronal lesions, as well asemain difficult to determine. It has beensignificant abnormalities in astrocytespeculated that the anti-epileptic effects of chloride-pilocarpine Neuralmorphology and function Importantlyscorpion are associated with inhibition ofRegen Res 2012: 7(626-433astrocytic functional changes could affect hippocampal astrocyte activityneuronalfunctionsastrocyteproliferationisToverifythishypothesisthepresentstudywww.nrronine.orga reliable and sensitive indicator foranalyzed the anti-epileptic effects of ethanolneuronal damage. The astrocyte activation extracts of scorpion(EES)and thedoi:10.3969/issn.1673537pathways and underlying mechanisms haveifluences on hippocampal GFAP proteinbeen the focus in recent studies, although and mRNA expression in rat models ofthe mechanisms of actions remain unclear. chronic epilepsy induced by lithiumGlial fibrillary acidic protein(GFAP)is achloride-pilocarpine. Results were thenspecific marker for astrocytes, and a small compared with valproic acid, a typicalnumber of GFAP-positive cells are visible in anti-epileptic drug used in the clinicthe hippocampus and cerebral cortex undernormal circumstances. However, GFAPRESULTSexpression significantly increases followingstatus epilepticus induced by a variety ofQuantitative analysis of experimentalchemical stimulations and electricalanimalsstimulations). Therefore it is possibleA total of 186 Sprague-Dawley rats werecerebral injury could be reduced byincluded in the present stuption of the control groYHa中国煤化工astrocytes following epilepsychronic epilepsy was inducCNMHGButhus [o]martensi Karsch, a widelychloride-pilocarpine injection in theLiang Y, et al. / Neural Regeneration Research 2012: 7(6 ): 426-433remaining 180 rats and model failure was appropriately seizures were observed. Normal behaviors graduallysupplemented. The 180 successful model rats wererecovered and some rats exhibited spontaneousrandomly assigned to five groups: model (intragastrical recurrent seizure(SRS)after the quiet period. SRSinjection of normal saline after modeling), valproic acid emerged as an appearance of localized seizure, such as(intragastrical injection of valproic acid after modeling), facial convulsion or unilateral forelimb clonus, whichlow-, medium-, and high-dose EES (intragastricaldeveloped into global stiffness and seizures, usuallyinjection of 290, 580, and 1 160 mg/kg EES afterlasting for 1 minute. However, all attacks weremodeling). Six rats were selected from each time point at self-terminated. The control rats exhibited no abnormal6 hours, 1, 3, 7, 14, and 30 days after emergence ofbehaviors during the observation perioepileptic symptoms. A 186 rats were included in the final Effects of varying doses of EEs on hippocampalanalysisGFAP expression in epileptic ratsBehavioral changes of epileptic ratsImmunohistochemical staining and light microscopyFollowing lithium chloride injection the rats did notanalysis revealed a brown-stained cytoplasmexhibit behavioral abnormalities and were freely activeGFAP-positive cells Staining in the nuclei was absent,However, following pilocarpine injection, some ratsand cells were rounded or irregular in shape and wereexhibited peripheral cholinergic activation(M-like effect) surrounded by radial processes. a small number ofithin 5 minutes, which were characterized by miosis GFAP-positive cells was observed in the hippocampus ofand erected hair, as well as limbic epileptic behaviorscontrol rats. Some hippocampal GFAP-positive cellssuch as erection, running, and wet dog-like tremblingwere visible at 6 hours after status epilepticus in theGrade k ll seizure occurred 515 minutes latermodel group, and the cells were primarily located in thecharacterized by convulsive seizure in the forelimbs and Ca region and dentate gyrus. By 30 days, the number ofstanding at a half-upright position. Continuous seizure positive cells significantly increased and reached a peakgrading of iv or greater was observed 17-27 minutes the cells were darkly stained and large, with manylater; the entire body and limbs experienced muscularbranches and processes. The cells appeared thickenedcontraction and loss of postural control, and each attack with a distorted alignment and clear boundaries. Thelasted for 30-120 seconds at intervals of 2-15 minutes. cells were typically star-shaped, especially in the dentateStatus epilepticus was determined by the appearance of gyrus. Changes in the number of GFAP-positive cells inpersistent seizure for 10 minutes; models of statusother treatment groups were consistent with the modelepilepticus were successfully induced in 85.65% of the group, although the number of GFAP-positive cells wasrats. The surviving rats entered a quiet periodsignificantly less than the model group Following(7-14 days), exhibiting the followtreatment, the gFAP-positive cells were smaller andappetite, and extreme irritability. During this period, no extended thin processes(Figure 1)igure 1 Glial fibrillary acidic protein(GFAP, arrows) expression in rat dentate gyrus at 30 days after model establishment(streptavidin biotin method; x 400). EES: Ethanol extracts of scorpion(A)Control group: GFAR-positive cells exhibit distinct boundaries and brown cytoplasm(B)Model group: GFAP-positive cells are shaped like a spider, surrounded by radial protrusions, and the number ofGFAP-positive cells is significantly increased(C)Valproic acid group: GFAP-positive cells exhibit distinct boundaries, typical star-shape appearance, and the number of cells issignificantly less than the model group(D)EES low-dose group: GFAR positive cells are shaped like a spider and surrounded by radial protrusions, but expression islighter and the number of protrusions is less than the model groupis significantly less and the number of protrusions is significantly less than the mode urn(E)EES moderate-dose group: GFAP-positive cells are shaped like a spider and(FEES high-dose group: GFAR positive cells exhibit clear boundaries and a typical starYHa中国煤化工out expressionCNMH GsS thanthe model groupLiang Y, et al. / Neural Regeneration Research 2012: 7(6 ): 426-433There was no significant difference in the number ofmodel rats at 3 days, which reached a peak at 7 daysGFAP-positive cells between the low-dose EES group GFAP mRNA expression gradually decreased, but didand model group, but the difference was statisticallynot reach normal levels, by 30 days( Figure 2). EESsignificant between other treatment groups and thetreatment at varying doses, as well as valproic acidmodel group(P< 0.05). The most significant difference treatment, resulted in significantly decreased GFAPwas in the high-dose EES group and valproic acid group, mRNA levels compared with the model group. Therewith no significant difference between them (Tables 1-3). were significant differences in EES high-and moderateEffects of varying eES doses on hippocampal GFaP dose groups and valproic acid group compared with themRNA expression in epileptic ratsmodel group(P<0.05). The GFAP mRNA expressionsReverse transcription-PCR analysis showed significantly returned to normal levels at 30 days in the EEsincreased GFAP mRNA levels in the hippocampus ofhigh-dose group and valproic acid group(Table 4)Table 1 Quantification of cells(cells/400-fold field of view )expressing glial fibrillary acidic protein in the hippocampal CA1 areaat different time pointsStatus epilepticus tim6 hours1 day3 days7 days30 days10.00±2.83B23.83±14723.83±3.0624.50±1.872600±4.0526.83±3.821283±24016.50±36227.33±26620.00±27621.00±303221.50±1.8723.67±2.1613.00±3.291600±4.47318.50±4.1421.00±2.1921.83±23222.67±2.161233±3.0116.83±512c17.67±3.2720.83±3.2521.67±3.98Data are expressed as mean+ SD from sixrats at each time point; aP<0.05, vs. group B; P<0.05, vs group C: P<0.05, vs group D(analysisof variance, pairwise comparison was tested using least significant difference test or g-test).(A)Controlgroup:(D-F)low, medium, and high-dose ethanol extracts of scorpion groups.Table 2 Quantification of cells(cells/400-fold field of view ) expressing glial fibrillary acidic protein in the hippocampal CA 3 areaat different time pointsroup6 hours1 day3 dar7 days1 4 dE30 days1150±2.66B9.50±1.3819.67±4.1323.17±3.662533±3.3927.50±4.8528.50±5.2410.50±3.0815.33±1.751733±28821.00±34122233±463221.50±38321267±2.6619.17±387020.17±3.7624.50±3.51026.33±44127.33±4088E13.17±3.661733±2581883±2.93221.83±2.14222.67±3.3323.83±3,191283±3921550±2171833±2.1621.33±1.8632233±4.932367±2.942Data are expressed as mean t SD of six rats at each time point; P<0.05, vs group B: P< 0.05, vs group C; P<0.05, vs group D(analysis ofvariance, pairwise comparison was tested using the least significant difference test or q-test). (A)Control group;(B)model group; (C)valproicacid group: (D-F)low, medium-, and high-dose ethanol extracts of scorpion groups.Table 3 Quantification of cells(cells/400-fold field of view )expressing glial fibrillary acidic protein in the hippocampal dentatedyrus at different time pointsStatus epilepticus timeGroup61 day3 days7 days14 days30 day10.50±1.971483±3.0626.17±5.4929.33±5.5030.50±3.893383±3.7635.00±8613.00±2371967±3613±3.08225.17±4.1726.83±4.262867±32021433±1.212567±4.1327.17±3.5429.00±64834.00±2.8334.33±6.7114.33±1.5121.83±2.5634.17±6.052433±4.3738.67±4.9729.50±3.7313.33±22521.67±30122350±4.3702717±52727.50±3.94Data are expressed as mean SD from sixrats at each time point; aP<0.05, vs. group B; P 1 hour exhibited collapse andlimb clonus within 4 minutes after diaz epam and atropineGroupSeizure grade(levels)injection. Status epilepticus disappeared at 3080 minutes, but seizures with grade IV and V repeatedlyoccurred In the control group, no seizures or SRS wereControl0Model630observed Model rats were treated with correspondingValproic acid3624treatments at 15 minutes after seizure treatment of eesLow-dose EESat varying doses significantly alleviated epileptic seizureMedium-dose EESand significantly reduced the frequency of epilepticHigh-dose EES3625izures with grade IV or greater and SRS in rats. ThereThere are 36 rats in each group, except six rats in the controlwere significant differences in these observed effects in0.0the EES moderate- and high-dose groups and valproicTYH中国煤化工eEs5group(chi-squanacid group compared with the model group(P< 0.05CNMHGables 5. 6Liang Y, et al. / Neural Regeneration Research 2012: 7(6): 426-433reached a peak at 7 days then gradually decreasedTable 6 Quantification of total number of spontaneousSteward et a/ performed dot blot assays, showing thatrecurrent seizures in ratsGFAP mRNA expression in the hippocampus is biphasicSeizure grade(levels) Totalfollowing brain injury; the first peak occurs at 1 -2 daysGroupIIIIV-Vnumber(10 times the control group) with a second peak at 6-8 days, whereas GFAP expression gradually increasesThe human GFAP gene promoter contains aModel126324Valproic acid132abCAMP-responsive binding protein, which correspond toLow-dose EEstranscription factors, such as Sp1, NF1, Apl, and Ap2 4Moderate-dose EES 361208280204Wang et al suggested that fos protein is expressed inHigh-dose EES102astrocytes of pentylenetetrazol-induced epileptic ratsThere are 36 rats in each group, except six rats in the controlFos protein binds with jun proteins, forming agroup. ap <0.05, vs. model group; bP< 0.05, vs low-dose EES transcription factor, and binding with Ap1 sites might leadgroup(chi-square test and rank sum test). EES: Ethanol extract ofto increased GFAP expression Besnard et al 4scorpiondemonstrated that the gfa gene modulates GFAPexpression, but the mechanisms underlying c-fos/c-junmodulation of the gfa gene and increased GFAP mRNADISCUSSIONtranscription remain poorly understoodWe speculated that early astrocyte responses and repairTemporal lobe epilepsy is the main type of refractorymechanisms were different from the pathophysiologicalepilepsy in adults, and typical pathological changesmechanisms occurring during the late period of damaginclude hippocampal sclerosis and atrophy that involves Early astrocyte activation is likely achieved via signalhippocampal and parahippocampal structures 4. Seizure transduction pathways during the process ofresults in the loss of hippocampal neurons, which isformation, GFAP mRNA transcription levels in brainthought to contribute to latency(epilepsy) gliosis andtissue are down-regulated via inherent negativeloop reconstruction -induced recurrentthereby feedback mechanisms, thereby controlling the formationleading to lasting brain damage, cognitive impairment, of the glial scarand increased risk of recurrent seizures. The rat model currently used anti-epileptic drugs exhibit efficacy viaof lithium chloride/pilocarpine-induced epilepsy isn channels, metabolic enzymes, andinternationally recognized as an ideal animal model forneurotransmitter transporters, thereby altering thestudying temporal lobe epilepsy in humans.Theischarge nature of neurons, inhibiting diffusion ofpresent study established a rat model of lithium chloride/ epileptic discharge reducing synchronous discharge 16)pilocarpine-induced chtilepsy to analyze the anti- and modulating learning, memory, and emotionalepileptic effect of EES is in a dose-dependent manner. behaviors / These results suggest that the use ofThe role of glial cells in epilepsy pathogenesis has been anti-epileptic drugs for preventing seizures are alsoassociated with the regulation of glutamateikely to induce damages to brain function andconcentrations and suppressed neuronaldevelopment", resulting in learning, memory, andhyperexcitability Neurons are more prone to damage by other cognitive functional deficits in patients, indiffuse excitation waves if normal functions of glial cells particular the fetus and newborn. Epilepsy treatmentare absent. Injured local tissue and neuronal loss are remains challenging in the clinic, and many treatmentsoften accompanied by astrocyte activation and increased for epilepsy are still in the exploration stageGFAP expression). Following pilocarpine-inducedScorpion is a dried arachnid from the Mesobuthusepilepsy, hippocampal astrocytes become activated, the martensii Karsch family and has been widely utilized as anumber of GFAP-positive cells increases, and astrocytes Chinese herb for treating epilepsy o Scorpion exhibitsproliferate 10). These changes are crucial for thestrong anti-epileptic effects, and the anti-epileptic peptidformation and maintenance of seizures. Increased GFaP isolated from the scorpion is more potent 9.Previousexpression is a hallmark for active protein synthesis and studies have not been performed to compare doses oftransportation in astrocytes, as well as indicator forEES and classic anti-epileptic drugs. It remains difficult toastrocyte activation; expression levels have beendistinguish between EES or monomers extracted fromassociated with reactive gliosis following neuronalthe scorpion and classical anti-epileptic drugs for use inhumans. In the present study, lithiumResults from the present study demonstrated that GFAP chloride/pilocarpine-induced models of chronic epilepsyprotein and mRNA expression changes were notwere treated with varying doses of EES, and results wereassociated with time in a rat model of lithiumcompared to the anti-epileptic effects of valproic acidchloride-pilocarpine-induced epilepsy, althoughResults showeo treat epilepsyexpression levels were dynamic. The number ofin lithium chlori中国煤化工des, indicatingGFAP-positive cells increased at 1 day after epilepsy,that the anti-epCNMHGcorpion isand GFAP mRNA levels significantly increased at 3 days, sufficient for preventing recurrent epllepsy. In addition, itLiang Y, et al. / Neural Regeneration Research 2012: 7(6): 426-433does not result in drug-dependence, making it an idealen filtrated and 25 mL was dried to a constant weightanti-epileptic drug 12, 20-22). We speculate that theand evaporated, followed by dehydration again in a wateranti-epileptic effects of EEs are equal to scorpionpath in the oven at 105C for 3 hours. The samples weremonomer(venom); it resulted in decreased numbers of then cooled in a dry dish for 30 minutes and preciselyhippocampal GFAP-positive cells and decreased GFAP weighedmRNA expression in epileptic rats. In addition,Evaporating dish constant weight: 49.746 6 g1 160 mg/kg of EES resulted in effects similar toEvaporating dish sample constant weight: 49.9067 g120 mg/kg of valproic acidExtract content =(evaporating dish sample constantPrevious studies have shown that scorpion extract plays weight- evaporating dish constant weight)/25 mLa neuroprotective role and inhibits neuronal apoptosis100 mL/tested sample weight x 100%=21. 23%following seizure 16, 18, 2, which is consistent with theDeterminations of extract content complied with requiredpresent study 24. In addition, scorpion extract has been standardsshown to prevent glial cell scar formation in epilepticExtraction steps: scorpions were crushed, a 2-kg samplerats 2, 2), and scorpion toxins in EES downregulatewas weighed and stored in 5 000-mL round-bottomexpression 12, thereby inhibiting gene transcription. The then filtered. The filtrates were heat-refluxed with3/ otranscription factors associated with gFaP geneflasks. heat refluxed with 4 L 60% ethanol for 1 hour, anpresent study confirmed the inhibition effect of EES on 60%ethanol for 30 minutes and filtered. The two filtrateshippocampal astrocyte proliferation Results suggested were mixed and ethanol was vacuum-recoveredthat EES prevented seizure-induced brain injury and(0.08 mPa, 70C). Finally, a 2 000-mL extractioncognitive impairment, and reduced the risk for seizuresolution was harvested and stored in the cold for furtherrecurrenceQuality standards( total nitrogen quality): 0.131 gMATERIALS AND METHODSammonium chloride was mixed with water to form a1.31 mg/mL ammonium chloride solution. HarvestedDesignsolution(0.05, 0.1, 0.2, 0.3, and 0. 4 mL) was added toA randomized, controlled, animal experiment20 mL polyvinyl alcohol solution(0. 1 g/L), 1 mLTime and settingnaphthalene reagent, and polyvinyl alcohol solution in aExperiments were conducted from May 2007 to April25-mL volumetric flask, which served as the control2008 at the Drug Research& Development Center,solution. A 25-mL flask containing 20 mL polyvinylKanghong Pharmaceutical, China and Department ofalcohol solution(0.1 g/L), 1 mL naphthalene reagent, andPathology, Sichuan Academy of Medical Sciencespolyvinyl alcohol solution was considered the blankSichuan Provincial People's Hospital, Chinasolution. the absorbance values of control and blankMaterialssolutions were read at 460 nm using colorimetricAnimalsdetermination. A standard curve was plotted to calculateA total of 186 clean, healthy, male, Sprague-Dawley rats, the regression equation as follows: y=0.542 4X+0.021 0,aged 3 months and weighing 200 20 g, were provided0.999 2(supplementary Table 1 online). According toy the Experimental Animal Center of Zhengzhouthe standard curve, total nitrogen quality in the scorpionUniversity, China(license No SCXK (Yu)2005-0001)extract measured 8.83% and total nitrogen content wasAll experimental use of animals were in strict4.49 ma/mLaccordance with the Guidance Suggestions for the Care Methodsand Use of Laboratory Animals, issued by the Ministry Medication preparationsof Science and Technology of ChinaAccording to Chinese Pharmacopoeia 2005 edition, theScorpion source and extractioncorpion clinical dosage is 512 g, and the conversionScorpions are present in Shandong Province of Chinafactor of equal effect between various animals andand are harvested from April to September each yearhuman was equivalent to 7(W). The daily dose in theChengdu Huasun Group(Chengdu City, Sichuanpresent rats was defined as follows: 120 mg/kg valproicprovince, China)provided the scorpions for the present acid, 290, 580, and 1 160 mg/kg for the low, medium-study". According to the Chinese Pharmacopoeia 2005 and high-dose EES (supplementary Table 2 onlineversion2, scorpion has been confirmed and qualifiedPreparation of epileptic modelsScorpion extract is utilized with the hot-dip methodEpileptic models were established in rats as previously(Appendix XA), according to the alcohol-soluble extract described -so. briefly, rats were intraperitoneallyassay in Pharmacopoeia of the People s Republic ofinjected with 127 mg/kg lithium chloride(Sigma, St Louis,China 2005 edition. In brief, scorpions were crushed and Mo, USA)on the first day, followed by 1 mg/kg atropineeighed, and 3.016 6 g samples were placed in 250-mL sulfate(Tianjin Jinyao Amino Acid Co Ltd, China)18-Erlenmeyer flask, and were mixed with 100 mL diluted 24 hours later,ethanol for 1 hour. Samples were extracted by heat reflux 30 minutes late中国煤化工cording tofor 1 hour, then cooled and weighed Sample weight loss Racine classificCNMH Gure: I levelas supplemented with diluted ethanol. Samples werefacial spasm and isolated myoclonus; Il level globalLiang Y, et al. / Neural Regeneration Research 2012: 7(6): 426-433clonic convulsions; Ill level: global clonic convulsions and performed. The sections were then mounted onto glassstanding; IV level: global stiffness-clonic convulsionsslides For the negative controls, phosphate bufferstanding and falling V level: IV-level performancesaline was used instead of primary antibody. Samplesrecurred, showing status epilepticus or seizures to death. were observed under a light microscope( Leica, SolmsThe models were considered successful uponGermany). Within each slice, eight fields of view wereappearance of level Iv global seizures, normal behavior randomly selected under 400 x magnification. Thedid not return between the two episodes 32, and status number of positively stained cells was calculated usingepilepticus lasted for>15 minutes. If the rats exhibited Image-pro Plus 6.0 software(Media Cybernetics,no grade IV seizures or greater by 30 minutes, andethesda, MD, USA)in accordance with stereologicalstatus epilepticus occurred within 15 minutes afterrinciples. The average number of GFAP-positiveintraperitoneal injection of 10 mg/kg pilocarpine, then the cells per unit in the CAl, CA3, and dentate gyrus ofinjection was terminated. If status epilepticus was not each slice was analyzed for statistical analysisobserved within 15 minutes after injection, the rats were Reverse transcription-PCR detection of GFAP mRNAtreated with additional pilocarpine(10 mg/kg)everyexpression in rat hippocampus15 minutes until status epilepticus was inducedTotal RNA was extracted from the rat hippocampusExperimental intervention and clinical efficacyaccording to instructions from the Trizol kit(InvitrogenRats were orally administered the corresponding drugs Carlsbad, CA, USA; 15596-018)and primers wereat 15 minutes after seizures valproic acid group: valproic designed using Primer Premier 5.0 software(Premieracid(120 mg/kg; 200 mg/tablet; Hunan XiangzhongBiosoft, Palo Alto, CA, USA) Primer sequencing wasPharmaceutical, China); EES groups: 290, 580, andsynthesized at Shanghai Gene Core Bio Technologies,1 160 mg/kg EES; control group and model group: equal China. Target gene GFAP upstream primer: 5'-CGA CCTdose of saline. If status epilepticus lasted >1 hour, or TGA GTC CTT GCG 3, downstream primer: 5'-CCGrats were endangered by convulsions, the rats wereTCT TTA CCA CGA TGT T3, amplified fragment lengthintraperitoneally injected with atropine sulfate(1 mg/kg) was 416 bp. Internal reference GAPDH upstream primerand diazepam (10 mg/kg; Tianjin Pharmaceutical, China) 5-ACC ACA GTC CAT GCC ATC AC-3, downstreanto terminate the seizure and improve survival rates. If the primer 5'-TCC ACC ACC CTG TIG CTG TA-3seizures were not alleviated, diazepam was repeatedly amplified fragment length was 485 bp PCR conditionsadministered until the seizures were controlled. At20 cycles total of 94oC for 2 minutes; 94oC for 15 seconds;8: 00 am daily, the rats were treated with the same drug 555C for 20 seconds; and 72 C for 20 seconds. Theintervention to observe the seizures. Samples wereabsorbance value of PCR products was calculated usingcollected at 4 hours after drug administrationTotalLab v2.01 software(TotalLab, Newcastle, UK) andObservations were performed over a course of 30 days. results represented the ratio of target genePreparation of rat hippocampal tissueroduct/GAPDHRats were conventionally anesthetized and perfused with Statistical analysisnormal saline. The left cerebral hemisphere wasData were analyzed using SPSS 16.0 statistical packageremoved and fixed with 4% paraformaldehyde solution, (SPSS, Chicago, IL, USA) Grouped data wereand the fixed paraffin sections were utilized forcompared using the chi-square test and rank sum test.immunohistochemical staining Hippocampal tissuesand measurement data were expressed as mean t SDwere separated from the right hemisphere and frozen in for analysis of unequal distance and repeatedliquid nitrogen for reverse transcription-PCR quantitative measurement data. Pairwise comparisons wereconducted using the least significant difference test orGFAP immunohistochemistry streptavidin bioting-test. P< 0.05 was considered statistically significantmethod in the rat hippocampusSections were conventionally de-waxed and hydrated; Author contributions: Hongbin Sun had full access to theendogenous peroxidase activity was eliminated bystudy design and revised the manuscript. All authorsincubating the sections in 3%H2O2. The sections were participated in experiment implementation and data collectionmicrowaved in 0.01 M sodium citrate buffer (pH 6.0)for Yi Liang drafted the manuscript and performed dataantigen retrieval, and then the sections were incubatedinterpretation and analysis in strict accordance with statisticalin rabbit anti-rat GFAP monoclonal antibody(1: 100Beijing Biosynthesis Biotechnology, China; bs-0199R) Conflicts of interest: None declaredat 4C overnight, followed by biotinylated goatFunding: The study was sponsored by the National Naturalanti-rabbit IgG(1: 500; Beijing Biosynthesisnce Foundation of China, No. 30740035; and Key ScientificBiotechnology )at 37C for 30 minutes and horseradish and Technological Project of Sichuan Province, No.05SG1672peroxidase-labeled avidin(Beijing BiosynthesisEthical approval: Animals protocols complied with nationalBiotechnology) at 37C for 30 minutes. DAB(DAB kit; ethics requireme中国大anre approved byBeijing Zhongshan Golden Bridge Company, Chinathe animal ethi煤化工 remy ofZLl-9031/90329033) was used as a chromogen for the Medical SciencesCN MHGHospitalstaining, and hematoxylin counterstaining wasLiang Y, et al. / Neural Regeneration Research 2012: 7(6): 426-433Acknowledgments: We would like to thank Dr Huarong Yang[14] Besnard F, Brenner M, Nakatani Y, et al. Multiple interacting sitesfrom Chengdu University of Traditional Chinese Medicine andulate astrocyte-specific transcription of the human gene forChengdu Huasun Group, China for guidance with the scorpionglial fibrillary acidic protein. J Biol Chem. 1991: 266(28)extraction process; Supervisor Xiaoping Gao, Dr Jianming Wu,[15] Wang Y, Chen LW, Rao ZR. Change in expression of gfap and fosand Yong Yang from Kanghong Pharmaceutical, China forprotein in forebrain of pentylenetetrazol( PTZ)-induced seizure inguidance with the lithium/pilocarpine-induced epilepsy modelChin J Clin Neurosci. 2004; 20(2): 159-16Supervisor Xiaoping Gao and Jiancheng San from the Drug[16] Rogawski MA, LO scher W. The neurobiology of antiepileptic drugsResearch Development Center, Kanghong PharmaceuticalNat Rev Neurosci. 2004: 5(7): 553-564[17]Kaindl AM, Asimiadou S, Manthey D, et al. Antiepileptic drugs andChina for guidance with reverse transcription-PCR; anhe developing brain Cell Mol Life Sci. 2006; 63(4): 399-413Professor Guizhen Chu from the Department of Pharmacology,[18 Zhao R, Zhang XY, Yang J, et al. Anticonvulsant effect of BmK IT2Sichuan University West China Medical Center and Longchuna sodium channel-specific neurotoxin, in rat models of epilepsy. 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