Overexpression of γ-aminobutyric acid transporter subtype I leads to susceptibility to Kainic acid-induced seizure in transgenic mice

γ-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter, and the GABAergic synaptic transmission is normally terminated by the rapid uptake through GABA transporters. With transgenic mice ubiquitously overexpressing GABA transporter subtype I (GAT1), the present study explored the pathophysiological role of GAT1 in epileptogenesis. Though displaying no spontaneous seizure activity, these mice exhibit altered electroencephalographic patterns and increased susceptibility to seizure induced by kainic acid. In addition, the GABAA receptor and glutamate transporters are up-regulated in transgenic mice, which perhaps reflects a compensatory or corrective change to the elevated level of GAT1. These preliminary findings support the hypothesis that excitatory and inhibitory neurotransmission, and seizure susceptibility can be altered by neurotransmitter transporters.

Temporal lobe epilepsy animal model established by stereotaxic microinjection of kainic acid

BACKGROUND： Kainic acid can be used to induce a model of epilepsy by systemic injection, such as intraperitoneal or subcutaneous injection. Individual rats have different responses to kainic acid, therefore high doses of drug are required and the success rate of model induction is low. It is necessary to develop an improved method to establish a temporal lobe epilepsy （TLE） animal model. OBJECTIVE： To explore an economic, stable and efficient method of establishing a TLE animal model. DESIGN, TIME AND SETTING： A completely randomized, controlled study. The experiments were performed in the Cellular Function Laboratory of the Physiology Department, Anhui Medical University from March to July 2007. MATERIALS： Twenty adult male Wistar rats, weighing 230-260 g, were provided by the Experimental Animal Centre of Nanjing Medical University. Kainic acid was purchased from Sigma in USA. Type SN-2 stereotaxic apparatus was made by Narishge in Japan. METHODS： Wistar rats were randomly divided into a kainic acid （KA） group （n = 12） and a normal saline （NS） group （n = 8）. For intrahippocampal microinjection, a burr hole was drilled in the skull at the following stereotaxic coordinates： anteroposterior （AP） 4.1 mm caudal to bregma; lateral （ML） 4.2 mm right lateral to the midline. Rats in the KA group were injected with 2.5 μL KA （0.4 g/L） into the center of the CA3 region, while in the NS group the same volume of NS was injected into the same site. MAIN OUTCOME MEASURES： Both groups were monitored under a video capture system for 12 weeks to record spontaneous seizures. Intracranial eletroencepholograph （IEEG） recordings in vivo were performed after the behavioral observations. After the IEEG recordings, hippocampi were processed into coronal sections. Nissl and Timm stainings were then performed to observe and confirm pathology. RESULTS： Twenty rats were involved in the final analysis. Behavioral observations： the eadiest spontaneous onset of epilepsy appeared 2 weeks after injection of KA. Eight rats had spontaneous onset of epilepsy 3-12 weeks after treatment. None of rats in the NS group had spontaneous onset of epilepsy. IEEG recordings： Epileptic-form waves, such as sharp waves and spike waves, were calculated by artificial analysis The number of epileptic-form waves in the KA group increased significantly compared to those of the NS group （P 〈 0.01）. Morphology results： In the KA group, Nissl staining and Timm staining revealed typical pathology in the hippocampal temporosphenoid lobe. In the NS group, no pathology was observed. CONCLUSION： Intrahippocampal microinjection of KA is a reliable method to establish a temporal lobe epilepsy animal model, requiring low doses of kainic acid and giving a high rate of success.

Gastrodin blocks neural stem cell differentiation into glial cells mediated by kainic acid

Kainic acid can simulate excitatory amino acids in vitro. Neural stem cells, isolated from newborn Wistar rats, were cultured in vitro and exposed to 100 4 000 #M kainic acid for 7 days to induce neuronal cell differentiation, causing the number of astrocytes to be significantly increased. Treatment with a combination of 0.5 mg/L gastrodin and kainic acid also caused the number of differentiated neurons to be significantly increased compared with treatment with kainic acid alone Experimental findings suggest that gastrodin reduces the excitability of kainic acid and induces neural stem cell differentiation into neurons.

Effects of rutin on oxidative stress in mice with kainic acid-induced seizure

Flavonoids are present in foods such as fruits and vegetables. Several studies have demonstrated a relationship between the consumption of flavonoid-rich foods and prevention of human disease, including neurodegenerative disorders. We assessed the effect of rutin （quercetin-3-O-rutinoside） on oxidative stress in kainic acid （KA）-induced seizure. METHODS： Thirty-six BALB/c mice were randomly divided into three groups. In the control group, saline （intra-peritoneal, i.p.） was administered for 7 d, and on the last day, KA （10 mg/kg, i.p.） was injected 30 min after administration of saline. In rutin groups, mice were pretreated with rutin （100 and 200 mg/kg, i.p.） for 7 d, and on the last day, KA （10 mg/kg, i.p.） was injected 30 min after administration of rutin. Subsequently, behavioural changes were observed in mice. Lipid peroxidation and oxidative stress were measured respectively in the early and late phases after KA-induced seizures. RESULTS： Seizure scores in the rutin groups were significantly lower than those in the control group （P 〈 0.01）. Furthermore, rutin dose-dependently inhibited the number of wet-dog shakes （WDS） （P 〈 0.05）. Malondialdehyde level in the hippocampus of the rutin groups was significantly lower than that in the hippocampus of the control group on days 1 and 21 after KA administration. In the rutin groups, the thiol levels observed on day 1 after KA administration were higher than that in the control group （P 〈 0.01）. CONCLUSION： These results indicate that rutin has potential anticonvulsant and antioxidative activities against oxidative stress in KA-induced seizure in mice.

Protective effects of gastrodine on dendritic spines in kainic acid-injured neurons

EphB2 affects neuronal cells by altering the dendritic spines. The present study analyzed the neu-roprotective effects of gastrodine by measuring EphB2 expression in rat neural cells cultured in vitro and injured by kainic acid. Gastrodine （12.5, 25, and 50 mg/L） improved morphology in kainic acid-injured neurons, reduced lactate dehydrogenase leakage, decreased neuronal apoptosis, and increased EphB2 expression in neuronal cells. A moderate dose of 25 mg/L gastrodine resulted in the most significant effects. These results suggested that gastrodine suppressed the neurotoxic effects of excitatory amino acids and provided neuroprotection by remodeling neuronal dendritic spines.

N-methyl-D-aspartate receptor subunit 1 protein expression in the hippocampus and temporal cortex of kainic acid-induced epilepsy rats

BACKGROUND： The N-methyl-D-aspartate receptor subunit 1 （NMDAR1） contributes to the incidence of epilepsy. However, the relationship between epilepsy-induced brain injury and NMDAR1 remains poorly understood. OBJECTIVE： To investigate changes in NMDAR1 protein expression in the hippocampus and temporal cortex of kainic acid-induced epilepsy rats. DESIGN, TIME AND SETFING： A randomized, controlled, animal experiment was performed at the Department of Physiology and Department of Pathology, Basic Medical College of Jilin University from March 2002 to March 2003. MATERIALS： Rabbit anti-NMDAR1 antibody was purchased from Wuhan Boster Biological Technology, China. METHODS： A total of 80 healthy, male, Wistar rats, aged 22 weeks, were randomly assigned to sham-surgery （n = 10） and model （n = 70） groups. Epilepsy models were established by injecting kainic acid （1μL） into the right amygdala, and rats were sacrificed at 2, 6, 24, 72 hours, and 7, 15, 30 days after surgery, with 10 animals at each time point. The rats in the sham-surgery group were injected with 1μL phosphate buffered saline into the right amygdala. MAIN OUTCOME MEASURES： NMDAR1 protein expression in the hippocampus and temporal cortex at 2, 6, 24, 72 hours and 7, 15, 30 days after epilepsy was detected using immunohistochemistry and flow cytometry analysis. RESULTS： In the sham-surgery group, a few NMDARl-positive cells were distributed in the hippocampus and temporal cortex. In the model group, NMDARl-positive cells were increased in the hippocampus and temporal cortex at 2 hours following kainic acid-induced epilepsy. They were significantly increased at 6 hours, and slightly decreased at 7 days （CA3 region and temporal cortex）, but remained greater than the sham-surgery group. This continued until day 30 （P 〈 0.01 ）. In addition, there were more NMDAR1 positive cells in the hippocampal CA3 and dentate gyrus than the temporal cortex （P 〈 0.01）. CONCLUSION： In epilepsy model rats, NMDAR1 protein expression was upregulated in the hippocampus and temporal cortex, and in particular in the hippocampal CA3 and dentate gyrus. NMDAR1 may participate in epilepsy and the excitation process of the epileptic brain.

Synthesis of both epimeric triacid ananlogs of kainic acid

A practical and cheap method for synthesis of C-4 carboxylic acid substituted kainic acid analogue 5 and its epimer 6 from trans-4-hydroxyproline is described. Using this method, more interesting intermediates and analogues could be obtained easily.

Mitochondrial and nuclear damages and caspase-3 expression in the hippicampal CA3 region of rats with kainic acid induced statuse pilepticus

BACKGROUND: Some scholars believed that the neuronal injury after status epilepticus is apoptosis, the main evidence is the changes of expressions of various apoptosis related genes, such as immediate-early gene, p53 gene and genes of bcl-2 family, etc. But there is still no ultrastructural evidence for apoptosis. OBJECTIVE: To observe the ultrastructural damages of mitochondrion and nucleus and the changes of caspase expression in neurons of hippocampal CA3 region in rats with status epilepticus induced by kainic acid. DESIGN: A randomized controlled study. SETTING: Department of Anesthesiology and Department of Neurology, Qilu Hospital of Shandong University. MATERIALS: Seventy-five adult male Wistar rats of 250-300 g, clean degree, were provided by the experimental animal center of Shandong University. Kainic acid was purchased from Sigma Company (USA); rabbit anti-rat polyclonal antibody caspase-3 from Santa Cruz Company (USA). METHODS: The experiments were carried out in the Department of Anesthesiology, Qilu Hospital of Shandong University from October 2005 to February 2006. ① The 75 rats were randomly divided into experimental group (n =45) and control group (n =30). ② Model establishment, convulsion grading and the judging standards for status epilepticus: Rats in the experimental group were given intraperitoneal injection of kainic acid (10 mg/kg), and those in the control group were injected with saline of the same volume. The time of seizure was recorded and their behavioral manifestations were observed, and the seizure was terminated by intraperitoneal injection of diazepam (10 mg/kg). ③ Observation under electron microscope: At 3, 12 and 24 hours after status epilepticus respectively, bilateral hippocampal tissues were taken out, semithin sections of about 75 nm were prepared after fixation, dehydration and embedding, and then observed under H-800 transmission electron microscope. ④ Immunohistochemical detection: Bilateral hippocampi were removed at 3, 12 and 24 hours after status epilepticus respectively, the fixation, dehydration, transparence, wax immersion and embedding were performed, then serial sections of CA3 region were immunohistochemically determined by the SABC method. Leica QWinV3 image analytical software was applied, then the average number and average gray value of positive cells were calculated. MAIN OUTCOME MEASURES: Results of observation under electron microscope, that of immunohistochemical staining of neurons in hippocampal CA3 region; Comparison of number of caspase-3 positive cells and gray value. RESULTS: All the 75 Wistar rats were involved in the analysis of results. ① Results of observation under electron microscope: At 3 hours after status epilepticus, swelling crista and membranous disintegration were observed under electron microscope. At 24 hours, obvious nuclear changes occurred, and manifested as the side-aggegation of chromatins. ② Results of immunohistochemical detection: In the experimental group, the number of caspase-3 positive cells at 3 hours after status epilepticus had no obvious difference as compared with that in the control group (P > 0.05); At 12 hours, the number and gray value of caspase-3 positive cells in the experimental group were higher than those in the control group (10.49±0.68 vs. 5.33±0.43; 45.57±2.27 vs. 19.79±0.33, P < 0.05), the same results were also observed at 24 hours (37.36±0.57 vs. 5.12±0.47; 115.24±1.22 vs. 18.73±0.42, P < 0.01). CONCLUSION: In the rat models of status epilepticus induced by kainic acid, mitochondrial damage was earlier than the increase of caspase-3 expression and nuclear changes, which suggested that mitochondrion was the key link for the neuronal death after status epilepticus.

A Study of Epileptiform Hippocampal Unit Activity Induced by Intracerebroventricular Injection of Kainic Acid in Rats

Hippocampal EEG and unit activities were recorded after seizures were initiated withintracerebroventricular injection of kainic acid (KA) in rats. Three kinds of hippocampal unit re-sponses to KA were found:1. Positive units: These units were characterized by high frequency burst firing temporally coincidentwith each hippocampal EEG spike.2. Negative units: These units showed a cessation of firing during each EEG paroxysm.3. Indifferent units: These units showed no evident chanses coincident with EEG paroxysms.Most positive units were hippocampal complex spike cells which correspond histologically tohippocampal pyramidal cells, and most complex spike cells fired in positive bursts after KA treat-ment. In the early period after KA injection, the positive units were concentrated in CA3 area. It was suggested that the activities of positive units may be considered as the typical epileptiformhippocampal unit activity induced by KA, and the firing features of negative units ma be the resultof the influence of hippocampal inhibitory interneurons or the result of excessive cellulardepolarization, and that hippocampal pyramidal cells were more sensitive to the epileptogenic ef-fect of KA than hippocampal intemeurons, and some pyramidal cells in CA3, in particular,may serve as "epileptic pacemaker neurons " in KA-induced epileptogenesis.

Dynamic changes of mG1uR1 mRNA expression in rat hippocampus during seizures induced by kainic acid

Objective: To investigate temporospatial changes of mG1uR1 mRNA expression in rat hippocampus during seizures induced by kainic acid. Methods: Using in situ hybridization with Dig-labeled cDNA probes to examine mG1uR1 mRNA expression. The relative number of hybridization signal was quantified by image analysis systems. EEG and behavior changes of the rats were simultaneously observed. Results: All rats of the KA-injected group exhibited severe limbic convulsions. The EEG recordings also showed electrical seizure activities. The mG1uR1 mRNA expression began to increase at 1 h after KA injection and reached its maximal level at 4 - 8 h, and then it began to decrease gradually and came to the lowest by the end of 72 h. Conclusion: The enhanced early expression of mG1uR1 mRNA in hippocampus suggested that mG1uR1 may play an important role in epilepogenesis and may be related to the subsequent neuronal damage.

Time-dependent changes of astrocytes following microinjection of kainic acid and irradiation by gamma-knife in adult rat brain

objective: Two groups of rats were microinjected with kainic acid (KA) and irradiated with gam ma knife respectively on unilateral nucleus caudate-putamen to compare the response of astrocytes. Methods: The astrocytes were identified with anti-GFAP immunohistochemical ABC method and the progress of their reaction to the 2 insults was examined from 3 h to 30 d after the lesion. Results: Both lesions could induce hyperplasia and hypertrophy of astrocytes and 2 types of GFAP-ir cells were found, one with small cell body and thin process, and the other with hypertrophic cell body and thick and long process. The timecourse of GFAP expression in the 2 groups was different. In KA microinjection group, large necrotic area was ob served in the target within 24 h. Three days later, a few astrocytes appeared around the necrosis. With in crease of the survival time, hyperplasia and hypertrophy of astrocytes began to increase. Whereas in gamma knife group, hyperplasia and hypertrophy were evident from 3 h to 7 d and necrotic dots could be seen in the target on day 14. On day 30, necrosis was tnore obvious with gradual variations in GFAP expression around the necrotic area. Conclusion: The above results indicated that GFAP could be used as a marker for CNS in jury; the difference in their timing and distribution pattern suggested different mechanisms in KA microinjec tion group and gamma-knife irradiation group.

Kainic Acid, NMDA and Bicuculline Induce Elevation in Concentrations of Glutathione and Amino Acids <i>in Vivo</i>: Biomarkers for Seizure Predisposition?

The present study was carried out to investigate the effect of NMDA, bicuculline and kainic acid (KA) on the extracellular concentration of glutathione, phosphoethanolamine (PEA) and taurine in rat hippocampus in vivo. Rats were implanted with intrahippocampal microelectrodes perfused with free-glucose Krebs-Ringer solution and allowed to recover for about 2 h. After assaying baseline concentrations of amino acids, NMDA or bicuculline was administered intrahippocampally, whereas KA was given systemically. Either treatment resulted in significant high extracellular concentrations of glutathione, but only NMDA or KA resulted in high concentrations of PEA and taurine. Interestingly, the increase in glutathione concentration due to KA was followed by a delayed increase of glutamate and PEA. Our results demonstrated that increased efflux of glutathione, a common consequence of different neuroexcitotoxic agents, occurs in vivo. Given that the agents used in the present study were also convulsunts, the implication of the findings on seizure predisposition was also considered.

Coexistence of nitric oxide synthase with γ-aminobutyric acid during seizure induced by kainic acid in SD rats

Objective: Functional significance of NO and central inhibitory neurotransmitter γ-aminobutyric(GABA) during seizures were investigated morphorlogically. Methods: A kainate-induced complex partialseizure model was used in our experiment. Twenty SD rats were randomly divided into KA 30, 60, 90, 200min and control groups. The brain sections were stained by NADPh (nicotinamide adenine dinucleotide phosphate ) diaphorase (Nd ) histochemically, and were further stained by GABA immunohistochemically.Results: Histological and immunohistochemical study revealed that in KA groups the number of Nd and GABA-positive double labelled neurons in CA3 region, CA3 region and dentate gyms was significantly reduced,compared with the control group. Conclusion: Nd coexisted with GABA in the brain. Reduction of GABA release led to relief of GABA-ergic inhibition and in the same way, reduction of NO release weakened its negative feedback modulation. Therefore neuronal synchronous paroxysmal discharges increased. GABA and NO,both having antiepileptic action, acted through different ways or different link in the same way. NO may involve in the effect of GABA-ergic neurons and play cooperative antiepileptic action with GABA.

Changes of Monoanine Levels in Different Brain Regionsof Rat with Ventral GIobus Pailidus Injured by KainicAcid Injection

Ventral globus pallidus-injured rats by kainic acid(10 mg) were used to derter-mine the monoamine levels in four different brain regions.In this model,a great decrease of NE con-centration was observed in both hippocampus and frontal cortex compared with nonnal contro.This result hints that a damage of noradrenergic neurons in this model has occurred. DA concentrations in the four brain zones after kainic acid injection were all reduced, but only in the frontal cortex and striatum the pronounced reductions were discovered while DA turnover rates in frontal cortex, stria-tum and meddullapons were significantly reduced. These results revealed a DA metabolic disorder occurring in these regions.However,5-HT concentrations as well as DBH activity, expressed by ratio of NE/DA,showed no marked difference in this model. In our study it is found that the changes of monoamine levels in this model basically reflect those discovered in AD patients.

中缝背核内微量注射海人酸(KAINIC ACID)对唇针镇痛效应的影响

我们以往的实验表明,脑内5-羟色胺能神经原系统在唇针镇痛中起着重要的作用。电解损毁5-羟色胺能神经原胞体集中的中脑中缝核,唇针镇痛效应降低;电刺激中缝背核,则唇针镇痛效应增高山。 电解损毁核团目前还是经常采用的研究脑功能的一种方法,但此法不仅损毁了所要研究的核团内的细胞体。

Effect of ketogenic diet on hippocampus mossy fiber sprouting and GluR_5 expression in kainic acid induced rat model

Ketogenic diet （KD） is a high fat, low protein, low carbohydrate diet. Its antiepileptic effect is certain but the underlying mechanism is unknown. Mossy fiber sprouting in the inner molecular layer of the dentate gyrus causes the synaptic reorganization in the hippocampus, which is an important cause of temporal lobe epilepsy in animals and humans. It is also essential to the genesis and development of epilepsy. As the predominant excitatory neurotransmitter in the central nervous system, glutamate plays a role in synaptic reorganization and development of epilepsy.

神经元限制性沉默因子REST/NRSF参与调控癫痫作用及机制研究

目的探究神经元限制性沉默因子(REST/NRSF)参与调控癫痫作用及分子机制。方法采用免疫组织化学染色、免疫荧光、Western blot和qPCR检测癫痫患者病灶组织和海人藻酸(kainic acid,KA)癫痫小鼠海马脑区REST/NRSF表达水平的变化;采用病毒注射,脑电图记录和行为学方法检测在海马CA1区分别敲低或过表达REST/NRSF后对小鼠癫痫发作的影响。结果癫痫患者病灶REST/NRSF表达水平相对于脑外伤患者脑组织明显升高;KA模型组小鼠海马CA1区REST/NRSF蛋白和mRNA水平均明显升高,Kv7.2、Kv7.3钾通道mRNA表达水平明显下调;脑内注射NMDA兴奋海马脑区小鼠REST/NRSF表达水平明显上调;海马CA1区敲低REST/NRSF明显升高Kv7.2、Kv7.3钾通道mRNA表达水平,明显降低小鼠脑电图棘波、尖波发放频率以及癫痫发作等级;海马CA1区过表达REST/NRSF明显降低Kv7.2、Kv7.3钾通道mRNA表达水平,明显升高小鼠棘波、尖波发放频率,癫痫症状明显加重。结论小鼠海马脑区REST/NRSF通过转录调控Kv7.2、Kv7.3钾通道参与癫痫疾病发生发展。

恒清Ⅴ号方干预海人藻酸致癫痫小鼠的作用机制研究

目的:探讨恒清Ⅴ号方干预海人藻酸致癫痫小鼠的作用机制。方法:选取雄性野生型C57BL/6J小鼠40只,随机分为正常对照组、模型组、恒清Ⅴ号方干预组及丙戊酸钠干预组,每组10只。恒清Ⅴ号方干预组给予恒清Ⅴ号方灌胃,丙戊酸钠干预组给予丙戊酸钠溶液腹腔注射,正常对照组及模型组给予生理盐水灌胃,每日1次,持续10 d。最后1次给药干预后,模型组、恒清Ⅴ号方干预组及丙戊酸钠干预组均给予海人藻酸腹腔注射以诱发小鼠癫痫发作,观察各组小鼠行为变化及空间工作记忆能力。观察结束后检测各组小鼠海马组织中丙二醛(MDA)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和总抗氧化能力(T-AOC)水平。结果:与正常对照组比较,模型组小鼠癫痫持续状态时间明显延长,癫痫潜伏期缩短,Ⅲ级以上惊厥发作次数增加,新异臂探索次数、新异臂探索时间及新异臂探索路程均减少,差异均有统计学意义(P<0.01)。与模型组比较,恒清Ⅴ号方干预组与丙戊酸钠干预组癫痫持续状态时间均明显缩短,癫痫潜伏期延长,Ⅲ级以上惊厥发作次数减少,新异臂探索次数、新异臂探索时间及新异臂探索路程均增加,差异均有统计学意义(P<0.01)。与丙戊酸钠干预组比较,恒清Ⅴ号方干预组癫痫持续状态时间缩短,癫痫潜伏期时间延长,Ⅲ级以上惊厥发作次数减少,新异臂探索次数、新异臂探索时间及新异臂探索路程均增加,差异均有统计学意义(P<0.05)。与正常对照组比较,模型组小鼠海马组织中MDA水平升高(P<0.01),SOD、CAT及T-AOC水平下降(P<0.01)。与模型组比较,恒清Ⅴ号方干预组、丙戊酸钠干预组小鼠海马组织中MDA水平均下降(P<0.01),SOD、CAT及T-AOC水平均升高(P<0.01)。与丙戊酸钠干预组比较,恒清Ⅴ号方干预组小鼠海马组织中MDA水平均下降(P<0.05),SOD、CAT及T-AOC水平均升高(P<0.05)。结论:恒清Ⅴ号方可以干预海人藻酸诱导小鼠癫痫发作,改善癫痫发作症状及其认知功能,其作用优于丙戊酸钠。恒清Ⅴ号方可能是通过抗氧化应激来发挥抗癫痫作用。

两种海人酸癫痫小鼠模型的建立及比较研究

目的比较分析海人酸(kainic acid,KA)侧脑室(intracerebroventricular,ICV)注射和腹腔注射(intraperitoneal injections,IP)两种给药路径建立的颞叶癫痫小鼠模型早期的行为学和病理学差异。方法100只C57BL/6N野生型(wild type,WT)雄性小鼠(体质量为20~22 g),按随机数字表法分为4组:ICV+normal saline(NS)对照组(n=10),ICV+KA模型组(n=40),IP+NS对照组(n=10),IP+KA模型组(n=40)。ICV+KA模型组使用600 nL的KA(0.5 mg/mL)进行侧脑室注射,IP+KA模型组按25 mg/kg剂量进行腹腔注射,对照组使用等量的生理盐水进行侧脑室和腹腔注射。建立模型3 d后,进行行为学、分子生物学(Western blot)和神经病理损伤评估(FJB染色,TUNEL染色,免疫荧光染色)。结果两对照组无痫性发作,两模型组均出现痫性发作。IP+KA组与ICV+KA组死亡率分别为47.50%和65.00%,造模成功率分别为80.00%与60.00%。与IP+KA组比较,ICV+KA组小鼠模型成功率明显增高而死亡率明显减少。FJB染色和TUNEL染色结果显示,与IP+KA组比较,ICV+KA组海马的神经变性和凋亡改变程度变化更加明显(P<0.05)。与IP+KA组比较,ICV+KA组海马凋亡蛋白表达差异也有统计学意义(P<0.05)。免疫荧光结果显示,ICV+KA和IP+KA组海马和皮层的星形胶质细胞和小胶质细胞较对照组明显激活(P<0.05),但是ICV+KA组海马和皮层的胶质细胞激活程度均强于IP+KA组(P<0.05)。ICV+KA组海马和皮层的GFAP和Iba-1蛋白表达均高于IP+KA组(P<0.05)。结论两种KA制备方法均可制备出成功的癫痫模型。但ICV路径制备癫痫小鼠具有更高的成功率和更少的死亡率,并且神经病理损害程度和胶质细胞激活程度更加明显。

DAPK1调控癫痫发作诱导的阿尔茨海默病样病变

目的 探讨死亡相关蛋白激酶1 (death-associated protein kinase 1,DAPK1)对小鼠癫痫发作诱导阿尔茨海默病(Alzheimer's disease,AD)样病变的调控。方法 C57BL/6小鼠经腹腔注射25 mg·kg^(-1)海人藻酸(kainic acid,KA),建立颞叶癫痫(temporal lobe epilepsy,TLE)模型,对照小鼠注射等量生理盐水,检测小鼠海马体中淀粉样代谢通路中关键蛋白的表达,可溶性β淀粉样蛋白(amyloidβ-protein,Aβ)的分泌,tau的高度磷酸化及DAPK1通路的活化;WT小鼠和Dapk1 KO小鼠经腹腔注射KA,考察DAPK1对KA诱导的AD样病变的影响。结果TLE小鼠海马体中可溶性Aβ的分泌、淀粉样代谢通路中关键蛋白的表达、tau的磷酸化水平,以及DAPK1的表达和活性均高于对照小鼠。相比WT小鼠,Dapk1 KO小鼠海马体中KA诱导的Aβ的分泌和tau的磷酸化水平均明显下降。结论 TLE诱导了小鼠AD样病变,而DAPK1敲除可缓解小鼠脑老化的特征。