缝隙连接蛋白介导的细胞间通讯与胶质瘤自杀基因治疗

1967年Kevel首次在大鼠心肌细胞间和肝细胞间发现了一种构造精妙的连接方式,并将之命名为"缝隙连接"(gap junction,CJ)。中枢神经系统细胞间存在有多种特异性连接蛋白(connexin,CX)的分布和表达并与中枢神经系统多种疾病的发生、发展密切相关,如胶质细胞瘤、脑膜瘤、癫痫、脑缺血缺氧性损伤及Alzheimer病等。CX、GJ及由此构成的细胞内信号转导(gap junction intercellular communication,GJIC)通路在肿瘤自杀基因治疗"旁观者效应"(bystander effect,BE)中发挥着极为重要的作用。GJ及GJIC的生理病理变化直接影响脑胶质瘤细胞的增殖、浸润及凋亡。     

胶质瘤转染Cx43cDNA与放疗的协同作用

目的探讨体外培养脑胶质瘤细胞转染Cx43cDNA与放疗是否存在协同作用及该作用与缝隙连接细胞间通讯功能(GJIC)的关系。方法LipofectAMINE2000介导质粒DNA转染,G418筛选,半定量RT-PCR及划痕荷载染料传输试验鉴定。分别以MTT法及流式细胞仪检测C6、C6-Non、C6-Cx43细胞放疗组和未放疗组的细胞增殖率及凋亡率,进而观察转染Cx43基因与放疗杀伤胶质瘤细胞是否有协同作用。结果①C6-Cx43细胞Cx43mRNA表达显著增加,染料传输能力较C6-Non明显增强;②转染Cx43cDNA,上调C6细胞GJIC功能后,放疗对脑胶质瘤细胞的毒性作用明显增强。结论①转染Cx43cDNA可上调C6细胞GJIC。②GJIC功能上调后,放疗对脑胶质瘤细胞的毒性作用明显增强,转染Cx43cDNA与放疗存在协同作用。③缝隙连接细胞间通讯可能是肿瘤转染Cx43cDNA与放疗存在协同作用的机制之一。     

反义miR-221/222上调Cx43恢复人胶质瘤细胞系U251细胞间缝隙连接通讯的体外研究

目的 探讨反义miR-221/222上调缝隙连接蛋白43(Cx43)以恢复人胶质瘤细胞系U251细胞间缝隙连接通讯的作用机制.方法 脂质体共转染反义寡核苷酸(AS-miR-221/222),下调U251细胞miR-221和miR-222表达水平,采用Northern blotting法检测U251细胞miR-221和miR-222表达水平;虫荧光素酶活性分析并获得靶基因;Western blotting法和免疫荧光法检测U251细胞Cx43表达水平;划痕标记染料示踪技术检测细胞间缝隙连接通讯.结果 AS-miR-221/222组U251细胞miR-221(t=1312.152,P=0.000)和miR-222(t=1226.031,P=0.000)表达水平明显降低;荧光活性明显高于其他各组(均P=0.000),证实Cx43基因为miR-221和miR-222靶基因;Cx43表达水平亦明显升高,且高于无义序列组(t=735.768,P=0.000)和对照组(t=686.252,P=0.000).对照组和无义序列组U251细胞细胞间缝隙连接通讯缺失,罗氏黄仅传递划痕细胞边缘单列细胞;AS-miR-221/222组U251细胞细胞间缝隙连接通讯明显恢复,罗氏黄传递至划痕细胞邻近7～8列细胞.结论 反义miR-221/222可通过上调Cx43表达水平恢复人胶质瘤细胞系U251细胞间缝隙连接通讯.     

癫痫大鼠海马缝隙连接蛋白43的表达及生胃酮的保护作用

目的研究癫痫大鼠海马星形胶质细胞缝隙连接蛋白43(Connexin43,CX43)表达及缝隙连接蛋白阻滞剂生胃酮对其表达的影响。方法用免疫组化法检测癫痫发作后各时间点大鼠海马星形胶质细胞CX43免疫反应阳性表达。同时观察致痫前给予不同剂量生胃酮对大鼠海马星形胶质细胞CX43免疫反应阳性表达的影响。结果对照组大鼠海马星形胶质细胞CX43可见少量散在表达。癫痫组大鼠海马星形胶质细胞CX43表达1h即可见增加,并随时间延长而增加,72h达高峰。生胃酮各组大鼠海马星形胶质细胞CX43免疫反应阳性表达较同一时间点癫痫组低(P<0.01),且与生胃酮剂量呈负相关(P<0.01)。结论癫痫大鼠海马缝隙连接蛋白43的表达与癫痫发病机制密切相关。缝隙连接蛋白阻滞剂生胃酮影响缝隙连接蛋白43表达,具有肯定的神经保护作用。     

BMSCs作为HSV-tk载体联合CX43增强旁效应治疗胶质瘤的实验研究

目的 以骨髓间充质干细胞(BMSCs)为自杀基因HSV-tk载体,联合连接蛋白CX43介导的旁效应,对以BMSCs肿瘤追踪特性为基础的胶质瘤进行治疗研究.方法 以CX43质粒转染C6胶质瘤细胞,以腺病毒介导HSV-tk转染BMSCs;建立C6胶质瘤模型,实验动物分为4组:C6对照组、CX43-C6组、tk-BMSCs/C6/GCV治疗组和tk-BMSCs/CX43-C6/GCV治疗组.观察动物生存期,MRI监测肿瘤体积;脑切片行PCNA、原位凋亡检测肿瘤中央与边缘部位细胞增殖与凋亡情况.结果 CX43组、tk/GCV组生存期长于对照组,二者联合治疗组生存期最长,MRI示部分肿瘤消失;肿瘤内部与边缘细胞增殖活性下降,凋亡明显.结论 tk-BMSCs/GCV体系可有效杀伤侵袭瘤细胞在内的胶质瘤灶,连接蛋白CX43可增强此治疗效果.     

人脑胶质瘤Cx43基因表达及其与肿瘤细胞增殖关系的研究

目的 探讨缝隙连接蛋白43(Cx43)基因表达与胶质瘤细胞增殖之间的关系,及其在胶质瘤发生发展中的作用,拟为手术后治疗及疗效观察提供客观依据.方法 采用原位杂交和免疫组织化学染色方法检测胶质瘤细胞Cx43 mRNA、Cx43 蛋白和增殖细胞核抗原的表达水平.结果 对照脑组织和胶质瘤细胞Cx43 mRNA 阳性表达率分别为100%(10/10)和61.70%(29/47),差异具有统计学意义(Z =- 5.407,P = 0.000);低度恶性(WHOⅠ～Ⅱ级)胶质瘤细胞Cx43 mRNA 阳性表达率为100%(7/7)和93.75%(15/16),高度恶性(WHOⅢ～Ⅳ级)为33.33%(6/18)和16.67%(1/6);Cx43 mRNA 阳性表达率与胶质瘤组织病理学分级呈负相关(rs = - 0.794,P = 0.000).对照脑组织和胶质瘤细胞Cx43 蛋白表达水平与Cx43 mRNA 基本一致.对照脑组织增殖细胞核抗原表达阴性;不同级别胶质瘤细胞增殖细胞核抗原阳性表达率依次为WHOⅣ级100%(6/6)、WHOⅢ级94.44%(17/18)、WHOⅡ级62.50%(10/16)和WHOⅠ级42.86%(3/7);增殖细胞核抗原阳性表达率与胶质瘤组织病理学分级呈正相关(rs = 0.589,P = 0.000);WHOⅢ～Ⅳ级与WHOⅠ～Ⅱ级之间差异具有统计学意义(H = 13.239,P = 0.000).Cx43 mRNA 与Cx43蛋白表达水平呈正相关(rs = 0.963,P = 0.000),Cx43 mRNA 及其蛋白质与增殖细胞核抗原表达水平呈负相关(rs = - 0.621,P = 0.000;rs = - 0.913,P = 0.000).结论 胶质瘤细胞Cx43 mRNA 及其蛋白质与增殖细胞核抗原表达水平和肿瘤组织病理学分级呈负相关.提示,Cx43 基因表达与肿瘤细胞增殖活性和胶质瘤恶性进展密切相关.     

连接蛋白拟似肽对海人酸致痫大鼠脑电活动的影响

目的观察针对连接蛋白43(CX43)合成的特异性的缝隙连接阻断剂-连接蛋白拟似肽对海人酸致痫大鼠脑电活动的影响。方法建立18只大鼠癫痫动物模型,分连接蛋白拟似肽组、甘珀酸组和对照组(每组6只),在在体上分别局部给予连接蛋白似似肽、甘珀酸和生理盐水,用脑电图仪观测用药前后每组大鼠皮层脑电活动的变化情况。结果连接蛋白拟似肽组及甘珀酸组给药后癫痫的发作次数明显比给药前发作次数减少,癫痫波的平均振幅也明显变小,给药前后比较有显著性差异(P<0.01),生理盐水组给药前后癫痫的发作次数及平均振幅几乎没有变化。连接蛋白拟似肽组给药前后癫痫的发作次数和波幅的变化值与甘珀酸组给药前后癫痫的发作次数和波幅的变化值相比无显著性差异。结论针对CX43合成的连接蛋白拟似肽可以特异性地抑制癫痫的发作。     

脂质体介导pBLAST2-hHGF质粒转染Lewis大鼠肝卵圆细胞

背景：在体外将外源基因导入肝细胞相当困难,而利用肝脏干细胞导入外源性基因较为容易。目前大鼠肝卵圆细胞增殖模型的建立已较为成熟,但作为原代细胞,肝卵圆细胞的稳定培养和传代是较为困难的。 目的：建立pBLAST2-hHGF质粒稳定转染大鼠肝卵圆细胞的细胞株,观察转染细胞的生物学特性。 方法：取稳定培养的第4代雄性Lewis大鼠肝卵圆细胞,采用脂质体介导DNA转染的方法将pBLAST2-hHGF质粒转染到肝卵圆细胞,然后在倒置显微镜下观察转染细胞的形态变化、细胞增殖情况,检测细胞转染后的增殖分化能力,确定转染细胞的稳定培养条件,使用western blot和ELISA方法检测转染细胞hHGF蛋白的表达。 结果与结论：第4代以脂质体介导成功转染pBLAST2-hHGF质粒的肝卵圆细胞在培养基中添加不同剂量和种类细胞因子的条件下转染细胞可稳定传代14代,其形态与未转染细胞比较无明显变化,但生长增殖速度明显快于未转染细胞,去除培养液中的生长因子后pBLAST2-Hhgf/肝卵圆细胞迅速分化为肝细胞和胆管上皮细胞,western blot方法检测转染细胞有hHGF蛋白表达,ELISA法检测培养液中hHGF蛋白含量高。结果提示,使用脂质体介导pBLAST2-hHGF质粒转染肝卵圆细胞方法可靠,转染细胞稳定培养并传代次数长于肝卵圆细胞,转染细胞具备分泌hHGF的能力,可用于后续研究。     

倒千里光碱对肝大部分切除小鼠肝脏损伤后再生修复的影响**☆

背景：目前大多数应用倒千里光碱造成肝损伤模型都是以大鼠为实验对象,有研究报道倒千里光碱并不能抑制小鼠肝细胞增殖,也有报道倒千里光碱对小鼠的肝细胞具有增殖抑制作用。 目的：观察单纯肝脏大部分切除及其联合应用倒千里光碱致小白鼠对肝损伤后再生修复的影响。 方法：40只C57BL/6J小鼠随机数字表法分为2组,每组20只。倒千里光碱/肝脏部分切除组：腹腔注射倒千里光碱溶液70 mg/kg,注射2次,每次间隔2周,4周后肝脏2/3切除。肝脏部分切除组：腹腔注射生理盐水70 mg/kg,注射2次,每次间隔2周,4周后行肝脏2/3切除。观察术后14 d肝脏大体结构恢复情况;苏木精-伊红染色观察术后第3,7天肝细胞损伤情况;BrdU染色观察术后第3天成熟肝细胞增殖情况;CK19和C-kit免疫组织化学方法观察术后第3,7,14天肝脏卵圆细胞增生情况。 结果与结论：肝脏部分切除组14 d肝大体结构基本恢复正常,而倒千里光碱/肝脏部分切除组肝脏大小没有明显的恢复。苏木精-伊红染色可见倒千里光碱/肝脏部分切除组明显的肝细胞变性改变;BrdU染色肝脏部分切除组术后第3天有明显肝细胞增殖,而倒千里光碱/肝脏部分切除组肝细胞增殖很少。CK19和C-kit免疫组织化学结果显示倒千里光碱/肝脏部分切除组术后第3天开始肝脏主要通过肝卵圆细胞增生并逐渐向肝细胞和小胆管分化,从汇管区开始向肝小叶内延伸以修复损伤。提示倒千里光碱可抑制小鼠肝脏损伤后肝细胞增殖再生。建立倒千里光碱/肝脏部分切除小鼠模型可诱导肝脏卵圆干细胞增生。     

辛醇对红藻氨酸致痫大鼠的保护作用及其对海马组织连接蛋白43的影响

目的 研究辛醇对红藻氨酸(kainic acid,KA)致痫大鼠海马组织连接蛋白43(connexin43,CX43)表达的影响及其抗痫效应.方法 采用行为学评分评价KA致痫及辛醇腹腔注射后致痫大鼠的痫性发作程度和潜伏期,应用免疫组织化学方法检测海马CX43的表达.结果 KA组CX43的表达较正常对照组明显增高(P<0.01);辛醇组CX43的表达也较正常对照组明显增高(P<0.01),但较KA组明显下降.致痫后3h内辛醇组痫性发作评分较KA组明显下降(P<0.01),潜伏期明显延长(P<0.01).结论 反复痫性发作后CX43的表达增加.辛醇能够减少CX43表达,降低痫性发作的评分,延长痫性发作的潜伏期,具有较明显的抗癫痫效应.

Abstract：

Objective To study the effects of octanol on the expression of connexin 43 in the brain of epilepsy-rats induced by kainic acid and the anti-epileptic effect of octanol. Methods The epilepsy-rats induced by kainic acid and treated by octanol was assessed by behavior score. The expression of CX43 in the hippocampal tissue was measured by immuno-histochemistry. Results The expression of CX43 in the group of epilepsy-rats induced by kainic acid was higher than that in control group( P <0. 01) ;the expression of CX43 in the group treated by octanol was higher than that in control group( P <0. 01) ,but lower than the group of epilepsy-rats induced by kainic acid;the Patel score of the rats in the group treated by octanol was lower than the group only induced by kainic acid within 3 hours after the induction (P <0. 01) ,and the latency was longer (P <0.01). Conclusions After the epileptic seizure repeatedly, the expression of CX43 was upregulated. Octanol could reduce the expression of CX43, decrease the Patel score,and extend the latency of epileptic seizure. It has obviously anti-epileptic effect.     

急性肝功能衰竭模型大鼠缝隙连接蛋白的表达*

背景：缝隙连接蛋白是组成相邻细胞间通道的主要结构,承担着细胞间的多种物质传输和信息交流的作用,可协助调节细胞的生长和分化。 目的：观察急性肝功能衰竭大鼠缝隙连接蛋白32表达与肝细胞增生的关系。 方法：采用乳果糖+庆大霉素灌胃和四氯化碳+橄榄油腹腔注射法建立大鼠急性肝功能衰竭模型。造模前7 d,苯巴比妥组大鼠用含体积分数0.08%苯巴比妥的水喂养,直至取材。对照组大鼠不造模,仅腹腔注射橄榄油与生理盐水的混合物。分别于造模后1,3,7,10,14 d取材。 结果与结论：大鼠肝功能衰竭后,部分大鼠出现死亡,存活大鼠肝细胞出现变性坏死,谷丙转氨酶明显升高,肝细胞间缝隙连接蛋白32 mRNA及蛋白表达明显降低。苯巴比妥可降低肝功能衰竭大鼠的死亡率,同时在一定程度上降低肝功能衰竭大鼠谷丙转氨酶水平及肝细胞间缝隙连接蛋白32 mRNA及蛋白的表达。说明通过苯巴比妥预先下调肝细胞间的缝隙连接蛋白32水平可以减轻急性肝功能衰竭大鼠急性期的肝脏损害,促进残存肝细胞增生和肝功能的好转,降低急性肝功能衰竭大鼠的病死率。     

Stages of status epilepticus in the developing brain

BACKGROUND: Adult rats undergo five distinct electrographic stages during status epilepticus (SE). Whether developing animals manifest those stages is not yet known. GOALS: Determine in the kainic acid (KA) model: (1) the EEG stages of SE in P15 and P35 rats; (2) the relative susceptibilities of these two age groups to develop SE; and (3) the effect of phenobarbital on SE stages. MATERIALS AND METHODS: Experiment 1: Three groups of P15, and three of P35 rats received intraperitoneally (i.p.) low (5 mg/kg), intermediate (10 mg/kg), or high (15 mg/kg) KA doses. Experiment 2: One group of P35 rats received KA (12 mg/kg), and one KA and phenobarbital (70 mg/kg i.p.). EEGs were recorded through intracranial electrodes and were reviewed and staged blindly. RESULTS: Both age groups manifested the five EEG stages of SE, but these occurred at the low dose in the P15 rats, and at the intermediate and high doses in the P35 rats. Unlike P35 rats, P15 rats were less likely to progress through all five stages, and had different behavioral manifestations that did not segregate into distinct stages. Phenobarbital caused an initial increase in paroxysmal beta discharges and in tonic activity and scratching. It subsequently resulted in less severe and shorter stages of SE. CONCLUSION: Both P15 and P35 rats can progress through the five distinct electrographic stages of SE. However, P15 rats are less likely to progress through all these stages. They also have a lower seizure threshold and different behavioral manifestations that do not segregate into separate stages. Phenobarbital shortens and modifies the behavioral and electrographic stages of SE.     

兔骨髓间充质干细胞体外向心肌样细胞诱导分化：缝隙连接蛋白43的

背景：骨髓间充质干细胞经过诱导因素刺激后,能够分化为心肌细胞,同时表达缝隙连接蛋白43,移植后可改善心功能或修复受损的心脏起搏传导系统。而缝隙连接蛋白43在维持心脏正常功能中发挥着重要作用。 目的：观察兔骨髓间充质干细胞体外诱导分化为心肌样细胞过程中缝隙连接蛋白43的表达变化,及诱导后骨髓间充质干细胞间隙连接通讯功能的改变。 方法：采用Percoll非密度梯度离心法与贴壁法分离培养兔骨髓间充质干细胞,正常组不进行任何干预,诱导组加入5-氮胞苷向心肌样细胞诱导,免疫荧光及流式细胞仪检验细胞诱导前后缝隙连接蛋白43的表达。将原代培养1 d的乳鼠心肌细胞分别接种于上述两组细胞爬片上,免疫荧光观察兔骨髓间充质干细胞与心肌细胞形成间隙连接的情况。划痕试验设立正常组、诱导组以及添加甘草次酸的阻滞剂组,观察兔骨髓间充质干细胞诱导前后细胞间隙连接的功能变化。 结果与结论：正常组骨髓间充质干细胞缝隙连接蛋白43呈弱表达,5-氮胞苷诱导2,4周后缝隙连接蛋白43的表达均显著增加(P < 0.01),细胞间隙连接通讯功能明显增强(P < 0.001),且随诱导时间的延长呈依赖性增强(P < 0.05)。骨髓间充质干细胞与心肌细胞共培养后,诱导组缝隙连接蛋白43明显呈线状表达在两个相邻细胞的接触面。与诱导4周时比较,阻滞剂组细胞间隙连接通讯功能明显受到抑制(P < 0.001)。提示骨髓间充质干细胞能够自发表达缝隙连接蛋白43,在移植后早期能与心肌细胞形成间隙连接,从而有利于移植后心肌电传导,并发挥骨髓间充质干细胞的旁分泌效应。 关键词：缝隙连接蛋白43;间隙连接;5-氮胞苷;诱导;心肌细胞;骨髓间充质干细胞     

Long‐term antiepileptic drug administration during early life inhibits hippocampal neurogenesis in the developing brain

Certain antiepileptic drugs (AEDs) that are commonly used to treat seizures in children also affect cognition, and these effects can persist into adulthood, long after drug withdrawal. Widespread enhancement of apoptosis may be one mechanism underlying these lasting cognitive changes. Whether AEDs affect other processes in brain development during early postnatal life has not, however, been systematically analyzed. Here we determined whether chronic administration of common AEDs during early life alters cell proliferation and neurogenesis in the hippocampus. Postnatal day 7 (P7) rats received phenobarbital, clonazepam, carbamazepine, valproate, topiramate, or vehicle for 28 days. Bromodeoxyuridine was administered on P34 to label dividing cells. Cell proliferation was assessed 24 hr later, and cell survival and differentiation were assessed 28 days later. Phenobarbital and clonazepam significantly inhibited cell proliferation by 63% and 59%, respectively, and doublecortin immunoreactivity (indicator of neurogenesis) in the dorsal hippocampus was also significantly decreased by 26% and 24%, respectively. Survival of new cells steadily decreased in phenobarbital and clonazepam groups over 28 days. Reduced cell proliferation and survival resulted in fewer new neurons in the dentate gyrus, as confirmed by neuronal counting on P62. There were, however, no differences in cell distribution pattern or differentiation toward neuron and glial cells when phenobarbital and clonazepam groups were compared with controls. There were no changes in rats exposed to carbamazepine, valproate, or topiramate. Thus, inhibiting cell proliferation, survival, and neurogenesis in the developing hippocampus may be another potential mechanism underlying brain impairment associated with certain AED therapies in early life. © 2009 Wiley‐Liss, Inc.     

Panax notoginseng saponins influence on transplantation of neural stem cell-derived dopaminergic neurons in a rat model of Parkinson’s disease *☆

BACKGROUND： Dopaminergic neurons differentiated from neural stem cells have been successfully used in the treatment of rat models of Parkinson＇s disease; however, the survival rate of transplanted cells has been low. Most cells die by apoptosis as a result of overloaded intracellular calcium and the formation of oxygen free radicals.
OBJECTIVE： To observe whether survival of transplanted cells, transplantation efficacy, and dopaminergic differentiation from neural stem cells is altered by Panax notoginseng saponins （PNS） in a rat model of Parkinson＇s disease.
DESIGN, TIME AND SETTING： Cellular and molecular biology experiments with randomized group design. The experiment was performed at the Animal Experimental Center, First Hospital of Sun Yat-sen University from April to October 2007.
MATERIALS： Thirty-two adult, healthy, male Sprague Dawley rats, and four healthy Sprague Dawley rat embryos at gestational days 14-15 were selected. The right ventral mesencephalon was injected with 6-hydroxydopamine to establish a model of Parkinson＇s disease. 6-hydroxydopamine and apomorphine were purchased from Sigma, USA.
METHODS： Neural stem cells derived from the mesencephalon of embryonic rats were cultivated and passaged in serum-free culture medium. Lesioned animals were randomly divided into four groups （n = 8）： dopaminergic neuron, dopaminergic neuron ＋ PNS, PNS, and control. The dopaminergic neuron group was injected with 3 μL cell suspension containing dopaminergic neurons differentiated from neural stem cells. The dopaminergic neurons ＋ PNS group received 3 μ L dopaminergic cell suspension combined with PNS （250 mg/L）. The PNS group received 3 μL PNS （250 mg/L）, and the control group received 3 μL DMEM/F12 culture medium.
MAIN OUTCOME MEASURES： The rats were transcardially perfused with 4% paraformaldehyde at 60 days post-grafting for immunohistochemistry. The rats were intraperitoneally injected with apomorphine （0.5 mg/kg） to induce rotational behavior. RESU     

Increased 5-HT2C receptor binding in the brain stem and cerebral cortex during liver regeneration and hepatic neoplasia in rats

In the present study, serotonin 2C (5-HT(2C)) receptor binding parameters in the brainstem and cerebral cortex were investigated during liver generation after partial hepatectomy (PH) and N-nitrosodiethylamine (NDEA) induced hepatic neoplasia in male Wistar rats. The serotonin content increased significantly (p<0.01) in the cerebral cortex after PH and in NDEA induced hepatic neoplasia. Brain stem serotonin content increased significantly (p<0.05) after PH and (p<0.001) in NDEA induced hepatic neoplasia. The number and affinity of the 5-HT(2C) receptors in the crude synaptic membrane preparations of the brain stem showed a significant (p<0.001) increase after PH and in NDEA induced hepatic neoplasia. The number and affinity of 5-HT(2C) receptors increased significantly (p<0.001) in NDEA induced hepatic neoplasia in the crude synaptic membrane preparations of the cerebral cortex. There was a significant (p<0.01) increase in plasma norepinephrine in PH and (p<0.001) in NDEA induced hepatic neoplasia, indicating sympathetic stimulation. Thus, our results suggest that during active hepatocyte proliferation 5-HT(2C) receptor in the brain stem and cerebral cortex are up-regulated which in turn induce hepatocyte proliferation mediated through sympathetic stimulation.     

Combined sub-threshold dosages of phenobarbital and low-frequency stimulation effectively reduce seizures in amygdala-kindled rats

Low-frequency stimulation (LFS) is a potential therapy utilized in patients who do not achieve satisfactory control of seizures with pharmacological treatments. Here, we investigated the interaction between anticonvulsant effects of LFS and phenobarbital (a commonly used medicine) on amygdala-kindled seizures in rats. Animals were kindled by electrical stimulation of basolateral amygdala in a rapid manner (12 stimulations/day). Fully kindled animals randomly received one of the three treatment choices: phenobarbital (1, 2, 3, 4 and 8 mg/kg; i.p.; 30 min before kindling stimulation), LFS (one or 4 packages contained 100 or 200 monophasic square wave pulses, 0.1-ms pulse duration at 1 Hz, immediately before kindling stimulation) or a combination of both (phenobarbital at 3 mg/kg and LFS). Phenobarbital alone at the doses of 1, 2 and 3 mg/kg had no significant effect on the main seizure parameters. LFS application always produced anticonvulsant effects unless applied with the pattern of one package of 100 pulses, which is considered as non-effective. All the seizure parameters were significantly reduced when phenobarbital (3 mg/kg) was administered prior to the application of the non-effective pattern of LFS. Phenobarbital (3 mg/kg) also increased the anticonvulsant actions of the effective LFS pattern. Our results provide an evidence of a positive cumulative anticonvulsant effect of LFS and phenobarbital, suggesting a potential combination therapy at sub-threshold dosages of phenobarbital and LFS to achieve a satisfactory clinical effect.