马桑内酯在大鼠离体海马脑片上引起的癫痫样放电活动

Coriaria lactone (CL) is an active constituent of a medicinal herb used as psychosolytic in traditional Chinese medicine. Recently it has been found that CL appears to act as a convulsant agent. Subsequently both acute and chronic epilepsy models caused by CL have been established successfully. In order to observe further the epileptogenic effects of CL in vitro, the hippocampal slice technique was used in experiments with 36 slices. The results are as follows: Addition of CL to the perfusion bathing the slices of rat hippocampus increased evoked-response in body layer of CA1 in a dose-dependent manner, and induced epileptiform burst. CL augmented population spike of CA1 pyramidal neurons triggered by either orthodromic stimulus (through synapses) or antidromic stimulus (direct effect on the axon of CA1 pyramidal cells) without alteration of presynaptic fiber volley and field-EPSP, and there was no significant change in latency of burst. Finally GABA had a weak effect on CL-induced epileptiform activity. These observations suggest that CL probably has a direct effect on the soma of pyramidal neurons, CL-induced epileptiform burst may be a result of altering the innate capacity of burst and increasing the intrinsic excitability of pyramidal cell membranes.     

串刺激诱发大鼠海马脑片痫样放电模型的建立及谷氨酸对它的影响

A train of pulses (60 Hz, pulse duration 100 microseconds, train duration 2s, 2-3 fold maximal intensity) were applied to the CA3 stratum radiatum fiber pathway in rat hippocampal slices in vitro. Recordings of the field potentials were made from the stratum of CA3 pyramidal cells in response to these pulses so that the model of stimulus train-induced bursting (STIB) could be studied. It was a new and valuable model of epileptiform activity in hippocampal slices in vitro and the effect of L-glutamate (Glu) on STIB was also studied. Results showed that: 1. The number of train stimulus for a stable STIB averaged 5.5 +/- 2.1; the induced number of population spikes (PS) increased with an average in 5.2; and the STIB activity was long-last, persisting for up to 3-4 hours following the last train. 2. Superfusions of Glu in concentrations of 1, 2, and 3 mmol/L in hippocampal slices produced inhibition of STIB in CA3, but the first antidromic PS was unaffected, suggesting the site of inhibition is postsynaptic. The mechanisms related to the genesis of STIB and its inhibition by superfusion of Glu were discussed.     

丙戊酸钠对荷包牡丹硷诱发大鼠海马脑片痫样放电的影响

Experiments were conducted on the rat hippocampal slices in vitro with the superfusion of bicuculline (Bcl, 10 mumol/L) and stimulation of Schaffer collaterals by single pulse to produce epileptiform activity of the pyramidal cells in CA1 area. In these experiments the effect of constant microinjection of sodium valproate onto the surface of slice on the epileptiform activity was studied. The results showed that the valproate, in concentration of 20, 30, 50 and 100 mmol/L, inhibited the Bcl-induced epileptiform activity in a manner of dose-dependence. It was deduced that the inhibition of epileptiform activity by the valproate is possibly related to the recovery of and increase in the inhibition mediated by GABAergic neurons and may be also to the non-synaptic mechanism of the valproate in rat hippocampus.     

丙戊酸钠对荷包牡丹硷诱发大鼠海马脑片痫样放电的影响

用荷包牡丹硷(Bicuculline,Bcl)10μmol/L灌流大鼠离体海马脑片,单脉冲刺激Schaffer侧支,激活 CAl区锥体细胞产生痫样放电。观察了丙戊酸钠(Sodium valproate,VPA)微量恒速推注到脑片表面对痫样放电的影响。结果表明,20、30、50和100mmol VPA抑制Bcl诱发的痫样放电,其抑制作用随剂量增加而增强。VPA这一抗痫样放电作用,可能与恢复和增强海马γ-氨基丁酸(GABA)能神经元介导的抑制功能有关,但也可能与非突触的作用机理有关。     

串刺激诱发大鼠海马脑片痫样放电模型的建立及谷氨酸对它的影响

作者用串脉冲(60Hz,波宽100μS,串长2S,2～3倍引起最大反应的刺激强度)刺激大鼠离体海马脑片CA3区辐射层,记录CA3区锥体细胞层产生的场电位,在国内首次成功地建立了串刺激诱发的暴发放电(STIB)模型——一种有价值的新型离体海马脑片痫样放电模型;并观察了L-谷氨酸(Glu)对该模型的影响;讨论了STIB的产生和Glu对其抑制的有关机理。     

膜片钳技术在成年大鼠海马脑片应用的初步研究

目的：制备成年大鼠海马脑片,使其适应于膜片钳全细胞记录,并观察海马锥体神经元的电生理特性。方法：选200 g左右的成年SD大鼠,雌雄不拘,10%水合氯醛腹腔注射麻醉,四肢固定,剪开胸腔,夹闭下腔静脉,以0 ℃切片液进行心脏灌注,断头,取下全脑,置入0 ℃切片液中冰镇3 min,再修块,切片,厚度约350 μm,移入37 ℃预热的人工脑脊液中孵育1 h后,转入23 ℃水浴中孵育30 min备用。倒置相差显微镜和红外微分干涉相差显微镜观察脑片的神经元形态;全细胞膜片钳记录脑片神经元的电生理学特性。结果：海马CA1区神经元光泽度好,立体感强且突起明显;在进行膜片钳全细胞记录时,封接顺利,并且可以记录到电流和动作电位的变化图形。结论：本方法制备的成年大鼠脑片,可以耐受离体后缺血缺氧造成的损伤,保持电生理活性而适应膜片钳全细胞记录模式。     

马桑内酯对大鼠海马脑片细胞外Na+、K+活度的影响

目的探讨癫痫过程中神经细胞Na     

离体培养大鼠海马脑片的形态学与细胞反应性研究

目的:探索离体大鼠海马脑片培养方法,观察培养脑片的形态变化和细胞反应性.方法:选择生后6～9d的Wistar鼠,分离海马,切成400ìm厚的脑片,转移至带有微孔膜插件的6孔培养板内,人37%的CO2培养箱中进行培养.通过肉眼、倒置相差显微镜观察培养海马脑片的形态学变化;用免疫组化染色检测脑片神经细胞内Fos蛋白表达变化,以判断培养脑片有无对外界伤害性刺激的应激反应能力;用膜片钳技术检测神经细胞的电生理活动,以判断培养海马脑片的活性和生理功能.结果:(1)随着体外培养时间延长,培养脑片内神经细胞数目增多,而脑片明显变薄,至培养4周时厚度约150ìm厚.(2)致癫痫样发作药物匹罗卡品作用于脑片后,导致培养海马脑片CA1区细胞内Fos蛋白表达增高.(3)利用膜片钳技术,在培养1、2、3、4周的4个时点对海马脑片CA1区锥体细胞作全细胞记录,皆记录到细胞电流变化图形.结论:本方法在体外培养的海马脑片至少可存活4周,且具备良好的活性和一定的生理功能.     

马桑内酯点燃大鼠海马脑片CA1区的双脉冲抑制观察

作者以逆向-顺向刺激双脉冲抑制为指标,首次观察了用马桑内酯点燃大鼠海马脑片CA1区,由γ-氨基丁酸(GABA)介导的回返性突触抑制的变化。结果表明:大鼠海马脑片CA1区双脉冲抑制持续时程约为40～60ms;马桑内酯点燃大鼠海马脑片CA1区双脉冲抑制与对照组比较无统计学差异(P=0.06),提示GABA介导的突触抑制在马桑内酯化学点燃中不起主要作用。     

马桑内酯点燃大鼠海马脑片CA1区的双脉冲...

Paired-pulse depression (PPD) technique was used to investigate the potency of recurrent synaptic inhibition mediated by GABA in area CA1 of 54 hippocampal slices from coriaria lactone (CL)-kindled and control rats. When paired stimuli were sent to the axon of CA1 pyramidal cell and Schaffer collaterals, the effect of population spike PPD lasted about 40-60ms; no significant change was observed on PPD potency between kindled and control groups (P = 0.06, 2-way ANOVA). The results indicate that the GABA-ergic synaptic inhibition seemed not to play a key role in the maintenance of the chemical kindling induced by CL.     

左乙拉西坦对马桑内酯致痫海马神经细胞钠电流的影响

目的 探讨左乙拉西坦 (Levetiracetam, LEV) 对马桑内酯(coriaria lactone, CL)致痫SD大鼠海马神经元钠电流增加的影响.方法 利用膜片钳全细胞模式, 记录急性分离后CL致痫的大鼠海马神经元钠电流,并给予LEV进行干预,分为对照组、LEV 150 μmol/L组、LEV 300 μmol/L组和未处理组.另用LEV 300 μmol/L预处理40 min后再给予CL致痫,观察钠电流的变化情况.结果 CL致痫后钠电流增加,加入LEV 150 μmol/L后最大电流峰值增加了36.92%±2.84%(P＜0.05),与未处理组比较差异无统计学意义(P＞0.05).LEV 300 μmol/L组钠电流增加了16.58%± 1.56%(P＞0.05);但与对照组相比,差异无统计学意义(P＞0.05).经LEV预处理后,CL仍使钠电流增加了32.86%±6.73%(P＜0.05),与未处理组比较差异无统计学意义(P＞0.05).结论 LEV未能抑制CL致痫海马细胞钠电流的增加,其抗癫痫作用可能不是通过抑制钠电流而产生的.     

马桑内酯对大鼠海马神经元钠电流的影响

目的 研究马桑内酯(coriaria lactone,CL)对大鼠海马神经元钠离子通道电流的影响,探讨该影响在CL致痫中的意义.方法 利用全细胞膜片钳技术,在急性分离的大鼠海马神经元上记录钠电流,观察CL对电流幅度的影响.结果 经0.1 mg/mL、0.2 mg/mL的CL作用后,海马神经元钠电流有不同程度的增加[CL 0.1 mg/mL组的最大峰值电流密度为(-90.11±14.02) pA/pF,增幅为17.32%±8.52%;CL 0.2 mg/mL组的最大峰值电流密度为(-111.52±6.65) pA/pF,增幅为37.98%±4.91%];经与对照组相比,CL 0.2 mg/mL组(P＜0.05)的变化较CL 0.1 mg/mL组(P＞0.05)明显.结论 CL使海马神经元电压依赖性钠电流的幅度增大,从而改变细胞的兴奋性,引发异常放电.     

钙通道阻滞剂维拉帕米对马桑内酯所致家兔海马点燃性癫痫发作的影响

作者用马桑内酯所致家兔海马点燃模型,观察了钙通道阻滞剂维拉帕米对痫性活动的影响。实验结果表明,海马内直接注入维拉帕米对家兔点燃性癫痫发作有对抗作用,但肌肉注射效果差,这可能与该药不易通过血脑屏障有关。     

SC1001钠盐对马桑内酯所致家兔海马点燃性癫痫发作的影响

作者用点燃效应模型对SC1001钠盐的抗痫作用进行了观察。实验结果表明,此药对马桑内酯所致家兔海马点燃性发作有一定的对抗作用,但肌肉注射SC1001钠盐100mg/kg的疗效不如肌肉注射苯巴比妥钠30mg/kg显著。     

马桑内酯体外诱导大鼠星形胶质细胞P-糖蛋白表达

目的马桑内酯体外诱导耐抗癫痫药物(AED)的星形胶质细胞,并探讨可能的耐药机制。方法培养新生大鼠的星形胶质细胞,传代后向其培养液中按体积1μL,3μL,9μL,15μL滴加马桑内酯,分别在加入药物后第7d,14d,21d,28d用免疫细胞化学和图像分析的方法检测并分析P-糖蛋白的表达。结果从第14d开始至28d,随诱导时间的延长,P-糖蛋白的表达明显增加(P<0.01);在21d,28d组,随马桑内酯剂量的增加,P-糖蛋白的表达无明显增加(P>0.05)。结论马桑内酯体外诱导的大鼠星形胶质细胞对AEDs有耐药性,P-糖蛋白的表达增强可能是其耐药性产生的重要机制。     

肾上腺素能药物对点燃大鼠海马CA1区群集锋电位的影响

在马桑内酯点燃早期和点燃达标7天后的大鼠海马CA1区,用玻璃微电极记录该区锥体细胞群集锋电位(PS),并观察滴加肾上腺素能受体激动剂至胞体层对PS的影响。实验结果表明:去甲肾上腺素(NE)5μmol/L使PS增高,NE50μmol/L使PS稍降低。使PS增高和降低的作用能分别被β和α受体拮抗剂阻断。α受体激动剂苯肾上腺素(PE)能使PS降低,β受体激动剂异丙肾上腺素(ISO)使PS增高。在点燃早期大鼠的海马脑片上,ISO5μmol/L仅使PS轻微增高,此效应明显低于对照组,提示β肾上腺素能受体激动后使PS增高的效应降低,可能有助于点燃的建立。     

马桑内酯局部癫痫持续状态大鼠大脑皮质的形态计量研究

用马桑内酯明胶淀粉微粒包埋于１０只大鼠左侧大脑半球的前肢运动区，诱发局部癫痫持续状态。发作高峰（６小时）时取材，用光镜、电镜和体现学方法研究大脑皮质运动区第Ⅴ层的形态、结构、细胞数量及神经毡成分的变化。与对照鼠比较，癫痫鼠大脑皮质第Ⅴ层神经元的数目减少，以灶区下降最多，灶旁区次之；胶质细胞数也明显减少，各区间下降程度相近。癫痫鼠皮质神经毡的突触性终末数及其面积分数明显下降，以灶区为甚，灶旁区次之；而胶质突起的面积分数却明显增加，以灶区为甚，镜灶区次之。