【作者】 王莉；

【导师】 余巨明；

【作者基本信息】 川北医学院 ， 内科学， 2011， 硕士

【摘要】 背景:癫痫是神经系统常见病、多发病,患病率约5‰,其中20%-40%的患者现有药物难治,属难治性癫痫。目前针对难治性癫痫的治疗策略可采取:多药联合治疗、迷走神经刺激治疗、脑深部电刺激治疗、生酮饮食治疗及外科手术治疗。但这些治疗存在要么疗效差、副作用大,要么有创、技术条件要求高、费用高等缺点,从而限制了它们在临床上的普遍使用。若能利用非药物、非创伤性的方法治疗难治性癫痫,则是癫痫病学家梦寐以求的事,也将为癫痫的治疗和机制研究开辟十分有前景的方向。经颅磁刺激(TMS)作为一种新兴的电生理技术,具有无痛、无创、穿透性强、安全性高、易于操作等优点,已被广泛运用于神经、精神领域,尤其在抑郁症和帕金森病的治疗上取得了明显的效果,展示出了诱人的应用前景。在癫痫方面,目前也有了初步的实验及临床研究报道,其结果令人鼓舞,这更引起了我们的浓厚兴趣,但有关TMS的抗痫效果报道并不一致,其机制也不清楚。分析其原因可能与研究者们所用的磁刺激参数不一致等因素有关。为此,我们提出如下研究假设:1、rTMS抗痫治疗有效并存在最佳磁刺激参数(如频率及强度)。2、rTMS抗痫作用的机制可能是影响到了癫痫发病机制的几个关键环节,如皮质兴奋抑制功能失衡、离子通道基因表达等。目的:①观察不同刺激频率及强度低频rTMS预处理大鼠2周对氯化锂-匹鲁卡品致痫作用的影响,并筛选出最佳的刺激频率及强度参数。②采用最佳频率及强度参数行rTMS预处理,观察rTMS预处理结束后不同时间海马CA3区Kca1.1、Na_v1.6、NMDAR1、GAD65表达的变化,以探讨rTMS抗痫的可能机制。③观察rTMS对致痫灶比较明确的局灶性难治性癫痫患者的治疗效果及安全性,并初步探讨其对大脑皮质兴奋性和脑血流的影响。我们希望本研究能为rTMS将来作为一种有效方法用于癫痫,尤其是难治性癫痫的临床治疗及机制研究提供帮助。资料和方法:①在其它磁刺激参数相同的情况下,分别采用不同频率(0 Hz、0.3 Hz、0.5 Hz、0.8 Hz及1.0Hz)对SD大鼠进行每日1次、连续14d的磁刺激治疗,然后制作氯化锂-匹鲁卡品急性癫痫模型,观察痫性发作潜伏期和发作程度的差异,以评价频率对癫痫发作的影响,并确定“最佳”刺激频率。②在其它磁刺激参数相同的情况下,分别采用不同强度(磁刺激最大输出强度的0%、10%、20%、30%及40%)对SD大鼠进行每日1次、连续14d的磁刺激治疗,然后制作氯化锂-匹鲁卡品急性癫痫模型,观察痫性发作潜伏期和发作程度的差异,以评价刺激强度对癫痫发作的影响,并确定“最佳”刺激强度。③采用确定的最佳频率与强度对SD大鼠进行每日1次、连续14d的磁刺激治疗,并采用免疫组织化学方法检测磁刺激后6h、24h、1w、3w和6w等不同时点海马CA3区Kca1.1、Na_v1.6、NMDAR1、GAD65蛋白的表达。④选择4例致痫灶比较明确的局灶性难治性癫痫患者,对其癫痫灶相应脑区给予每日1次、连续10d的磁刺激治疗,采用癫痫发作日志、EEG、SPECT和静息运动阈值(RMT)等方法分别观察rTMS治疗前后临床发作、癫痫波数量、脑血流及大脑皮质兴奋性的变化。结果:①0.3-1.0HZ频率范围内的rTMS均可延长氯化锂-匹鲁卡品致痫大鼠痫性发作潜伏期,并不同程度降低癫痫发作程度,与假刺激组比较潜伏期延长均有统计学意义(P<0.01或P<0.05),但发作程度减轻仅0.5HZ和0.8HZ组有统计学差异(P<0.05);不同频率组间比较,0.5HZ及0.8HZ组大鼠痫性发作潜伏期延长及癫痫发作程度降低最明显(P<0.05)。②与假刺激组比较,30%、40%最大输出强度的低频rTMS均明显延长了氯化锂-匹鲁卡品致痫大鼠痫性发作潜伏期(P<0.01),40%最大输出强度的低频rTMS还明显降低了大鼠癫痫发作程度(P<0.01)。③Kca1.1、Na_v1.6、NMDAR1、GAD65在海马各区锥体细胞及齿状回颗粒细胞表达。在CA3区,阳性神经元密集分布于锥体细胞层,在始层和分子层仅有零星分布。阳性细胞胞浆、胞膜呈棕黄色,胞核无染色。--- rTMS后6h,海马CA3区锥体细胞层Kca1.1阳性神经元密度即明显增高,24小时达到顶峰,随后呈下降趋势,但直至3周,其增高程度与较假刺激组相比仍有统计学差异(P<0.01);核浆比值于6h即显著降低,1周时达低谷,随后核浆比值逐步上升,但直至6周,与假刺激组相比仍有统计学意义(P<0.01)。--- rTMS后6周内,CA3区锥体细胞层Na_v1.6阳性神经元密度随时间变化差异不明显,与假刺激组相比无统计学意义;核浆比值仅rTMS后6小时短暂性上升(与假刺激组比P<0.05),其余时点无统计学差异。--- rTMS后CA3区锥体细胞层NMDAR1阳性神经元密度呈短暂性降低,仅6小时点与假刺激组比差异有显著性(P<0.01);而核浆比值6周内各时点与假刺激组相比均无显著性差异(P>0.05)。--- rTMS后6小时,CA3区锥体细胞层GAD65阳性神经元密度即明显增加(P<0.05);在24小时-1周达到高峰(P<0.01);此后呈下降趋势,但直至3周,增高程度与假刺激组相比仍有差异(P<0.01);与假刺激组比,核浆比值于rTMS后6小时即有下降趋势,并在1周时达显著性差异水平(P<0.05),此后,核浆比开始回升。④4例局灶性难治性癫痫患者经10d rTMS治疗后,3个月内癫痫发作频率由总共19次减少到1次,其中3例无发作,1例发作1次;EEG观察,1例癫痫波减少了20%;SPECT图像半定量分析显示rTMS后4例患者兴趣区放射线摄取比值(ROI值)均进一步降低;所有患者RMT均增高。结论:1 rTMS对实验动物及癫痫患者均有确切的抗痫作用,并具有频率及强度依赖性,以0.5HZ及40%最大输出强度的参数最佳(本实验条件下)。2低频rTMS可提高癫痫患者皮层RMT、进一步降低局部脑组织血流灌注、影响实验动物海马CA3区Na_v1.6、Kca1.1、NMDAR1、GAD65蛋白的表达,从而推测低频rTMS的抗痫机制是复杂的,可能涉及到与癫痫发病相关的多个环节。3低频rTMS将有望成为癫痫、尤其是难治性癫痫治疗及机制研究的新手段。更多还原

【Abstract】 Background:Epilepsy is the second largest disease in neurological disorders. Its prevalence rate is up to 5‰. Of these patients, about 20-40% are unresponsive to currently used drugs, which is termed as refractory epilepsy. Currently, the therapeutic strategies for refractory epilepsy include combinations of antiepileptic drugs, vagus nerve stimulation, deep brain stimulation, responsive cortical stimulation and surgery.However, several drawbacks such as poor efficacy, side effects, invasive procedures , demanding medical equipment and high cost exist in these strategies and hinder their wide use in clinical practice. Therefore, epileptologists never stop to seek for a non-drug, non-invasive method to treat refractory epilepsy. If this comes true, it will open up a promising road for therepay and insight into the epileptogenesis.As a new electrophysiological technique, transcranial magnetic stimulation ( TMS ) has many advantages , such as painless, non-invasive, easy to operate. This merits have made TMS be widely applied in neurology and psychiatry. The good therapeutic effects especifically in patients with depression and parkinsonism have already presaged a brilliant future for TMS. As far as epilepsy is concerned, several preliminary studies experimentally and clinically have shown encouraging results:TMS has antiepileptic effect. However, these results are not totally consistent. The disparity probably manily resulted from different parameters adminstered during rTMS. In addition, the underlying mechanism of TMS on epilepsy has not been established. With regard to this status quo, we assume that:1、rTMS is effective in epilepsy and there are optimal parameters (e.g. frequency and intensity). 2、The mechanism of antiepileptic effect of rTMS may involve in several key links close to epileptogenesis, such as imbalance of cortical excitability and inhibition.Objective:①To observe the effects of different frequency and intensity rTMS pretreatment on the epileptogenity of lithium- pilocarpine in rats, and try to find the optimal frequency and intensity which have the best antilepileptic effects.②To investigate the dynamic expressions of Kca1.1、Na_v1.6、NMDAR1、GAD65in the CA3 region of hippocampus whin 6 weeks after the end of 2-week daily rTMS pretreatment, which delivered with the optimal frequency and intensity, and explore the possible mechanisms of rTMS on epilepsy.③To evaluate the effect and safety of rTMS on patients with refractory focal epilepsy, whose epileptogenic zones are well demarcated, and to explore the effects of rTMS on cortical excitability and cerebral blood perfusion.Materials and methods:①Except for frequency, all other stimulus parameters were established. The SD rats were treated with rTMS trains at one of five frequencies: 0, 0.3, 0.5, 0.8 or 1.0Hz for 14 consecutive days , followed by an intraperitoneal injection of lithium chloride and pilocarpine to make acute epilepsy mode. Subsequently, the latencies and severity of seizures were determined and compared within different frequency groups, hoping to evaluate the effect of rTMS frequency on seizures and find the optimum stimulus frequency.②Except for stimulus intensity, all other stimulus parameters were established. The SD rats were treated with rTMS trains at one of five stimulator maximum outputs: 0%, 10%, 20%, 30% or 40% for 14 consecutive days , followed by an intraperitoneal injection of lithium chloride and pilocarpine to establish acute epilepsy mode. Subsequently, the latencies and severity of seizures were observed and compared within different intensity groups, hoping to evaluate the effect of rTMS stimulus intensity on seizures and select the optimum stimulus intensity.③The SD rats were administered with already determined optimum frequency and intensity rTMS for 14 consecutive days. After finished the rTMS protocol, the animals were killed at 6h, 24h, 1w, 3w and 6w respectively, and the expressions of Kca1.1,Na_v1.6,NMDAR1,GAD65 in the CA3 region of hippocampus were examined by immunohistochemistry.④4 patients with intractable focal epilepsy, whose epileptogenic zones are well demarcated, were enrolled. All patients were adminstered with rTMS treatment for 10 consecutive days. The seizure frequency, the number of interictal spikes, the cerebral blood flow, and the cortical excitability were determined before and after rTMS by keeping diary, EEG, SPECT and RMT , respectively.Result:①0.3-1.0Hz rTMS dramatically prolonged seizure latencies and also reduced seizure severity to some extent. Compared with the sham group (0Hz), all other group(0.3-1.0Hz) had significant prolongation in seizure latencies(P<0.05 or P<0.01), but for seizure severity, only 0.5Hz and 0.8Hz group had much lighter seizures, with seizure severity scores significantly lower than those in the other groups (P<0.05). Compared among frequency groups, the prologation of seizure latencies and the reduction of seizure severity in the 0.5Hz and 0.8Hz group were more pronounced than in the 0.3Hz and 1. 0 Hz group , and the differences reached statistically significant level (P<0.05).②Compared with the sham group (0%), only groups of 30% and 40% stimulator maximum output had significant prolongation in seizure latencies(p<0.01), and only group of 40% stimulator maximum output had significantly reduced seizure severity(P<0.01).③The expressions of Kca1.1、Na_v1.6、NMDAR1、GAD65 protein could be observed in all regions of hippocampus in all groups of rats. In CA3, positive neurons were densely distributed in the stratum pyramid, but scattered in the molecular layer. Positive cells characterized as brown in the cytoplasm and membrane, but no staining in the nucleus.---The Kca1.1-positive neuron density was dramatically increased in the stratum pyramid of CA3 at 6h after rTMS, reached the peak at 24h, then took on a decline tendency. Compared with the sham group, the Kca1.1- positive neuron densities were significantly increased for at least 3w after rTMS(P<0.01). The nucleus/cytoplasm ratio of the Kca1.1-positive neuron was decreased significantly at 6h after rTMS, reached the lowest at 1w, then demonstrated a tendency to increase. Compared with the sham group, The decreased nucleus/cytoplasm ratios had significant differences in the whole period of 6w after rTMS(P<0.01). --- The Na_v1.6 -positive neuron density in the hippocampal CA3 pyramid layer,compared with the sham group, had no significant difference at 6h、24h、1w、3w、6w following rTMS protocol. The nucleus/cytoplasm ratio of the Na_v1.6 -positive neuron , compared with the sham group, was transiently increased only at 6h point after rTMS(P<0.05),no significant changes at the other time points--- NMDAR1-positive neuron density in the hippocampal CA3 pyramid layer, compared with the sham group, was transiently decreased at 6h after rTMS protocol(P<0.01), but this change was not obvious at other time points. The nucleus/cytoplasm ratios had no significant difference at all time points of the whole 6-week observation compared with the sham group(P>0.05).--- The GAD65- positive neuron density in the stratum pyramid of hippocampus CA3, Compared with the sham group, was obviously elevated at 6h following rTMS(P<0.05), went up to the peak at 24h and maintained at this level for1w(P<0.01), then declined gradually. Nevertheless, the increased GAD65- positive neuron density kept at a statistically different level even after the end of rTMS protocol for 3w . The nucleus/cytoplasm ratio demonstrated a tendency to decrease at 6h and reached the significant level at 1w(P<0.05)after rTMS protocol, then rose gradually.④The seizure frequency in 4 patients with refractory focal epilepsy decreased from 19 (3-month basal period) to 1 (3-month follow-up period after rTMS) after 10 consecutive days’rTMS treatment, that is, 3 patients were seizure free , 1 patient had one seizure in the 3-month follow-up period after rTMS. EEG analysis displayed one patient had less interictal spikes post- rTMS than pre-rTMS: it decreased from 16 to 12, a 20% reduction. The other 3 patients had no spikes on EEG before and after rTMS. Sngle photon emission computed tomography (SPECT) semi-quantitative analysis showed the ROI values further decrease in all of the 4 patients after rTMS. The RMT was clearly elevated in all patients after rTMS treatment.Conclusion:①rTMS has definite antiepileptic effect on both experimental rats and epileptic patients, and this effect is stimulus frequency and intensity dependent, with the best choice of 0.5 Hz and 40% stimulator maximum output , respectively(under our experimental conditions).②Low-frequency rTMS can elevate RMT, further decrease the focal cerebral blood flow, and affect the expressions of Kca1.1、Na_v1.6、NMDAR1、GAD65 proteins in the hippocampal CA3 of rats. It is therefore reasonably infered that the antiepileptic mechanism underlying low frequency rTMS is complex: it probably involves several links related to epileptogenesis.③Low-frequency rTMS is hopeful to be a new tool in the treatment of epilepsy, especially refractory epilepsy, and the study of epileptogenic mechanisms.更多还原