- 學位論文
深層腦刺激對PTZ誘導癲癇與睡眠障礙的影響
Effects of deep brain stimulation on PTZ-induced seizure and sleep disruption
摘要
癲癇是一種常見的神經系統疾病，病患約佔世界人口的1%。其中約70％的癲癇患者可以使用抗癲癇藥物進行治療。而剩下的30%無法使用抗癲癇藥物做治療的類型被稱為頑固型癲癇。深部腦部電刺激(DBS)通過電極傳遞電流以刺激目標區域，在近期已被許可作為癲癇的替代治療選擇，而在目標區域選擇中，視丘前核(the anterior nucleus of thalamus, ANT)因其可向扣帶迴(cingulate gyrus)傳遞訊號再投射至海馬旁迴(parahippocampal gyrus)進而輸送訊號至大範圍的大腦皮質(entorhinal cortex)，成為具良好發展性之抗癲癇效果目標核區。迄今為止，DBS的作用機制仍不是非常清楚。癲癇形成過程(epileptogenesis)為致使癲癇的重要過程，影響epileptogenesis的主要因素為發炎反應，它在受傷的大腦中迅速且持續地誘發後續下游作用。當中第一介白素(interleukin-1 beta, IL-1β)與腫瘤壞死因子 (tumor necrosis factor-α, TNFα)為重要指標。根據本實驗室先前的研究，IL-1β濃度的變化可幫助N-甲基-D-天冬氨酸受體(NMDAR)的活化使癲癇的發生率增加。在本研究中，我們使用戊四氮(pentylenetetrazol, PTZ)誘導的小鼠評估ANT-DBS的抗癲癇作用與對癲癇引起之睡眠障礙的影響，並分析是否藉由調節促炎因子濃度達到抗癲癇之目的。本篇研究將實驗動物分為五組：單純給予saline之控制組、單純給予PTZ之致癲癇組、於PTZ藥物注射前10分鐘給予ANT-DBS持續30分鐘之治療組、單純植入ANT-DBS電極給予PTZ藥物注射之致癲癇組與單純植入ANT-DBS電極給予saline注射之控制組。首先分析在低劑量PTZ誘導癲癇發作的持續時間，ANT-DBS治療組明顯低於PTZ誘導組。在interictal spike (IIS) 部分，研究認為IIS的頻率與Epileptogenesis相關，我們發現在ANT-DBS治療組中，儘管仍有IIS的出現，但數量明顯低於PTZ誘導組，且出現的時間晚於PTZ致癲癇組。接著分析ANT-DBS對睡眠障礙的影響，首次注射PTZ後， PTZ致癲癇組的NREM與控制組相比明顯下降。第六次注射PTZ後，發現PTZ致癲癇組於NREM(non-rapid eye movement, NREM)的時間與PFS控制組相比明顯減少，ANT-DBS治療組則與PFS控制組沒有顯著差異。在REM (rapid eye movement, REM)的睡眠時間則是在光暗期(ZT19-24)發現ANT-DBS治療組明顯高於PTZ致癲癇組。為了解ANT-DBS是否也對癲癇引起的睡眠片段化產生影響，我們分析在光亮周期(ZT0-12)中的階段轉換率，第一次注射PTZ後，兩組的轉換率並沒有明顯差別，而在第六次注射PTZ後發現，PTZ致癲癇組的轉換率比起PFS控制組與ANT -DBS治療組有明顯上升，且ANT-DBS治療組與PFS控制組相比則沒有明顯變化，表明ANT-DBS可能有助於改善睡眠片段化，進而提升睡眠品質。再進一步了解ANT -DBS對睡眠的改善是由於睡眠階段次數(bouts number)的增加或是睡眠持續時間(bouts duration)的延長，於NREM中，三組在次數與時間皆無明顯差異，而在REM中ANT -DBS不論是持續次數或是持續時間都明顯高於PTZ組別，說明其是藉由提升REM的次數與時間來達到改善睡眠效果。實驗室先前研究已發現IL-1β能幫助活化NMDAR致使神經興奮性提高進而使癲癇發作率提高，以及文獻指出TNFα為癲癇進程的重要指標之一，因此我們使用酵素結合免疫吸附分析法測定海馬迴與下視丘中的IL-1β與TNFα表現量，結果顯示在海馬迴與下視丘中的IL-1β表現量沒有明顯變化，海馬迴中的TNFα也沒有明顯改變，而在下視丘中發現PTZ誘導組之TNFα表現量與控制組比有明顯提升。綜上所述，ANT-DBS在PTZ誘導癲癇小鼠中具有抗癲癇的作用，並有助於改善睡眠片段化，且可能能夠延緩epileptogenesis。
並列摘要
Epilepsy, which affects about 1% of the world’s population, is a common neurological disease. About 70% of patients with epilepsy can use anti-epileptic drugs, AEDs, for treatment. Furthermore, 30% of patients that cannot be treated with AED called refractory epilepsy. Deep brain stimulation (DBS), which delivers an electrical current through an electrode to stimulate the target region(s), has been recently licensed as an alternative treatment option for epilepsy. In the selection of the target area, the anterior nucleus of thalamus (ANT) can transmit the signal to the cingulate gyrus then project to the parahippocampal gyrus and then send the signal to a large cortex of the brain through entorhinal cortex which becomes the target area for the antiepileptic effect of DBS. To date, the mechanism of DBS is still unclear. Epileptogenesis, which develops the progression in epilepsy, is mediated by inflammation in the injured brain. Among them, the proinflammatory factor interleukin-1 (IL-1β) and tumor necrosis factor-α (TNFα) are important mediators. Based on our previous research, the raise of IL-1β concentrations can activate N-methyl-D-aspartate receptor (NMDAR), which has a markedly increase in epilepsy. In our study, we used pentylenetetrazol (PTZ)-induced epileptic mice to evaluate the anti-epileptic effects of ANT-DBS, and understood the influence on epilepsy-induced sleep disruption. In this study, the experimental animals were divided into five groups: the control group given pyrogen-free saline (PFS) alone (the PFS group), the epilepsy group given PTZ alone (the PTZ group), the treatment group given ANT-DBS 10 minutes before PTZ drug injection and continue for 30 minutes (the ANT-DBS+PTZ group), the epilepsy group implanted with the ANT-DBS electrode and given PTZ alone (the sham surgery+PTZ group), and the control group implanted with the ANT-DBS electrode and given PFS alone (the sham surgery+PFS group). We analyzed the duration of seizure, the ANT-DBS+PTZ group had significantly lower seizure duration than that of PTZ group. The number of interictal spikes (IIS) is related to epileptogenesis, and we found that the IIS obtained from the ANT-DBS+PTZ group is significantly lower than that of PTZ group, and the time to appear the IIS was later than the PTZ group. Next, we analyzed the effect of ANT-DBS on sleep disruption. After the first time injection of PTZ, NREM sleep was significantly decreased in the PTZ group. After the sixth injection of PTZ, the NREM sleep of the PTZ group decreased significantly. The ANT-DBS+PTZ group is not significantly different from the PFS group. The PTZ group significantly decreased REM sleep during the light period. In order to understand whether the ANT-DBS also affects the fragmentation of sleep caused by epilepsy, we analyzed the transition between different vigilance states during the light cycle (ZT0-12). After the sixth injection of PTZ, the transition rate was significantly increased, indicating that PTZ-induced epilepsy caused sleep fragmentation. The ANT-DBS improved the fragmentation of sleep induced by PTZ. We further analyzed sleep architecture to understand whether the improvement of sleep by ANT-DBS is due to the increase of bouts number or the extension of bouts duration. In NREM, the three groups have no significant difference in number and duration, while in REM, both the number and duration in the ANT-DBS+PTZ group are significantly higher than those of the PTZ group, indicating that it improves sleep by increasing the number and duration of REM. Previous studies found that IL-1β can activate NMDAR to increase nerve excitability and thus increases the susceptibility of seizures. The literature points out that TNFα is also one of the important indicators of epileptogenesis. Therefore, we used an enzyme‐linked immunosorbent assay to measure IL-1β and TNFα concentrations in the hippocampus and hypothalamus. The results showed that the expression of TNFα in the PTZ group was significantly increased when compared with the PFS group in the hypothalamus, and ANT-DBS could decrease the PTZ-induced enhancement of TNF-. In summary, ANT-DBS has antiepileptic effects in PTZ-induced epilepsy mice and helps to improve the effect of sleep disruption, and it might be helpful to suppress the epileptogenesis.