評估腦部深層電刺激術對於癲癇動物模式之抗癲癇功效

Abstract

癲癇為常見的神經疾病，大約影響世界1%的人口，而其中三分之一的病患為所謂的頑固型癲癇，是無法被抗癲癇藥物控制的癲癇類型。手術切除癲癇中心是一種治療頑固型癲癇的方式，但並不是所有病患適合這項手術。因此，其他治療方式，像是腦部深層電刺激，迫切地需要應用在頑固型癲癇的治療上。腦部深層電刺激 (DBS) 最開始用於治療運動失調的疾病，像是巴金森氏症。由於視丘前核 (The anterior nucleus of the thalamus, ANT) 可經由扣帶迴 (Cingulate gyrus)投射訊息到大範圍的大腦皮質，所以目前ANT是被看好具有良好的抗癲癇效果之目標核區。在本篇研究中，我們將評估ANT DBS對於經由戊四氮(Pentylenetetrazol, PTZ) 所誘導的癲癇大鼠之抗癲癇功效、對睡眠的影響，以及探討DBS抑制癲癇之機制為何。實驗室先前的研究指出在利用毛果芸香鹼(pilocarpine) 誘導癲癇產生之前一小時給予ANT DBS的抗癲癇效果勝過前半小時給予電刺激治療。本篇研究將大鼠分為三組：單純給予PTZ之癲癇組、給予PTZ並搭配藥物注射前10分鐘給予ANT DBS持續30分鐘之治療組以及給予PTZ並搭配藥物注射前60分鐘給予ANT DBS持續3小時之治療組。我們分析了PTZ注射後到癲癇發作的時間，以及癲癇發作的持續時間。在PTZ注射前四次的結果中，DBS 30分鐘組別之大鼠有較晚發作癲癇以及癲癇持續時間降低的情形。然而在第14次注射PTZ之後，PTZ組別跟給予DBS治療之組別在癲癇發作時間以及持續時間上並沒有明顯差異。ANT DBS的抗癲癇效果在前面幾次的PTZ注射較為顯著。接著我們分析ANT DBS對於癲癇大鼠睡眠的影響。光亮週期之非快速動眼期 (Non-rapid eye movement, NREM) 以及快速動眼期 (Rapid eye movement, REM) 睡眠時間在第一次接受PTZ注射並且搭配30分鐘ANT DBS後有顯著的提升，但這提升的效果只有REM到了第14次注射PTZ並搭配ANT DBS後還能看到顯著差異，而NREM睡眠時間在三組並無差異。ANT DBS 3小時的組別相較於PTZ組別以及ANT DBS 30分鐘組別對於睡眠時間並沒有達到顯著差異。我們發現多次注射PTZ之大鼠會隨著施打PTZ次數增加，其NREM delta power也相對提升，而有接受ANT DBS治療之大鼠其NREM delta power提升的變化量與單純接受PTZ的組別相比有顯著的降低。根據其他研究指出NREM睡眠會促使癲癇產生但REM睡眠則會抑制癲癇產生。ANT DBS能降低NREM delta power，增加癲癇產生的閾值，並且增加REM睡眠時間進而抑制癲癇在睡眠中發生的機率。實驗室先前研究指出PTZ會增加N-methyl-d-aspartate (NMDA) 受體表現量，而NMDA受體在神經興奮性扮演十分重要的角色。我們利用西方墨點法測量NMDA受體其次單位NR1, NR2以及p-NR2在ANT DBS 30分鐘組和PTZ組大腦皮質、海馬迴及視丘的表現量。我們發現在NR2表現量在ANT DBS 30分鐘組的大腦皮質及海馬迴有下降的趨勢，其中大腦皮質下降趨勢甚多，在NR1以及p-NR2的表現量並沒有明顯差異。結果說明ANT DBS可能可以降低興奮性離子通道NMDA受體之表現量而達到抑制癲癇的效果。此篇研究最終目標希望能將實驗結果應用於與台大電機系以及台大醫院合作研發之晶片，並且成功抑制癲癇的傳遞。

Epilepsy is a neuronal disorder and affects 1% population of the world. One-third of patients have refractory epilepsy, which is hard to be controlled with anti-epileptic drugs (AEDs). Resective surgery is an alternative treatment for refractory epilepsy but not all of patients with refractory epilepsy are suitable for this surgery. Therefore, other alternative therapy, such as an electrical stimulation of deep brain structure is a pressing need for refractory epilepsy. Deep brain stimulation (DBS) has used in the treatment of movement disorders, like Parkinson’s disease. The anterior nucleus of the thalamus (ANT) may be a promising brain region for DBS in suppressing epilepsy, because the ANT has a wide projection to neocortex via the cingulate gyrus. In our study, we are going to evaluate the anti-epileptic efficacy of ANT DBS in pentylenetetrazol (PTZ)-induced epilepsy rat model, assess the effects of ANT DBS on sleep in epileptic rats, and determinate the mechanisms of DBS. Our previous study shows that pre-treatment of DBS for 1 hour prior to the epilepsy onset exhibits better anti-epilepsy efficacy than 30 minutes DBS in pilocarpine-induced epilepsy rats. In current study, rats were divided into three groups: the PTZ group, PTZ with 30-min DBS group (stimulation starting 10 mins prior to the PTZ injection) and PTZ with 3-h DBS group (stimulation starting 1 hr prior to the PTZ injection). We analyzed the latency to seizure onset and the duration of seizure after PTZ injections. Rats in the 30-min DBS group showed a longer seizure onset latency and shorter duration of seizure than the PTZ group in the first four times of PTZ injections. However, after 14th time of PTZ injection, there was no significant difference between PTZ group and PTZ with DBS treatment group in the amplitude and the duration of seizure. DBS prolonged the latency to seizure onset and reduce the duration of seizure at the first few times of PTZ injections. We then analyzed the effects of ANT DBS on sleep in the epileptic rats. Both non-rapid eye movement (NREM) sleep and rapid eye movement (REM) sleep time in light period showed significantly increase in rats received the 1st 30-min DBS, but only the amount of REM sleep remained increasing in rats treated with 30-min DBS after the 14-time PTZ injection. Both NREM sleep and REM sleep in PTZ with 3-h DBS group after the 14-time PTZ injection showed no significant change compared with both PTZ and PTZ with 30-min DBS group. We also found that the delta power during NREM sleep was decreased in epileptic rats with DBS when compared with those without receiving DBS. According to other researches, seizure was facilitated during NREM sleep and suppressed during REM sleep. DBS seems to decrease the power of synchronized brain wave in NREM sleep to increase epilepsy threshold and to increase the amount of REM sleep to suppress epilepsy. Our previous research demonstrated that PTZ increases the expression of the subunits of N-methyl-d-aspartate (NMDA) receptors, which plays important roles in neuronal excitability. The expression of NMDA receptor subunits, NR1, NR2 and p-NR2, in cortex, hippocampus and thalamus after 15 times of ANT DBS and PTZ injection were measured by the Western blot. NR2 was decreased in cortex and hippocampus of ANT DBS 30-min group. NR1 and p-NR2 had no significant difference in ANT DBS 30-min group. The reduction of NMDA receptor expression was involved in the mechanism of ANT-DBS anti-epileptic efficacy. Last, our goal is to apply a close-loop DBS microchip, designed by NTU Department of Electrical Engineering, to the epileptic animals and determine whether the microchip successfully suppresses the seizure propagation.