| 摘要: | 抽搐性癲癇重積狀態(convulsive status epilepticus, CSE)是最常見之兒童青少年神經學急症。但有三成CSE病童對抗癲癇藥物反應不佳，因此必須發展非藥物新療法。已知經顱光生物調節(transcranial photobiomodulation, tPBM) 能降低大鼠於癲癇重積狀態所上升之興奮性神經傳導物質，但tPBM是否能減緩癲癇行為在仍未知。臨床上抗癲癇藥物帝拔癲(Depakine, sodium valproate, valproic acid, VPA)常用於治療兒童癲癇重積狀態，然而帝拔癲具有劑量依賴性肝毒性。若能發展輔助性療法合併帝拔癲使用好降低帝拔癲劑量並且仍維持甚至增加抗癲癇療效，將會是頑固型癲癇病童的一大福音。基於tPBM與VPA之機制相似性，tPBM具發展為輔助性療法合併帝拔癲使用之潛力。因此本博士論文研究致力於探討tPBM作為單一療法、輔助性療法合併使用VPA，以及前處置對青春前期大鼠癲癇之療效。本研究以出生後30-39天大之Sprague-Dawley大鼠為研究對象，以皮下注射pentylenetetrazole (PTZ, 90 mg/kg)誘發癲癇以及癲癇重積狀態。大鼠頭頂去毛後接收近紅外線波長808 nm之雷射照射。在第一部分—tPBM單一療法實驗中，在tPBM後立即注射PTZ；而在第二部分—tPBM輔助性療法實驗中，則是在注射VPA後30分鐘給予tPBM，並且在tPBM後立即注射PTZ。其中，VPA再分為低劑量(100 mg/kg)、中劑量(200 mg/kg)，以及高劑量(400 mg/kg)：在第三部分—tPBM前處置，tPBM則是在注射PTZ前60分鐘給予。在tPBM單一療法實驗當中進一步作tPBM機制或現象探討，包括將鼠腦冷凍切片進行Hematoxylin‐eosin (H&E)染色, 免疫螢光染以及terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL) assay；至於tPBM輔助性療法以及tPBM前處置實驗，僅分析癲癇行為。第一部分的結果顯示tPBM減緩平均癲癇分數並且降低癲癇重積狀態的發生率以及死亡率。根據H&E染色，tPBM減少與癲癇形成(epileptogenesis)相關腦區之神經損傷。根據免疫螢光染色合併TUNEL assay，tPBM減少parvalbumin‐positive interneurons (PV‐INs)之細胞凋亡比例，並且保留PV-INs軸突環繞於principal cells所形成的perisomatic inhibitory network之完整性。tPBM亦減緩神經發炎、 星狀膠細胞增生，以及微膠細胞增生。在本研究中，tPBM透過海馬迴細胞色素c氧化酶作用之現象也被確立。而在第二部分的結果，tPBM合併使用低劑量VPA減少嚴重癲癇重積狀態之發生率，並且具縮短stage 4–7 seizures的total duration之趨勢。然而，tPBM合併使用高劑量VPA卻會增加maximum seizure stages並且延長stage 4–7 seizures的total duration。tPBM前處置具縮短PTZ所誘發之stage 4–7 seizures的total duration及延長第一次癲癇大發作(generalized tonic clonic seizures)的latency之趨勢，但並沒有顯著差異。總結，使用808奈米之經顱光生物調節對PTZ所致癲癇具有抗癲癇效果。其中tPBM單獨使用能減緩癲癇與癲癇重積狀態，而tPBM作為輔助性療法合併使用帝拔癲的效應因帝拔癲劑量不同而異。至於tPBM前處置於注射PTZ前1小時給予tPBM並無法減緩PTZ所致癲癇大發作。這些數據證明了tPBM對小兒癲癇有療效。
Convulsive status epilepticus (CSE) is the most common neurological emergency in children and adolescents, yet about 30% patients had poor response to antiepileptic drugs (AEDs). Due to the refractory pediatric CSE to AEDs, developing new non-pharmacological treatments for pediatric CSE is urgently needed. It was known that transcranial photobiomodulation (tPBM) could reduce the elevated excitatory neurotransmitters after SE. However, whether tPBM could alleviate seizure behaviors was unknown. Clinically, Depakine (sodium valproate, valproic acid, VPA) is one of the most frequent used AEDs for pediatric CSE. Nevertheless, there is the dose-dependent hepatotoxicity in VPA. Therefore, it would be a good news for patients suffered from pediatric CSE if add-on therapy to VPA could reduce the dose of VPA while maintaining or even increase the anticonvulsive effects so that the hepatotoxicity would be reduced. Considering the mechanic similarities between tPBM and VPA, tPBM is a potential candidate for add-on therapy to VPA. Therefore, in this study of PhD dissertation, I applied near infrared laser with a wavelength 808 nm transcranially, and the tPBM was served as monotherapy, add-on therapy to valproic acid, and preconditioning to evaluate their effects on peripubertal rats with seizures. In this study, Sprague-Dawley rats at postnatal day 30-39 received subcutaneous pentylenetetrazole (PTZ, 90 mg/kg) to induce seizures and status epilepticus (SE). Rat scalp with hair removed received irradiation of near-infrared with wavelength 808 nm。In Part I—tPBM monotherapy experiments, the PTZ was injected right after tPBM. In Part-II —tPBM add-on therapy to VPA, the tPBM was performed 30 min after VPA injection and the PTZ was also injected right after tPBM. There were low-dose (100 mg/kg), medium-dose (200 mg/kg), and high-dose (400 mg/kg) VPA in tPBM add-on therapy experiments. In part III—tPBM preconditioning, tPBM was performed 60 min before PTZ injection. Further mechanisms or phenomena were elucidated in tPBM monotherapy experiments. The evaluation methods include Hematoxylin‐eosin (H&E) staining, immunofluorescence, and terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL) assay on frozen sections of rat brains. As for tPBM add-on therapy and tPBM preconditioning, only seizure behaviors were analyzed. In Part I, I showed that tPBM is capable of attenuating PTZ-induced seizures by significantly reducing mean seizure scores, incidence of severe status epilepticus (SE), and reduced the incidence of mortality. According to results of H & E staining, tPBM lessened neuronal damage in multiple brain regions involved in epileptogenesis. The TUNEL assay and PV immunofluorescence staining showed that tPBM reduced apoptotic ratio of parvalbumin-positive interneurons (PV-INs) in the hippocampus and preserved perisomatic inhibitory network of PV-INs surrounding principle cells. tPBM also attenuated neuroinflammation, astrogliosis and microgliosis. The phenomenon that therapeutic actions of tPBM through hippocampal cytochrome c oxidase (CCO) was also validated. In Part II, I demonstrated that tPBM added-on to valproic acid had synergistic anticonvulsive effect, but it was restricted to low-dose VPA, yet off-set effects were observed at medium and high-level VPA. In part III, I examined the effects of tPBM preconditioning on PTZ-induced seizures, and found that tPBM had trends of shortening the total duration of severe seizures and prolonged the latency of stage 4-7 seizures which contributes to SE, but without statistical significance. In conclusion, tPBM with wavelength 808 nm had anticonvulsive effects to PTZ-induced seizures. In which, tPBM served as monotherapy attenuates PTZ-induced seizures and SE. Particularly, the effects of tPBM add-on therapy differed from the doses of VPM which tPBM add-on with. Lastly, tPBM preconditioning in the setting of tPBM 1hr before PTZ injection could not alleviate PTZ-induced generalized tonic-clonic seizures. These data suggested that tPBM with wavelength 808 nm had therapeutic effect to pediatric epilepsy. |
