Pilocarpine引發之癲癇對於腦部齒迴GABA神經細胞之研究

Abstract

在癲癇症中，最常見亦為最難以治療者為顳葉型癲癇症，其病理特徵主要為海馬迴的病變，在動物模型中可見到神經細胞在海馬迴大量減少。位於齒迴的hilus，包含mossy cells以及γ-氨基丁酸中間神經元，是癲癇症中主要受到傷害的腦區之一。γ-氨基丁酸中間神經元又分成許多不同種類的亞型，其中calretinin-immunoreactive (CR-IR)中間神經元是被認為扮演癲癇產生的關鍵性角色，其會選擇性地支配其他的中間神經元並對癲癇症有特別敏感性，而neuropeptide Y-IR (NPY-IR)中間神經元一般被認為具有扮演抑制癲癇活性的功能。為探討CR-IR與NPY-IR這二類中間神經元在癲癇後的數目變化及相關機制，我們以免疫螢光染色法，對同年齡的控制組與以pilocarpine引發癲癇之癲癇組大鼠為動物模式，針對海馬迴齒迴區域之GAD67-IR、NPY-IR與CR-IR中間神經元細胞的分布以及數量上的變化做長期比較，更進一步利用DCX免疫螢光染色觀察新生的細胞是否會發展成NPY-IR、CR-IR 中間神經元，以進一步了解此二類中間神經元在癲癇症之病理機制中扮演的可能角色。 研究結果發現： 1) GAD67-IR與NPY-IR 細胞在控制組和癲癇後一週到八週之癲癇組大鼠齒迴之的數量相較均沒有顯著差異 2) 然而CR-IR 細胞在癲癇組大鼠齒迴之數目自癲癇引發後第一週到第八週均顯著低於控制組。 3) 在pilocarpine引發癲癇老鼠中連續一到八週NPY-IR cells在第一週數量與控制組沒有差異，到第二週NPY-IR cells數量會明顯較控制組減少，然而在第八週細胞數目有回升的現象，而在之間不同週數，NPY也會異位的表達在顆粒細胞上，inner molecular layer也會出現NPY-IR dendrite大量增生的情形。 4) 在pilocarpine引發癲癇連續一到八週老鼠中GAD67+ NPY-IR cells數量上控制組與癲癇組數量上皆無顯著差異。 5) 在pilocarpine引發癲癇連續一到八週老鼠中GAD67+ CR-IR cells數量上癲癇組比較控制組減少，其中第二週有達到統計上顯著差異。 6) 在pilocarpine引發癲癇第八周時，DCX-IR cells會和NPY-IR以及CR-IR cells出現colocalized的情形。無論在引發癲癇老鼠或是控制組都有此情形出現。 總結本篇實驗結果我們可以了解在癲癇引發後再轉變成慢性癲癇這段期間腦部細胞組織層次發生的現象，有助於了解癲癇症產生後可能因為神經保護胜肽NPY以及鈣離子結合蛋白CR的減少，使得腦部抑制性與興奮性的神經傳遞出現不平衡的狀態，有可能導致再發性癲癇的發生；然而我們也發現在慢性癲癇期，海馬迴的新生細胞有可能分化成為這些分泌神經保護胜肽NPY-IR以及鈣離子結合蛋白CR-IR的細胞，這極可能是癲癇症後出現的自我保護機制。

Temporal lobe epilepsy (TLE) is the most common epilepsy syndrome with medical refractory characteristics. The specific neuronal loss in the hippocampus is considered to be involved in the development of TLE. Hilus, which is composed of mossy cells and GABAergic interneurons, is a vulnerable areas in the hippocampus after suffering from TLE. Different subtypes of GABAergic interneuron are founds in this area. Among thses subtypes, calretinin-immunoreactive (CR-IR) interneurons which selectively innervate other interneuron are related to epileptogenesis, while neuropeptide Y-immunoreactive (NPY-IR) interneurons exert inhibitory effects on seizure activities . Our aim is to investigate the longitudinal change in the number of the CR-IR and NPY-IR interneurons and the underlying mechanism after seizure onset, immunohistochemistry is used to observe the distribution and the number of these two interneurons between control and epileptic state. Wistar rats were treated with pilocarpine to induce TLE, The alteration was studied from 1-8 weeks after seizure induction. In addition, neurogenesis occurs after seizure induction, therefore we intended to figure out if new-born neuron differentiate into GABAergic interneurons by CR or NPY co-stained with doublecortin (DCX), a marker of new-born neurons Our finding is described as follows: 1) The number of GAD67-IR cells was not significantly changed between control and seizure rats during 1st -8th week. 2) The number of CR-IR neurons was decreased in seizure rats than in control during 1st -8th week after seizure onset. 3) NPY-IR neuron number of seizure rats were more increase than control at the 1st and 8th week, the condition was opposite at the other time point. 4) The number of GAD67+ NPY-IR cells was not significantly changed between control and seizure rats during 1st -8th week. 5) The number of GAD67+ CR-IR cells was decreased in seizure rats than in control during 1st -8th week after seizure onset. 6) At the 8th week, there were DCX+ CR-IR cells and DCX+ NPY-IR cells in the hilus and subgranule zone. In conclusion, we suggest that recurrent seizure development by observing histology in rats brain, it may result from neuropeptide Y and calretinin reduced. Therefore, there was a imbalance condition in hippocampus area and may leading to recurrent seizure. We also found that the new born cell may become the CR-IR and NPY-IR cells, it may be a self-protecting mechanism in the epilepsy.