D-cycloserine 對 MPTP 所誘發之細胞介白素－2 與行為缺陷之效果：麩胺酸神經系統在巴金森氏症誘發失智症的可能角色

Effects of D-cycloserine on MPTP-induced Changes of Interleukin-2 and Behavioral Deficit: Possible Role of Glutamatergic System in Dementia in Parkinson’s disease

陳昶豪(Chang-Hao Chen)

指導教授 ： 黃淑桂 ； 何應瑞

中山醫學大學/醫學院/醫學研究所/碩士(2007年)

https://doi.org/10.6834/CSMU.2007.00006

摘要

MPTP（1-methyl-4-phenyl-1,2,3,6-tetrahydropyrindine）會專一性破壞多巴胺神經細胞，其所引發的神經退化及行為缺陷與巴金森氏症患者相似，因此常被用來建立巴金森氏症的動物模型。文獻指出，麩胺酸神經系統（glutamatergic system）之 N-methyl-D-aspartate（NMDA）受體參與神經發炎退化、及學習、認知等功能。D-cycloserine（DCS）是 NMDA 受體上甘胺酸結合位（glycine binding site）的部份致效劑（partial agonist），可以調節 NMDA 受體的活性。本研究探討 DCS 對於 MPTP 所誘發巴金森氏症大鼠的神經退化與行為缺陷的影響。實驗ㄧ：將 MPTP（1 μmole）注射到大鼠中腦黑質體（substantia nigra pars compact, SNc），再觀察 MPTP 處理後 2 週與 4 週內大鼠的行為變化與腦組織內的生化反應。實驗二：在 MPTP 處理後隔天起連續 13 天每天給大鼠施予 DCS (30, 100 及 200 mg/kg, i.p.）處理，再觀察大鼠的行為變化與腦組織內的生化反應。本研究的行為測驗分別以支架測試與滾輪儀測量大鼠的運動功能，以高腳十字迷宮評估類焦慮行為，以 T 型迷宮測量記憶功能，以開放空間中的新物件探索試驗評估物件辨識能力；腦組織內的生化反應是以免疫組織化學染色法（immunohistochemistry; IHC）測量 tyrosine hydroxylase 染色評估多巴胺神經細胞，以 OX-6 染色評估微膠細胞活化狀態，以酵素聯結免疫分析（enzyme-linked immuno sorbent assay; ELISA）測量細胞介白素－2（interleukin-2; IL-2）的含量。實驗ㄧ的結果顯示：大鼠經 MPTP 處理後，大鼠會出現運動功能障礙，但是手術 7 天後該行為缺陷會逐漸消失。於 MPTP 處理後 2 週內大鼠之類焦慮程度增高，在 T 型迷宮中的正確選擇率與開放空間中的新物件探索的時間百分比皆下降；再者大鼠經 MPTP 處理 2 週後，其 SNc 與紋狀體中的多巴胺神經細胞均大量減少，腦中微膠細胞也大量活化，紋狀體、杏仁核與大腦皮質內的 IL-2 含量顯著增加。實驗二的結果顯示，MPTP 合併 DCS 處理可以恢復巴金森氏症大鼠的焦慮程度並且改善 MPTP 所導致在 T 型迷宮與物件探索試驗中的行為缺陷。DCS 可以降低微膠細胞的活性，並且可以使腦組織內的 IL-2 含量恢復正常，並減少 MPTP 所造成的神經細胞損害。本研究結果推論：神經發炎反應與麩胺酸神經系統興奮性毒性所引起的神經退化可能在巴金森氏症所誘發的行為缺陷的病理生理學上扮演重要的角色，DCS 可能可以作為上述神經與行為疾病的治療用藥。

關鍵字

巴金森氏症誘發失智症 ； 大鼠動物模型 ； MPTP ； 行為研究 ； 活化態微膠細胞 ； IL-2 ； D-cycloserine ； 麩胺酸神經系統 ； NMDA 受體 ； 免疫組織化學染色法

並列摘要

This study was carried out to test the effects of D-cycloserine (DCS), a partial agonist of the glycine binding site located on the glutamatergic N-methyl-D-aspartate (NMDA) receptors , on the changes of behavior and neurodegeneration in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease (PD) animal model in Wistar rats. Behavior was monitored for 2 or 4 weeks after MPTP (1 μmole ) lesion that was injected into the substantia nigra pars compacta (SNc) of the rats. Another group was treated with DCS (30, 100, and 200 mg/kg/day, i.p.) that was taken a day after the day of MPTP lesion, for 13 days. Motor function was measured by bar test or rotarod; anxiety level was evaluated in an elevated plus-maze (EPM); learning and memory function was analyzed with the correct response in T-maze; recognitive function was assessed by using the novel object exploration test in an open field (OF). The brain tissue was taken for analyzing IL-2 by enzyme-linked immuno sorbent assay (ELISA). A serial brain sections were taken for detecting dopamine neuron and activated microglia by immunohistochemistry (IHC), stained by tyrosine hydroxylase and OX-6, respectively. The results showed that MPTP lesion caused the catalepsy that was seen in the first week after the lesion, high anxiety-like behavior in EPM, deficits in performance in T-maze, and impairment in the novel object exploration in OF. The IHC and ELISA demonstrated that MPTP treatment resulted in destruction of dopaminergic neurons in SNc and striautum, elevation of activated microglia and the level of IL-2 in the striatum, amygdala, and cortex, 2 weeks after the lesion. However, administration with DCS reversed the MPTP-induced deficits in motor function, anxiety, learning, and recognition. Co-administration DCS also reduced the IL-2 level and glial reaction in the brain tissue. In conclusion, DCS was able to prevent the MPTP-induced impairment of behavioral and immune functions, suggesting that immune and excitatory mechanisms of neurodegeneration may contribute to the neuronal damage and behavior change following PD.

並列關鍵字

Parkinson’s disease associated dementia ； rat animal model ； MPTP ； behavior research ； activated microglia ； IL-2 ； D-cycloserine ； Glutamatergic system ； NMDA receptor ； IHC

參考文獻

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