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研究生:郭曉縈
研究生(外文):Hsiao-Ying Kuo
論文名稱:豐富環境介入對中大腦動脈阻斷後大鼠於缺血邊緣區細胞凋亡之神經保護作用
論文名稱(外文):The Neuroprotective Effects of Enriched Environment on Apoptosis in Penumbra Area after Brain Ischemia in Rats
指導教授:王瑞瑤
指導教授(外文):Ray-Yau Wang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:物理治療暨輔助科技學系
學門:醫藥衛生學門
學類:復健醫學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:74
中文關鍵詞:豐富環境介入神經保護作用腦衍伸神經生長因子PI3K/Akt 存活路徑缺血邊緣區缺血性腦中風
外文關鍵詞:Enriched environmentNeuroprotectionBDNFPI3K/Akt pathwayPenumbra areaBrain ischemia
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研究背景與目的:暫時性局部大腦缺血會造成缺血中心細胞死亡並引發缺血邊緣區接續的細胞凋亡。若是能及時給予適當的治療介入,可以避免缺血邊緣區細胞凋亡,減少梗塞區域並增進功能性回復。豐富環境是一種治療介入,可以提供運動、認知、群居社交的刺激。過去研究發現豐富環境介入除了可以降低正常大鼠老化過程中海馬迴附近細胞的自發性凋亡,也可以減少患有帕金森氏症小鼠其黑質紋狀系統之細胞凋亡,然而尚未有研究探討豐富環境介入對於腦部缺血後細胞凋亡的情形。另一方面,在缺血性中風之大鼠模型中,也發現於豐富環境介入後,中樞神經一種含量豐富的神經營養素,腦衍伸神經生長因子的含量會上升。而腦衍伸神經生長因子在活化PI3K/Akt存活路徑中扮演重要的角色。因此推測豐富環境介入可能會透過增加腦衍伸神經生長因子量提高PI3K/Akt存活路徑活性,以減少缺血邊緣區細胞凋亡。因此,本研究探討豐富環境介入對大腦缺血性中風後大鼠於缺血邊緣區路徑神經保護作用之效果與機制。
研究方法:Sprague-Dawley大鼠隨機分配至四個組別(每組15隻):豐富環境介入兩天組(E2)為接受暫時性中大腦動脈阻斷術休息一天後介入兩天然後犧牲;E2的控制組(C2)則為術後休息三天不做任何介入。豐富環境介入六天組(E6)為接受暫時性中大腦動脈阻斷術休息一天後介入六天然後犧牲;E6的控制組(C6)則為術後休息七天不做任何介入。本研究中,先利用脫氧核糖核苷酸末端轉移酶介導缺口末端標記法計算缺血邊緣區細胞凋亡總數,再以酵素結合免疫分析法定量腦衍伸神經生長因子的濃度。最後使用蛋白質轉漬法計算Akt蛋白量,表示PI3K/Akt存活路徑活化程度。此外,動作功能則利用滾輪式跑步機執行測試。將介入組收取的實驗結果與其相對應的控制組比較。凋亡細胞數目與總細胞數之比值(%)、腦衍伸神經生長因子(pg/g)、Akt蛋白量(相對濃度%)皆以無母數曼-惠特尼U考驗法比較之。共變數分析用以比較實驗組與控制組之組內兩個測量時間點間,腦衍伸神經生長因子以及磷酸化Akt與Akt蛋白量之平均值差異。大鼠滾輪式跑步機之動作功能表現則以單因子多變量分析比較之。統計顯著水準α定為0.05。
研究結果:介入組於兩天介入或六天介入後,缺血邊緣區之凋亡細胞數目明顯減少(p = 0.009),而且動作功能也有明顯的進步(C2 vs. E2, p = 0.003; C6 vs. E6, p = 0.014)。BDNF濃度僅有在兩天介入組高於其控制組(p = 0.008),並且六天控制組的腦衍伸神經生長因子濃度明顯高於兩天控制組(p < 0.001),而六天介入組與其控制組間則無明顯差異(p = 0.548)。兩天介入組的磷酸化Akt含量明顯高於其控制組(p = 0.047),但是六天介入組與其控制組間則無顯著差異(p = 0.917)。此外,除了控制組的磷酸化Akt蛋白量沒有差異外,磷酸化Akt與Akt的蛋白量會隨著時間明顯下降。
討論與總結:急性期豐富環境介入可以減少缺血性腦中風後,大鼠在缺血邊緣區的細胞凋亡,並且增進動作功能表現。腦衍伸神經生長因子或許可以透過活化磷酸化Akt蛋白量,促使存活路徑執行以及時挽救缺血邊緣區即將凋亡的細胞。然而,腦衍伸神經生長因子在較晚期主要功能似乎不是神經保護作用。總結以上,由本研究發現在腦部缺氧短期內,給予至少兩天的豐富環境介入,可以挽救缺血邊緣區即將凋亡的細胞,並且促進動作功能回復。
Background and Purpose: Transient focal cerebral ischemia induces necrosis in core area followed by apoptosis in penumbra area mostly. Enriched environment (EE) is a complex surrounding that can provide physical, cognitive, and social stimulations. Previous studies showed that EE can reduce spontaneous apoptosis in the normal rats’ hippocampus and apoptotic cells in nigrostriatal system in rats with Parkinson’s disease. However, the effects of EE on apoptosis in penumbra area during acute cerebral ischemia are not known. Additionally, the highly abundant neurotrophin, brain-derived neurotrophic factor (BDNF), has a critical role in cell PI3K/Akt survival pathway. The purpose of this study was to explore the neuroprotective effects of EE and possible role of BDNF on penumbra area in transient focal ischemic rat brain.
Methods: Adult male SD rats were randomly assigned into 4 groups (n=15 for each group). Rats in group E2 received EE post middle cerebral artery occlusion (MCAO) for 2 days, and rats in group C2 stayed in the regular cage for 2 days as the control group. Rats in group E6 received EE post MCAO for 6 days, and rats in group C6 stayed in the regular cage for 6 days as the comparable control group. The TUNEL assay was used to measure the apoptotic cells in penumbra area and BDNF was quantified by ELISA. The amount of pAkt, an indicator of the PI3K/Akt activity, was measured by immunoblotting. Furthermore, rota-rod test was used to evaluate the motor performance. All data from EE groups were compared with their comparable control groups. Mann-Whitney U test was used to analyze the ratio of TUNEL positive cells over total cells (%), BDNF (pg/mg) and activation of Akt (%). Analysis of covariate (ANCOVA) was used to analyze the mean concentration of BDNF and activation of Akt between day in experimental and control groups. One-way multivariance ANOVA (MANOVA) was used to analyze the duration of rota-rod test. An alpha level of .05 indicated the significance.
Results: After intervention, the apoptotic cells in the penumbra area of EE groups were less than its comparable control groups (p = 0.009) and the motor performance was better in EE groups (C2 vs. E2, p = 0.003; C6 vs. E6, p = 0.014). The level of BDNF in group E2 was higher than C2 (p = 0.008), and group C6 was higher than C2 (p < 0.001), but there was no significant difference between C6 and E6 (p = 0.548). The level of pAkt in group E2 was significant higher than C2 (p = 0.047) but not significant between C6 and E6 (p = 0.917). Furthermore, pAkt and Akt levels decreased over time except pAkt level in the control group.
Discussion and conclusion: The EE intervention during acute brain ischemia may rescue the penumbra cells from apoptosis and improve motor performance. BDNF may protect cells from apoptosis by triggering the survival pathway, associated with upregulation and activation of Akt, in the penumbra area timely. However, BDNF may not involve in neuroprotection in late phase. Our findings indicate that EE for at least 2 days has neuroprotective function on apoptosis in penumbra area and enhance motor performance in acute focal ischemic stroke.
THESIS APPROVAL FORM i
ACKNOWLEDGMENTS ii
CONTENTS iv
LIST OF FIGURES vii
ENGLISH ABSTRACT viii
CHINESE ABSTRACT xi
ABBREVIATION 1
CHAPTER I INTRODUCTION 3
1.1 Background 3
1.2 Purpose 4
CHAPTER II REVIEW OF THE LITERATURE 5
2.1 Cerebral Ischemia 5
2.2 Molecular Mechanisms of Apoptosis 8
2.3 Neuroprotective Strategies in Ischemic Stroke 12
2.4 Phosphoinositide 3-Kinase/Akt (PI3K/Akt) Survival Signal Pathway 13
2.5 Enriched Environment 15
2.6 Summary 18
CHAPTER III MATERIALS AND METHODS 20
3.1 Animals 20
3.2 Experimental Design 20
3.3 Model of Transient Focal Ischemia: Middle Cerebral Artery Occlusion (MCAO) 22
3.4 Intervention Protocol: Enriched Environment 23
3.5 Outcome Measurements 23
3.6 Statistical Analysis 29
CHAPTER IV RESULTS 30
4.1 Amount of Apoptotic Cells in the Penumbra Area 30
4.2 Concentration of BDNF in the Penumbra Area 30
4.3 Level of Akt Activity in the Penumbra Area 31
4.4 The Motor Performance: Duration on the Rota-Rod 31
CHAPTER IV DISCUSSION 33
5.1 Enriched Environment Intervention Prevents Ischemic Penumbra Cells from Apoptosis during Acute Phase 33
5.2 Possible Mechanisms of Enriched Environment for Neuroprotective Effects 34

5.3 Enriched Environment Promote Motor Performance Recovery During Acute Phase 37
5.4 Limitations and Directions of Further Researches 38
CHAPTER VI CONCLUSIONS 39
REFERENCES 40
APPENDICES 57
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