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系統識別號 U0026-0812200911564616
論文名稱(中文) 評估神經保護劑褪黑激素於大小鼠腦缺血性中風模式之神經保護效益
論文名稱(英文) The efficacy of melatonin in experimental models of cerebral ischemia
校院名稱 成功大學
系所名稱(中) 醫學工程研究所碩博士班
系所名稱(英) Institute of Biomedical Engineering
學年度 94
學期 2
出版年 95
研究生(中文) 李宜堅
研究生(英文) E-Jian Lee
電子信箱 ejian@mail.ncku.edu.tw
學號 P88881043
學位類別 博士
語文別 英文
論文頁數 96頁
口試委員 口試委員-張憲彰
口試委員-吳天賞
口試委員-鍾高基
指導教授-張冠諒
口試委員-張志涵
口試委員-陳翰容
口試委員-蔡明世
口試委員-邱文達
口試委員-賴國安
中文關鍵字 缺血性腦中風  腦血管通透性屏障  神經電氣生理.  褪黑激素  神經保護 
英文關鍵字 blood-brain barrier  gray and white matter pathology  Neuroprotection  melatonin  electrophysiological recovery  stroke 
學科別分類
中文摘要   本研究係探討褪黑激素對於實驗鼠缺血性腦中風之神經保護特性與治療之相關機轉. 近年來褪黑激素為神經科學中神經保護劑之一熱門話題. 研究發現褪黑激素是一種強而有效之自然抗氧化劑與自由基截取劑. 另外有潛力降低缺血後能量流失,增強受傷神經存活. 本研究進一步擴展追求褪黑激素於缺血性腦中風腦電生理回復之急性期保護效益. 另外我們假設中風後之電生理與功能回復的保護作用須要同時降低大腦灰質與白質病變, 亦或也須要於中風之初期降低腦血管通透性的屏障之損壞, 以利於神經元恆定性, 功能和神經血管回復, 進而降低缺血性腦中風之轉化出血, 最後我們將利用褪黑激素於大腦灰白質與神經血管屏障之保護效益與延長中風事後治療之效益.

  首先本研究利用大白鼠進行中大腦動脈縫線栓塞, 同時我們探討褪黑激素於組織壞死, 神經電氣, 與神經行為之保護效益. 本研究發現褪黑激素是一種強而有效之神經保護劑,且無明顯副作用. 我們接著利用小白鼠進行可逆轉之中大腦動脈縫線栓塞以逐步探討缺血後期治療, 評估其進一步灰白質之神經保護特性與其神經保護潛能., 應用影像處理系統計算染色腦切片之栓塞大小並評估栓塞後個別神經運動感覺功能; 腦血流,腦電生理與體重回復. 另外利用染色法Nissl , Neu-N, APP, and tau-1等加以評估中風誘發之神經元, 寡突細胞和軸突傷害. 經由實驗,證明褪黑激素於可逆轉中大腦動脈縫線栓塞後至少60-90 min仍具有效神經保護作用. 它是一種強有效之神經保護劑值得進一步研究探討. 本研究接著探討褪黑激素對於腦可逆轉中大腦動脈縫線栓塞後缺血後期治療對於氧化壓力, 脂酯過氧化作用, 去氧核醣核酸傷害改善機制之評估. 我們接著研究於中大腦動脈栓塞後之急性初期, 施以褪黑激素可扮演降低組織血纖維蛋白溶原活化劑給藥後之轉化性出血, 保護腦血管通透性屏障與降低發炎之角色. 經由實驗, 我們發現褪黑激素於可逆轉中大腦動脈縫線栓塞後至少60-90 min仍具有效神經保護, 保護腦血管通透性屏障與舒緩組織血纖維蛋白溶原活化劑所產生之轉化性出血, 我們認為它是一種強有力之神經保護劑.基於上述研究, 我們發現褪黑激素是一種深具潛力的神經保護劑.


英文摘要   The neuroprotective properties of melatonin were evaluated in Sprague-Dawley rats and B6 mice subjected to middle cerebral artery (MCA) occlusion. A series of experiments with delayed treatment of melatonin have been employed to examine whether exogenous melatonin offers neuroprotective action against MCA occlusion which includes ischemic brain damage not only caused by necrotic but also by apoptotic processes. Postmortem infarct volumes will be determined by quantitative image analysis of Nissl-stained brain sections and TTC-staining method. In addition, postischemic electrophysiological recovery was evaluated. The protective efficiency of melatonin against ischemia- and reperfusion-induced neuronal perikarya, axonal, and oligodendrocyte pathology and increases in oxidative stress was also evaluated. The data provides a potential outlook of melatonin to treat ischemic stroke patients.

  Firstly, neuroprotective properties of melatonin were evaluated in rats subjected to transient middle cerebral artery (MCA) occlusion. The study examined electrophysiological, histological and neurobehavioral outcomes following transient focal cerebral ischemia caused by intraluminal suture occlusion of the middle cerebral artery. Postmortem infarct volumes will be determined by quantitative image analysis of TTC-stained brain sections. The effects of melatonin on cortical blood perfusion were also examined in the model. Our results showed that melatonin could effectively reduce brain infarction and improve electrophysiological and functional outcome.

  Secondarily, we examined the effect of melatonin on gray and white matter damage after ischemic stroke. The changes in the post-ischemic expression in gray and white matter pathology as well as oxidative stress to cell membrane and DNA by which the neuroprotective effects of melatonin may be mediated were also evaluated. The results showed that melatonin is a good candidate for protecting against gray and white matter pathology after ischemic stroke.

  Finally, we examined the possible role of melatonin to reduce postischemic damage to late-onset rise in the blood-brain barrier and the t-PA-associated hemorrhagic transformation after ischemic stroke. We have observed that melatonin decreases the late-onset rise in blood-brain barrier and improves the hemorrhagic transformation associated with t-PA therapy after ischemic stroke. Additionally, we examined the possible role of melatonin in neurovascular unit protection and its effects on postischemic neurovascular oxidative and nitrosative damage. We found that melatonin effectively reduces postischemic neurovascular oxidative and nitrosative damage and improves early rise in the blood-brain barrier after stroke.

  These neuroprotective actions observed with melatonin will further solidify the possible role of the neuroprotective agent for clinical use and provide a potential outlook to treat patients against ischemic stroke. We suggest a need for a pilot clinical trial of melatonin for acute ischemic stroke patients and our data may also benefit neurosurgeons to perform a planned cerebrovascular surgery.


論文目次 中文摘要 I
Abstract III
誌謝 VI
目 錄 VII
List of Figures IX
List of Tables X
Chapter 1 Experimental background and introduction 1
1.1 Pathophysiology of ischemic brain damage 1
1.2 Multifactorial pathogenicity of ischemic stroke 1
1.3 The need of neuroprotectant targeting at gray and white matter 2
1.4 The need of neuroprotectant targeting at the blood-brain barrier as well 3
1.5 The importance of neuroprotectant acting to decrease hemorrhagic transformation associated with t-PA-induced thrombolysis 4
1.6 Melatonin’s potential neuroprotection in the field of ischemic stroke 4
Chapter 2 Materials and Methods 7
2.1 Animals’ preparation and anesthesia 7
2.1.1 For electrophysiological studies 7
2.1.2 For gray and white matter and bood-brain barrier studies 7
2.1.3 For photothrombotic ischemic studies 8
2.2 Experimental models 8
2.3 Measurements of local cortical blood perfusion (LCBF) 10
2.4 Somatosensory evoked potential (SSEP) recordings 11
2.5 Animal sacrifice and quantification of ischemic damage 12
2.5.1 Using 2, 3, 5-triphenyltetrazolium chloride (TTC) 12
2.5.2 Cresyl violet stain and immunohistochemistry 12
2.6 Quantification of Gray Matter Damage 14
2.7 Cell Counting 14
2.8 Quantification of Oxidative Stress, Oligodendrocyte Pathology and Axonal Damage 15
2.9 Measurement of Evans Blue Leakage 16
2.10 Quantification hemorrhage volume 17
2.11 Detection of oxyradicals using hydroethidine in situ 17
2.12 Immunohistochemistry for nitrotyrosine 19
2.13 Neurobehavioral testing and body weight measurements 19
2.14 Statistical analysis 20
Chapter 3 Results 21
3.1 Delayed treatment with melatonin enhances electrophysiological recovery following transient focal cerebral ischemia 21
3.2 Melatonin attenuates grey and white matter damage in a mouse model of transient focal cerebral ischemia 34
3.3 Melatonin attenuates postischemic increase in blood-brain barrier permeability and decreases hemorrhagic transformation of tissue-plasminogen activator therapy following ischemic stroke 45
3.4 Melatonin decreases neurovascular oxidative/nitrosative damage and protects against early increases in the blood-brain barrier permeability after transient focal cerebral ischemia 54
Chapter 4 Discussion 62
4.1 summary 62
4.2 Anatomical and clinical implications 62
4.3 Mechanisms clarified and other potential mechanisms of action 63
4.4 Potential clinical use 66
Chapter 5 Conclusion 68
Reference 69
自 述 74
論文著述 76
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