應用雷射光化學與超快雷射技術建立斑馬魚中風模型

Abstract

腦中風為腦部因血液循環出問題而引起腦部功能損傷之疾病，可分為缺血性與出血性中風。根據世界衛生組織的統計，腦中風為十大死因的第二名。傳統研究中風的動物模型需要仰賴複雜手術，因此重複性較低。此外，除可能有出血的風險外，也不易在特定腦血管誘發中風，以及觀察血管及細胞在中風後的變化。近年，斑馬魚已成為熱門的動物模型之一，其優點包含繁殖周期短，以及基因型已被完整解出、方便大量藥物篩選等優點，且幼魚的身體透明，便於應用雷射及光學顯微鏡技術。組織胞漿素原活化劑（tissue plasminogen activator）目前仍為唯一核准用於治療急性缺血性中風的溶血栓藥物，但使用上有許多限制及副作用。在此研究的第一部份，我應用實驗室建立的光化學技術在斑馬魚背部動脈形成血栓，探討應用斑馬魚測試溶血栓藥物的可行性。透過三維影像軟體分析血塊體積變化，我們發現未注射藥物的控制組在光化學誘發凝血後因血球持續附著，血塊會繼續增大。相反的，注射溶血栓藥物的實驗組血塊體積則會顯著減少。此結果展現應用光化學於斑馬魚誘發血栓的方法可應用於測試溶血栓藥物。在第二部份，我將光化學誘發血栓的方法應用於建立斑馬魚腦部缺血性中風模型。透過調整雷射照射時間，能夠控制部分性與完全性的腦血管栓塞，並且能選擇性的在不同部位的腦血管誘發血栓生成。應用運動神經細胞表現綠色螢光蛋白的轉基因魚，發現在中風區域附近運動神經細胞的綠色螢光強度有顯著下降，也經由碘化丙啶染色證實神經細胞死亡。此外我也利用飛秒雷射技術破壞單一腦血管，建立斑馬魚出血性中風模型。最後，我也嘗試觀察阻塞或出血型中風後損傷血管的新生與重塑，以及血流復流的情形。我們預期此研究未來除了能幫助腦中風及腦血管病變的研究之外，也能夠應用於開發新穎的溶血栓藥物或是治療中風的方法。

Stroke, which occurs as a result of deficiency in blood supply to the brain by occlusion (ischemic stroke) or bleeding (hemorrhagic stroke), is a leading cause of death. The use of animal models is an indispensable component for biomedical research and pharmaceutical development. With its well-characterized genome, high reproductive rate, translucency in the body and high conservation relative to human beings, the zebrafish has recently emerged as a popular model organism. In this research, I evaluate the feasibility to test thrombolytic agents using our zebrafish model of photochemical thrombosis. For zebrafish injected with tissue plasminogen activator (tPA), the blood clot became lysed gradually and the clot size decreased significantly. In contrast, the clot continued to grow in size for the control group that was not injected with tPA. I extended this photochemical approach to develop the zebrafish model of ischemic stroke. Through the control of laser exposure, partial or complete occlusion at selected cerebral blood vessels was induced reproducibly. Facilitated with a transgenic fish line that expresses green fluorescence protein in motor neurons, an infarction area near the occluded vessel was identified with decreased cellular fluorescence and positive stain of propidium iodide. I demonstrate also the application of multiphoton induced ablation to induce hemorrhagic stroke in zebrafish. Finally, I characterized the remodeling and altered hemodynamics of cerebral vasculature after ischemic and hemorrhagic stroke. We anticipate that our approach will facilitate not only fundamental research of stroke but also investigation of new therapeutic strategies.