應用光化學誘發生成血栓建立斑馬魚中風模型

Abstract

依據世界衛生組織的統計，腦中風在已開發國家中為十大死因之一，其死亡率僅次於心臟疾病。腦中風源自於供應腦部的血液受到了阻礙而造成了腦部細胞的損傷，進而影響腦部功能。腦中風可分為兩類，一種是血塊阻塞腦血管造成的缺血性中風；另外一種是血管破裂造成的出血性中風。傳統上研究腦中風的動物實驗多利用哺乳類的動物模型，其中以大鼠和小鼠最為普遍。而動物實驗上造成中風的方法有將血管暴露於氯化鐵 (FeCl3) 溶液中造成腐蝕進而產生血栓, 利用止血鉗夾住血管造成缺血，或者利用電流刺激血管造成血栓產生。這些傳統實驗方法的缺點為再現性較低，而且需要複雜的實驗手法才能完成。近年斑馬魚 (Danio rerio) 已成為常用的動物模型，其優點包括繁殖週期短, 基因型已被完整解出，還有幼魚的身體呈透明狀方便利用光學儀器觀察。在此研究中，我利用光化學誘發血栓生成的方法應用在建立人類中風的斑馬魚模型。第一部分我們首先最佳化實驗參數。我們利用螢光強度決定斑馬魚體內光化學染劑的濃度，接著建立了光化學染劑濃度與血栓生成時間的實驗參數。我們在斑馬魚背部主動脈 (dorsal aorta) 展現光化學誘發產生血塊，並利用血小板 (platelet) 和血纖維蛋白 (fibrin) 的螢光抗體標定血塊成分，並應用影像軟體分析其三維結構。第二部分的工作則是將此方法應用於造成斑馬魚的腦部缺血性中風。我們發現了在腦部基底動脈 (basilar artery) 分支血管缺血時，斑馬魚的生理狀況包括心功能及血液動力學和行為模式都有受到影響，呈現出相似於人類腦中風的症狀。根據以上的結果顯示出斑馬魚可以利用於人類中風的研究，而且比起以往的實驗方法來的簡易且具重複性。我們預期此動物模型之後可應用在快速藥物篩選，開發新的中風治療方法，或者對於中風疾病的研究有更深一層的了解。

According to World Health Organization (WHO), stroke is the primary cause of adult disability in developed countries and ranks only behind cardiac diseases as a cause of death. Stroke could lead to a rapid loss of brain function as a result of disturbance of the cerebral blood flow. Stroke can be caused by ischemia or hemorrhage; the former is caused by an interruption of the blood supply while the latter results from a rupture of blood vessels. A variety of animals such as rats or mice have been employed as model animals in the basic research of stroke. Common methods to induce stroke in the model animals include corrosion of blood vessels with FeCl3, ligation an artery by introducing a nylon intraluminal suture or applying an electrical current to the vessel. However, these methods suffer from drawbacks such as irreproducibility or complicated procedures of surgery. Recently, zebrafish (Danio rerio) has become a popular model organism of both fundamental and applied biomedical research. The zebrafish is a well-characterized vertebrate with a short cycle of reproduction, and transparent embryos and early larvae, hence allowing observation of blood circulation with light microscopy. In this study, I report a novel zebrafish model of stroke by photochemically induced thrombosis. I first established experimental parameters at the dorsal aorta of zebrafish larvae. To determine the intravascular concentration of photosensitizer in vivo, we developed a method based on fluorescence detection. We then determined the time to form thrombus at varied doses of photosensitizers. We demonstrated photochemically induced thrombosis in the dorsal aorta and showed the three-dimensional structure of thrombus labeled with fluorescent antibody of platelets and fibrins. We then applied this approach and successfully induced thrombosis in selected cerebral blood vessels of zebrafish larvae. We found that there was a drastic change in the hemodynamics, cardiac function, and swimming behaviors. We also observed rerouting of some cerebral vessels and neovasculature after stroke in zebrafish. The phenomena conform to the characteristics of human stroke. To our knowledge, this is the first demonstration of photochemically induced thrombosis and stroke in zebrafish larvae. In contrast to conventional method, our approach does not require complicated procedures of surgery. We anticipate that this new stroke animal model might not only facilitate development of therapeutic interventions against stroke but also improve our understanding of the pathophysiology of stroke.