建立分析斑馬魚心律與胎動的自動化程序及其應用

斑馬魚由於易於維護，需要的實驗室空間小，後代數量多，胚胎發育過程快，繁殖週期短，因此逐漸成為廣泛使用的低等脊椎動物模型。此外，斑馬魚基因組已完全測序，並且在反映在藥物反應中的蛋白質水平上顯示出與人類基因具有 70% 的相似性，因為包含藥物結合靶標的蛋白質是相似的。與其他脊椎動物模型相比，斑馬魚在早期發育階段具有透明度高的優勢，在心血管研究中，可以方便進行非侵入性的觀察。當前斑馬魚心臟相關性能評估的方法多半只獲取心率參數。然而，還有其他重要的心臟生理參數，例如每搏輸出量、射血分率、縮短分率、心輸出量和心跳規律性等。在本研究論文中，我們開發了一個基於 kymograph 的巨集指令，可以在 ImageJ軟體平台上來量測這些珍貴的心臟生理參數。此外，我們也在水蚤上測試了我們的工具的通用性，發現的確能夠用來研究較為快速的心臟跳動與相關生理。我們也開發了一種定量魚類胚胎尾部擺動活力的TCMacro巨集指令，利用監測像素強度差異變化，並撰寫excel VBA 腳本，簡化數據處理過程並獲得尾部擺動次數、持續時間和間隔三個主要參數，可以用來量測魚類胚胎發育早期的神經與肌肉的反射運動。總之，本研究論文開發出兩種操作方便且應用性高的ImageJ巨集指令，分別是kymograph與TCMacro，可以一站式，高精度與方便地獲取多個心臟與神經性能參數。對於研究人員利用魚類胚胎進行藥物或環境毒物之心臟與神經生理評價，帶來非常大的方便性與高再現性。

關鍵字

斑馬魚； ImageJ ；影像分析；巨集指令；心臟生理；尾部擺動

並列摘要

Zebrafish is a widely used animal model due to being easy to maintain, requiring little laboratory space, have a high offspring number, fast embryonic development process and short reproductive cycle. Additionally, zebrafish genome is fully sequenced and showed 70% similarity with human genes at protein level reflected in drug response, as the protein comprising drug binding targets is similar. During its early stage, zebrafish has several advantages compared to other popular animal models, such as body transparency and high locomotor activity. In cardiovascular study, its body transparency during larval stage enable non-invasive observation of cardiovascular system in zebrafish. Current methods in cardiac performance assessment are mostly limited to obtaining heart rate. However there are other important cardiac endpoints such as stroke volume, ejection fraction, fractional shortening, cardiac output, and heartbeat regularity which mainly have to use third-party software to obtain. In this study, we developed an ImageJ macro script of kymograph to conduct cardiac physiology endpoint assessment. Additionally, we also tested our tool utility on Daphnia, which have transparent body with extremely faster heart rate. Next, we developed a tool, TCMacro, to observe tail coiling, a reflex response in fish embryos mainly used for neurotoxicity assessment. Previous methods to analyze this activity is largely based on paid third-party software. TCMacro is an ImageJ-based macro script that able to reduce operational complexity. The basic concept of TCMacro is on monitoring the pixel intensity changes during tail coiling movement. In addition, excel VBA scripts were also included to ease the process of data processing and obtaining three major endpoints of tail coiling counts, tail coiling duration, and tail coiling interval. In conclusion, two ImageJ-based macros of kymograph and TCMacro were developed to provide researchers an easy to use inexpensive, one-stop method to obtain multiple cardiac performance and tail coiling activity endpoints in fish embryos with high accuracy and convenience.

並列關鍵字

zebrafish ； cardiovascular ； kymograph ； tail coiling ； macro ； ImageJ

參考文獻

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建立分析斑馬魚心律與胎動的自動化程序及其應用

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