飛秒脈衝雷射消融引發細胞凋亡於斑馬魚心肌傷害模型

Abstract

心臟受傷後的組織再生 (regeneration) 、腔室的形貌變化 (cardiac remodeling) 及心功能的改變是重要的研究課題。由於具有發育快速、基因改造技術成熟、飼養費用低等優點，斑馬魚近年來已成為一受歡迎的實驗動物。在此研究我們應用飛秒脈衝雷射消融技術 (femtosecond-pulsed laser ablation)，嘗試建立心肌傷害的斑馬魚模型。我們用近紅外光飛秒脈衝雷射，在斑馬魚幼魚心室上的特定區域掃描，可造成心肌傷害。我們也應用「共軛焦螢光顯微鏡」及「擬動態三維影像技術」觀察心室受傷後形貌及功能的變化。結果顯示，雷射消融區域內的心肌細胞已死亡。心室受傷後約兩小時，照射區域的傷口明顯向腔室內部塌陷，腔室的體積縮小，但腔室之截面積則維持不變。在12到24小時後，心室腔室逐漸地呈現更扁平化，但這段期間之腔室體積則無明顯改變。在此階段，受傷區域之面積略為縮小，顯示心肌已開始再生。雖然在斑馬魚幼魚心臟上觀察到的心室腔室之結構變化和心肌再生現象與人類心肌梗塞後呈現的特徵不同，但心功能參數例如心輸出量 (stroke volume) 及射出分率 (ejection fraction) 則呈現類似的下降。我們進一步利用老鼠的肌肉細胞，探討飛秒脈衝雷射消融引發的細胞死亡機制。結果顯示飛秒脈衝雷射消融可造成細胞凋亡 (apoptosis)，細胞內的粒線體在脈衝雷射照射後會斷裂為片段，綜合其他證據顯示此細胞死亡為受粒線體調控的內生性 (intrinsic) 細胞凋亡機制，我們也發現脈衝雷射照射後細胞內的鈣離子及活性含氧物質 (reactive oxygen species) 會增加，但是鈣離子增加並非飛秒脈衝雷射消融引發細胞凋亡的主要啟動因素。此研究幫助了解飛秒脈衝雷射消融引發的組織傷害和細胞死亡的機制。和傳統以刀片切除或低溫造成斑馬魚心肌受傷的方法，應用飛秒脈衝雷射消融可在心臟的任意位置及控制的範圍引發局部的心肌損傷，未來預期可應用於心肌受傷後再生的研究。

Cardiac regeneration and remodeling after myocardial injury has received much research attention. Zebrafish has recently emerged as another popular model organism because of its numerous attractive features such as rapid development, ease of genetic manipulation and low cost of maintenance. Here we report the application of femtosecond (fs)-laser ablation to develop a zebrafish model of myocardial injury. Illumination of the zebrafish heart with a fs-laser beam (800 nm, 80 MHz, ~100 fs) for a short duration consistently created a lesion on selected target regions of myocardium. Cell viability stains confirmed cardiomyocyte death near the target region after ablation. Confocal fluorescence microscopy and pseudodynamic imaging were employed to observe the morphological and functional changes of the injured heart. Approximately two hours after ablation at the ventricular myocardium, the chamber showed a slight inward deformation and its volume decreased significantly while the lateral dimension remained similar. The chamber remained unchanged in size with time (till 12 to 24 hours) while became flattened with the lateral dimension being significantly enlarged. Besides, the lesion seemed to decrease in size indicating the onset of regeneration. While the general feature of such structural deformation and regenerative process is distinct from that in human heart after myocardial infarction, the ejection fraction, a crucial parameter of cardiac function, consistently became decreased. To gain more mechanistic insight, we investigated also fs-laser ablation on a mouse myoblast cell line. Our results show that ablation of cells in vitro could induce apoptotic death, and such cell death was mediated by a mitochondria-dependent pathway; mitochondria consistently became fragmented. Both intracellular calcium ion and reactive oxygen species (ROS) increased significantly after ablation while the former was found not likely to be the major determinant of apoptotic death. In comparison to other methods such as resection with scissors or cryoinjury with a cooled probe, our approach uniquely allows creation of myocardial injury on selected sites of the heart with controlled lesion size. This study advances our understanding of tissue injury and cell death induced by fs-laser ablation, which is expected to facilitate fundamental study on cardiac regeneration after injury.