我们感谢V. Zimmermann的鱼类护理，以及Thomas Bise博士，Catherine Pfefferli博士和Lea Gigon启动该项目及其初步结果。这项工作得到了瑞士国家科学基金会的支持，资助号为310030_208170。

这项研究是根据所有相关的道德法规进行的。斑马鱼的繁殖、饲养和维护符合欧洲实验动物科学协会联合会 （FELASA） 准则25。动物舍和所有实验程序均由瑞士弗里堡州兽医办公室批准。
1. 设备和设置
<ol><li>安排在可以制造研究仪器的技术车间生产不锈钢冷冻探头。 注意：提供具有特定尺寸的仪器设计（图 1A）。</li>
	<li>为避免在操作过程中处理探头时冻伤，请将手柄插入移液器吸头，并用胶带包裹。</li>
	<li>准备一个用于麻醉工作溶液的烧杯，一个用于处理鱼的勺子，一个潮湿的海绵和一个装有系统水的水箱，让鱼在手术后恢复。</li>
	<li>在每次实验前新鲜准备工作溶液。要制备工作溶液，请将 4 mL 三卡因储备溶液加入烧杯中的 100 mL 系统水中。 注意：麻醉储备液由溶解在 980 mL 蒸馏水中的 4 g 三卡因组成。用1M Tris-HCl，pH 9将pH调节至7后，用蒸馏水将溶液填充至1L。储备溶液对光敏感，应储存在4°C的琥珀色瓶中。</li>
</ol>2. 肌肉冷冻损伤手术
<ol><li>通过将探针浸入液氮中至少3分钟开始该过程。将海绵在系统水中弄湿，然后将其放在平坦的表面上。</li>
	<li>将一条成年斑马鱼转移到三卡因工作溶液中，并在 1 分钟或 2 分钟后用勺子轻轻触摸鱼以确认其无反应。如果鱼仍然有反应，请再等一会儿。</li>
	<li>将麻醉过的鱼放在湿海绵上。将尾柄定位在臀鳍后方和尾鳍前方。</li>
	<li>从液氮中取出冷冻探针。轻轻摇动探头，确保尖端上没有残留的液氮。</li>
	<li>将刮刀的边缘垂直于身体放在尾柄上（图1C）。将探头保持在此位置6秒，不要施加压力（图1D）。 注意：冷冻探头的重量足以确保仪器与组织之间的接触。</li>
	<li>从组织中释放冷冻探针，并将鱼转移到装有系统水的水箱中。</li>
	<li>在鱼从麻醉中醒来后恢复呼吸和游泳时监测它。如果30秒后没有发生眼球运动，通过将系统水移入鳃中来刺激鱼，直到动物自行开始呼吸。 注意：鱼应在几分钟内在回收水箱中恢复游泳。 注意：在接下来的几天里，可以拍摄鱼以监测它们的游泳活动（视频1 和 视频2）。在视频录制之前，将对照和受伤的鱼转移到半透明的交配缸中，让它们习惯至少 1 分钟。</li>
</ol>3.尾柄采集固定
<ol><li>准备 2 mL 4% 福尔马林或其他适合后续测定的固定剂、培养皿、镊子和手术剪刀。</li>
	<li>根据地区兽医办公室给予的法律许可，在冷冻损伤后的选定时间点对鱼实施安乐死。 注意：安乐死是通过过量服用三卡因溶液（300mg / L）约10分钟进行的。鳃运动丧失和尾鳍夹反射证实死亡。安乐死的时间点应根据再生阶段选择：例如，从冷冻损伤后 1 天 （dpci） 到 3 dpci 进行伤口清除;从3 dpci到10 dpci，用于启动肌肉再生;从 10 dpci 到 30 dpci 进行再生;并在30 dpci后完成组织修复。因此，为了评估肌肉退化和再生的不同步骤和生物学过程，需要在冷冻损伤后的不同时间点对鱼群实施安乐死。</li>
	<li>将安乐死的鱼放入含有去离子水的培养皿中。使用剪刀在尾柄的前后进行切口（图1E）。让组织在培养皿的去离子水中流血。</li>
	<li>收集尾柄，并用镊子将其转移到微量离心管中制备的固定溶液中。 注意：小心地将试管倒置几次，并在4°C下过夜。</li>
</ol>4.安装尾柄
<ol><li>在摇臂上用1x PBS洗涤固定组织10分钟。然后，将其转移到2mL微量离心管中，其中预冷的30%蔗糖在4°C的去离子水中，并轻轻倒置管数次。将微量离心管在4°C直立位置放置至少24小时。 注意：尾柄将漂浮在蔗糖溶液的顶部，并随着组织脱水而开始下沉。</li>
	<li>用 5 mm 的 O.C.T. 封片剂层填充嵌入模具。用镊子调整培养基中的尾柄。将其放在模具底部，并调整其横向或冠状截面的方向（图1F）。</li>
	<li>让培养基在一盒干冰中冷冻。一旦组织稳定在所需位置，在 O.C.T. 完全冻结之前填充模具的其余部分。将模具在-80°C下储存至少1小时。 注意：在这些条件下，组织可以保存数月。</li>
</ol>5. 用低温恒温器切割切片
<ol><li>设置切割厚度为 25 μm 的低温恒温器。将腔室温度调节为 −26°C，将试样温度调节至 −24°C，将切割角度调整至 12°。</li>
	<li>将冷冻块与标本一起放入低温恒温器中，并固定其方向以平行于块的底部切割。切割直到到达标本，然后修剪块以简化样本收集。</li>
	<li>准备六张粘合玻片。用连续的数字标记载玻片，以准备样品的重复。</li>
	<li>开始切割，并在标记的粘附载玻片上收集组织切片。根据进一步的实验需求排列幻灯片上的部分。</li>
	<li>让载玻片在室温下干燥1小时。将它们储存在-20°C的封闭盒子中。 注意：载玻片可以在此条件下安全存放长达 1 年。</li>
</ol>

成年斑马鱼的尾柄冷冻损伤

<ol>
	<li>Muneoka, K., Allan, C. H., Yang, X., Lee, J., Han, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+regeneration+and+regenerative+medicine.">Mammalian regeneration and regenerative medicine.</a> Birth Defects Research Part C: Embryo Today: Reviews. 84 (4), 265-280 (2008).</li><li>Carlson, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+principles+of+regeneration+in+mammalian+systems.">Some principles of regeneration in mammalian systems.</a> Anatomical Record. Part B, New Anatomist. 287 (1), 4-13 (2005).</li><li>Dumont, N. A., Bentzinger, C. F., Sincennes, M. -. C., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+cells+and+skeletal+muscle+regeneration.">Satellite cells and skeletal muscle regeneration.</a> Comprehensive Physiology. 5 (3), 1027-1059 (2015).</li><li>Relaix, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+on+skeletal+muscle+stem+cells.">Perspectives on skeletal muscle stem cells.</a> Nature Communications. 12 (1), 692 (2021).</li><li>Tulenko, F. J., Currie, P., Cartner, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+myology.">Zebrafish myology.</a> The Zebrafish in Biomedical Research. , 115-121 (2020).</li><li>Siegel, A. L., Gurevich, D. B., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+myogenic+precursor+cell+that+could+contribute+to+regeneration+in+zebrafish+and+its+similarity+to+the+satellite+cell.">A myogenic precursor cell that could contribute to regeneration in zebrafish and its similarity to the satellite cell.</a> The FEBS Journal. 280 (17), 4074-4088 (2013).</li><li>Corona, B. T., Wenke, J. C., Ward, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+volumetric+muscle+loss+injury.">Pathophysiology of volumetric muscle loss injury.</a> Cells Tissues Organs. 202 (3-4), 180-188 (2016).</li><li>Pfefferli, C., Ja&#378;wi&#324;ska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+art+of+fin+regeneration+in+zebrafish.">The art of fin regeneration in zebrafish.</a> Regeneration. 2 (2), 72-83 (2015).</li><li>Sehring, I. M., Weidinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advancements+in+understanding+fin+regeneration+in+zebrafish.">Recent advancements in understanding fin regeneration in zebrafish.</a> WIREs Developmental Biology. 9 (1), 367 (2020).</li><li>Poss, K. D., Wilson, L. G., Keating, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+regeneration+in+zebrafish.">Heart regeneration in zebrafish.</a> Science. 298 (5601), 2188-2190 (2002).</li><li>Sanz-Morej&#243;n, A., Mercader, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+insights+into+zebrafish+cardiac+regeneration.">Recent insights into zebrafish cardiac regeneration.</a> Current Opinion in Genetics &amp; Development. 64, 37-43 (2020).</li><li>Chassot, B., Pury, D., Ja&#378;wi&#324;ska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+fin+regeneration+after+cryoinjury-induced+tissue+damage.">Zebrafish fin regeneration after cryoinjury-induced tissue damage.</a> Biology Open. 5 (6), 819-828 (2016).</li><li>Chablais, F., Veit, J., Rainer, G., Ja&#378;wi&#324;ska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+heart+regenerates+after+cryoinjury-induced+myocardial+infarction.">The zebrafish heart regenerates after cryoinjury-induced myocardial infarction.</a> BMC Developmental Biology. 11, 21 (2011).</li><li>Schnabel, K., Wu, C. C., Kurth, T., Weidinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+cryoinjury+induced+necrotic+heart+lesions+in+zebrafish+is+associated+with+epicardial+activation+and+cardiomyocyte+proliferation.">Regeneration of cryoinjury induced necrotic heart lesions in zebrafish is associated with epicardial activation and cardiomyocyte proliferation.</a> PLoS One. 6 (4), e18503 (2011).</li><li>Gonzalez-Rosa, J. M., Martin, V., Peralta, M., Torres, M., Mercader, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+scar+formation+and+regression+during+heart+regeneration+after+cryoinjury+in+zebrafish.">Extensive scar formation and regression during heart regeneration after cryoinjury in zebrafish.</a> Development. 138 (9), 1663-1674 (2011).</li><li>Ryan, R., Moyse, B. R., Richardson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+cardiac+regeneration-Looking+beyond+cardiomyocytes+to+a+complex+microenvironment.">Zebrafish cardiac regeneration-Looking beyond cardiomyocytes to a complex microenvironment.</a> Histochemistry and Cell Biology. 154 (5), 533-548 (2020).</li><li>Ja&#378;wi&#324;ska, A., Sallin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+versus+scarring+in+vertebrate+appendages+and+heart.">Regeneration versus scarring in vertebrate appendages and heart.</a> Journal of Pathology. 238 (2), 233-246 (2016).</li><li>Alexander, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+orientation+of+muscle+fibres+in+the+myomeres+of+fishes.">The orientation of muscle fibres in the myomeres of fishes.</a> Journal of the Marine Biological Association of the United Kingdom. 49, 163-290 (1969).</li><li>Morin-Kensicki, E. M., Melancon, E., Eisen, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segmental+relationship+between+somites+and+vertebral+column+in+zebrafish.">Segmental relationship between somites and vertebral column in zebrafish.</a> Development. 129 (16), 3851-3860 (2002).</li><li>Berberoglu, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite-like+cells+contribute+to+pax7-dependent+skeletal+muscle+repair+in+adult+zebrafish.">Satellite-like cells contribute to pax7-dependent skeletal muscle repair in adult zebrafish.</a> Developmental Biology. 424 (2), 162-180 (2017).</li><li>Montandon, M., Currie, P. D., Ruparelia, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examining+muscle+regeneration+in+zebrafish+models+of+muscle+disease.">Examining muscle regeneration in zebrafish models of muscle disease.</a> Journal of Visualized Experiments. (167), e62071 (2021).</li><li>Pipalia, T. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+dynamics+of+regeneration+reveals+role+of+two+distinct+Pax7+stem+cell+populations+in+larval+zebrafish+muscle+repair.">Cellular dynamics of regeneration reveals role of two distinct Pax7 stem cell populations in larval zebrafish muscle repair.</a> Disease Models &amp; Mechanisms. 9 (6), 671-684 (2016).</li><li>Ratnayake, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+provide+a+transient+muscle+stem+cell+niche+via+NAMPT+secretion.">Macrophages provide a transient muscle stem cell niche via NAMPT secretion.</a> Nature. 591 (7849), 281-287 (2021).</li><li>Kaliya-Perumal, A. -. K., Ingham, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Musculoskeletal+regeneration:+A+zebrafish+perspective.">Musculoskeletal regeneration: A zebrafish perspective.</a> Biochimie. 196, 171-181 (2022).</li><li>Alestr&#246;m, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+Housing+and+husbandry+recommendations.">Zebrafish: Housing and husbandry recommendations.</a> Laboratory Animals. 54 (3), 213-224 (2019).</li><li>Saera-Vila, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocyte+dedifferentiation+drives+extraocular+muscle+regeneration+in+adult+zebrafish.">Myocyte dedifferentiation drives extraocular muscle regeneration in adult zebrafish.</a> Investigative Ophthalmology &amp; Visual Science. 56 (8), 4977-4993 (2015).</li><li>Baust, J. G., Gage, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+of+cryosurgery.">The molecular basis of cryosurgery.</a> BJU International. 95 (9), 1187-1191 (2005).</li><li>Galluzzi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+cell+death:+recommendations+of+the+Nomenclature+Committee+on+Cell+Death+2018.">Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.</a> Cell Death and Differentiation. 25 (3), 486-541 (2018).</li><li>Chablais, F., Ja&#378;wi&#324;ska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+myocardial+infarction+in+adult+zebrafish+using+cryoinjury.">Induction of myocardial infarction in adult zebrafish using cryoinjury.</a> Journal of Visualized Experiments. (62), e3666 (2012).</li><li>Chablais, F., Ja&#378;wi&#324;ska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regenerative+capacity+of+the+zebrafish+heart+is+dependent+on+TGFbeta+signaling.">The regenerative capacity of the zebrafish heart is dependent on TGFbeta signaling.</a> Development. 139 (11), 1921-1930 (2012).</li><li>Bise, T., Sallin, P., Pfefferli, C., Ja&#378;wi&#324;ska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+cryoinjuries+modulate+the+efficiency+of+zebrafish+heart+regeneration.">Multiple cryoinjuries modulate the efficiency of zebrafish heart regeneration.</a> Scientific Reports. 10 (1), 11551 (2020).</li></ol>

作者没有什么可透露的。

斑马鱼提供了一种脊椎动物模式生物来研究肌肉再生的机制。大多数现有的损伤方法，如激光消融或刺伤，导致相对较小的组织破坏20,21,22,23。已对眼外肌进行了大切除术26。然而，由于切割体壁对健康有害，这种手术方法可能不太适合侧向肌肉组织。为了避免这种侵入性手术，该协议描述了一种较轻的损伤形式，但会对尾柄造成严重损害。这种方法依赖于表面操作，允许非常精确地瞄准身体一侧的几个子宫肌层。冷冻损伤模型的优势在于其可重复性和产生广泛肌肉退化的能力;基于这些优势，该模型为研究身体如何对显着肌肉损失做出反应提供了新的途径。
极端寒冷的应用导致热休克，其破坏受影响肌肉组织中的质膜和细胞器27。结果，受伤的肌纤维经历了“意外”细胞死亡28。因此，受损组织可以通过伤口清除的自然机制被吸收。斑马鱼对冷冻损伤手术的耐受性很好，因为这项研究的存活率接近 100%，因为预冷探针在确切的持续时间内正确定位在身体上。但是，如果伤口太宽（例如，如果施加的压力太大或冷冻损伤的持续时间太长），鱼可能会在手术后不久表现出异常的游泳运动，并且动物应该被安乐死作为人道终点。对于其他鱼类，冷冻探针的暴露时间应根据身体的大小进行调整。
冷冻损伤后，鱼可以恢复游泳活动，没有任何异常运动的症状。然而，冷冻受伤的鱼游泳的动态性不如对照鱼，这表明有一些轻微的损伤。需要进一步量化冷冻损伤后不同时间点的鱼类行为，以确定游泳性能的时间变化。
冷冻损伤方法对尾柄其他非肌肉组织的影响仍有待阐明。显然，最外层的身体层（即皮肤）因手术而受损。在这种情况下，冷冻损伤方法可以为研究伤口愈合、鳞屑再生和色素沉着模式的恢复提供新的策略。此外，子宫肌层的脉管系统和神经支配也可能受到冷冻损伤的影响，这些主题需要进一步研究。
冷冻损伤模型以前已用于研究斑马鱼心脏再生13,14,15,29。与心室切除方法10相比，该方法显示出一些优势，因为富含胶原蛋白的疤痕的瞬时沉积，可以更好地模拟人类的梗死愈合反应30。值得注意的是，斑马鱼可以在多次冷冻损伤后再生心脏31。有趣的是，冷冻损伤也被应用于斑马鱼鳍，导致组织溶解过程12。与经典的鳍截肢相反，剩余的冷冻损伤残肢包含一个扭曲的边缘，其中有死物质和健康细胞的混合物。对斑马鱼器官（心脏和鳍）的研究表明，即使在广泛的组织损伤后，斑马鱼也能恢复其原始功能成分的强大能力。冷冻损伤的骨骼肌是否激活修复和再生过程之间的相互作用值得未来的研究。

在脊椎动物中，各种组织的受损部分在生命周期中经历稳态更新和恢复。这种更新和恢复能力通常取决于感受态干细胞的存在或成熟细胞的增殖能力1,2。骨骼肌包括有丝分裂后肌纤维，其与局部干细胞相关，称为卫星细胞3,4,5,6。因此，该组织包含用于有效密封中断连续性区域或修复小伤口的细胞源。然而，哺乳动物骨骼肌中较大的体积损失通常伴随着非再生修复，例如纤维化7。动物模型可以为促进广泛受损器官再生的生物学机制提供新的见解。
斑马鱼是一种成熟的模式生物，具有很高的再生能力。成年斑马鱼可以再生其尾鳍的截肢部分或心室的切除顶点8,9,10,11。此外，以前已将冷冻损伤方法应用于研究斑马鱼的鳍和心脏再生12,13,14,15。在内脏的情况下，冷冻损伤方法具有诱导细胞死亡而不破坏器官完整性的优点，从而模仿生理条件16,17。组织碎片在伤口愈合过程中通过自然清除分解，然后是修复过程。然而，这种方法是否可以应用于骨骼肌仍有待确定。
在鱼类中，横向肌肉组织使躯干在游泳过程中能够左右弯曲18.骨骼肌被组织成同色异构体，称为肌粒，它们被结缔组织隔开5,19。斑马鱼可以在轻微的组织破坏后再生肌肉，例如由激光消融或刺伤引起的组织破坏20,21,22,23,24，但整个肌层是否可以在广泛损伤后再生仍然未知。这种知识上的差距可能是由于缺乏合适的伤害模型。该协议建立了一种新的方法来诱导骨骼肌的广泛损伤，跨越多个肌层。所描述的冷冻损伤方法基于用预冷的不锈钢仪器快速冷冻和解冻肌纤维。尽管受到广泛的破坏，但鱼类的健康并没有受到严重损害。整个肌粒可以恢复，因此，这项工作为研究成年斑马鱼肌肉组织再生机制提供了新的模型系统。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Program</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health (NIH)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Photoshop Version 23.5.3</td>
			<td>Adobe</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Material/ Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35/10 mm Petri Dish</td>
			<td>Greiner Bio-one</td>
			<td>Item No.: 627102</td>
			<td></td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Sony</td>
			<td>/</td>
			<td>HDR-PJ410</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Histcom</td>
			<td>HRA C50</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde ~36%</td>
			<td>Sigma-Aldrich</td>
			<td>47630</td>
			<td></td>
		</tr>
		<tr>
			<td>Macro 50 mm f/2.8 EX DG lens</td>
			<td>Sigma</td>
			<td>/</td>
			<td>Discontinued lense</td>
		</tr>
		<tr>
			<td>Peel-A-Way Embedding Truncated Molds T8</td>
			<td>Polyscience, Inc.</td>
			<td>18985</td>
			<td></td>
		</tr>
		<tr>
			<td>Slides Superfrost Plus</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Sponge</td>
			<td>any</td>
			<td>any</td>
			<td>flat sponge, c.a. 7cm x 3 cm x 1 cm</td>
		</tr>
		<tr>
			<td>Stainless steel cryoprobe</td>
			<td>Custom-made</td>
			<td>/</td>
			<td>specifics in the article</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>84100</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Any</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>TCS SP2</td>
			<td>Leica</td>
			<td>/</td>
			<td>Discontinoued product</td>
		</tr>
		<tr>
			<td>Tissue-Tek O.C.T. compound</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine (Anestethic)</td>
			<td>Sigma</td>
			<td>E10521</td>
			<td></td>
		</tr>
		<tr>
			<td>Dyes and Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dapi</td>
			<td>Sigma</td>
			<td>10236276001</td>
			<td>Concentration: 1/2000</td>
		</tr>
		<tr>
			<td>Phalloidin-Atto-565 (F-actin)</td>
			<td>Sigma</td>
			<td>94072</td>
			<td>Concentration: 1 / 500</td>
		</tr>
		<tr>
			<td>Tropomyosin (TPM1)</td>
			<td>DHSB</td>
			<td>CH1</td>
			<td>Concentration: 1 / 50</td>
		</tr>
		<tr>
			<td>Recipies/Solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>123 mM NaCl</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2.7 mM KCl</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>10 mM Na2HPO4</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>1.8 mM KH2PO4</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>AFOG solution</td>
			<td>3 g Fuchsin</td>
			<td>Fisher Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2 g Orange G</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>1 g Anilin blue</td>
			<td>Fulka AG</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>200 ml acifidied distilled H2O (pH 1.1)</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a cryoinjury model for studying skeletal muscle regeneration of the caudal peduncle in adult zebrafish

骨骼肌在轻微损伤后通过激活卫星样干细胞进行更新和恢复。肌肉组织的严重损伤通常会导致人类纤维化。与哺乳动物相比，斑马鱼具有更高的先天器官再生能力，为研究器官广泛损伤后的组织修复提供了强大的模型。在这里，描述了一种冷冻损伤模型，以诱导成年斑马鱼尾柄的四个肌层严重损伤。定制的冷冻探头设计用于贴合体型，并可重复地损伤从皮肤到中线的外侧肌肉组织。重要的是，身体完整性保持不变，鱼继续游泳活动。通过组织切片上肉瘤蛋白的组织学染色和荧光染色来评估骨骼肌的变化。这种方法将开辟新的研究途径，旨在了解骨骼肌的退化如何诱导修复反应，从而重新激活成年斑马鱼的肌源程序。

冷冻损伤后监测鱼 为了确定肌损伤对动物的影响，对冷冻损伤后1天（dpci）和5 dpci的对照和冷冻损伤鱼进行了视频记录。每组有五条鱼。在1 dpci时，冷冻受伤的鱼游泳不太活跃，但它们没有表现出任何异常运动，例如旋转，卷积或平衡降低（视频1）。在畜牧系统中，它们在水箱中的位置和食物摄入量与未受伤的鱼相似。在接下来的几天里，正常行为一直持续存在，如 5 dpci 的视频（视频 2）所示。总之，尾柄的冷冻损伤程序并未严重影响动物的健康。
尾柄切片的组织学分析 为了评估损伤的程度，选择了4 dpci的时间点，因为这是肌纤维碎片在伤口中完全被吸收的时间点。为了分析沿身体背腹轴和前后轴的冷冻损伤的影响，使用了两组鱼（即分别是尾柄的冠状和横向部分）（图1F）。
通过由苯胺蓝、酸性紫红色和橙色 G （AFOG） 组成的三色染色分析切片。使用这种试剂组合，完整的肌肉显示为橙色，脊髓显示为深红色，胶原基质显示为蓝色。为了确定受损肌层的数量，即鱼肌肉组织的同色异构体单位，分析了一系列切片（图2）。肌细胞边界，称为肌瘤，通过胶原沉积识别，如蓝色检测。受损区域由没有橙色染色确定。对具有明显肌瘤的标本进行仔细检查后发现，大约四个连续的肌粒受损，这是从缺乏橙色染色推断的那样（n，鱼的数量= 4;图3A，A'）。同一条鱼的未受伤侧作为内部参考。
为了检查垂直于身体轴线的伤口深度，使用斑马鱼在4 dpci和7 dpci下制备横向切片。后一个时间点对应于肌源程序的激活，从而对应于肌肉再生的开始。这些标本的AFOG染色显示，在身体的冷冻损伤侧腹中广泛缺乏橙色染色，划定了退化骨骼肌的区域（图3B，C）。在 4 dpci 和 7 dpci 时，伤口区域从皮肤向垂直隔膜跨越组织。这表明冷冻损伤方法深深地针对尾蒂的外侧半部分，该蒂在手术后 7 天内仍然没有功能性肌肉。总之，四个子宫肌层在尾柄的一侧严重受损。
横向切片的免疫荧光分析 为了评估肌肉再生的动力学，实验组的鱼在4 dpci，7 dpci，10 dpci和30 dpci下被安乐死。尾柄的横截面使用鬼笔环肽（与丝状肌动蛋白[F-肌动蛋白]结合）、检测肌节蛋白的原肌球蛋白-1抗体和标记细胞核的DAPI进行多色荧光染色标记。在所有时间点，未受伤的身体一半提供了内部控制;在未受伤的对照部位强烈检测到F-肌动蛋白和原肌球蛋白1，表明组织未受损（图4）。
在4 dpci时，尾柄的损伤侧含有丰富的DAPI阳性细胞，但几乎没有观察到F-肌动蛋白和原肌球蛋白1免疫荧光，表明伤口区域肌肉退化（图4A-B'）。在7 dpci时，可以在靠近垂直体中线的伤口部分检测到原肌球蛋白1和F-肌动蛋白（图4C-D'）。这种表达模式划定了新肌纤维形成开始在尾柄中的位置。在10 dpci时，两个肌肉标志物都向身体表面扩展，表明骨骼肌正在再生（图4E-F'）。在30 dpci时，身体两侧显示出相似的F-肌动蛋白染色分布（图4G-H'）。这一发现表明，在尾柄冷冻损伤后，骨骼肌得到了有效的恢复。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65491/65491fig01.jpg"> 图1：肌隐损伤的实验装置 。 （A） 由不锈钢制成的定制冷冻探头的尺寸。仪器的远端部分由一把刮刀组成，铲子的边缘深度为1毫米，以解释斑马鱼体的曲率。该工具的中间部分包括一个用作重物的圆柱体和一个储液罐，以在手术过程中保持刮刀的低温。仪器的近端是薄金属手柄的形式。（乙，丙）麻醉的成年鱼在潮湿的海绵上，冷冻探针在尾柄上。探头在室温下。（B）将探针的边缘水平放置在尾柄附近，以显示鱼和工具之间的相对大小。（C）对于冷冻损伤，工具的尖端垂直于鱼。（D）从鱼的腹侧冷冻损伤程序的示意图，以全面的方式显示操作。将冷冻探针在液氮中预冷，并立即放置在鱼的一侧6秒。 （E）在冷冻损伤后的特定时间点，对鱼实施安乐死，并收集其尾柄进行固定。（F）固定材料经组织学处理，沿冠状面或横面切片。 <a href="https://www.jove.com/files/ftp_upload/65491/65491fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65491/65491fig02.jpg"> 图2：从身体背侧到腹侧位置的尾柄受损肌层的组织学分析。 冷冻损伤后 4 天对一系列冠状切片进行 AFOG 染色 （dpci）。这些部分从背侧到腹侧，如第一和最后一面板的顶部所示。这些部分是不相邻的，它们之间的间隔约为150μm。未受伤的肌肉通过肌肉的橙色染色来检测，而受伤的组织缺乏这种染色并且呈现灰色（用虚线包围的区域）。含胶原蛋白的组织，如皮肤，被染成蓝色。脊髓呈杆状结构，呈红色。鱼的数量，n = 4。 <a href="https://www.jove.com/files/ftp_upload/65491/65491fig02large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65491/65491fig03.jpg"> 图 3：使用 AFOG 染色评估尾柄损伤深度。 （A，A'）冠状部分位于脊髓水平（红色染色的水平杆）。下图显示了一个放大区域，上面图像中有一个框架。连续的肌肌边界显示为斜向脊髓的胶原条纹（蓝色）（放大图像A'中的红色箭头）。（乙，丙）横截面显示未受伤的侧翼有橙色染色的肌肉和冷冻损伤的侧面有灰色染色。受损区域用黑色虚线包围。垂直隔膜（用红色虚线表示）将身体细分为对照侧和冷冻损伤侧。鱼的数量，n = 4 每个时间点。<a href="https://www.jove.com/files/ftp_upload/65491/65491fig03large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65491/65491fig04.jpg"> 图 4：冷冻损伤后肌肉蛋白的免疫荧光检测。4 dpci、7 dpci、10 dpci 和 30 dpci 的横截面荧光染色，如面板左侧和顶部 （A-B'） 所示。在 4 dpci 时，损伤组织（用虚线包围）为 DAPI 阳性（蓝色），但无鬼笔环肽染色（绿色）或原肌球蛋白-1 免疫反应性（红色），提示冷冻损伤后肌纤维退化。（中德）在 7 dpci 时，两个肌肉标志物逐渐出现在受伤区域，表明再生过程。在新形成的纤维中，原肌球蛋白-1似乎比F-肌动蛋白更强烈。（E-F'）在 10 dpci 时，损伤区充满新的肌纤维，与 F-肌动蛋白相比，这些肌纤维显示出更高强度的原肌球蛋白-1 免疫反应性。（G-H'）在30 dpci时，在身体两侧检测到类似的肌纤维模式。面板 A、C、E 和 H 中的帧包含在右侧相邻图像中放大的区域。使用Adobe Photoshop从图像中擦除在子宫肌层外发出荧光的真皮鳞片。鱼的数量，n = 4 每个时间点。<a href="https://www.jove.com/files/ftp_upload/65491/65491fig04large.jpg" target="_blank">请点击此处查看此图的大图。</a>

Watch this Scientific Journal Video about A Cryoinjury Model for Studying Skeletal Muscle Regeneration of the Caudal Peduncle in Adult Zebrafish at JoVE.com

作者聚焦：用于研究成年斑马鱼尾柄骨骼肌再生的冷冻损伤模型  

该协议描述了一种冷冻损伤模型，以诱导成年斑马鱼中几个尾部肌层的严重损伤。该方法为研究非哺乳动物脊椎动物严重丧失组织后的骨骼肌再生提供了一种新方法。

研究成年斑马鱼尾柄骨骼肌再生的冷冻损伤模型

Skeletal muscle undergoes renewal and restoration after minor injury through the activation of satellite-like stem cells. Severe injuries of the ...

斑马鱼模型可以帮助我们了解有效器官再生的自然机制。我们的实验室之前已经建立了一种冷冻损伤方法，用于研究斑马鱼如何恢复心脏和健康。通过这个新协议，我们的目标是将我们的研究扩展到另一个器官，即肌肉组织。我们还没有理解为什么斑马鱼的器官再生比人类更有效。该协议提供了一种有前途的方法，以研究肌肉组织的成功恢复，尽管鱼体中的伤口相对较大。这种冷冻损伤模型至少具有三个优点。首先，肌肉组织迅速受损，而不会破坏身体的完整性。其次，伤口是可复制的，并通过自然愈合清除。最后，鱼在冷冻损伤后可以正常游泳，这表明这个过程对动物有轻微的影响。我们的研究结果将斑马鱼尾柄引入再生生物学领域。这项研究路径将有助于破译指导肌生成程序重建成年斑马鱼复杂肌肉组织的分子机制。

a-cryoinjury-model-for-studying-skeletal-muscle-regeneration-caudal

Research

JoVE Journal

Biology

A Cryoinjury Model for Studying Skeletal Muscle Regeneration of the Caudal Peduncle in Adult Zebrafish

1.9K 次观看.  University of Fribourg. 斑马鱼模型可以帮助我们了解有效器官再生的自然机制。我们的实验室之前已经建立了一种冷冻损伤方法，用于研究斑马鱼如何恢复心脏和健康。通过这个新协议，我们的目标是将我们的研究扩展到另一个器官，即肌肉组织。我们还没有理解为什么斑马鱼的器官再生比人类更有效。该协议提供了一种有前途的方法，以研究肌肉组织的成功恢复，尽管鱼体中的伤口相对较?...

视频: 作者聚焦：用于研究成年斑马鱼尾柄骨骼肌再生的冷冻损伤模型  

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

成人和胚胎骨骼的肌肉Microexplant文化和骨骼肌肉干细胞的分离

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

科研

生物学

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

在体内电穿孔Morpholinos到成年斑马鱼视网膜

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

激光伤害的斑马鱼胚胎骨骼肌

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavaging成年斑马鱼

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo&#39;s swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

对于侧线再生成年斑马鱼的含量

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

In Vivo Surface Electrocardiography for Adult Zebrafish

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of zebrafish not only as a research model for human cardiac electrophysiology and myopathies but also as a surrogate model in high throughput pharmaceutical screening for potential cardiotoxicities to humans, such as QT prolongation. As such, in vivo electrocardiography for adult zebrafish is an electrical phenotyping tool that is necessary, if not indispensable, for cross-sectional or longitudinal in vivo electrophysiological characterizations. However, too often, the lack of a reliable, practical, and cost-effective recording method remains a major challenge preventing this in vivo diagnostic tool from becoming more readily accessible. Here, we describe a practical, straightforward approach to in vivo electrocardiography for adult zebrafish using a low-maintenance, cost-effective, and comprehensive system that yields consistent, reliable recordings. We illustrate our protocol using healthy adult male zebrafish of 12-18 months of age. We also introduce a rapid real-time interpretation strategy for quality validation to ensure data accuracy and robustness early in the electrocardiogram recording process.

Here, we present a reliable, minimally invasive, and cost-effective method to record and interpret electrocardiograms in live anesthetized adult zebrafish.

在 Vivo 表面心电图中成人斑马鱼

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of ...

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the zebrafish cardiovascular tissues have been limiting in studying their electrophysiological properties. Previous methods have been described for dissection of zebrafish hearts and isolation of ventricular cardiac myocytes. However, the isolation of zebrafish atrial and vascular myocytes for electrophysiological characterization was not detailed. This work describes new and modified enzymatic protocols that routinely provide isolated juvenile and adult zebrafish ventricular and atrial cardiomyocytes, as well as vascular smooth muscle (VSM) cells from the bulbous arteriosus, suitable for patch-clamp experiments. There has been no literary evidence of electrophysiological studies on zebrafish cardiovascular tissues isolated at embryonic and larval stages of development. Partial dissociation techniques that allow patch-clamp experiments on individual cells from larval and embryonic hearts are demonstrated.

The present protocol describes the acute isolation of viable cardiac and vascular smooth muscle cells from adult, juvenile, larval, and embryonic zebrafish (Danio rerio), suitable for electrophysiological studies.

从成年、幼年、幼虫和胚胎斑马鱼中分离心脏和血管平滑肌细胞以进行电生理研究

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the ...

Methods Article

研究成年斑马鱼尾柄骨骼肌再生的冷冻损伤模型