The authors have nothing to disclose.

所有的程序都按照"实验动物福利原则"所述的准则进行（卫生出版没有。85-23国家研究院​​，1985年修订）和经批准的罗莎琳德富兰克林大学实验动物管理和使用委员会动物协议08-19。 1，庆大霉素诱导的毛细胞坏死<ol><li>制备硫酸庆大霉素生理盐水在0.004％的终浓度（4.32毫摩尔）。 </li><li>在含有0.004％（4.32毫米）庆大霉素溶液的容器放置成鱼（ 斑马鱼 ，4-6个月的婴儿）。任何容器均可使用，但我们使用鱼容器从医药品水产系统是在宽7所示，在高6，和在长7。将容器鱼在孵化器设定在28℃，24小时。在足够的电平设置流体在罐的总容积在一个可行的状态保持鱼的24小时期间。注:庆大霉素液的曝气不是NECEssary如果有足够的容积用于鱼被处理的数量。 </li></ol>毛细胞的2。活体染色<ol><li>制备的荧光活体染料[4 - 4 - 二乙基氨基）-N-甲基吡啶鎓碘化物（485nm激发λ和甲醇603 nm发射λ）从15毫克/毫升的工作原液的0.08％的浓度（在生理盐水中）乙醇。 </li><li>以确定是否庆大霉素治疗是有效的控制和庆大霉素的一个子集处理的鱼是由在含有活体染料6孔培养板的孔放置鱼立即染色。根据需要为要达到统计学显着性使用鱼的足够数量（培养板，基于神经丘计数检查者的速度，将鱼在所述板以交错的方式随着时间的推移，这样的鱼不染色，在75分钟按照步骤2.3中所述。 </li><li>通过荧光显微镜放置板从一个抽屉里板凳2.2步可用于检查神经丘染色的。关掉房间的灯，以防止活体染料的猝灭过在室温下1小时，染色时间。 </li><li>准备两种染料洗出和麻醉水箱。染料洗出的水是正常的鱼和水的麻醉水，补充足2 - 苯氧基乙醇，这样一1:1000稀释在正常的鱼水的实现。 </li><li>将鱼超出正常的鱼水冲洗多余的活体染料，并继续执行步骤3.1观察生命染料染色的鱼。 </li><li>为了研究神经丘的再生，转移在28°C洗涤正常的鱼水的孵化8-16小时之间的庆大霉素处理的鱼</li><li>在8-16小时之间的不同时间，鱼从培养箱中取出，洗涤，染色按照步骤2.1-2.4所示。继续执行步骤3.1观察生命染料染色的鱼。 </li></ol> 3，麻醉测定鱼和神经丘的荧光计数<ol><li&gt;吸干纸巾上每条鱼去除多余的液体，然后将其放置在一个湿润的滤纸是集中在塑料培养皿的盖子。 </li><li>放置在荧光立体显微镜阶段的盖子，得到的中间体线圈的活体染料染色的神经丘的数字图像。 </li><li>使用数码相机放置在荧光立体显微镜载2X的放大率捕获图像用于后续的定量分析。注:立体显微镜的放大倍数设定可能取决于显微镜使用的品牌，但该设置应该让身体中缝内便于查看和个别神经丘的计数。 </li><li>通过计数在鱼刚好接近右胸鳍的最底层的腹侧四个指定的线圈内的可见神经丘的数量（参见图1）确定再生的量。统计分析使用适当的测试，如方差分析Ør中的学生T检验。实验应利用最少的每个时间点5鱼和所有的实验应重复至少3倍的。 </li><li>基于神经丘再生时间曲线（ 见图3），数8-16小时庆大霉素后洗出的神经丘是再生曲线的线性相位内。注意:使用线性时间阶段允许控制组和实验组之间的正确定量分析。 </li></ol>个别毛细胞4。为获得更高的分辨率定量分析的，如果神经丘分析无统计学意义荧光计数<ol><li>如果在神经丘水平的定量分析是不显著，分析在单个毛细胞的电平也可以被用来获得较高程度的分辨率。在特定的时间点庆大霉素后洗出（时间点的基础上，早期的神经丘研究），活体染料ST选择鱼泉如在方案2中所述的鱼，然后用2 - 苯氧基乙醇以1:500稀释为1-5分钟安乐死的鱼。 </li><li>在柔和的光，以防止淬火，使4切口，使得正方形皮瓣制备是如下进行的。做一个切口沿鱼的上肋，直到它与肛门鳍对准，然后使整个腹部切口，最后，这样的方皮瓣被创建使这些切口的每一侧上的两个垂直切口。注意:这种皮肤准备工作将纳入在神经丘实验中使用的身体中针。 </li><li>将皮肤标本在载玻片上，然后将一个圆形玻璃盖片在皮肤切除标本帮锚杆和扁平化的组织进行后续的数字成像。 </li><li>利用皮肤标本从步骤4.3，获得毛细胞的中间体的线圈的每个神经丘内的数字图像。拍摄图像在至少60X的放大倍数再算上头发CE单个神经丘内LLS为对照组和实验组的比较定量分析（ 见图4）。 </li></ol>

<ol>
	<li>Dambly-Chaudire, C., Sapde, D., Soubiran, F., Decorde, K., Gompel, N., Ghysen, 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+Lateral+Line+of+Zebrafish:+a+Model+System+for+the+Analysis+of+Morphogenesis+and+Neural+Development+in+Vertebrates.">The Lateral Line of Zebrafish: a Model System for the Analysis of Morphogenesis and Neural Development in Vertebrates.</a> Biol. Cell. 95 (9), 579-587 (2003).</li><li>Montgomery, J., Carton, G., Voigt, R., Baker, C., Diebel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+Processing+of+Water+Currents+by+Fishes.">Sensory Processing of Water Currents by Fishes.</a> Phil. Trans. Royal Soc. London B Biol. Sci. 355 (1401), 1325-1327 (2000).</li><li>Buck, L. M., Winter, M. J., Redfern, W., Whitfield, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ototoxin-Induced+Cellular+Damage+in+Neuromasts+Disrupts+Lateral+Line+Function+in+Larval+Zebrafish.">Ototoxin-Induced Cellular Damage in Neuromasts Disrupts Lateral Line Function in Larval Zebrafish.</a> Hearing Res. 284 (1-2), 1-2 (2012).</li><li>Engelmann, J., Hanke, W., Mogdans, J., Bleckmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+Stimuli+and+the+Fish+Lateral+Line.">Hydrodynamic Stimuli and the Fish Lateral Line.</a> Nature. 408 (6808), 51-52 (2000).</li><li>Olszewski, J., Haehnel, M., Taguch, M., Liao, J. 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+Larvae+Exhibit+Rheotaxis+and+Can+Escape+a+Continuous+Suction+Source+Using+Their+Lateral+Line.">Zebrafish Larvae Exhibit Rheotaxis and Can Escape a Continuous Suction Source Using Their Lateral Line.</a> PloS One. 7 (5), e36661 (2012).</li><li>Raible, D. W., Kruse, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+the+Lateral+Line+System+in+Embryonic+Zebrafish.">Organization of the Lateral Line System in Embryonic Zebrafish.</a> J. Comp. Neurol. 421 (2), 189-198 (2000).</li><li>Coffin, A. B., Reinhart, K. E., Owens, K. N., Raible, D. W., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Extracellular Divalent Cations Modulate Aminoglycoside-Induced Hair Cell Death in the Zebrafish Lateral. 253 (1-2), 1-2 (2009).</li><li>Harris, J. A., Cheng, A. G., Cunningham, L. L., MacDonald, G., Raible, D. W., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neomycin-Induced Hair Cell Death and Rapid Regeneration in the Lateral Line of Zebrafish (Danio. 4 (2), 219-234 (2003).</li><li>Ma, E. Y., Rubel, E. W., Raible, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+Signaling+Regulates+the+Extent+of+Hair+Cell+Regeneration+in+the+Zebrafish+Lateral+Line).">Notch Signaling Regulates the Extent of Hair Cell Regeneration in the Zebrafish Lateral Line).</a> J. Neurosci. 28 (9), 2261-2273 (2008).</li><li>Brignull, H. R., Raible, D. W., Stone, 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=Feathers+and+Fins:+Non-Mammalian+Models+for+Hair+Cell+Regeneration.">Feathers and Fins: Non-Mammalian Models for Hair Cell Regeneration.</a> Brain Res. 1277, 12-23 (2009).</li><li>Bibliowicz, J., Tittle, R. K., Gross, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+Better+Understanding+of+Human+Eye+Disease+Insights+From+the+Zebrafish,+Danio+Rerio.">Toward a Better Understanding of Human Eye Disease Insights From the Zebrafish, Danio Rerio.</a> Prog. Mol. Biol. Transl. Sci. 100, 287-330 (2011).</li><li>Mione, M. C., Trede, N. S. <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+As+a+Model+for+Cancer.">The Zebrafish As a Model for Cancer.</a> Dis. Model. Mech. 3 (9-10), 9-10 (2010).</li><li>Norton, W., Bally-Cuif, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+Zebrafish+As+a+Model+Organism+for+Behavioural+Genetics.">Adult Zebrafish As a Model Organism for Behavioural Genetics.</a> BMC. Neurosci. 11, (2010).</li><li>Mathur, P., Guo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Zebrafish+As+a+Model+to+Understand+Mechanisms+of+Addiction+and.">Use of Zebrafish As a Model to Understand Mechanisms of Addiction and.</a> Complex Neurobehavioral Phenotypes. Neurobiol. Dis. 40 (1), 66-72 (2010).</li><li>Ignatius, M. S., Langenau, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+As+a+Model+for+Cancer+Self-Renewal.">Zebrafish As a Model for Cancer Self-Renewal.</a> Zebrafish. 6 (4), 377-387 (2009).</li><li>Milan, D. J., MacRae, C. 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+Genetic+Models+for+Arrhythmia.">Zebrafish Genetic Models for Arrhythmia.</a> Prog. Biophys. Mol. Biol. 98 (2-3), 2-3 (2008).</li><li>Van Trump, W. J., Coombs, S., Duncan, K., McHenry, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gentamicin+Is+Ototoxic+to+All+Hair+Cells+in+the+Fish+Lateral+Line+System.">Gentamicin Is Ototoxic to All Hair Cells in the Fish Lateral Line System.</a> Hear. Res. 261 (1-2), 1-2 (2010).</li><li>Littleton, R. M., Hove, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+a+Nontraditional+Model+of+Traditional+Medicine.">Zebrafish: a Nontraditional Model of Traditional Medicine.</a> J. Ethnopharmacol. 145 (3), 677-685 (2013).</li><li>Harris, J. A., Cheng, A. G., Cunningham, L. L., MacDonald, G., Raible, D. W., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neomycin-Induced Hair Cell Death and Rapid Regeneration in the Lateral Line of Zebrafish (Danio. 4 (2), 219-234 (2003).</li><li>Olszewski, J., Haehnel, M., Taguchi, M., Liao, J. 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+Larvae+Exhibit+Rheotaxis+and+Can+Escape+a+Continuous+Suction+Source+Using+Their+Lateral+Line).">Zebrafish Larvae Exhibit Rheotaxis and Can Escape a Continuous Suction Source Using Their Lateral Line).</a> PLoS One. 7 (5), 36661-36 (2012).</li><li>Liang, J., Wang, D., Renaud, G., Wolfsberg, T. G., Wilson, A. F., Burgess, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Stat3/Socs3a+Pathway+Is+a+Key+Regulator+of+Hair+Cell+Regeneration+in+Zebrafish+[Corrected.">The Stat3/Socs3a Pathway Is a Key Regulator of Hair Cell Regeneration in Zebrafish [Corrected.</a> J. Neurosci. 32 (31), 10662-10673 (2012).</li><li>Nakae, M., Asaoka, R., Wada, H., Sasaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Dye+Staining+of+Neuromasts+in+Live+Fishes:+An+Aid+to+Systematic+Studies.">Fluorescent Dye Staining of Neuromasts in Live Fishes: An Aid to Systematic Studies.</a> Ichthyol Res. , 286-290 (2012).</li><li>Magrassi, L., Purves, D., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Probes+That+Stain+Living+Nerve+Terminals.">Fluorescent Probes That Stain Living Nerve Terminals.</a> The J. Neurosci. 7 (4), 1207-1214 (1987).</li><li>Owens, K. N., Coffin, A. B., Hong, L. S., Bennett, K. O., Rubel, E. W., Raible, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+Mechanosensory+Hair+Cells+of+the+Zebrafish+Lateral+Line+to+Aminoglycosides+Reveals+Distinct+Cell+Death+Pathways.">Response of Mechanosensory Hair Cells of the Zebrafish Lateral Line to Aminoglycosides Reveals Distinct Cell Death Pathways.</a> Hear. Res. 253 (1-2), 1-2 (2009).</li><li>Namdaran, P., Reinhart, K. E., Owens, K. N., Raible, D. W., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Identification of Modulators of Hair Cell Regeneration in the Zebrafish Lateral. 32 (10), 3516-3528 (2012).</li><li>Herrera, A. A., Banner, L. 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+Use+and+Effects+of+Vital+Fluorescent+Dyes:+Observation+of+Motor+Nerve+Terminals+and+Satellite+Cells+in+Living+Frog+Muscles.">The Use and Effects of Vital Fluorescent Dyes: Observation of Motor Nerve Terminals and Satellite Cells in Living Frog Muscles.</a> J. Neurocytol. 19 (1), 67-83 (1990).</li><li>Hickey, P. C., Jacobson, D., Read, N. D., Louise Glass, ., L, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-Cell+Imaging+of+Vegetative+Hyphal+Fusion+in+Neurospora+Crassa.">Live-Cell Imaging of Vegetative Hyphal Fusion in Neurospora Crassa.</a> Fungal. Genet. Biol. 37 (1), 109-119 (2002).</li><li>Olsen, A. S., Sarras, M. P., Intine, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limb+Regeneration+Is+Impaired+in+an+Adult+Zebrafish+Model+of+Diabetes+Mellitus.">Limb Regeneration Is Impaired in an Adult Zebrafish Model of Diabetes Mellitus.</a> Wound Repair Regen. 18 (5), 532-542 (2010).</li><li>Olsen, A. S., Sarras, M. P., Leontovich, A., Intine, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heritable+Transmission+of+Diabetic+Metabolic+Memory+in+Zebrafish+Correlates+With+DNA+Hypomethylation+and+Aberrant+Gene+Expression.">Heritable Transmission of Diabetic Metabolic Memory in Zebrafish Correlates With DNA Hypomethylation and Aberrant Gene Expression.</a> Diabetes. 61 (2), 485-491 (2012).</li></ol>

This work was supported by a research grant from the Iacocca Family Foundation, National Institutes of Health Grant DK092721 (to R.V.I.), and Rosalind Franklin University start-up funds. No potential conflicts of interest relevant to this article were reported. G.C.P., S.M.M, and N.D. researched data. M.P.S. Jr. and RI oversaw the project, contributed to the discussion of the data, and oversaw the writing and editing of the manuscript.

基于已建立的侧线（LL）再生的胚胎和幼虫斑马鱼8,24,25分析文献的广泛身体，我们的研究目标是开发在斑马鱼的定量检测横向线条的再生可能被应用到被研究最好在成年鱼的疾病模型。我们发现，应用到成鱼开发的胚胎/仔鱼过程时，某些关键点是很重要的。最重要的这些点被认为:1）侧线的数量沿鱼的纵向轴线神经丘，神经丘毛细胞染色的2），3）治疗的氨基糖苷的浓度和持续时间，和4）的持续时间侧线再生以下氨基糖苷类治疗时机。这些点将会在下面的讨论中加以解决。 对于神经丘沿斑马鱼的​​侧线的数目，胚胎和幼虫斑马鱼有因为一个给定的线迹相比，成鱼在其下数的显着优点，该神经丘有一个简单的图案。这种低数量已经允许调查人员清楚地识别每个神经丘，并分配一个名称给它6。以这种方式，再生的研究可以在任何时间通过量化名的特定神经丘的再现以下从一种氨基糖苷类剂停药。更大数量的成年人每针神经丘的介绍时相比，胚胎或幼虫的再生神经丘的再现过程中准确计数和定量显著困难。如图所示，我们评估了成人的内针侧线神经丘的模式和确定中体（Figures. 1和2）提供针的最佳区域进行分析。 神经丘与毛细胞的染料，例如2的染色 - [4 - （二甲基氨基）苯乙烯基]-N-ethylpyridi鎓碘化物（DASPEI）或4 -二-2-天冬氨酸允许一个幼体和稚鱼用荧光立体显微镜26,27可视化的侧线神经丘。这些相同的污渍也是有效的成年鱼和我们的定量分析表明，该中间体区域内的神经丘的数量没有显著差异，观察其中正常对照斑马鱼（与正常对照组成鱼平均61.45神经丘）。 已经报道了毛细胞的染料，例如2 - [4 - （二甲基氨基）苯乙烯基]-N-乙基吡啶鎓碘化物（DASPEI）或4 -二-2 -天冬氨酸本身可以是有毒的神经丘细胞24，和鱼不能反复沾上这些荧光剂如果是监测氨基糖苷类诱导再生。同鱼的反复染色引入了多种毒性事件（由染色和氨基糖苷类两者），使实验未可解释23。因此，所有的实验在我们的研究中多套鱼，才能要求并行4 - 二-2-天冬氨酸染色表明，神经丘是:1）目前在未处理庆大霉素控制条件，2）完全消融即刻以下庆大霉素曝光，和3）在一些再生小时后庆大霉素治疗停药之后洗出这个氨基糖苷类。以这种方式，所有的鱼用的神经丘染料染色只有一次。 应当指出，虽然范王牌等人 17的程序建立在成年鱼毛细胞消融的情况下，它们不建立的标准曲线而导致需要对对照组和实验组之间的定量比较神经丘再生。因此，我们接着范王牌等人 17的毛细胞消融（0.004％使用24小时暴露时间庆大霉素浓度，参见图2对毛细胞消融的结果的程序在24hr），但扩大了他们的工作，以建立神经丘再生的标准曲线。这允许使用4针，我们建立了我们的实验条件下，中间主体区域的再生LL在成年斑马鱼的比较分析（参见图1和图2）。为了确定是否能够获得的毛细胞消融在较短期间内，我们还测试了硫酸铜，已在仔鱼被有效地用于周期短至2小时的效果。我们的研究表明，硫酸铜（5-50 mM的各种暴露时间长达48小时如先前报道Liang 等 21对幼体）没有被发现是有效的成鱼作为毛细胞消融的试剂。这凸显了一个事实，即用于毛细胞胚胎消融和幼虫条件不能总是直接转移与成鱼使用。 为属于该侧线再生的成人，我们发现其胚胎/幼虫和成鱼之间的神经丘再生相似的时间框架。至于其他人，斑马鱼的胚胎和幼虫显示神经丘再生在12-24小时后氨基糖苷类先前报道洗出8。我们观察到神经丘再生中出现的8-12时间帧（不被视为完整的线圈为8小时的时间点， 如图所示2）与在16 HPG达到平稳的线性相位。没有发现，直到24 HPG针神经丘内完全控制一样的外观作为报道的胚胎和幼虫。完全控制般的外观是指无论在成年斑马鱼的所有四针中间体区域内的数量和神经丘的强度。此外，如果是在神经丘的电平获得的定量结果没有统计学显著，研究者可以在他们的研究中神经丘里延伸到各个毛细胞的水平利用共聚焦显微镜所描述的我们的程序。 在这篇文章中所描述的神经丘/毛细胞再生法可以应用到那些最好的表现在成年斑马鱼，而不是在早期幼体/萌芽阶段的疾病状态。该测定的一个限制涉及的实验条件的影响，无论是1）的转基因品系，模仿特定疾病状态或在成年斑马鱼的侧线系统内的干细胞2）的药理学诱导的疾病状态。在这方面，成年斑马鱼的特定疾病状态可能会或可能不会影响毛发细胞谱系的干细胞，并且要注意的是很重要的神经丘再生是完全依赖于这些干细胞的增殖/分化处理8,24,25 。 由于这一限制的一个例子，我们将描述在I型糖尿病的成年斑马鱼模型进行实验。这particul芳疾病模型，以研究诱导的高血糖28的长期继发并发症发展成年斑马鱼。对于一些以前28,29描述的原因，这些研究只能使用成年斑马鱼进行。因为随着专业化的细胞结构，他们受神经支配末梢神经的糖尿病患者有不利影响，我们想确定是否神经丘/毛细胞的侧线的再生也受损在糖尿病斑马鱼。使用侧线再生法无统计学显著延迟是在神经丘再生检测。为了证实这一点否定结果时，实验重复在各个毛细胞的更精细的水平。同样，没有统计学显著差异，观察控制和糖尿病组之间的毛细胞再生。因此，该数据是与高血糖妨碍神经丘/毛细胞再生的假设不一致; possibly由于毛细胞谱系的干细胞，以高血糖状态的电阻。有了这个限制考虑，本文中所描述的神经丘/毛细胞再生法确实提供了测试任何特定的成年斑马鱼疾病模型是否涉及功能障碍中的毛细胞再生的过程，使用侧线系统监控的手段。积极的结果将意味着干细胞的参与和进一步的研究将因此被担保。

侧线（LL）系统是鱼类和两栖类，它负责听觉，平衡，rheotaxis调处行为，如教育和捕食者回避1-5找到了mechanosensory器官。这两者它是由簇毛细胞，支持细胞包围，被定位在结构，称为神经丘6。这些神经丘通常组织成垂直线（称为针）沿主体和尾部与一些在鱼的头部观察到的水平线圈的纵向轴线。在成人中，神经丘是显著更大数目的线圈相比，胚胎或仔鱼6内。在斑马鱼生物医学研究都集中在抗生素治疗，噪音引起的创伤，慢性感染等的影响。对毛细胞，试图7,8，以便更好地了解他们在人体的影响。 与大多数脊椎动物，德硬骨鱼，如斑马鱼（ 斑马鱼 ），必须重新生成丢失的毛细胞的能力。因为它们的快速开发时间和高再生能力的斑马鱼是特别有用的。迄今为止，然而;侧线上的开发和/或再生斑马鱼研究主要利用的胚胎和幼虫阶段的鱼，由于侧线神经丘的数量减少可以较容易计数和分析6,9,10。 然而，神经系统和非神经系统疾病11-16尽可能多的斑马鱼模型研究了成鱼，而不是幼虫，我们专注于开发在成年斑马鱼用庆大霉素横向线再生法（以前在斑马鱼幼虫用一种氨基糖苷类和最近与成年鱼17用），这样的测定法是可用的，可以被应用到当前的成年斑马鱼的疾病模型。而此前公布的程序由Van特朗普ét铝，17设立在成鱼的毛细胞消融的条件，他们没有建立标准曲线这是需要控制组和实验组利用转基因斑马鱼线或药物诱发的疾病状态时，如之间的定量比较神经丘再生在斑马鱼18。因此，我们跟着范·特朗普等人 17的毛细胞消融的程序，但建立在他们的工作，以建立神经丘再生的标准曲线，以使研究者比较对照组和实验组在使用我们的数据，如与成年斑马鱼疾病模型。该测定也被设计为允许扩展的分析对个体毛发细胞时，需要解决的一个更高的水平。

<table border="0" cellpadding="0" cellspacing="0" width="431">
	<tbody>
		<tr>
			<td>Gentamicin sulfate solution (50mg/ml)</td>
			<td>Sigma Aldrich</td>
			<td>G1397</td>
			<td></td>
		</tr>
		<tr>
			<td>2 Phenoxyethanol</td>
			<td>Sigma Aldrich</td>
			<td>P1126</td>
			<td></td>
		</tr>
		<tr>
			<td>4-4-diethylaminostryryl-N-methylpyridinium iodide (4-Di-2-Asp)</td>
			<td>Aldrich</td>
			<td>D-3418</td>
			<td>485 nm excitation &#955; and 603 nm emission &#955;</td>
		</tr>
		<tr>
			<td>in methanol</td>
		</tr>
		<tr>
			<td>6 well plates</td>
			<td>Mid Sci</td>
			<td>TP92006</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td>08-757-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom Microwell Dishes</td>
			<td>Matek Corporation</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting&#160; Microscope</td>
			<td>Nikon</td>
			<td>TMZ-1500</td>
			<td>Any dissecting microscope is fine.</td>
		</tr>
		<tr>
			<td>Camera for Imaging</td>
			<td>Nikon</td>
			<td>Q imaging</td>
			<td>Any camera is suitable.</td>
		</tr>
		<tr>
			<td>Image J software</td>
			<td>National Institutes of Health</td>
			<td>NIH Image</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS Elements</td>
			<td>Nikon</td>
			<td></td>
			<td>Any imaging software is suitable.</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus</td>
			<td>FV10i</td>
			<td>Any high resolution fluorescent microscope is suitable</td>
		</tr>
		<tr>
			<td>Aquatic System</td>
			<td>&#160;KG Aquatics ZFS Rack System.</td>
			<td></td>
			<td>Any aquatic system can be used</td>
		</tr>
	</tbody>
</table>

an assay for lateral line regeneration in adult zebrafish

由于听力和平衡紊乱在人的临床重要性，模式生物如斑马鱼已被用于研究横向线的开发和再生。斑马鱼是由于其快速的开发时间和它的高再生能力为这些研究特别有吸引力的。迄今为止，侧线再生斑马鱼的研究主要是利用，因为在这几个阶段的较小的数字神经丘的胚胎和幼虫的鱼。这使侧线再生/和或更早发育阶段的开发更容易进行定量分析。由于神经系统和非神经系统疾病的许多斑马鱼模型研究了成鱼，而不是在胚胎/幼虫，我们专注于开发一个定量的侧线试验再生的成年斑马鱼，这样的分析是可用的，可以被应用到当前成年斑马鱼疾病模型。由Van TRUM建立在前人研究所描述的毛细胞在成年墨西哥洞穴盲鱼和斑马鱼（ 斑马鱼 ）的消融过程P 等17中 ，我们分析的目的是让控制组和实验组之间的定量比较。这是通过开发一种基于神经丘再现过以下毛细胞在限定的横向线的区域庆大霉素引起的坏死一个24小时时间段的百分比的再生神经丘标准曲线来实现。该测定也被设计为允许扩展的分析，以当需要的分辨率更高水平的个体毛发细胞的水平。

优化的程序，在量化成年斑马鱼的侧线神经丘再生。 斑马鱼幼虫的神经丘是容易量化;然而，成年斑马鱼的侧线具有更大数目的每针脚神经丘作出定量分析更加困难6,17,19,20的。就像在图1A中 ，头部有许多神经丘相比，任一中间部分或尾部的显著更高;用具有如图所示的一维数最少的神经丘的尾部区域。因为线圈中的头柄是复杂和显著更大的神经丘的数量，也没有借给本身作为用于定量分析的区域。此外，无论我们测试的庆大霉素浓度，神经丘的整个头部完全消融是很难达到;神经丘的观察点离开<html>如先前报道的范王牌等 17相反庆大霉素治疗后，尾部太少神经丘，正因为如此，我们选择了中间体区域（ 图1B），以定量分析神经丘再生的成人。在这个地区，我们确定了四针正后方的侧胸鳍说是一致的神经丘数量在所有成年人[61.45（N = 95）]（ 图1B和1E）。重要的是，我们可以通过一个24小时的0.004％庆大霉素治疗始终如一，完全消融这一地区的神经丘（如前面的范特朗普等人 17日报道），允许随后准确测定神经丘再生（比较图1B和1E以及分段与图2,0小时）。 p_upload/51343/51343fig1highres.jpg"宽度="500"/&gt; 图1。神经丘的成年斑马鱼的线圈内的荧光图案沿纵向轴线如图所示。面板A是头部区域， 面板B是中间主体区域具有用于与所述盒定量分析了4针。 面板C是后牙体区域。 面板D是尾鳍区域。 4针用于定量分析的中体区的高倍放大显示在1X和2X的E组 。放大功率。 已经报道了硫酸铜治疗是一种有效的化学方法诱导毛细胞快速坏死胚胎和幼虫21。在这里，我们测试硫酸铜治疗，希望它可以缩短时间，诱发神经丘消融。硫酸铜的浓度范围从5-50毫米长达48小时，已动用先前报道Liang 等 21研究发现，硫酸铜是致命的高浓度，而不是有效的在成鱼低浓度的不同的曝光时间（数据未显示） 。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51343/51343fig1highres.jpg" target="_blank">请点击此处查看此图的放大版本。</a> 用于侧线再生的成年斑马鱼的荧光分析参数。 再生监测后四中体针内的所有神经丘下面的24小时庆大霉素治疗（ 图2，比较0小时与对照组）和阳性再生是由至少三个神经丘的线圈内的外观决定了烧蚀。 （ 图2,0小时）。 8 HPG，鱼大约三分之一有一些复苏的迹象（N = 34）;虽然神经丘的强度微弱的再生针（ 图2，8小时，按箱概述昏针）。神经丘的数目和它们的强度持续增加以线性方式，直到再生达到在16 HPG高原（与图2中 ，8小时，比较图2中 ，16小时）。但直到至少24 HPG，与庆大霉素治疗的所有鱼都等于侧线缝合相比，控件中的神经丘的数量和强度（ 图2，24小时）已经完全康复。以下庆大霉素停药时线为神经丘再生显示在图3中示出了恢复曲线的线性和高原阶段。我们注意到，在箱子的小于5％时，再生线圈并未作为单独的实体，而是表现为荧光的涂抹。 <i毫克ALT ="图2"FO:内容宽度="3英寸"SRC ="/ files/ftp_upload/51343/51343fig2highres.jpg"宽度="300"/&gt; 图2。神经丘的荧光图像。控制鱼，0人才招聘鱼（紧接在0.004％庆大霉素治疗24小时以下），8小时鱼（8 HPG）与用于定量分析的4针内神经丘的一些微弱的染色（仅30％的鱼表现出的这种图案染色在8 HPG; 70％没有表现出神经丘染色在这个时间点，将白框概括了被认为在鱼的30％这表明在8 HPG一定程度再生的微弱染色神经丘），16小时的鱼，和24小时的鱼。 4针关于1），观察24 HPG神经丘的数量和神经丘的染色2）强度内神经丘完全再生。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51343/51343fig2highres.jpg" target="_blank">请点击此处</a>查看该图的放大版本。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51343/51343fig3highres.jpg" width="500" /> 图3示出图形的神经丘再生的时间过程以下从成年斑马鱼与0.004％庆大霉素24小时治疗停药。如图所示，恢复开始在8 HPG时间点和达到平台期在16 HPG时间点。这个定义该线性相位内8-16小时，时间点间神经丘再生的衬垫相应该被用来定量比较对照组和实验组。 神经丘毒性长期接触荧光染色染料引起的。 在我们的估计来执行这些实验中一个理想的方法是将用荧光染料再在0小时弄脏鱼在治疗之前，染色，然后再弄脏吨再生的时间点。然而，我们遇到了并发症这些研究这是事实，毛细胞荧光染色的染料，如4 -二-2 -天冬氨酸，如也可以是与其他线粒体渍22可以对毛细胞23毒性影响的情况。这一事实要求我们使用鱼分成几组，因为同样的鱼不能被聘用的反复染色。在所有情况下，实验的鱼包括控制并行进行处理，以消除实验变异性。 共聚焦分析在个别毛细胞的水平。 如果是从神经丘分析得到的结果没有统计学对照组和实验组之间显著，1可能延长这些研究中的个体冰雹细胞的水平，以获得较高程度的分辨率的定量比较。正如在图4中 ，从对照组神经丘（一桅在12小时再生的是该图中所示）可以通过皮肤制剂从中间体区的共聚焦显微镜进行观察。 8小时，10小时和12小时的再生时间，我们发现，对照组（7只/组）有一系列的0-4毛细胞/神经丘。如预期的对照组中，当定量分析，神经丘之间无统计学差异，检测在每个神经丘毛细胞的数目方面在上面所指出的（P值从0.230-0.472范围）的时间点。这种方法可以在任何对照组与实验组之间需要扩展或确认从神经丘研究的第一阶段获得的数据时，应考虑。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51343/51343fig4highres.jpg" width="500" /> 我描述的图4。分析使用定义的侧线区域的皮肤准备毛细胞/神经丘再生N协议的步骤3.3。从斑马鱼的皮肤准备获得一个神经丘内毛细胞的荧光共聚焦图像。两个活体染料染色的毛细胞，示出了控制鱼的神经丘（ 图4）内。这张照片是获得12小时后去除庆大霉素（线性再生阶段）。白支架符号（）表示单个神经丘而白色箭头表示邻近的毛细胞的支撑单元。支持细胞没有这些条件下染色，显示为黑色的空间。放大倍率，60X。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 1 </td><td> 1，庆大霉素治疗[0.004％（4.32毫米）]对照组和实验鱼24小时，在28℃下使用孵化器。 </td></tr><tr><td> 2 </td><td> 2，洗出庆大霉素发起毛细胞再生。返回鱼到28℃培养箱中培养8-16小时之间研究者选定的时间段。 </td></tr><tr><td> 2 </td><td> 3，活体染料染色[0.08％4 - 4 - 二乙氨基-N-甲基碘化物（4 - 二-2-ASP）]对照组和实验鱼为1小时，在室温，然后洗出污渍与鱼水荧光成像。 </td></tr><tr><td> 1或2个</td><td> 4，如果必要的，因为从神经丘的分析不显着的结果，重复1-3协议与对照组和实验鱼的一个单独的组，但随后得到皮肤准备单独的毛细胞的共聚焦分析。 </td></tr></tbody></table> 表1。协议的概要上文所述。

Watch this Scientific Journal Video about An Assay for Lateral Line Regeneration in Adult Zebrafish at JoVE.com

An Assay for Lateral Line Regeneration in Adult Zebrafish

由于神经和非神经系统疾病的许多斑马鱼模型研究在成年鱼，而不是胚胎/幼虫，我们开发了可应用于成年斑马鱼疾病模型的定量侧线再生试验。该法涉及的分辨率在1）神经丘和2）单个毛细胞的水平。

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 ...

an-assay-for-lateral-line-regeneration-in-adult-zebrafish

Nanyang Technological University

Research

JoVE Journal

Biology

12.5K 次观看.  Dr. William M Scholl College of Podiatric Medicine, Rosalind Franklin University of Medicine and Science. 由于神经和非神经系统疾病的许多斑马鱼模型研究在成年鱼，而不是胚胎/幼虫，我们开发了可应用于成年斑马鱼疾病模型的定量侧线再生试验。该法涉及的分辨率在1）神经丘和2）单个毛细胞的水平。 

视频: An Assay for Lateral Line Regeneration in Adult Zebrafish

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

在成年斑马鱼的全肾骨髓移植

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

科研

生物学

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

在成年斑马鱼的眼窝射出

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

腹腔注射到成年斑马鱼

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

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 ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

再生的成年斑马鱼尾鳍在吗啉代入体内电穿孔

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

在成年斑马鱼的生化分析采血车

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

颅面部发育中的SOX10可视化:使用枫转基因斑马鱼线时间推移共聚焦显微镜

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

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 ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

恢复成年斑马鱼的心脏的高通量应用

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel &#8220;repeated&#8221; blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be&#160;&#8804;0.4% of body weight every week or &#8804;1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

重复采血车在成年斑马鱼验血

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...