笔者想感谢米歇尔·德奇为完善这一眼消融技术，泰勒·伯克霍尔茨与功能测试援助，迈克尔·勒文的抗抑制蛋白抗体，而顺治Morokuma为珀尔帖板信息。这项工作是由西密歇根大学WSB的SFSA资助。

1.动物文化和处理注:此协议使用Schmidtea地中海 ，二倍体涡物种，基因组测序16，17是常用的再生研究。然而，该测定法是与其他物种，诸如Girardia虎纹和Girardia dorotocephala（它们是可商购）同样成功。 <ol><li> 保持"蠕虫水"为0.5g / L海盐超纯（或过滤的去离子）水制成蠕虫。使用无菌聚丙烯或玻璃容器来保存蠕虫水。见<a href="http://ecsource.jove.com/files/ftp_upload/55594/Supplementary_File_1.docx" target="_blank">补充文件1</a>对蠕虫水制备的细节。 注意:不要使用肥皂清洗容器（或其它电源）如肥皂是有毒的蠕虫;代替用70％乙醇擦拭，并允许风干。 &lt;OL&gt; <li>戴上手套，用涡虫的工作，以保护他们免受污染的同时。 注:蠕虫是环境毒素和化学品，包括肥皂，漂白剂，洗发水，护发素，和洗手液非常敏感。 </li></ol></li><li>房子菌落在聚丙烯食品容器，与盖子部分打开在约20℃（室温）下的空气交换。在任一培养皿商店实验蠕虫（每100mm培养皿20个蠕虫）或未经处理的组织培养板（每孔1个蠕虫，对于12孔板）。为了减轻压力，保持双方的殖民地和实验在黑暗中。 注:殖民地应该有每升水虫不超过500-1000蠕虫。对于实验，让完全到位盖子，由于气流是专为这些菜。 </li><li>通过从移液管滴加果泥，放置食物的液滴在整个容器订阅与泥有机牛肉或鸡肉肝脏非实验蠕虫每周一次。允许蠕虫2个小时以消耗食物在清理容器在天黑前（参见步骤1.4）。在使用之前，储存在-20℃下短期（周）原浆或-80℃长期（数月）;不重新冻结泥再利用（如细菌可以开花损害蠕虫）。 注意事项:肝应该不含激素或抗生素，最好不预先冻结。冷冻前（前馈或）离心机原浆以除去空气并防止食物浮动。饮食宜沉到容器的底部。原浆的1毫升足以用于500-1,000蠕虫。 </li><li>兑水每周一次用新鲜水虫两个殖民地和实验。菌落的容器也应与未漂白纸巾擦拭（以除去粘液残基陷阱废物）每周一次（供给和再次2天后后2小时）饥饿蠕虫，或每周两次用于馈送蠕虫。为了留住生物膜，不抹容器的80％以上。 </li><li> 移动容器（或手术SETU之间蠕虫p）的使用移液管。到附着于容器表面自由蠕虫，喷出的水过/蜗杆以从表面释放它的后面。然后吸取蜗杆到移液管，而试图保持在移液管的底部英寸（较薄）部分内的蜗杆。 <ol><li>后送吸管吮吸起来之前，有少量虫水。 </li><li>尝试移动水绕的蠕虫病毒，而不是病毒本身，以帮助防止用吸管触及他们撕裂软体蠕虫。 </li><li>保持覆盖，除非转让蠕虫或更换水实验菜肴。 </li></ol></li></ol> 2.准备工作<ol><li> 停止至少一周之前的实验喂养蚯蚓。 <ol><li>选择已没喂食了≥1周并且至少5-7毫米长度蠕虫。转移蠕虫至培养皿2/3满蠕虫水的，并通过滑动盘下方的标尺确认虫长度。测量w ^奥姆斯同时充分延伸和移动。 注:虽然小（5-7毫米）蠕虫执行后续免疫荧光原位杂交分析，更大的蠕虫（8-10毫米）时，更容易与工作更好地工作-尤其是当学习消融。 </li><li>确保蠕虫是整体，无破损或最近再生。 注意:作为相对于正常蠕虫蠕虫再生将在头部和/或尾部区轻得多（或没有）颜料。 </li><li>如果蠕虫进行RNAi或药物治疗，在这一点上做到这一点。如果蠕虫被馈送作为处理（作为RNA干扰）的一部分，等待最后喂食后至少7天继续到步骤2.2之前。 </li></ol></li><li> 清洁用70％乙醇的工作空间和解剖显微镜基，并允许它完全干燥。选择蠕虫的盘放置到所述范围的左边。到的范围的权利，放置一对＃5钳子的，一个31克5 / 16-英寸胰岛素针用1毫升的注射器，和一个干净的移液管。 <ol><li>确保器械接触到工作台（其可以引入污染物）保持。使用硬质物品（如筷子休息），以保持镊子，针和吸管干净。可替换地，在新鲜棕色（未漂白的）纸巾的顶部位置的仪器。 注:因为它们与右手这些个人和随后的指令写入。左手个人应该扭转左/右方向。 </li></ol></li><li>旁边的工作空间，将无绒织物擦巾的盒子，新鲜水虫的来源，以及一些额外的移液管（从台式保护）。地方虫水的塑料洗瓶便于分配的。还放置一个标记的12孔板（或100毫米培养皿）的范围的权利，用于收集被消融的动物。 </li><li> 如果使用定制的珀耳帖板来固定蠕虫，珀耳帖板定位到在叔凹陷他解剖显微镜的基部和直到珀耳帖板的工作表面是充分地冷却调整直流电源上的输出（通常为〜5 V）。可选地，通过填充100毫米培养皿使冷板½充分用水和冷冻至少24小时。丢弃该盖板，将解剖范围的基础上的100mm培养皿的底部，然后将60毫米的培养皿的盖颠倒直接在冰（图1A）的表面上。 <ol><li>水在100mm培养皿已经冻结后，冰的"火山"可以出现在板的中心。如果发生这种情况，用一个刀片刮冰平，以从表面除去任何不规则性。 注:在手术的冰会融化，使得消融困难得多。预先准备几个冰盘，并更换冻结的用于尽快60毫米培养皿的盖开始浮动在测定过程中熔化的。 </li></ol></li><li> 准备手术的表面。切5厘米×10厘米的塑料片石蜡膜（4cm 2的如使用培养皿冷板）并将其放置在固定装置的中心。折叠无绒布纸巾擦拭成方形大致2 cm 2并且将其放置在膜的顶部。切下一块的白色滤纸1.5-2厘米2，将其上的擦拭顶部。参见图1B-1E。 <ol><li>按住折叠处用戴手套的手指擦拭，并用虫水轻轻挫伤擦拭，以确保它保持折叠。使用移液管的一侧滚动抹平。 注意:冷的温度有助于保持蠕虫移动。工作"干"也有助于固定。组织擦拭行为通过滤纸芯吸水，手术过程中保持蜗杆湿润而不使蠕虫完全风干（这是致命的）。 </li></ol></li></ol> 3.手术消融<ol><li> 用移液管上放置È蠕虫背侧上到滤纸上。在关闭光源和引导鹅颈，使光线集中在蠕虫。调整解剖范围聚焦和放大倍率使眼睛清晰可见（整个蠕虫并不需要在图）。放大5倍是一个很好的起点。通过使得头部指向朝向研究员（朝向显微镜的前面）倾斜30-40度的石蜡膜旋转，向右定向蠕虫。 <ol><li>避免使用明亮的光线，以防止多余的蠕虫运动。涡虫显示负趋光性，并会尝试逃避耀眼的光芒。 </li><li>如果蠕虫被定位朝上（含咽口可见）腹侧，使用产钳无光泽的一面轻轻地重新定位蠕虫背侧了（眼睛可见）。避免接近动物用钳子的尖端，以尽量减少重新定位动物时通过软组织撕裂的可能性。 </li><li&gt;调整显微镜焦点使眼睛显然聚焦。此外，还要确保两个眼睛和周围的乳头组织在视野中。 </li></ol></li><li> 保持新鲜针/注射器在右手就好像它是一个笔（拇指和食指之间）。做好防珀尔帖板（或培养皿冷板）的左拇指，并使用左拇指为支点在其上的注射器的底部将休息（图2A）。通过显微镜观察，并确保针的斜面是可见的。 注:针是非常尖锐的。操作时他们一定要小心。戴乳胶或丁腈手套可以提供一些保护。 <ol><li>使用左手拇指，以帮助保持针/注射器在整个手术过程平稳，避免握手。 </li><li>成大约40°用左手拇指和左手食指（与手术表面上的食指）的角度，以帮助保持手术的表面稳定。 </li></ol></li><li> 同朝上（如调羹）所述针的斜面，定位在垂直于眼（图2B）的针。使用针的尖端轻轻穿透组织覆盖在眼睛的视杯（白色，未着色区域）的薄层，由右至左舀。非常温和地去除位于视杯内的任何色素斑组织，小心不要刺破底部或破坏视杯边界。 <ol><li>如果蠕虫一直为&gt; 1-2分钟的滤纸在该过程期间的任何时刻，添加蠕虫水一滴再水合蜗杆。 注:脱水会增加病毒的易感性翻录受伤。 </li></ol></li><li>重复步骤3.3，直到所有的黑色颜料的组织细胞（色素细胞）的，以及所有的视杯（感光细胞的树突）的白色组织的，被除去。用纸巾擦拭小心的擦拭取出切除组织粘在针的末端。如果双眼睛阿夫拉和灰是期望的，在这一点上烧蚀第二只眼睛。 注:为避免受伤，针可抓住用干净的纸巾针擦拭举行的拇指和食指，然后用另一只手通过擦去拇指和食指拉针进行清洗。可替代地，针可以被擦拭到一个湿纸巾擦拭的表面并检测清洁度。 </li><li> 当完成后，可使用移液管到蜗杆移动到含有新鲜水虫标记的菜。如果分析组数据，放置最多20个蠕虫在一个100毫米的培养皿。如果随着时间的推移在同一个人的跟踪再生，放置1个蜗杆成12孔板的每个孔中。一旦所有蠕虫被转移，通过去除从餐具/孔所有蠕虫水中，并用更多的新鲜水蠕虫更换冲洗蠕虫。 <ol><li>从过滤器除去蠕虫，后送与蜗杆水少量移液管并释放水到所述蠕虫。这应该解除ŧ他蠕虫关闭滤纸，立即被吸进吸管转移。 </li></ol></li><li>孵育约20℃（室温）下避光实验并按照再生过程。 注:蠕虫可被存储在一个温度控制的恒温箱中保持恒定的温度，但是这不是严格必需的。大部分房间的温度只有+ 5°C，这将不会影响该蠕虫的再生能力有所不同。 </li><li>如果需要的话，固定用于免疫组织化学蠕虫（参见图3-4）和/或基因表达的原位杂交分析。不同的固定方法为免疫组织化学涡虫18，19，20和原位杂交21，22，23个协议存在。 </li><li>当实验结束后，sacrifiCE由从培养皿中除去蠕虫水中，并用70％的乙醇代替活蠕虫。蠕虫孵育3-5分钟，检查蠕虫病毒裂解和转灰。 </li><li>发生一次牺牲了正常的废物流中未经处理的蠕虫。避免将活虫体进入环境，特别是非本地物种。 </li></ol>

<ol>
	<li>Gentile, L., Cebria, F., Bartscherer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+planarian+flatworm:+an+in+vivo+model+for+stem+cell+biology+and+nervous+system+regeneration.">The planarian flatworm: an in vivo model for stem cell biology and nervous system regeneration.</a> Dis Model Mech. 4 (1), 12-19 (2011).</li><li>Elliott, S. A., Sanchez Alvarado, 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+history+and+enduring+contributions+of+planarians+to+the+study+of+animal+regeneration.">The history and enduring contributions of planarians to the study of animal regeneration.</a> Wiley Interdiscip Rev Dev Biol. 2 (3), 301-326 (2013).</li><li>Emili Sal&#243;, R. B., Tsonis, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+3.">Chapter 3.</a> Animal Models in Eye Research. , 15-26 (2008).</li><li>Lapan, S. W., Reddien, 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=dlx+and+sp6-9+Control+optic+cup+regeneration+in+a+prototypic+eye.">dlx and sp6-9 Control optic cup regeneration in a prototypic eye.</a> PLoS Genet. 7 (8), e1002226 (2011).</li><li>Lapan, S. W., Reddien, 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=Transcriptome+analysis+of+the+planarian+eye+identifies+ovo+as+a+specific+regulator+of+eye+regeneration.">Transcriptome analysis of the planarian eye identifies ovo as a specific regulator of eye regeneration.</a> Cell Rep. 2 (2), 294-307 (2012).</li><li>Inoue, T., 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=Morphological+and+functional+recovery+of+the+planarian+photosensing+system+during+head+regeneration.">Morphological and functional recovery of the planarian photosensing system during head regeneration.</a> Zoolog Sci. 21 (3), 275-283 (2004).</li><li>Pineda, 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=The+genetic+network+of+prototypic+planarian+eye+regeneration+is+Pax6+independent.">The genetic network of prototypic planarian eye regeneration is Pax6 independent.</a> Development. 129 (6), 1423-1434 (2002).</li><li>Paskin, T. R., Jellies, J., Bacher, J., Beane, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planarian+Phototactic+Assay+Reveals+Differential+Behavioral+Responses+Based+on+Wavelength.">Planarian Phototactic Assay Reveals Differential Behavioral Responses Based on Wavelength.</a> PLoS One. 9 (12), e114708 (2014).</li><li>Raffa, R. B., Martley, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine-induced+increase+in+planarian+locomotor+activity+and+block+by+UV+light.">Amphetamine-induced increase in planarian locomotor activity and block by UV light.</a> Brain Res. 1031 (1), 138-140 (2005).</li><li>Sandmann, T., Vogg, M. C., Owlarn, S., Boutros, M., Bartscherer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+head-regeneration+transcriptome+of+the+planarian+Schmidtea+mediterranea.">The head-regeneration transcriptome of the planarian Schmidtea mediterranea.</a> Genome Biol. 12 (8), R76 (2011).</li><li>Vasquez-Doorman, C., Petersen, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NuRD+complex+component+p66+suppresses+photoreceptor+neuron+regeneration+in+planarians.">The NuRD complex component p66 suppresses photoreceptor neuron regeneration in planarians.</a> Regeneration (Oxf). 3 (3), 168-178 (2016).</li><li>Deochand, M. E., Birkholz, T. R., Beane, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+regulation+of+planarian+eye+regeneration.">Temporal regulation of planarian eye regeneration.</a> Regeneration. 3 (4), 209-221 (2016).</li><li>Sakai, F., Agata, K., Orii, H., Watanabe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+regeneration+ability+of+spontaneous+supernumerary+eyes+in+planarians+-eye+regeneration+field+and+pathway+selection+by+optic+nerves.">Organization and regeneration ability of spontaneous supernumerary eyes in planarians -eye regeneration field and pathway selection by optic nerves.</a> Zoolog Sci. 17 (3), 375-381 (2000).</li><li>Asano, Y., Nakamura, S., Ishida, S., Azuma, K., Shinozawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhodopsin-like+proteins+in+planarian+eye+and+auricle:+detection+and+functional+analysis.">Rhodopsin-like proteins in planarian eye and auricle: detection and functional analysis.</a> J Exp Biol. 201 (Pt 9), 1263-1271 (1998).</li><li>Cross, S. 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=Control+of+Maintenance+and+Regeneration+of+Planarian+Eyes+by+ovo.">Control of Maintenance and Regeneration of Planarian Eyes by ovo.</a> Invest Ophthalmol Vis Sci. 56 (12), 7604-7610 (2015).</li><li>Robb, S. M., Gotting, K., Ross, E., Sanchez Alvarado, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SmedGD+2.0:+The+Schmidtea+mediterranea+genome+database.">SmedGD 2.0: The Schmidtea mediterranea genome database.</a> Genesis. 53 (8), 535-546 (2015).</li><li>Robb, S. M., Ross, E., Sanchez Alvarado, ., A, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SmedGD:+the+Schmidtea+mediterranea+genome+database.">SmedGD: the Schmidtea mediterranea genome database.</a> Nucleic Acids Res. 36, D599-D606 (2008).</li><li>Beane, W. S., Tseng, A. S., Morokuma, J., Lemire, J. M., Levin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+planar+cell+polarity+extends+neural+growth+during+regeneration,+homeostasis,+and+development.">Inhibition of planar cell polarity extends neural growth during regeneration, homeostasis, and development.</a> Stem Cells Dev. 21 (12), 2085-2094 (2012).</li><li>Forsthoefel, D. J., Waters, F. A., Newmark, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+cell+type-specific+monoclonal+antibodies+for+the+planarian+and+optimization+of+sample+processing+for+immunolabeling.">Generation of cell type-specific monoclonal antibodies for the planarian and optimization of sample processing for immunolabeling.</a> BMC Dev Biol. 14, 45 (2014).</li><li>Ross, K. 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=Novel+monoclonal+antibodies+to+study+tissue+regeneration+in+planarians.">Novel monoclonal antibodies to study tissue regeneration in planarians.</a> BMC Dev Biol. 15, 2 (2015).</li><li>Cardona, A., Fernandez, J., Solana, J., Romero, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+situ+hybridization+protocol+for+planarian+embryos:+monitoring+myosin+heavy+chain+gene+expression.">An in situ hybridization protocol for planarian embryos: monitoring myosin heavy chain gene expression.</a> Dev Genes Evol. 215 (9), 482-488 (2005).</li><li>King, R. S., Newmark, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+protocol+for+enhanced+detection+of+gene+expression+in+the+planarian+Schmidtea+mediterranea.">In situ hybridization protocol for enhanced detection of gene expression in the planarian Schmidtea mediterranea.</a> BMC Dev Biol. 13, 8 (2013).</li><li>Pearson, B. J., 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=Formaldehyde-based+whole-mount+in+situ+hybridization+method+for+planarians.">Formaldehyde-based whole-mount in situ hybridization method for planarians.</a> Dev Dyn. 238 (2), 443-450 (2009).</li></ol>

作者什么都没有透露。

此眼消融技术提高了对当前方法（如打孔）通过排除脑组织和主要限制切除到眼组织中。通过实践，这项技术可以被大多数人来执行，技术人员在显微外科经验不足，但认真的本科生经历。因此建议该技术使用在实验的消融，包括通过免疫组织化学或在眼睛标志物4，5，13 原位杂交（如果可能）完全去除所有眼部?...

涡虫是研究成体干细胞介导再生一个功能强大的模式生物。这些非寄生的扁虫淡水拥有再生的任何及所有丢失的组织，包括他们的中枢神经系统和大脑1的能力。研究早在1700年2，技术在涡领域在过去10 - 15年的进展（如基因组测序， 原位杂交，免疫组织化学，RNA干扰（RNAi），和转录）已经更新了这个历史的模式生物。具体来说，涡虫最近获得了普及作为眼科研究3一个新兴的模式。 涡具有只有两种组织类型，在感光体的神经元和色素细胞原型眼睛;这使得它的眼干细胞群的鉴定和证明，许多相同的基因调节脊椎动物的眼睛去velopment是保守的涡虫4,5。光学杯位于背侧和包括感光体神经元的白色，无颜料的树突和所述半月形黑色素细胞，眼睛经由视交叉支配脑。除了作为用于阐明再生过程6所述的模型，涡虫眼是非常适合于学习的视觉机构7的演变，行为反应的光（涡显示负趋光）8，和行为9的神经学基础。 在涡眼再生大部分已经研究主要有两种情况:作为头再生的一部分以下断头4，图 10和以下只是眼部组织11的切除，12 </SUP&gt;。眼睛上再生涡虫的大多数研究都使用了断头方法，因为它是简单明了。最常见的涡眼切除方法迄今通过打孔器是用细玻璃毛细管13，14，但也有一些研究也已经进行截肢仅次于眼（局部断头）15。然而，所有的这些方法涉及的不仅仅是眼睛许多其他组织（如脑，肠，肾管）的损失，潜在的复杂结果的解释。这里介绍的眼睛消融协议限制切除到更特定于眼内的眼组织（特别排除脑），导致数据。另外，称取7-14天，开始馈送断头蠕虫不同，眼烧蚀蠕虫将消融12的24小时内进料，允许RNAi实验（其中的RNAi经由食物递送）被performeð兼任。 虽然眼睛消融术在技术上是很难比斩首成功执行，涉及眼切除目前的研究尚未包括在他们的程序的详细说明。这个视频文章的目的是为了使研究人员能够一致地删除涡视杯，而不会干扰底层的脑组织和撤除其他一些组织成为可能。此方法可用于单和双眼消融，并适用于广泛的调查。像大多数再生试验中，眼消融非常适合结合两者的药理学和遗传学（RNAi）的屏幕，以及行为研究。这里，我们介绍的方法蠕虫的处理，保持了涡虫的殖民地，和眼睛消融技术本身。

<table><tbody><tr><td>Instant Ocean sea salts</td><td>Spectrum Brands&amp;nbsp;</td><td>SS15-10</td><td>&quot;10 Gallon&quot; box&amp;nbsp; (net weight 3 lbs)</td></tr><tr><td>Kimwipes EX-L lint-free tissue wipe</td><td>Kimberly-Clark</td><td>34155</td><td>4.5 x 8.5 in&amp;nbsp;</td></tr><tr><td>Whatman #2 filter paper</td><td>Sigma&amp;nbsp;</td><td>WHA1002125</td><td>Circles, 125 mm diameter, white</td></tr><tr><td>Easy Touch Insulin syringe (with needle)</td><td>Pet Health Market</td><td>17175-04</td><td>&amp;nbsp;U-100 1 cc syringe, 31 G 5/16 in needle</td></tr><tr><td>100 mm Petri dish</td><td>VWR</td><td>25384-342</td><td>100 mm x 15 mm</td></tr><tr><td>60 mm Petri dish</td><td>VWR</td><td>25384-092</td><td>60 mm x 15 mm</td></tr><tr><td>Dumont #5&amp;nbsp; forceps&amp;nbsp;</td><td>Fine Science Tools</td><td>11254-20</td><td>Inox, straight tip , 11 cm</td></tr><tr><td>Transfer pipettes&amp;nbsp;</td><td>Samco Scientific&amp;nbsp;</td><td>225</td><td>Graduated, large bulb, 7.5 mL, non sterile</td></tr><tr><td>Parafilm M paraffin film</td><td>Brand&amp;nbsp;</td><td>701606</td><td>&amp;nbsp;4 in x 125 ft roll</td></tr><tr><td>12-well untreated tissue culture plate</td><td>VWR</td><td>15705-059</td><td>Untreated, flat bottom, sterile, Falcon brand</td></tr><tr><td>Plastic food containers (for colony)&amp;nbsp;</td><td>Ziploc</td><td>Large rectangle</td><td>2.25 qt (2.12 L), 10&quot; x 6 -3/4 &quot; x 3 -3/16&quot;&amp;nbsp;</td></tr><tr><td>Planaria (&lt;em&gt;Girardia tigrina&lt;/em&gt;)</td><td>Carolina Biological</td><td>132954</td><td>Sold as &quot;Brown&quot; Planaria; most often they are &lt;em&gt;G. tigrina&lt;/em&gt; (aka &lt;em&gt;Dugesia tigrina&lt;/em&gt;), but sometimes are &lt;em&gt;G. dorotocephala&lt;/em&gt; (aka &lt;em&gt;Dugesia dorotocephala&lt;/em&gt;); either will work.</td></tr><tr><td>Planaria (&lt;em&gt;Schmidtea mediterranea&lt;/em&gt;)</td><td>n/a</td><td>n/a</td><td>&lt;em&gt;S. mediterranea&lt;/em&gt; are not commercially available. At this time animals are only obtainable from laboratories that use them and have extra animals.</td></tr><tr><td>Brown paper towels&amp;nbsp;</td><td>Grainger</td><td>2U229</td><td>9-3/16 x 9-3/8&quot; 1-Ply Multifold Paper Towel, UNBLEACHED</td></tr><tr><td>Wash bottle (for worm water), optional</td><td>VWR</td><td>16650-275</td><td>Wash Bottles, Low-Density Polyethylene, Wide Mouth, 500 mL</td></tr><tr><td>Anti-synapsin antibody, optional</td><td>Developmental Studies Hybridoma Bank</td><td>3C11</td><td>Supernatant</td></tr><tr><td>Anti-arrestin antibody, optional</td><td>n/a</td><td>n/a</td><td>Not commercially available. Kind gift from Michael Levin, Tufts University</td></tr><tr><td>Nalgene Lowboy carboy with spigot (for storing worm water), optional</td><td>Nalge Nunc International Corporation</td><td>2324-0015</td><td>15 L,&amp;nbsp; polypropylene, low profile makes it easier to fill plastic colony containers&amp;nbsp;</td></tr><tr><td>Custom Peltier plate, optional</td><td>Williams Machine, Foxboro, MA&amp;nbsp;</td><td>n/a</td><td>Design specifics courtesy of Junji Morokuma, Tufts University:&amp;nbsp; Peltier plate is constructed of a standard thermoelectric heat pump (for example, All Electronics Corp Catalog # PJT-1, 30 mm&lt;sup&gt;2&lt;/sup&gt;).&amp;nbsp; The square heat pump is covered with a thin mirrored surface, then placed inside a 30 mm&lt;sup&gt;2&lt;/sup&gt; square hole in a circular plexiglass form (~50 mm in diameter). This form is of similar thickness to the heat pump, and fits flush into a well tooled in the center of a round heat sink (~115 mm in diameter). The form/heat pump is&amp;nbsp; &quot;anchored&quot; to the sink with silicone base heat sink compound. The leads are threaded through holes drilled through both the form and the the heat sink. The bottom half of the heat sink is tooled into a &quot;foot&quot; that fits into the opening of your microscope&#039;s base plate.&amp;nbsp;</td></tr><tr><td>DC power source (for Peltier plate), optional</td><td>B &amp;amp; K Precision</td><td>1665</td><td>Regulated Low Voltage DC Power Supply, 1-18 V (DC), 1-10 amps.</td></tr><tr><td>Other common supplies</td><td></td><td></td><td></td></tr><tr><td>Gloves</td><td></td><td></td><td></td></tr><tr><td>Razor blade&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>Dissecting scope with gooseneck lighting</td><td></td><td></td><td></td></tr><tr><td>Chopstick rests, optional</td><td></td><td></td><td></td></tr></tbody></table>

surgical ablation assay for studying eye regeneration in planarians

在成体干细胞和再生机制的研究中，涡虫扁虫是在体内模型系统中的缝钉。这是因为在很大程度上他们丰富的多能干细胞群和伤害，这将是灾难性的大多数动物后再生的所有细胞和组织类型的能力。近日，涡已经得到普及，作为眼睛再生的典范。其再生整个眼睛能力（包括两种组织类型:色素细胞和光感受器）允许用于调节视觉系统再生机制的清扫。眼消融具有用于检查眼睛特定的途径和机制，其中最重要的是，再生在很大程度上仅仅局限于眼组织上的其他技术（如断头或打孔）几个优点。这个视频本文的目的是演示如何可靠地除去涡视杯，而不会干扰脑或周围组织。蠕虫和已建立的细胞集落的维护的处理也被描述。这种技术使用一个31克，5 / 16-英寸胰岛素针手术舀出固定化在冷板涡虫的视杯。此方法既包括单，双眼烧蚀，与眼睛在1-2周内再生，从而允许一个宽范围的应用。特别地，该消融技术可以很容易地与为了更好地理解的再生机制及其演进，眼干细胞和它们的维护，和phototaxic行为反应和它们的神经学基础的药理学和遗传学（RNA干扰）屏结合。

对于第1-2小时手术后，动物可能比完整的蠕虫（但是他们仍然会移动）表现出下降的运动。如果需要的话，蠕虫将在外科手术后24个小时内吃（例如，RNAi的馈送）。在之后一段时间内同一个人的眼睛再生时，确保每次取虫的照片既手术（完好）之前，在1个小时后消融（HPA）。 4天后消融（DPA）再生色素细胞应该是可见的，并且通过14 DPA整个眼睛将有完全再生（ 图3A-B...

Watch this Scientific Journal Video about Surgical Ablation Assay for Studying Eye Regeneration in Planarians at JoVE.com

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

该协议示出如何一致切除涡眼（光杯），而不干扰周围的组织。使用胰岛素针和注射器，任一个或两个的眼睛可以被烧蚀以促进调查调节眼再生，再生视觉的演变，和光诱导行为的神经基础的机制。

手术消融分析在研究涡虫再生眼

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their ...

surgical-ablation-assay-for-studying-eye-regeneration-in-planarians

Research

JoVE Journal

Developmental Biology

10.9K 次观看.  Western Michigan University. 该协议示出如何一致切除涡眼（光杯），而不干扰周围的组织。使用胰岛素针和注射器，任一个或两个的眼睛可以被烧蚀以促进调查调节眼再生，再生视觉的演变，和光诱导行为的神经基础的机制。 

视频: Surgical Ablation Assay for Studying Eye Regeneration in Planarians

Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea

Planarians are flatworms that are extremely efficient at regeneration. They owe this ability to a large number of stem cells that can rapidly respond to any type of injury. Common injury models in these animals remove large amounts of tissue, which damages multiple organs. To overcome this broad tissue damage, we describe here a method to selectively remove a single organ, the pharynx, in the planarian Schmidtea mediterranea. We achieve this by soaking animals in a solution containing the cytochrome oxidase inhibitor sodium azide. Brief exposure to sodium azide causes extrusion of the pharynx from the animal, which we call &#34;chemical amputation.&#34;&#160;Chemical amputation removes the entire pharynx, and generates a small wound where the pharynx attaches to the intestine. After extensive rinsing, all amputated animals regenerate a fully functional pharynx in approximately one week. Stem cells in the rest of the body drive regeneration of the new pharynx. Here, we provide a detailed protocol for chemical amputation, and describe both histological and behavioral methods to assess successful amputation and regeneration.

Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea

The planarian Schmidtea mediterranea is an excellent model for studying stem cells and tissue regeneration. This publication describes a method to selectively remove one organ, the pharynx, by exposing animals to the chemical sodium azide. This protocol also outlines methods for monitoring pharynx regeneration.

真涡虫的化学截肢和咽部再生Schmidtea mediterranea

Planarians are flatworms that are extremely efficient at regeneration. They owe this ability to a large number of stem cells that can rapidly respond to ...

科研

发育生物学

A Planarian Motility Assay to Gauge the Biomodulating Properties of Natural Products

A straightforward, controllable means of using the non-parasitic planarian, Dugesia tigrina, a free-living aquatic flatworm, to study the stimulant and withdrawal properties of natural products is described. Experimental assays benefitting from unique aspects of planarian physiology have been applied to studies on wound healing, regeneration, and tumorigenesis. In addition, because planarians exhibit sensitivity to a variety of environmental stimuli and are capable of learning and developing conditioned responses, they can be used in behavioral studies examining learning and memory. Planarians possess a basic bilateral symmetry and a central nervous system that uses neurotransmitter systems amenable to studies examining the effects of neuromuscular biomodulators. Consequently, experimental systems monitoring planarian movement and motility have been developed to examine substance addiction and withdrawal. Because planarian motility offers the potential for a sensitive, easily standardized motility assay system to monitor the effect of stimuli, the planarian locomotor velocity (pLmV) test was adapted to monitor both stimulation and withdrawal behaviors by planarians through the determination of the number of grid lines crossed by the animals with time. Here, the technique and its application are demonstrated and explained.

Planarian motility is used to gauge the stimulant and withdrawal properties of natural products when compared to the movement of the animals in spring water alone.

测量天然产品生物调节特性的平面运动分析

A straightforward, controllable means of using the non-parasitic planarian, Dugesia tigrina, a free-living aquatic flatworm, to study the stimulant and ...

生物化学

Confocal Microscope-Based Laser Ablation and Regeneration Assay in Zebrafish Interneuromast Cells

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally replenished in non-mammalian vertebrates such as zebrafish. The zebrafish lateral line system is a useful model for characterizing sensory hair cell regeneration. The lateral line is comprised of hair cell-containing organs called neuromasts, which are linked together by a string of interneuromast cells (INMCs). INMCs act as progenitor cells that give rise to new neuromasts during development. INMCs can repair gaps in the lateral line system created by&#160;cell death. A method is described here for selective INMC ablation using a conventional laser-scanning confocal microscope and transgenic fish that express green fluorescent protein in INMCs. Time-lapse microscopy is then used to monitor INMC regeneration and determine the rate of gap closure. This represents an accessible protocol for cell ablation that does not require specialized equipment, such as a high-powered pulsed ultraviolet laser. The ablation protocol may serve broader interests, as it could be useful for the ablation of additional cell types, employing a tool set that is already available to many users. This technique will further enable the characterization of INMC regeneration under different conditions and from different genetic backgrounds, which will advance the understanding of sensory progenitor cell regeneration.

Laser ablation is a widely applicable technology for studying regeneration in biological systems. The presented protocol describes use of a standard laser-scanning confocal microscope for laser ablation and subsequent time-lapse imaging of regenerating interneuromast cells in the zebrafish lateral line.

斑马鱼内胚细胞中基于共体显微镜的激光消融和再生测定

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally ...

Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis support continuous growth of the entire visual system, both in the eye and the brain. Coordinated growth between the retinae and the optic tectum ensures accurate retinotopic mapping as new neurons are added in the eyes and brain. To address whether retinal axons provide crucial information for regulating tectal stem and progenitor cell behaviors such as survival, proliferation, and/or differentiation, it is necessary to be able to compare innervated and denervated tectal lobes within the same animal and across animals. 
Surgical removal of one eye from living larval zebrafish followed by observation of the optic tectum achieves this goal. The accompanying video demonstrates how to anesthetize larvae, electrolytically sharpen tungsten needles, and use them to remove one eye. It next shows how to dissect brains from fixed zebrafish larvae. Finally, the video provides an overview of the protocol for immunohistochemistry and a demonstration of how to mount stained embryos in low-melting-point agarose for microscopy.

The article explains how to surgically remove eyes from living zebrafish larvae as the first step toward investigating how retinal input influences optic tectum growth and development. In addition, the article provides information about larval anesthetization, fixation, and brain dissection, followed by immunohistochemistry and confocal imaging.

活斑马鱼幼虫的眼睛去除以检查视觉系统的神经支配依赖性生长和发育

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

激光烧蚀斑马鱼Pronephros研究肾小管上皮再生

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

生物学

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

药理和功能的遗传分析处理涡虫的再生日本三角涡虫</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

激光诱导的斑马鱼视网膜变性及再生模型中的 m ü ller 胶质细胞活化

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

神经科学

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

涡固定，局部照射，组织移植

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

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 ...

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform (RpEGEN) for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent retinal and vascular tissues. At present, the limited reparative capacity of mammalian RPE, which is restricted to small injuries, has hindered progress to understanding in vivo RPE regenerative processes. Here, a detailed methodology is provided to facilitate the study of in vivo RPE repair utilizing the zebrafish, a&#160;vertebrate model capable of robust tissue regeneration. This protocol describes a transgenic nitroreductase/metronidazole (NTR/MTZ)-mediated injury paradigm (rpe65a:nfsB-eGFP), which results in ablation of the central two-thirds of the RPE after 24 h treatment with MTZ, with subsequent tissue recovery. Focus is placed on RPE ablations in larval zebrafish and methods for testing the effects of pharmacological compounds on RPE regeneration are also outlined. Generation and validation of RpEGEN, a MATLAB script created to automate quantification of RPE regeneration based on pigmentation, is also discussed. Beyond active RPE repair mechanisms, this protocol can be expanded to studies of RPE degeneration and injury responses as well as the effects of RPE damage on adjacent retinal and vascular tissues, among other cellular and molecular processes. This zebrafish system holds significant promise in identifying genes, networks, and processes that drive RPE regeneration and RPE disease-related mechanisms, with the long-term goal of applying this knowledge to mammalian systems and, ultimately, toward therapeutic development.

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform RpEGEN for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

This protocol describes the methodology to genetically ablate the retinal pigment epithelium (RPE) using a transgenic zebrafish model. Adapting the protocol to incorporate signaling pathway modulation using pharmacological compounds is extensively detailed. A MATLAB platform for quantifying RPE regeneration based on pigmentation was developed and is presented and discussed.

硝基还原酶/甲硝唑介导的消融和MATLAB平台（RpEGEN）研究斑马鱼视网膜色素上皮的再生

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent ...