作者感谢露丝Zelkha，MS，她在成像慷慨援助。这项研究是由临床科学家发展计划奖K12EY021475到ME支持，授予R01 EY024349-01A1到ARD，从国家眼科研究所，美国国立卫生研究院，并从研究的无限制资助防盲核心补助EY01792。 ARD是一个职业发展奖从研究到防止失明的收件人。

在动物身上进行的所有程序符合视觉与眼科声明在视觉研究使用动物研究协会。十四四到六个月大的男/女C57BL / 6J野生型小鼠用于:对损伤模型十只小鼠和四个对照动物（未受损角膜）。 1.动物的制备<ol><li>麻醉小鼠用100毫克/千克氯胺酮腹腔注射5毫克/公斤甲苯噻嗪混合物。麻醉生效后，捏脚趾确认麻醉深度;动物不应该反应。 </li><li>在进行实验来验证没有任何先前的角膜损伤的前检查裂隙灯下眼。 </li><li>灌输1％盐酸丁卡因的到眼睛上的下降。经过60 - 90秒，测试角膜反射，确认没有感觉。盐酸丙美卡因0.5％，可替代使用。 </li><li>当角膜感觉的损失一定，轻轻地放在5％碘伏对眼睛和眼睛周围的头发的下降。擦去降1分钟后，或它涂在眼部周围的头发，以防止头发从与刀尖干扰。 </li></ol> 2.打磨角膜上皮<ol><li>戴上外科手套和隔离衣。准备所需的工具:无菌旋转毛刺和一对无菌镊子。为了更好的控制，使用弯曲的镊子。放置在过程中的无菌片的工具。 </li><li>使用弯曲镊子，通过从鼻侧抓住盖子，轻轻proptosing眼稳定的眼睛。避免用太多的压力。 </li><li>在手术显微镜，刮胡子360°左右的角膜缘区使用无菌旋转毛刺的两倍。周围的角膜缘去5的速度 - 每回合持续6秒。 注:毛刺有一个固定的速度。然而，它在既定的速度转动，在"端螺母"应被完全收紧。 </li><li>从角膜缘删除整个角膜上皮与旋转毛刺了limbus。为更好的效果，移动以循环方式的工具和保持它稍微平行于角膜表面到其尖端的侧面工作。小心不要弄伤基质没有重新涂刷，其中上皮细胞已被移除的区域。不要使用对眼睛的压力。根据需要清洁画笔笔尖。 </li><li>灌输无菌磷酸盐缓冲盐水的对角膜的下降和擦去用软清洁组织的任何已分离的上皮片。 </li><li>刮剩下的上皮细胞，如果有的话。 </li><li>在手术过程中需要注意的是鼠标尾部。 注:如果发生任何动静，麻醉深度已经消退，需要进一步注入。三分之一到主注入量的三分之二应该足够了。不要过量使用麻醉药物的动物。 </li><li>使用它们的另一只眼睛前消毒所有的工具。 </li></ol> 3.确认完全去除上皮<ol><li>申请为1mg / ml荧光素钠一滴在角膜上。清洁角膜30秒后，并检查其与钴蓝色滤色器，在显微镜下，以验证完全去除上皮。 注:具有上皮缺损的角膜区用绿色显示。 </li></ol> 4.手术后护理<ol><li>放在一个加热毯动物并将其保存在一个安全的地方（ 例如，在笼子里），而下恢复。不要让一个无意识的动物在该公司有意识的人的。 </li><li>应用1％红霉素眼药膏。这也将防止眼睛干涩。继续申请了一个星期抗生素的日常。 </li><li>注入0.1毫克/手术后公斤丁丙诺啡皮下和后再次12小时。继续注射每天两次两天。 </li><li>观察动物30 - 45分钟，直到从麻醉中完全恢复。 注意:腹面对呼吸肌轻度运动表明动物的健康，而下恢复。 </li></ol>

<ol>
	<li>Afsharkhamseh, N., 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=Stability+of+limbal+stem+cell+deficiency+after+mechanical+and+thermal+injuries+in+mice.">Stability of limbal stem cell deficiency after mechanical and thermal injuries in mice.</a> Exp. Eye Res. 145, 88-92 (2015).</li><li>Dor&#224;, N. J., Hill, R. E., Collinson, J. M., West, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+tracing+in+the+adult+mouse+corneal+epithelium+supports+the+limbal+epithelial+stem+cell+hypothesis+with+intermittent+periods+of+stem+cell+quiescence.">Lineage tracing in the adult mouse corneal epithelium supports the limbal epithelial stem cell hypothesis with intermittent periods of stem cell quiescence.</a> Stem Cell Res. 15 (3), 665-677 (2015).</li><li>Ahmad, S., Osei-Bempong, C., Dana, R., Jurkunas, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+culture+and+transplantation+of+human+limbal+stem+cells.">The culture and transplantation of human limbal stem cells.</a> J. Cell Physiol. 225 (1), 15-19 (2010).</li><li>Kolli, S., Ahmad, S., Lako, M., Figueiredo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+clinical+implementation+of+corneal+epithelial+stem+cell+therapy+for+treatment+of+unilateral+limbal+stem+cell+deficiency.">Successful clinical implementation of corneal epithelial stem cell therapy for treatment of unilateral limbal stem cell deficiency.</a> Stem Cells. 28 (3), 597-610 (2010).</li><li>Ahmad, S., Figueiredo, F., Lako, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+epithelial+stem+cells:+characterization,+culture+and+transplantation.">Corneal epithelial stem cells: characterization, culture and transplantation.</a> Regen. Med. 1 (1), 29-44 (2006).</li><li>He, H., Yiu, 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=Stem+cell-based+therapy+for+treating+limbal+stem+cells+deficiency:+A+review+of+different+strategies.">Stem cell-based therapy for treating limbal stem cells deficiency: A review of different strategies.</a> Saudi J. Ophthalmol. 28 (3), 188-194 (2014).</li><li>Pal-Ghosh, S., Pajoohesh-Ganji, A., Tadvalkar, G., Stepp, 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=Removal+of+the+basement+membrane+enhances+corneal+wound+healing.">Removal of the basement membrane enhances corneal wound healing.</a> Exp Eye Res. 93 (6), 927-936 (2011).</li><li>Stepp, 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=Wounding+the+cornea+to+learn+how+it+heals.">Wounding the cornea to learn how it heals.</a> Exp. Eye Res. 121, 178-193 (2014).</li><li>Amirjamshidi, H., 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=Limbal+fibroblast+conditioned+media:+a+non-invasive+treatment+for+limbal+stem+cell+deficiency.">Limbal fibroblast conditioned media: a non-invasive treatment for limbal stem cell deficiency.</a> Mol. Vis. 17, 658-666 (2011).</li><li>Li, F. 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=Evaluation+of+the+AlgerBrush+II+rotating+burr+as+a+tool+for+inducing+ocular+surface+failure+in+the+New+Zealand+White+rabbit.">Evaluation of the AlgerBrush II rotating burr as a tool for inducing ocular surface failure in the New Zealand White rabbit.</a> Exp. Eye Res. 147, 1-11 (2016).</li><li>Ti, S. E., Anderson, D., Touhami, A., Kim, C., Tseng, S. C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+outcome+following+transplantation+of+ex+vivo+expanded+limbal+epithelium+on+amniotic+membrane+for+total+limbal+deficiency+in+rabbits.">Factors affecting outcome following transplantation of ex vivo expanded limbal epithelium on amniotic membrane for total limbal deficiency in rabbits.</a> Invest. Ophthalmol. Vis. Sci. 43 (8), 2584-2592 (2002).</li><li>Ma, Y., 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=Reconstruction+of+chemically+burned+rat+corneal+surface+by+bone+marrow-derived+human+mesenchymal+stem+cells.">Reconstruction of chemically burned rat corneal surface by bone marrow-derived human mesenchymal stem cells.</a> Stem Cells. 24 (2), 315-321 (2006).</li><li>Bu, 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=Effects+of+activated+omental+cells+on+rat+limbal+corneal+alkali+injury.">Effects of activated omental cells on rat limbal corneal alkali injury.</a> Exp. Eye Res. 121, 143-146 (2014).</li><li>Luengo Gimeno, F., Lavigne, V., Gatto, S., Croxatto, J. O., Correa, L., Gallo, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+corneal+stem-cell+transplantation+in+rabbits+with+severe+ocular+alkali+burns.">Advances in corneal stem-cell transplantation in rabbits with severe ocular alkali burns.</a> J. Cataract Refract. Surg. 33 (11), 1958-1965 (2007).</li><li>Lin, Z., 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=A+mouse+model+of+limbal+stem+cell+deficiency+induced+by+topical+medication+with+the+preservative+benzalkonium+chloride.">A mouse model of limbal stem cell deficiency induced by topical medication with the preservative benzalkonium chloride.</a> Invest. Ophthalmol. Vis. Sci. 54 (9), 6314-6325 (2013).</li><li>Huang, A. J., Tseng, 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=Corneal+epithelial+wound+healing+in+the+absence+of+limbal+epithelium.">Corneal epithelial wound healing in the absence of limbal epithelium.</a> Invest. Ophthalmol. Vis. Sci. 32 (1), 96-105 (1991).</li><li>Liu, C. Y., 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=Cornea-specific+expression+of+K12+keratin+during+mouse+development.">Cornea-specific expression of K12 keratin during mouse development.</a> Curr. Eye Res. 12 (11), 963-974 (1993).</li><li>Nakatsu, M. N., Gonz&#225;lez, S., Mei, H., Deng, S. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+limbal+mesenchymal+cells+support+the+growth+of+human+corneal+epithelial+stem/progenitor+cells.">Human limbal mesenchymal cells support the growth of human corneal epithelial stem/progenitor cells.</a> Invest. Ophthalmol. Vis. Sci. 55 (10), 6953-6959 (2014).</li><li>Pajoohesh-Ganji, A., Pal-Ghosh, S., Tadvalkar, G., Stepp, 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=Corneal+goblet+cells+and+their+niche:+implications+for+corneal+stem+cell+deficiency.">Corneal goblet cells and their niche: implications for corneal stem cell deficiency.</a> Stem Cells. 30 (9), 2032-2043 (2012).</li><li>McCloy, R. A., Rogers, S., Caldon, C. E., Lorca, T., Castro, A., Burgess, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+inhibition+of+Cdk1+in+G+2+phase+overrides+the+SAC+and+decouples+mitotic+events.">Partial inhibition of Cdk1 in G 2 phase overrides the SAC and decouples mitotic events.</a> Cell Cycle. 13, 1400-1412 (2014).</li></ol>

The authors declare that they have nothing to disclose.

本文介绍了一种可重复的和相对简单的技术来创建LSCD的小鼠模型。但是也有一些值得一提的这个模型的几个重要方面。第一，使用的化学物质11，12不同的机型，损伤主要涉及表面上皮（和基底膜），与底层角膜基质或其它人工结构的损害最小。因此，仅存在有限炎症和疤痕，这两者都可以变得复杂的程序，并使其更难以评估旨在角膜缘干细胞的任何干预的结果。一个有趣的最近研究由李等人。 10，其比较使用与手术和化学处理的不同组合的旋转毛刺，还发现毛刺更安全和兔创建LSCD更有效。其次，模式似乎至少长达三个月1是稳定的。创建这个模型包括使用旋转毛刺，仪器是熟悉的人谁与角膜的工作，以及为那些谁做鼠标伤人研究临床医生。它比使用钝锅铲优点是损伤更均匀和可重复性1。 为了更好地破坏角膜缘干细胞，角膜缘区域进行处理两次。基于对老鼠的眼睛的工作经验，待遇超过两回合的经常擦伤角膜缘血管，引起出血。万一该技术在其他动物中使用，这个步骤可能依赖于角膜上皮厚度进行修改。在这项研究中，用0.5毫米的毛刺尖端的机动工具是利用;在其它形状和尺寸毛刺提示也可与可用于代替，这取决于角膜的规格和在眼睛尺寸。 Li 等人 。 10报道了2.5毫米毛刺以适于一致地除去兔角膜和角膜缘上皮细胞。 为了获得最佳结果，角膜缘和角膜上皮细胞中以循环方式被剃光并使用了毛刺的侧面。以防止动物毛发从与刀尖干扰时，聚维酮碘滴散布在眼睛周围的头发，而不是被擦掉。该工具不应该在一个地方经营静态，因为这可能会导致穿孔。在一个非常缓慢的步伐可能会导致出血治疗角膜缘;每一轮近6秒的速度是适当的C57BL / 6J小鼠的眼睛。关键是要避免重新涂刷裸露的基质，因为这将导致更多的炎症和间质混浊。而研磨上皮压力不应施加到眼球。在手术后，角膜用荧光素染料染色，以确保完全的上皮去除。它防止动物恢复期间眼球干燥是很重要的。限制是，这种技术无法去除干细胞的100％。这也是有些运营商依赖和需要实践给予合作nsistent结果。 当研究角膜上皮表型，整装染色，以评估该模型的结果的最佳方法。这是因为它允许整个角膜上皮被一次评估，而横截面只会给每个特定部分的信息。 这种控制技术可以在其他动物诱发LSCD小鼠，而且很可能也是如此。它提供了LSCD一个合适的模型来研究新的治疗方法，并发现本病的病理生理的更广泛的方面。与一些改变，也可以是在学习角膜伤口愈合，血管发生和淋巴管生成以及有用。

角膜上皮是必需的，以保持角膜的清晰度和完整性。它是不断在整个寿命通过在角膜和结膜的交界处驻留在角膜缘-一个狭窄区域的上皮干细胞更新。这些自我更新角膜缘干细胞在再生角膜上皮至关重要的作用，这两个正常和损伤后。这些干细胞的部分或完全耗尽将导致复发性角膜糜烂，疼痛，角膜瘢痕和新血管形成，杯状细胞的外观，并且如果不进行治疗，角膜失明。这种情况被称为角膜缘干细胞缺陷（LSCD），并且可以是特发性;遗传;或获得由于化学或热损伤，长期佩戴隐形眼镜，和慢性炎症1-6。 研究LSCD需要一个合适的动物模型，不仅模仿人类的疾病，但是可重复的和可持续的，与至少一个伤害到其他角膜和眼结构的坐骑。这种模式是必要的，以评估治疗和澄清在分子和细胞水平的疾病机制。如1如前所述，通过使用一个旋转毛刺，人们可以很容易地开发LSCD的小鼠模型，具有上述优点并持续至少三个月。这项研究的目的是提出LSCD简单，重复性好，可持续的小鼠模型。 旋转毛刺是一个方便的工具，均匀地去除上皮细胞不损伤下面的基质1。它已被用于诱导伤口愈合研究中央角膜糜烂7,8。兹-呈现技术在小鼠创建LSCD以前尚未报道。此前推出了一个不太均匀伤病特别是在角膜缘和一个更多变型1,9用钝刮刀刮结果上皮细胞，与更多的修复邻方法F普通角膜上皮1。不同于钝刮，旋转毛刺去除上皮基底膜以及7，8，10。其他报道的方法以切断干细胞涉及使用化学物质，如氢氧化钠，正 - 庚醇，和苯扎氯铵，不仅可以诱导不想要的损伤底层眼构造，但也可能导致显著炎症和随后的角膜混浊或上皮鳞状上皮化生11-15。旋转毛刺不会与这些严重并发症。手术去除角膜缘上皮细胞，单独或与化学品的使用10，11，16，更难以进行，而不是在动物细眼和薄角膜缘上皮细胞（如小鼠）的最好的选择。此外，奇怪的是，limbectomy仍可能留下一些落后10角膜缘上皮。 将导致LSCD与新生血管形成的下述技术ð结膜模仿患者完全LSCD的介绍，并持续至少三个月1。它适用于那些谁旨在研究小鼠LSCD或伤口愈合病理生理学，免疫学，和潜在的治疗方法。可能的话，有一些修改，这个程序可以在大鼠或家兔10执行。作为旋转毛刺有效地除去上皮细胞，它可以在有关角膜上皮磨损/伤人及在任何相关的治疗或分子生物学研究中的任何研究中使用。

<table><tbody><tr><td>Rotating burr: AlgerBrush II rust ring remover&amp;nbsp;</td><td>Rumex International Co, Clearwater, FL</td><td>16-141</td><td>0.5 mm burr. Also available from other companies.</td></tr><tr><td>Surgical microscope</td><td>Wild Heerbrugg, Switzerland</td><td>Wild M691</td><td></td></tr><tr><td>Digital camera</td><td>Nikon, Thailand</td><td></td><td></td></tr><tr><td>Nikon FS-2 slit lamp</td><td>Nikon, Japan</td><td></td><td></td></tr><tr><td>Polyclonal anti-CK12 antibody</td><td>Santa Cruz Blotechnology, CA,</td><td>SC-17101</td><td>1:100 concentration</td></tr><tr><td>Monoclonal anti-CK8 antibody</td><td>TROMA-I-s, Iowa City, IA</td><td>AB_531826</td><td>1:50 concentration</td></tr></tbody></table>

a simple mechanical procedure to create limbal stem cell deficiency in mouse

角膜缘干细胞缺陷（LSCD）是故障或角膜缘上皮干细胞，然后将角膜上皮被替换结膜损失的状态。患有复发性角膜上皮缺损，疼痛，炎症，视力遭受损失。 先前，LSCD的鼠模型进行了说明，并相对于其他两个模型。的目标是产生LSCD的一致小鼠模型，这两个模仿人类的表型和持续足够长以使得可以研究疾病的病理生理学和评估新的治疗方法。这里，该技术进行更详细的说明。 用旋转毛刺的电动工具被设计为从角膜表面除去锈环或平滑翼状胬肉床的患者。它是一个合适的装置以产生所需的LSCD模型。这是一个现成的，易于使用的工具，用细尖，使得它适合于小眼睛的工作，如小鼠。它的应用可以防止不必要的眼创伤，它不会导致不必要的伤害，由于经常与化学损伤模型的情况。相对于钝刮，它消除了与基底膜上皮。在这个协议中，角膜缘区域被研磨两次，然后整个角膜上皮被剃光从角膜缘到角膜缘。为了避免基质伤害，呵护被带到曾经的上皮细胞已经删除不要刷角膜表面。

在执行此技术后一个月，角膜的100％开发浅表血管新生（ 图1A。照片拍摄的同时，在明场和钴蓝色的过滤器连接到裂隙灯摄像机）。在眼中的18/20（90％），新生血管介入整个角膜表面（ 图1A）。在2/20（10％），在三四个角膜象限观察新血管形成，但是它仍然达到角膜中心。 为了验证LSCD的发展，整个装载免疫进行1。这个步骤不是该协议的一部分，并且可以根据目标和利益而改变。三个变化被认为是LSCD指标:无细胞角蛋白（CK）12，具体到正常角膜上皮细胞17，18中间丝; CK8的外观，简单的上皮细胞标记<s向上&gt; 20，其不存在于正常的角膜;和角膜表面上杯状细胞的存在。为了评估角膜上皮表型，对于CK12和CK8双重免疫染色上整装样品损伤三个月后进行，如先前1,9所述。杯状细胞存在被MUC5AC-评估杯状细胞特异性粘蛋白免疫染色1，15，1945天以下上皮去除。杯状细胞的出现时间减少。对于最好的结果，它在受伤后进行染色不到两个月是重要的。 正常，未受损伤的角膜明示CK12-一个成熟的角膜上皮细胞17 18 -throughout角膜表面的特异性标记物，但它们是没有任何CK8的或杯状的整装样品的角膜部分细胞（MUC5AC）（ 图1B） 。在比较正常的眼睛，CK12表达上万人受伤几乎不存在<html> Ÿ模型角膜，而他们显著显示CK8阳性和杯状细胞（ 图1B）。这些观察表明LSCD的发展。 如果有兴趣的定量免疫荧光结果，使用图像-J对通过在显微镜下相同的设置拍摄的原始图像不变量化分别免疫染色20密度。简言之，选择整装样品的角膜部分，测量该区域的集成密度和面积，并计算经校正的密度为背景20。 CK12的至CK8密度比以前提出了关于样品1的更大的数目。相比于对照的减少率（正常角膜）和杯状细胞的角膜表面上的外观表明LSCD的发展。 658 / 54658fig1.jpg"/&gt; 图1: 角膜表型小鼠创建LSCD后。裂隙灯检查表明，所有角膜个月（A，比例尺为0.5mm）产生某种程度的新生血管。正常角膜表达CK12遍布角膜，但没有任何CK8或杯状细胞（MUC5AC）的。 CK12是在LSCD模型不存在，而CK8和杯状细胞上出现的角膜（B，比例尺:200微米）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54658/54658fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse at JoVE.com

A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse

下面的文章提供了一个简单，可重复的方法来有效地创建角膜缘干细胞缺乏（LSCD）的一个可持续发展的小鼠模型。该动物模型是在测试和比较的用于角膜缘干细胞的疾病的治疗的效力是有用的。

一个简单的机械过程来创建在小鼠角膜缘干细胞缺乏症

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with ...

a-simple-mechanical-procedure-to-create-limbal-stem-cell-deficiency

Nanyang Technological University

Research

JoVE Journal

Medicine

8.3K 次观看.  University of Illinois at Chicago. 下面的文章提供了一个简单，可重复的方法来有效地创建角膜缘干细胞缺乏（LSCD）的一个可持续发展的小鼠模型。该动物模型是在测试和比较的用于角膜缘干细胞的疾病的治疗的效力是有用的。 

视频: A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

在免疫缺陷小鼠的建立和传播人类视网膜母细胞瘤肿瘤

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

科研

医学

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

方法对角膜细胞的谱系追踪使用多色荧光记者小鼠

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

发育生物学

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

腺病毒基因治疗对糖尿病性角膜病变:对伤口愈合和干细胞标记表达的人体器官培养角膜和角膜缘上皮细胞

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

基因治疗的载体和干细胞视网膜下注射到老鼠的眼睛

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

在中央小鼠中枢神经系统的干细胞移植的多模态成像

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

神经科学

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

病毒载体的基因治疗的小鼠视网膜色素上皮缘方针，视网膜下注射

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

一种用于小鼠精确碱烧伤的改良活检穿刺法

Corneal and Limbal Alkali Injury Induction Using a Punch-Trephine Technique in a Mouse Model

The cornea is critical for vision, and corneal healing after trauma is fundamental in maintaining its transparency and function. Through the study of corneal injury models, researchers aim to enhance their understanding of how the cornea heals and develop strategies to prevent and manage corneal opacities. Chemical injury is one of the most popular injury models that has extensively been studied on mice. Most previous investigators have used a flat paper soaked in sodium hydroxide to induce corneal injury. However, inducing corneal and limbal injury using flat filter paper is unreliable, since the mouse cornea is highly curved. Here, we present a new instrument, a modified biopsy punch, that enables the researchers to create a well-circumscribed, localized, and evenly distributed alkali injury to the murine cornea and limbus. This punch-trephine method enables researchers to induce an accurate and reproducible chemical burn to the entire murine cornea and limbus while leaving other structures, such as the eyelids, unaffected by the chemical. Moreover, this study introduces an enucleation technique that preserves the medial caruncle as a landmark for identifying the nasal side of the globe. The bulbar and palpebral conjunctiva, and lacrimal gland are also kept intact using this technique. Ophthalmologic examinations were performed via slit lamp biomicroscope and fluorescein staining on days 0, 1, 2, 6, 8, and 14 post-injury. Clinical, histological, and immunohistochemical findings confirmed limbal stem cell deficiency and ocular surface regeneration failure in all experimental mice. The presented alkali corneal injury model is ideal for studying limbal stem cell deficiency, corneal inflammation, and fibrosis. This method is also suitable for investigating pre-clinical and clinical efficacies of topical ophthalmologic medications on the murine corneal surface.

作者聚焦:推进角膜混浊治疗和再生的研究 

This protocol describes a method to induce an accurate and reproducible corneal and limbal alkali injury in a mouse model. The protocol is advantageous as it allows for an evenly distributed injury to the highly curved mouse cornea and limbus.

在小鼠模型中使用 Punch-Trephine 技术诱导角膜和角膜缘碱损伤

The cornea is critical for vision, and corneal healing after trauma is fundamental in maintaining its transparency and function. Through the study of ...

焕活视力:干细胞疗法治疗视网膜变性

Sub-Retinal Delivery of Human Embryonic Stem Cell Derived Photoreceptor Progenitors in rd10 Mice

Regeneration of photoreceptor cells using human pluripotent stem cells is a promising therapy for the treatment of both hereditary and aging retinal diseases at advanced stages. We have shown human recombinant retina-specific laminin isoform matrix is able to support the differentiation of human embryonic stem cells (hESCs) to photoreceptor progenitors. In addition, sub-retinal injection of these cells has also shown partial restoration in the rd10 rodent and rabbit models. Sub-retinal injection is known to be an established method that has been used to deliver pharmaceutical compounds to the photoreceptor cells and retinal pigmented epithelial (RPE) layer of the eye due to its proximity to the target space. It has also been used to deliver adeno-associated viral vectors into the sub-retinal space to treat retinal diseases. The sub-retinal delivery of pharmaceutical compounds and cells in the murine model is challenging due to the constraint in the size of the murine eyeball. This protocol describes the detailed procedure for the preparation of hESC-derived photoreceptor progenitor cells for injection and the sub-retinal delivery technique of these cells in genetic retinitis pigmentosa mutant, rd10 mice. This approach allows cell therapy to the targeted area, in particular the outer nuclear layer of the retina, where diseases leading to photoreceptor degeneration occur.

作者聚焦:推进视力恢复 - 基于干细胞的视网膜疾病疗法 

We describe a detailed protocol for the preparation of post-cryopreserved hESC-derived photoreceptor progenitor cells and the sub-retinal delivery of these cells in rd10 mice.

rd10小鼠中人胚胎干细胞衍生的光感受器祖细胞的视网膜下递送

Regeneration of photoreceptor cells using human pluripotent stem cells is a promising therapy for the treatment of both hereditary and aging retinal ...

一个简单的机械过程来创建在小鼠角膜缘干细胞缺乏症

摘要

探索更多视频

此视频中的章节

在免疫缺陷小鼠的建立和传播人类视网膜母细胞瘤肿瘤

方法对角膜细胞的谱系追踪使用多色荧光记者小鼠

腺病毒基因治疗对糖尿病性角膜病变:对伤口愈合和干细胞标记表达的人体器官培养角膜和角膜缘上皮细胞

基因治疗的载体和干细胞视网膜下注射到老鼠的眼睛

在中央小鼠中枢神经系统的干细胞移植的多模态成像

病毒载体的基因治疗的小鼠视网膜色素上皮缘方针，视网膜下注射

在小鼠模型中使用 Punch-Trephine 技术诱导角膜和角膜缘碱损伤

在小鼠模型中使用 Punch-Trephine 技术诱导角膜和角膜缘碱损伤

在小鼠模型中使用 Punch-Trephine 技术诱导角膜和角膜缘碱损伤

rd10小鼠中人胚胎干细胞衍生的光感受器祖细胞的视网膜下递送