我们要感谢凯撒 Ikkala 在实施这一方法以及后来在执行我们的中心研究问题时提供的宝贵的技术援助和深刻的帮助。我们还要感谢实验动物中心和安娜 Meller 在规划兽医工作指南方面的帮助。

所有实验均经国家动物实验委员会批准。
1. 筹备工作
<ol><li>准备所有的解决方案并保持室温, 除非另有说明。遵循废弃物处理条例, 处理废旧材料和解决方案。</li>
	<li>使用 NMRI 和 outbred 的股票, 年龄介于4-12 周和任何性别之间。如果使用 C57BL/6 菌株, 请按照 medetomidine 的方法在步骤1.3.2 中执行氯胺酮。有关其他步骤, 请按照 1.3. 3.-1.3. 5 中描述的说明进行操作。</li>
	<li>在开始手术前, 及时准备兽医药品。卡洛芬将在以后使用, 因此没有必要准备它在这一点上。<ol>
<li>为了准备麻醉的氯胺酮 medetomidine 的解决方案, 混合0.375 毫升 (50 毫克/毫升) 氯胺酮 (Ketaminol 兽医) 与0.25 毫升 (库存浓度1毫克/毫升) 的 medetomidine (Cepetor 兽医) 和添加1.25 毫升无菌0.9% 氯化钠。管理 0.15 mL/20 g 的鼠标重量 (7.5 毫克/千克, 氯胺酮和1毫克/千克, medetomidine)。</li>
		<li>为了在 C57BL/6 小鼠身上制备一种更稀的氯胺酮-medetomidine 麻醉溶液, 将氯胺酮的0.375 毫升 (浓度50毫克/毫升) 与0.25 毫升 (浓度1毫克/毫升) medetomidine, 加入2.5 毫升无菌0.9% 氯化钠。管理 0.15 mL/20 g 的鼠标重量 (3.75 毫克/千克, 氯胺酮和0.5 毫克/千克, medetomidine)。 注意: 这是一个替代的步骤, 只为成年 C57BL/6 小鼠。</li>
		<li>要准备 buprenorfin 镇痛, 添加0.1 毫升 (0.3 毫克/毫升) 的 buprenorfin 到1.9 毫升无菌0.9% 氯化钠。管理 0.2 mL/20 g 鼠标重量 (0.15 毫克/千克)。</li>
		<li>为停止麻醉准备 atipamezole, 添加0.1 毫升 (5 毫克/毫升) 的 atipamezole 到4.9 毫升0.9% 氯化钠。管理 0.15 mL/20 g 鼠标重量 (0.75 毫克/千克)。</li>
		<li>麻醉后使用卡洛芬镇痛。添加0.1 毫升 (库存浓度50毫克/毫升) 的卡洛芬到4.9 毫升0.9% 氯化钠。管理 0.15 mL/20 g 的鼠标重量 (7.5 毫克/千克)。</li>
	</ol>
</li>
	<li>准备荧光染色液<ol>
<li>为了可视化钴蓝光下的磨损 (图 1), 请使用荧光溶液。准备一个0.1% 荧光素溶液, 首先测量10毫克的荧光素盐与精细的规模, 然后添加到10毫升磷酸盐缓冲盐水溶液 (PBS)。</li>
		<li>保护荧光素溶液从光和震动5分钟在一个振动筛。 注意: 荧光素溶液对光敏感;将解决方案从光中覆盖。此解决方案可存储在 +4 °c 2-3 天。使用时, 将荧光素溶液放在用于眼药水的滴管瓶中是很有帮助的。将溶液移至滴管瓶时, 使用无菌过滤器。</li>
	</ol>
</li>
	<li>使用前后清洁眼部毛刺提示;首先与 PBS 然后与70% 乙醇。如果在同一操作过程中对几个小鼠进行磨损, 请在每个鼠标之间进行清洗步骤。</li>
	<li>把加热的盘子放在 (37 摄氏度) 上, 然后放上纸巾。</li>
</ol>2. 角膜磨损
注意事项: 在处理小鼠时, 使用防护服 (手套、实验室大衣)。称量鼠标估计用药量。
<ol><li>使用单手 scruffing 方法处理鼠标23。管理氯胺酮-medetomidine 混合腹腔 (ip) 到左下腹部。将鼠标放到一个单独的笼子里, 等待它入睡。这通常需要少于5分钟。然后, 管理第一 buprenorfin, 然后 atipamezole 通过 ip 注射。使用相同的处理技术。 注: 一旦麻醉被给予, 该议定书不应暂停在任何时刻。</li>
	<li>在继续之前, 检查加热板是否温暖。将麻醉鼠标放在加热板上。如果没有尾部反射, 麻醉的水平很好, 但是脚趾反射是存在的。用手指或镊子捏紧尾巴和脚趾来检查这些反射。不要过度使用武力。麻醉将持续20-25 分钟。</li>
	<li>要使磨损, 使用清洁的眼毛刺。旋转毛刺的底部, 以打开振动, 这是必不可少的, 使磨损。当毛刺正常工作时, 感觉到手上的振动。<ol>
<li>一次睁开一只眼睛, 用手指分别握住眼睑。然后牢固地触碰毛刺到角膜并且移动仪器前后并且侧身在眼睛表面。毛刺振动将执行划痕;不要按压、摇动或撕裂角膜。在角膜中央, 20 研磨在表面上的运动足以诱发伤口。不要提起毛刺, 尽量留在角膜的区域内进行擦伤。要获得标准尺寸的磨损, 需要几轮练习。</li>
	</ol>
 注意: 使用 betadine 眼科溶液可达到角膜无菌磨损。</li>
</ol>3. 对磨损的成像
<ol><li>使用钴蓝笔光 (图 1) 和荧光素溶液照亮磨损区域。眼部表面在周围光线中呈无色。荧光素应用后, 在用笔光照射眼睛时, 擦伤区域荧光绿色。<ol>
<li>如果需要, 同样打开擦伤的眼睛, 如在2.3 和管理一滴荧光素溶液从滴管瓶。用 PBS 或0.9% 氯化钠冲洗眼睛, 以减少荧光素的背景。用吸管瓶或吸管清洗。如果需要的话, 用柔软的擦拭和清洁睫毛来吸掉液体。过量的液体通常收集到眼睛的鼻边界, 靠近泪道的开口。 注意: 避免接触角膜, 而 cleaining 可能诱发轻微擦伤。</li>
	</ol>
</li>
	<li>拍摄角膜磨损的照片。<ol>
<li>要在每个成像会话期间获得相似的图像, 请使用钴蓝钢笔灯和单反相机的稳定附件工具 (图 2)。此协议提供了一个易于重复的设置 (图 2)。<ol>
<li>要连接钢笔灯和照相机, 请分别使用带有挠性手臂和夹具的台灯和可调节的相机臂。电缆-将钢笔灯与台灯和鼠标眼睛上方的位置系在一起。以不同的方式定位光线可能会改变磨损的可视化。图 2描述了定位这些工具的建议。</li>
		</ol>
</li>
		<li>使用单反相机拍照的眼睛。将动物置于所需的距离和位置, 并将相机 (1:12) 放在室内光线中。之后, 关闭所有其他的灯, 除了钴蓝钢笔灯。 注: 在成像过程中, 将钢笔灯手柄向下或使用橡皮筋保持灯亮起。如果图像变得模糊, 这可能会有帮助。与同事一起执行成像步骤是最容易的。</li>
	</ol>
</li>
</ol>4. 角膜磨损后醒来
<ol><li>管理第一 buprenorfin, 然后 atipamezole 通过 ip 注射。使用与2.1 相同的处理技术。</li>
	<li>将一滴抗菌剂、fucidin 酸眼膏 (Isathal) 放在磨擦和不擦伤的眼睛上, 在麻醉后保持眼部表面清洁。</li>
	<li>当老鼠在加热板上5-20 分钟内醒来时, 观察老鼠在麻醉后苏醒的时间。当它变成移动, 把它放在一个单独的笼子里。 注意: 如果醒来需要更长的时间, 把鼠标放在笼子里用加热的垫子, 或者把整个笼子放在加热的盘子上, 定期监视鼠标。当麻醉逐渐消退时, 老鼠通常会摇晃并向上与前体接触。此行为应在3-4 小时后恢复正常, 然后您可以将鼠标放回共享的笼子。</li>
	<li>在磨损后连续两天管理卡洛芬的镇痛和眼膏, 类似于步骤2.2。和4.2。</li>
</ol>5. 伤口愈合过程中的影像学
<ol><li>在本议定书中, 在磨损后使用时间点18小时和72小时观察伤口闭合。<ol>
<li>为连续成像, 麻醉老鼠解释在2.2。</li>
		<li>采取图片解释在3。</li>
		<li>唤醒动物与 atipamezole ip 和监测唤醒期, 如4.3 所述。 注: 如果不继续随访, 弄死鼠标右5.1.2。安乐死在6.1 被描述。</li>
	</ol>
</li>
</ol>6. 角膜收集和石蜡嵌入
<ol><li>在最后的时间点后, 牺牲动物。把鼠标放在可以用 CO2填充的房间里。用 CO 2 填充室内空气.当动物不动时, 将颈椎从尾部的底部牢牢拉起, 同时按下颈部区域, 使其脱臼。 注意: 始终遵循当地动物福利机构的指导方针。</li>
	<li>用解剖剪刀把皮肤上的开口切开到眼睛侧面, 收集整个眼球。把剪刀放在眼球下, 剪下眼神经, 将眼球从轨道上弹出。使用镊子, 如果眼睛不容易出来。<ol>
<li>在眼睛的背面做一个洞, 在视网膜一侧, 用26G 针让眼睛内的溶液自由穿透。 注意: 不要让眼睛变得干燥, 而是将它放在 PBS 中, 直到继续使用该协议。</li>
	</ol>
</li>
	<li>用 PBS 在2毫升管中采集眼球。此时不要暂停实验。</li>
	<li>删除 PBS 和固定眼球在4% 多聚甲醛 (粉煤灰) 在 +4 °c 4-5 小时。</li>
	<li>将固定眼球移到纸巾盒中进行组织处理。使用组织处理机和以下程序:<ol>
<li>用 PBS 冲洗。</li>
		<li>室温下在70% 乙醇中孵化 2 x 45 分钟。</li>
		<li>室温下在94% 乙醇中孵化 2 x 1 小时。</li>
		<li>室温下在绝对乙醇中孵育 3 x 45 分钟。</li>
		<li>在二甲苯中孵化 3 x 1 小时, 室温和最后二甲苯在37摄氏度。</li>
		<li>在石蜡60摄氏度孵育 3 x 1 小时。</li>
	</ol>
</li>
	<li>组织处理后, 使用组织嵌入块将眼球嵌入液体石蜡 (+ 60 °c)。把眼睛放在块里, 这样它就会朝侧面看, 外围的边界朝向向上。让方块凝固在一个凉爽的盘子里5-10 分钟。</li>
</ol>7. 角膜石蜡切片
<ol><li>根据机构或制造商的指示, 准备切片的切片。 注意: 处理锋利的切片刀片时要小心。</li>
	<li>安置一水浴与室温超纯水在切片旁边和另一个与被加热到 +50 °c 的超纯水。</li>
	<li>使眼球的5µm 厚的部分。 注意: 在切片过程中镜片容易折断。为避免这种情况, 每次开始新的一排节时, 都要用湿润的纸巾擦拭切口表面。用自来水滋润组织。</li>
	<li>在室温水浴中放置各节, 然后在玻璃滑梯的帮助下将它们移动到热水浴中。伸展热水浴中的部分, 直到所有的皱纹放松和部分变成哑光。</li>
	<li>用 +37 摄氏度一夜之间的玻璃片干燥。</li>
	<li>在 +60 摄氏度的加热板上保持1分钟的滑动, 将各部分连接到幻灯片上。在此之后, 切片可以直接处理染色, 免疫组织化学方法或储存在 +4 °c。</li>
</ol>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt-7a+up-regulates+matrix+metalloproteinase-12+expression+and+promotes+cell+proliferation+in+corneal+epithelial+cells+during+wound+healing.">Wnt-7a up-regulates matrix metalloproteinase-12 expression and promotes cell proliferation in corneal epithelial cells during wound healing.</a> Journal of Biological Chemistry. 280 (22), 21653-21660 (2005).</li><li>Nagata, M., Nakamura, T., Hata, Y., Yamaguchi, S., Kaku, T., Kinoshita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JBP485+promotes+corneal+epithelial+wound+healing.">JBP485 promotes corneal epithelial wound healing.</a> Science Reports. 5, 14776 (2015).</li><li>Stepp, M. A., Zhu, L., Cranfill, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+beta+4+integrin+expression+and+localization+in+vivo+in+response+to+corneal+epithelial+injury.">Changes in beta 4 integrin expression and localization in vivo in response to corneal epithelial injury.</a> Investigative Ophthalmology &amp; Visual Science. 37 (8), 1593-1601 (1996).</li><li>Stepp, M. A., Zhu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upregulation+of+alpha+9+integrin+and+tenascin+during+epithelial+regeneration+after+debridement+in+the+cornea.">Upregulation of alpha 9 integrin and tenascin during epithelial regeneration after debridement in the cornea.</a> Journal of Histochemistry &amp; Cytochemistry. 45 (2), 189-201 (1997).</li><li>Pal-Ghosh, S., Tadvalkar, G., Jurjus, R. A., Zieske, J. D., 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=BALB/c+and+C57BL6+mouse+strains+vary+in+their+ability+to+heal+corneal+epithelial+debridement+wounds.">BALB/c and C57BL6 mouse strains vary in their ability to heal corneal epithelial debridement wounds.</a> Experimental Eye Research. 87 (5), 478-486 (2008).</li><li>. Lab Animal Research. Rodent Handling and Restraint Techniques Available from: <a target="_blank" href="https://www.jove.com/science-education/10221/rodent-handling-and-restraint-techniques">https://www.jove.com/science-education/10221/rodent-handling-and-restraint-techniques</a> (2018)</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> Experimental Eye Research. 93 (6), 927-936 (2011).</li><li>Suzuki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-matrix+and+cell-cell+interactions+during+corneal+epithelial+wound+healing.">Cell-matrix and cell-cell interactions during corneal epithelial wound healing.</a> Progress in Retinal and Eye Research. 22 (2), 113-133 (2003).</li><li>Sato, Y., Seo, N., Kobayashi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+background+differences+between+FVB+and+C57BL/6+mice+affect+hypnotic+susceptibility+to+pentobarbital,+ketamine+and+nitrous+oxide,+but+not+isoflurane.">Genetic background differences between FVB and C57BL/6 mice affect hypnotic susceptibility to pentobarbital, ketamine and nitrous oxide, but not isoflurane.</a> Acta Anaesthesiologica Scandinavica. 50 (5), 553-556 (2006).</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=K14+++Compound+niches+are+present+on+the+mouse+cornea+early+after+birth+and+expand+after+debridement+wounds.">K14 + Compound niches are present on the mouse cornea early after birth and expand after debridement wounds.</a> Developmental Dynamics. 245 (2), 132-143 (2016).</li><li>Boote, C., 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=Quantitative+Assessment+of+Ultrastructure+and+Light+Scatter+in+Mouse+Corneal+Debridement+Wounds.">Quantitative Assessment of Ultrastructure and Light Scatter in Mouse Corneal Debridement Wounds.</a> Investigative Ophthalmology &amp; Visual Science. 53 (6), 2786 (2012).</li><li>Pal-Ghosh, S., 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=MMP9+cleavage+of+the+&#946;4+integrin+ectodomain+leads+to+recurrent+epithelial+erosions+in+mice.">MMP9 cleavage of the &#946;4 integrin ectodomain leads to recurrent epithelial erosions in mice.</a> Journal of Cell Science. 124 (Pt 15), 2666-2675 (2011).</li><li>Pal-Ghosh, S., Pajoohesh-Ganji, A., Brown, M., 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=A+mouse+model+for+the+study+of+recurrent+corneal+epithelial+erosions:+alpha9beta1+integrin+implicated+in+progression+of+the+disease.">A mouse model for the study of recurrent corneal epithelial erosions: alpha9beta1 integrin implicated in progression of the disease.</a> Investigative Ophthalmology &amp; Visual Science. 45 (6), 1775-1788 (2004).</li></ol>

作者没有什么可透露的。

伤人方法是研究角膜稳态和病理不同方面的常用工具。磨损模型为解决眼科相关问题提供了一种良好的控制方法。然而, 议定书中的某些关键点值得强调。值得注意的是, 有关兽医医学、伤口愈合时间线和结果的详细说明被优化为与 outbred NMRI 和代表种群一起使用, 但在小鼠26株中可能有所不同。通过这个协议, 实验动物将保持麻醉大约20分钟。这给了一个短但足够的时间窗口来执?...

许多器官的上皮层受到损伤。然而, 它们也包含通过伤口愈合来补偿组织损失的能力。角膜提供了一个很好的模型来研究伤口愈合。它形成眼睛的外部表面并且为敏感的眼部机器提供保护层。因此, 角膜的作用是对病原体和水分损失的物理屏障。它由三层组成;上皮, 基质和内皮。角膜上皮构成角膜的最外层。上皮细胞通过紧密连接1,2,3维持角膜屏障功能。一种脱细胞角膜基底膜, 鲍曼膜, 分离上皮与广泛的基质, 其中含有顽固性基质。在基质下, 内皮细胞将养分、水和氧气输送到上层。
角膜擦伤是非常普遍的在诊所4。角膜损伤是多种多样的, 但主要是由尘埃、沙子、划痕或其他异物等细小微粒引起的。此处描述的协议旨在再现临床上相关的角膜上皮磨损类型。在这样做的时候, 该协议为临床医生和角膜科学家在自己的研究中实施提供了一种可控和精性的方法。我们对小鼠角膜进行了体内损伤修复实验, 用钝性眼毛刺、Algerbrush ii. 研磨组织。在这里, 我们的目标是只对角膜中央上皮进行擦伤, 并使器官的其他部位不受损伤。因此, 该协议是理想的研究角膜上皮细胞动力学或基底膜在重新上皮, 细胞迁移, 增殖和分化在体内5。近年来, 该模型用于分析小鼠角膜祖细胞动力学, 揭示分化的角膜上皮细胞在损伤6、7后重建角膜干细胞位的能力。随着磨损, 角膜恢复正常的透明度和抗拉强度。有趣的是,体外研究表明, 再上皮发生不增加细胞增殖8。该协议描述了小鼠角膜不间断愈合的时间线。因此, 该方法适用于测试药物对愈合模式和速度的影响。
角膜已广泛用于伤口愈合研究。然而, 许多研究都依赖于其他的损伤模型。一种建立良好的角膜损伤模型是在角膜表面9应用氢氧化钠 (氢氧化钠) 或无滤纸的碱性烧伤。碱性照射会导致大而弥漫性损伤, 不仅影响角膜上皮, 而且还对结膜和基质9,10。强烈的碱性溶液已经被证明能诱发角膜溃疡、混浊和新生血管9。炎症细胞通常侵入6小时内的基质, 并保持在那里直到 24 h11。因此, 碱性损伤是与基质活化有关的研究中的一种可取的方法。另一种化学损伤可以通过应用二甲基亚砜 (亚砜) 在角膜9,10。其他常用的损伤模型包括通过基质和角膜伤口穿透的切口伤口, 这仅限于基质14,15的上部。这些方法也有助于回答有关间质伤口愈合的问题。不同的伤害模式有各自的优缺点。角膜上皮的磨损, 或清创, 首先开发使用钝手术刀或叶片在体外角膜16。该方法后来在鼠、鼠、兔17、18、19、20、21、22的体内应用。使用眼球毛刺 (图 1), 我们只删除一个选定的区域的上皮, 留下的其余的上皮不受影响。这样, 就可以精确靶向角膜不同部位的上皮切除。此外, 还可以用荧光素染色法评定磨损尺寸。此外, 在这里, 我们遵循磨损关闭在愈合期。
该方法具有多种优点, 包括磨损部位的精确定位, 不能与化学损伤一起进行, ii) 磨损快, iii) 无侵入性。在此, 我们描述了使用 outbred NMRI 鼠标作为模型的方法, 但这可以应用于大量的小鼠遗传模型, 以及大鼠和兔, 这是用来研究人类角膜破坏的常用模型。

<table>
	<tbody>
		<tr>
			<td>NMRI mouse</td>
			<td>Envigo</td>
			<td>275</td>
			<td></td>
		</tr>
		<tr>
			<td>0.9% NaCl</td>
			<td></td>
			<td></td>
			<td>use sterile</td>
		</tr>
		<tr>
			<td>Medetomidine</td>
			<td>Vetmedic</td>
			<td>Vnr087896</td>
			<td>Market name: Cepetor Vet</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Intervet</td>
			<td>Vnr511485</td>
			<td>Market name: Ketaminol Vet</td>
		</tr>
		<tr>
			<td>Buprenorfin</td>
			<td>Invidior</td>
			<td>3015248</td>
			<td>Market name: Temgesic</td>
		</tr>
		<tr>
			<td>Atipamezol</td>
			<td>Orion Pharma</td>
			<td>Vnr471953</td>
			<td>Market name: Antisedan Vet</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Norbrook</td>
			<td>Vnr027579</td>
			<td>Market name: Norocarp Vet</td>
		</tr>
		<tr>
			<td>1% fucidin acid eye ointment</td>
			<td>Dechra</td>
			<td>Vnr080899</td>
			<td>Market name: Isathal</td>
		</tr>
		<tr>
			<td>Fluorescein salt</td>
			<td>Sigma-Aldrich</td>
			<td>F6377</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline solution</td>
			<td></td>
			<td></td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Algerbrush ii ocular burr (0.5 mm tip)</td>
			<td>Algerbrush</td>
			<td>6.39768E+11</td>
			<td></td>
		</tr>
		<tr>
			<td>Cobalt Blue pen light</td>
			<td>SP Services</td>
			<td>DE/003</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>Kunz Instruments</td>
			<td>2007-0217</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital SLR camera</td>
			<td>Nikon</td>
			<td>D80</td>
			<td></td>
		</tr>
		<tr>
			<td>Adjustable camera arm and clamp</td>
			<td>Neewer</td>
			<td>10086132</td>
			<td>Height 28 cm</td>
		</tr>
		<tr>
			<td>Table lamp with a flexible arm and a clamp</td>
			<td>Prisma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Soft wipe</td>
			<td>KimtechScience</td>
			<td>7552</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 chamber</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection toolset</td>
			<td>Fine Science Tools</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td>Beckton Dickinson</td>
			<td>303172</td>
			<td></td>
		</tr>
		<tr>
			<td>26G needles</td>
			<td>Beckton Dickinson</td>
			<td>303800</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL Eppendorf tube</td>
			<td>Sarstedt</td>
			<td>689</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue casette</td>
			<td>Sakura Finetech</td>
			<td>4118F</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue processing machine ASP200S</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>VWR</td>
			<td>UN1307</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin wax</td>
			<td>Millipore</td>
			<td>K95523361</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue embedding mold 32 x 25 x 6 mm</td>
			<td>Sakura Finetech</td>
			<td>4123</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Microm</td>
			<td>HM355</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath for sectioning</td>
			<td>Orthex</td>
			<td>60591</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath for sectioning</td>
			<td>Leica</td>
			<td>HI1210</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome blade</td>
			<td>Feather</td>
			<td>S35</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>Th.Geyer GmbH &#38; Co.</td>
			<td>7,695,019</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure water</td>
			<td>Millipore</td>
			<td>MPGP04001</td>
			<td>MilliQ</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td>PFA</td>
		</tr>
	</tbody>
</table>

corneal epithelial abrasion with ocular burr as a model for cornea wound healing

小鼠角膜为研究创面愈合提供了良好的模型。角膜是眼睛最外层的一层, 因此是第一次伤害的防御。事实上, 在临床上发现的最常见的眼外伤是角膜磨损。在这里, 我们利用眼部毛刺诱发的磨损, 从而消除了在体内的角膜上皮细胞在麻醉小鼠。这种方法允许有针对性的和可再生的上皮中断, 使其他区域完好无损。此外, 我们还描述了荧光素染色的擦伤上皮细胞的可视化, 并就如何直观地观察角膜的擦伤提供了具体建议。然后, 我们遵循伤口愈合的时间线 0, 18 和72小时后, 磨损, 直到伤口重新表皮。角膜损伤的上皮磨损模型是研究角膜上皮细胞增殖、迁移和上皮的理想方法。然而, 在创面愈合过程中, 由于眼毛刺不渗透到基质细胞层, 这种方法不是最佳的研究。该方法也适用于临床应用, 如药物疗效前临床试验。

本协议描述了一种对小鼠角膜造成磨损损伤的模型, 并提出了在磨损后如何跟踪和可视化愈合过程的方法。最近, 我们使用这种方法来研究角膜上皮祖细胞在伤口愈合过程中的作用6。使用既定的工具是一个成功的磨损实验的关键。我们和其他人使用了 Algerbrush II 眼毛刺 (图 1) 来执行擦伤6,7...

Watch this Scientific Journal Video about Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing at JoVE.com

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

眼毛刺角膜上皮磨损与角膜创面愈合的模型研究

该协议描述了一种方法, 使磨损的眼睛表面的鼠标, 并遵循伤口愈合过程后。该协议利用眼部毛刺部分去除麻醉小鼠眼中的表面上皮。

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

corneal-epithelial-abrasion-with-ocular-burr-as-model-for-cornea

Research

JoVE Journal

Medicine

20.3K 次观看.  University of Helsinki. 该协议描述了一种方法, 使磨损的眼睛表面的鼠标, 并遵循伤口愈合过程后。该协议利用眼部毛刺部分去除麻醉小鼠眼中的表面上皮。

视频: Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

一个小鼠模型角膜血管生成研究掌上含量

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

科研

医学

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

伤口愈合的小鼠模型

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The etiology of IBD remains unknown and current therapeutic strategies are based on the unspecific suppression of the immune system. The development of treatments that specifically target intestinal inflammation and epithelial wound healing could significantly improve management of IBD, however this requires accurate detection of inflammatory changes. Currently, potential drug candidates are usually evaluated using animal models in vivo or with cell culture based techniques in vitro. Histological examination usually requires the cells or tissues of interest to be stained, which may alter the sample characteristics and furthermore, the interpretation of findings can vary by investigator expertise. Digital holographic microscopy (DHM), based on the detection of optical path length delay, allows stain-free quantitative phase contrast imaging. This allows the results to be directly correlated with absolute biophysical parameters. We demonstrate how measurement of changes in tissue density with DHM, based on refractive index measurement, can quantify inflammatory alterations, without staining, in different layers of colonic tissue specimens from mice and humans with colitis. Additionally, we demonstrate continuous multimodal label-free monitoring of epithelial wound healing in vitro, possible using DHM through the simple automated determination of the wounded area and simultaneous determination of morphological parameters such as dry mass and layer thickness of migrating cells. In conclusion, DHM represents a valuable, novel and quantitative tool for the assessment of intestinal inflammation with absolute values for parameters possible, simplified quantification of epithelial wound healing in vitro and therefore has high potential for translational diagnostic use.

Accurate assessment of anti-inflammatory effects is of utmost importance for the evaluation of potential new drugs for the treatment of inflammatory bowel disease. Digital holographic microscopy provides assessment of inflammation in murine and human colonic tissue samples as well as automated multimodal evaluation of epithelial wound healing in vitro.

多式联运定量相成像数字全息显微准确评估肠道炎症和上皮伤口愈合

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

创作和移植的糖尿病伤口愈合模型中脂肪源性干细胞 (ASC) 表

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

We describe a step-by-step method of performing direct pulp capping on mice teeth for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

伤口的牙髓评价一个直接盖髓模型的开发和治疗小鼠中修复性牙本质形成

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

This manuscript describes how incision-like lesions made on cultured epithelial cell monolayers conveniently model wound healing in vitro, allowing for imaging by confocal or laser scanning microscopy, and which can provide high-quality quantitative and qualitative data for studying both cell behavior and the mechanisms involved in migration.

以显微镜为基础的方法评估上皮细胞迁移过程中的体外创面愈合

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

创面愈合研究的前活体角膜器官培养模型

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

Targeting Gray Rami Communicantes in Selective Chemical Lumbar Sympathectomy

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the lower extremities, hyperhidrosis, etc. It is commonly practiced to position the puncture needle tip in front of the anterior fascia of the psoas major muscle and inject the inactivating agent around the sympathetic trunk, which is defined as conventional CLS. Although relatively rare, ureteropelvic damage is the most frequently reported complication of conventional CLS and can cause serious harm to patients. We found that injecting the inactivating agent behind the anterior fascia, which only targets gray rami communicantes, helped achieve therapeutic efficacy in&#160;vasodilation, sweat reduction, and pain relief comparable to conventional CLS, and serious complications were largely reduced. We define this procedure as selective CLS. Here, we present a protocol of selective CLS. The precise needle tract and accurate evaluation of the spreading of the contrast agent are critical to ensure that the drug is injected behind the anterior fascia of the psoas major muscle. The needle tip is at approximately one-third the dividing line of the vertebral body in the lateral view of a lumbar X-ray. The contrast is mainly confined around the needle tip and spreads outward and downward along the psoas muscle fibers. In this way, the anterior fascia provides a natural barrier for the ureteropelvic area, and the psoas major muscle provides a natural barrier for the lumbar nerve root. There are several highlights of this article, including 1) a detailed description of the selective CLS procedures, 2) an explanation of the anatomical basis for the implementation of selective CLS, and 3) an explanation of the differences between selective and conventional CLS.

We present a protocol for selective chemical lumbar sympathectomy (CLS), which inactivates only gray rami communicantes and not the sympathetic trunk. Selective CLS can help achieve therapeutic efficacy in vasodilation, sweat reduction, and pain relief that are comparable to conventional CLS, and serious complications, particularly ureteropelvic damages, can be reduced.

选择性化学腰椎交感神经切除术中灰色拉米关节的靶向性

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the ...

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

使用分裂的角质按钮的孔膜内皮器官培养模型

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

角膜内皮细胞损失非侵入性激光辅助实验模型的发展

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...