眼毛刺角膜上皮磨损与角膜创面愈合的模型研究
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Declarações
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| NMRI mouse | Envigo | 275 | |
| 0.9% NaCl | use sterile | ||
| Medetomidine | Vetmedic | Vnr087896 | Market name: Cepetor Vet |
| Ketamine | Intervet | Vnr511485 | Market name: Ketaminol Vet |
| Buprenorfin | Invidior | 3015248 | Market name: Temgesic |
| Atipamezol | Orion Pharma | Vnr471953 | Market name: Antisedan Vet |
| Carprofen | Norbrook | Vnr027579 | Market name: Norocarp Vet |
| 1% fucidin acid eye ointment | Dechra | Vnr080899 | Market name: Isathal |
| Fluorescein salt | Sigma-Aldrich | F6377 | |
| Phosphate-buffered saline solution | PBS | ||
| Algerbrush ii ocular burr (0.5 mm tip) | Algerbrush | 6.39768E+11 | |
| Cobalt Blue pen light | SP Services | DE/003 | |
| Hot plate | Kunz Instruments | 2007-0217 | |
| Digital SLR camera | Nikon | D80 | |
| Adjustable camera arm and clamp | Neewer | 10086132 | Height 28 cm |
| Table lamp with a flexible arm and a clamp | Prisma | ||
| Soft wipe | KimtechScience | 7552 | |
| CO2 chamber | |||
| Dissection toolset | Fine Science Tools | ||
| Syringes | Beckton Dickinson | 303172 | |
| 26G needles | Beckton Dickinson | 303800 | |
| 2 mL Eppendorf tube | Sarstedt | 689 | |
| Tissue casette | Sakura Finetech | 4118F | |
| Tissue processing machine ASP200S | Leica | ||
| Xylene | VWR | UN1307 | |
| Paraffin wax | Millipore | K95523361 | |
| Tissue embedding mold 32 x 25 x 6 mm | Sakura Finetech | 4123 | |
| Microtome | Microm | HM355 | |
| Water bath for sectioning | Orthex | 60591 | |
| Water bath for sectioning | Leica | HI1210 | |
| Microtome blade | Feather | S35 | |
| Glass slide | Th.Geyer GmbH & Co. | 7,695,019 | |
| Ultrapure water | Millipore | MPGP04001 | MilliQ |
| Paraformaldehyde | Sigma-Aldrich | 158127 | PFA |
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