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Medicina

研究角膜伤口愈合的上皮擦伤模型

Published: December 29, 2021
doi:
10.3791/63112
, , , ,
1College of Optometry,University of Houston, 2Children’s Nutrition Research Center,Baylor College of Medicine, 3Center for Translational Research on Inflammatory Diseases (CTRID),Michael E. DeBakey Veterans Affairs Medical Center

Summary

这里，描述了使用环锯和钝高尔夫球杆螺杆在小鼠中形成中央角膜上皮擦伤伤口的方案。这种角膜伤口愈合模型具有高度可重复性，现在正用于评估疾病背景下受损的角膜伤口愈合。

Abstract

角膜对视力至关重要，约占眼睛屈光力的三分之二。角膜在视觉中的作用至关重要的是其透明度。然而，由于其外部位置，角膜极易受到各种损伤的影响，这些损伤可能导致角膜透明度丧失并最终失明。针对这些损伤，有效的角膜伤口愈合对于维持角膜稳态和保持角膜透明度和屈光能力至关重要。在角膜伤口愈合受损的情况下，角膜变得容易受到感染，溃疡和瘢痕形成。鉴于角膜伤口愈合对保持角膜透明度和视力的根本重要性，更好地了解正常的角膜伤口愈合过程是了解与感染和疾病相关的角膜伤口愈合受损的先决条件。为了实现这一目标，角膜损伤的小鼠模型已被证明有助于进一步了解在正常生理条件下运作的角膜伤口愈合机制。这里，描述了使用环锯和钝高尔夫球杆螺杆在小鼠中产生中央角膜上皮磨损的方案。在该模型中，一个直径为2毫米的圆形环锯，以角膜为中心，用于划定伤口区域。高尔夫球杆螺杆经过精心使用，以清创上皮并形成圆形伤口，而不会损坏角膜上皮基底膜。由此产生的炎症反应作为表征良好的细胞和分子事件的级联进行，这些事件对于有效的伤口愈合至关重要。这种简单的角膜伤口愈合模型具有高度可重复性和良好的出版性，现在正用于评估疾病背景下受损的角膜伤口愈合。

Introduction

角膜是眼睛前三分之一的透明角膜。角膜具有多种功能，包括保护眼睛的内部结构和形成保护眼睛免受感染的结构屏障1。更重要的是，角膜对视力至关重要，提供约三分之二的眼睛屈光力2，3。角膜在视觉中的作用至关重要的是其透明度。然而，由于其向外的位置，角膜每天都会暴露在各种各样的损伤中，这可能导致其屏障功能的破坏，透明度的丧失以及最终的失明。角膜透明度丧失是全球视力障碍的主要原因4，5.角膜擦伤是急诊室（ER）就诊的常见原因，占ER6上眼部相关病例的一半。据估计，美国每年有超过100万人遭受眼部相关伤害7.针对这些损伤，有效的角膜伤口愈合对于维持角膜稳态和保持其透明度和屈光能力至关重要。在角膜伤口愈合受损的情况下，角膜变得容易受到感染，溃疡和瘢痕形成8，9。此外，屈光手术的日益普及给角膜10带来了独特的创伤性挑战。鉴于角膜伤口愈合对保持角膜透明度和视力的根本重要性，更好地了解正常的角膜伤口愈合过程是了解与感染和疾病相关的角膜伤口愈合受损的先决条件。

为此，已经开发了几种角膜伤口愈合的动物模型11，12，13，14，15。角膜伤口愈合的小鼠模型已被证明有助于进一步了解在正常生理条件下起作用的角膜伤口愈合机制。不同类型的角膜伤口已被用于研究角膜伤口愈合，每种伤口都适用于研究伤口愈合过程的不同方面。角膜伤口愈合研究中使用的最常见类型的伤口模型是机械和化学伤口模型。化学角膜伤口，主要涉及角膜上碱性烧伤的产生，可用于研究角膜溃疡，混浊和新生血管形成13。机械性角膜伤口涉及清创（擦伤）伤口和角膜切除术伤口14，15，16。完整或破裂的角膜上皮基底膜分别定义了清创术和角膜切除术伤口。在清创伤口中，上皮基底膜保持完整，而在角膜切除术伤口中，基底膜被破坏，主要渗透到前基质中。清创伤口对于研究角膜损伤后的再上皮化、上皮细胞增殖、免疫反应和神经再生最有用。另一方面，角膜切除术伤口对于研究角膜瘢痕形成最有用14，15。

这里，描述了使用环锯和钝高尔夫球杆螺杆在小鼠中形成中央角膜上皮擦伤伤口的方案。这种简单的角膜伤口愈合模型具有高度可重复性和良好的出版性，现在正用于评估疾病17背景下受损的角膜伤口愈合。

Protocol

所有动物协议均由休斯顿大学和贝勒医学院的机构动物护理和使用委员会批准。在处理和使用小鼠时，遵循了视觉和眼科研究协会（ARVO）关于在视觉和眼科研究中使用动物的声明中概述的指南。 1. 准备工作 荧光素溶液的制备 通过将10mg荧光素钠盐溶解在1mL无菌盐水或无菌1x磷酸盐缓冲盐水（PBS）中来制备1%荧光素溶液。注意：在使用当天或前一天…

Representative Results

图3 显示了用钝性高尔夫球杆孢子形成的角膜伤口的透射电子显微镜照片，表明上皮基底膜在损伤后确实完好无损。 图3：角膜擦伤后上皮基底膜保持完整。 用钝性高尔夫球杆刺形成的角膜伤口的透射电子显微照片。箭头指向伤口?…

Discussion

该方法论文的目的是描述一种方案，用于使用环锯和钝高尔夫球杆在小鼠中形成中央角膜上皮擦伤伤口。该小鼠模型已被用于研究角膜炎症及其对伤口愈合的贡献。这种类型的模型可用于研究正常生理条件下角膜伤口愈合机制和病理学17，28，29，41，42。该模型已被用于研究?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

资助：NIH EY018239（A.R.B.，C.W.S.和R.E.R.），P30EY007551（A.R.B.）和Sigma Xi资助研究（P.K.A.）。内容完全由作者负责，不代表美国国立卫生研究院或Sigma Xi的官方观点。

Materials

Anti-CD31 antibody BD Bioscience, Pharmingen 550274
Anti-CD41 antibody BD Bioscience, Pharmingen 553847
Anti-Ly6G antibody BD Bioscience, Pharmingen 551459
Bovine serum albumin (BSA) ThermoFisher scientific B14
C57BL/6 mice Jackson Laboratories 664
DAPI Sigma Aldrich D8417
DeltaVision wide-field deconvolution fluorescence microscope GE Life Sciences
Dissecting microscope Leica microsystems
Electronic Toploading Balances (Weighing scale) Fisher Scientific
Ethanol ThermoFisher scientific T038181000CS
Golf-club spud Stephens instruments S2-1135
Iris curve scissors Fisher Scientific 31212
Isoflurane Patterson veterinary 07-893-1389
Ketamine Patterson veterinary 07-890-8598
Phospate buffered saline (PBS) ThermoFisher scientific AM9624
Sodium fluorescein salt Sigma Aldrich 46970
Surgical blade (scapel blade) Fine Science tools 10022-00
Trephine Integra Miltex 33-31
TritonX -100 Fisher Scientific 50-295-34
Forcep Fine Science tools 11923-13
Xylazine Patterson veterinary 07-808-1947
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Tags

Epithelial AbrasionCorneal Wound HealingCorneal InflammationCorneal Wounding ModelFluorescein StainingWound ClosureRe-epithelializationImage Analysis
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Akowuah, P. K., De La Cruz, A., Smith, C. W., Rumbaut, R. E., Burns, A. R. An Epithelial Abrasion Model for Studying Corneal Wound Healing. J. Vis. Exp. (178), e63112, doi:10.3791/63112 (2021).
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