角膜上皮机械和化学损伤的离体和体内动物模型
Summary
在这里，开发了基于小鼠和兔的动物模型，用于角膜上皮的机械和化学损伤，以筛选新的治疗方法和潜在机制。
Abstract
眼表角膜损伤，包括化学烧伤和创伤，可能导致严重的瘢痕形成、眼睑、角膜角膜缘干细胞缺乏，并导致大的、持续的角膜上皮缺损。伴有以下角膜混浊和外周新生血管的上皮缺损会导致不可逆的视力损害，并阻碍未来的治疗，尤其是角膜移植术。由于动物模型可以作为有效的药物开发平台，因此本文开发了小鼠角膜损伤和兔角膜上皮碱烧伤模型。新西兰白兔用于碱烧模型。在肌内和局部麻醉下，可以将不同浓度的氢氧化钠施加到角膜的中心圆形区域30秒。在大量等渗生理盐水冲洗后，残留的松散角膜上皮被去除，角膜毛刺深入到该圆形区域内的Bowman层。通过在钴蓝光下荧光素染色记录伤口愈合。C57BL / 6小鼠用于小鼠角膜上皮的创伤模型。使用直径为2mm的皮肤冲头标记小鼠中央角膜，然后在体视显微镜下用0.5mm毛刺的角膜锈环去除器进行清创。这些模型可用于前瞻性地验证滴眼液或混合药物（如干细胞）的治疗效果，这些药物可能促进角膜上皮再生。通过使用体视显微镜和成像软件观察角膜混浊、外周新生血管形成和结膜充血，可以监测这些动物模型中的治疗效果。
Introduction
人类角膜由五个主要层组成，在眼部屈光中起着关键作用，以保持视力和结构完整性，以保护眼内组织1。角膜的最外层是角膜上皮，由五到六层细胞组成，这些细胞依次与基底细胞分化并向上移动以从眼表脱落1。与人类和新西兰兔的角膜相比，小鼠角膜具有相似的角膜结构，但由于上皮和基质2的厚度减少，其外围比中央部分薄。由于其在眼科系统中的独特位置，许多外部损伤如机械损伤、细菌接种和化学制剂等可能容易危及上皮完整性，进而导致威胁视力的上皮缺损、感染性角膜炎、角膜融化，甚至角膜穿孔。
尽管润滑剂、抗生素、抗炎剂、自体血清产品和羊膜等各种治疗剂已被用于改善再上皮形成和减少疤痕形成，但其他可以实现伤口愈合、减少炎症和抑制疤痕形成的潜在治疗方式仍在不同的平台上开发和测试。已经提出了用于角膜上皮伤口愈合的各种动物模型，包括糖尿病小鼠3使用角膜锈环去除器去除角膜上皮，通过无菌25 G针头对小鼠角膜上皮进行线性划痕以进行细菌接种4，环钻辅助通过角膜锈环去除器去除角膜上皮5，超过一半角膜和角膜缘的上皮烧灼6，环钻通过钝的手术刀刀片7擦伤兔角膜，以及通过液氮8快速冷冻对牛角膜损伤。
除了对角膜上皮的机械损伤外，化学制剂也是对眼表的常见侮辱，尤其是酸性和碱性物质。氢氧化钠（NaOH，0.1-1 N，持续30-60秒）是角膜化学烧伤9，10，11，12，13的小鼠和兔模型中常用的化学物质之一。在大鼠化学烧伤模型中，还将100%乙醇施用于角膜，然后使用手术刀片14进行额外的机械刮擦。由于维持健康的眼表依赖于功能单位，包括眼睑、睑板腺、泪系统、结膜和角膜，因此体内动物模型比离体培养的角膜上皮细胞或角膜组织具有一些优点。本文演示了角膜擦伤创面的小鼠模型和角膜碱烧伤的兔模型。
Protocol
Representative Results
Discussion
角膜损伤的小鼠和兔模型为监测伤口愈合、测试新疗法以及研究伤口愈合和治疗途径的潜在机制提供了一个有用的 离体 和 体内 平台。根据研究目的，不同的动物模型可用于短期或长期实验。例如，在 体内小鼠角膜上产生上皮缺陷后，局限性上皮缺陷可用于监测少量液体治疗剂。同时，周围的功能单位，如眼睑、泪系统和结膜，可以在体内 条件下进行评估，而不是细?…
Declarações
The authors have nothing to disclose.
Acknowledgements
该研究由台湾原子能委员会（批准号A-IE-01-03-02-02），科学技术部（批准号NMRPG3E6202-3）和长庚医学研究项目（批准号CMRPG3H1281）资助。
Materials
| 6/0 Ethicon vicryl suture | Ethicon | 6/0VICRYL | tarsorrhaphy |
| Barraquer lid speculum | katena | K1-5355 | 15 mm |
| Barraquer needle holder | Katena | K6-3310 | without lock |
| Barron Vacuum Punch 8.0 mm | katena | K20-2108 | for cutting filter paper |
| C57BL/6 mice | National Laboratory Animal Center | RMRC11005 | mouse strain |
| Castroviejo forceps 0.12 mm | katena | K5-2500 | |
| Corneal rust ring remover with 0.5 mm burr | Algerbrush IITM; Alger Equipment Co., Inc. Lago Vista, TX | CHI-675 | for debridement of the corneal epithelium |
| Filter paper | Toyo Roshi Kaisha,Ltd. | 1.11 | |
| Fluorescein sodum ophthalmic strips U.S.P | OPTITECH | OPTFL100 | staining for corneal epithelial defect |
| Ketamine hydrochloride | Sigma-Aldrich | 61763-23-3 | intraperitoneal or intramuscular anesthetics |
| New Zealand White Rabbits | Livestock Research Institute, Council of Agriculture,Executive Yuan | Rabbit models | |
| Normal saline | TAIWAN BIOTECH CO., LTD. | 100-120-1101 | |
| Proparacaine | Alcon | ALC2UD09 | topical anesthetics |
| Skin biopsy punch 2mm | STIEFEL | 22650 | |
| Sodium chloride (NaOH) | Sigma-Aldrich | 1310-73-2 | a chemical agent for alkali burn |
| Stereomicroscope | Carl Zeiss Meditec, Dublin, CA | SV11 | microscope for surgery |
| Westcott Tenotomy Scissors Medium | katena | K4-3004 | |
| Xylazine hydrochloride 23.32 mg/10 mL | Elanco animal health Korea Co., LTD. | 047-956 | intraperitoneal or intramuscular anesthetics |
Referências
- Sridhar, M. S. Anatomy of cornea and ocular surface. Indian Journal of Ophthalmology. 66 (2), 190-194 (2018).
- Henriksson, J. T., McDermott, A. M., Bergmanson, J. P. G. Dimensions and morphology of the cornea in three strains of mice. Investigative Ophthalmology & Visual Science. 50 (8), 3648-3654 (2009).
- Wang, X., et al. MANF promotes diabetic corneal epithelial wound healing and nerve regeneration by attenuating hyperglycemia-induced endoplasmic reticulum stress. Diabetes. 69 (6), 1264-1278 (2020).
- Ma, X., et al. Corneal epithelial injury-induced norepinephrine promotes Pseudomonas aeruginosa keratitis. Experimental Eye Research. 195, 108048 (2020).
- Chan, M. F., Werb, Z. Animal models of corneal injury. Bio Protocol. 5 (13), 1516 (2015).
- Lan, Y., et al. Kinetics and function of mesenchymal stem cells in corneal injury. Investigative Ophthalmology & Visual Science. 53 (7), 3638-3644 (2012).
- Watanabe, M., et al. Promotion of corneal epithelial wound healing in vitro and in vivo by annexin A5. Investigative Ophthalmology & Visual Science. 47 (5), 1862-1868 (2006).
- Murataeva, N., et al. Cannabinoid CB2R receptors are upregulated with corneal injury and regulate the course of corneal wound healing. Experimental Eye Research. 182, 74-84 (2019).
- Carter, K., et al. Characterizing the impact of 2D and 3D culture conditions on the therapeutic effects of human mesenchymal stem cell secretome on corneal wound healing in vitro and ex vivo. Acta Biomaterialia. 99, 247-257 (2019).
- Sanie-Jahromi, F., et al. Propagation of limbal stem cells on polycaprolactone and polycaprolactone/gelatin fibrous scaffolds and transplantation in animal model. Bioimpacts. 10 (1), 45-54 (2020).
- Sun, M. M., et al. Epithelial membrane protein (EMP2) antibody blockade reduces corneal neovascularization in an In vivo model. Investigative Ophthalmology & Visual Science. 60 (1), 245-254 (2019).
- Yang, Y., et al. Cannabinoid receptor 1 suppresses transient receptor potential vanilloid 1-induced inflammatory responses to corneal injury. Cell Signal. 25 (2), 501-511 (2013).
- Bai, J. Q., Qin, H. F., Zhao, S. H. Research on mouse model of grade II corneal alkali burn. International Journal of Ophthalmology. 9 (4), 487-490 (2016).
- Oh, J. Y., et al. Anti-inflammatory protein TSG-6 reduces inflammatory damage to the cornea following chemical and mechanical injury. Proceedings of the National Academy of Sciences of the United States of America. 107 (39), 16875 (2010).
- Wang, T., et al. Evaluation of the effects of biohcly in an in vivo model of mechanical wounds in the rabbit cornea. Journal of Ocular Pharmacology and Therapeutics. 35 (3), 189-199 (2019).
- Gong, Y., et al. Effect of nintedanib thermos-sensitive hydrogel on neovascularization in alkali burn rat model. International Journal of Ophthalmology. 13 (6), 879-885 (2020).
- Yao, L., et al. Role of mesenchymal stem cells on cornea wound healing induced by alkali burn. PLoS One. 7 (2), 30842 (2012).
