SSR-Stem Cell Biology Laboratory, Center for Regenerative Ophthalmology, L V Prasad Eye Institute, Hyderabad, India; (b).Manipal Academy of Higher Education, Manipal, Karnataka, India.
SSR-Stem Cell Biology Laboratory, Center for Regenerative Ophthalmology, L V Prasad Eye Institute, Hyderabad, India.
Exp Eye Res. 2019 Aug;185:107665. doi: 10.1016/j.exer.2019.05.005. Epub 2019 May 13.
Limbal stem cell deficiency (LSCD) is one of the serious cause of visual impairment and blindness with loss of corneal clarity and vascularization. Factors such as ocular burns (acids, lime, thermal), genetic disorders or infections results in the loss of limbal stem cells leading to LSCD. Reliable animal models of LSCD are useful for understanding the pathophysiology and developing novel therapeutic approaches. The purpose of the present study was to validate small and large animal models of LSCD by immunohistochemcal, clinical and histopathological comparison with human. The animal models of LSCD were created by topical administration of sodium hydroxide on the ocular surface of C57BL/6 mice (m, n = 12) and New Zealand white rabbits (r, n = 12) as per the standard existing protocol. Human corneal specimens (h, n = 12) were obtained from tissue bank who had chemical burn-induced LSCD. All samples were either paraffin embedded or frozen in cryogenic medium and the sections were processed for Hematoxylin-Eosin and Periodic Acid-Schiff staining to analyse the morphology and histopathological features of the corneal surface such as vascularization, inflammation, presence of goblet cells, epithelial hyperplasia and keratinization. Immunofluorescence was performed to distinguish between corneal (CK3+), conjunctival (CK19+) and epidermal (CK10+) epithelial phenotype. Histological analysis of corneal specimens from the three groups showed the presence of goblet cells (h:83%, m:50%, r:50%, p = 0.014), epithelial hypertrophy (h:92%, m:50%, r:66.6%, p = 0.04), epithelial hyperplasia (h:50%, m:17%, r:17%, p = 0.18), intra epithelial edema (h:42%, m:33%, r:100%, p = 0.02), stromal inflammation (h:100%, m:67%, r:67%, p = 0.01) and stromal vascularization (h:100%, m:50%, r:67%), in varying proportions. Immunostaining showed presence of total LSCD (CK19 + and/or CK10+, CK3-) in 92% of human and 50% of animal specimens. While partial LSCD (CK19 + and/or CK10+, CK3+) was seen in 8% of human and 50% of animal specimens. Our study shows the significant differences in the extent of vascularization, inflammation, epithelial thickness and goblet cell formation in mice and rabbit models of LSCD when compared to post-chemical burn LSCD in human corneas. In both mice and rabbit models complete LSCD developed in only 50% of cases and this important fact needs to be considered when working with animal models of LSCD.
角膜缘干细胞缺乏症（LSCD）是导致视力障碍和失明的严重原因之一，其特征为角膜混浊和血管化丧失。眼部烧伤（酸、石灰、热）、遗传疾病或感染等因素会导致角膜缘干细胞丧失，从而引发 LSCD。可靠的 LSCD 动物模型有助于理解其病理生理学，并开发新的治疗方法。本研究旨在通过与人类进行免疫组织化学、临床和组织病理学比较，验证 LSCD 的小动物和大动物模型。LSCD 动物模型通过在 C57BL/6 小鼠（m，n=12）和新西兰白兔（r，n=12）的眼表面局部给予氢氧化钠来创建，根据现有的标准方案进行。从组织库中获得了化学烧伤诱导的 LSCD 人类角膜标本（h，n=12）。所有标本均进行石蜡包埋或在低温介质中冷冻，并对切片进行苏木精-伊红和过碘酸希夫染色，以分析角膜表面的形态和组织病理学特征，如血管化、炎症、杯状细胞存在、上皮增生和角化。进行免疫荧光染色以区分角膜（CK3+）、结膜（CK19+）和表皮（CK10+）上皮表型。三组角膜标本的组织学分析显示存在杯状细胞（h：83%，m：50%，r：50%，p=0.014）、上皮肥大（h：92%，m：50%，r：66.6%，p=0.04）、上皮增生（h：50%，m：17%，r：17%，p=0.18）、上皮内水肿（h：42%，m：33%，r：100%，p=0.02）、基质炎症（h：100%，m：67%，r：67%，p=0.01）和基质血管化（h：100%，m：50%，r：67%），比例不同。免疫染色显示，92%的人类和 50%的动物标本存在总 LSCD（CK19+和/或 CK10+，CK3-）。而在 8%的人类和 50%的动物标本中则出现部分 LSCD（CK19+和/或 CK10+，CK3+）。本研究表明，与人类化学烧伤后 LSCD 相比，LSCD 小鼠和兔模型在血管化、炎症、上皮厚度和杯状细胞形成程度方面存在显著差异。在小鼠和兔模型中，仅 50%的病例发展为完全 LSCD，在使用 LSCD 动物模型时需要考虑到这一重要事实。
