Clinical features and corneal optical anisotropies in a rabbit model of limbal stem cell deficiency

ABSTRACT Purposes: To investigate the intra-laboratory reproducibility of clinical features and to evaluate corneal optical anisotropies in a rabbit model of limbal stem cell deficiency. Methods: Limbal injury was induced in the right eye of 23 adult New Zealand White rabbits using a highly aggressive protocol that combined 360 degrees limbal peritomy, keratolimbectomy, alkaline chemical burn, and mechanical removal of the epithelium. Clinical evaluation of the injured eyes was performed for 28 days and included corneal impression cytology. Corneas with a severe clinical outcome set typical of limbal stem cell deficiency were then collected, subjected to a histopathological examination, and examined for optical anisotropies. Corneas from healthy rabbit eyes were used as controls. Differences in optical path due to stromal collagen birefringence, as well as linear dichroism related to the expression and spatial orientation of glycosaminoglycan chains from proteoglycans, were measured from cross-sections under a quantitative polarized light microscope. Results: One eye showed signs of hypopyon and was excluded. Signs of ocular inflammation were observed in all eyes studied (n=22). Corneal impression cytology did not detect goblet cells. Twelve of the 22 corneas presented a clinical outcome set typical of limbal stem cell deficiency, which is characterized by the presence of epithelial defects, inflammatory cells, moderate-to-severe opacity, and neovascularization. Microscopic studies under polarized light revealed that relative to controls, limbal stem cell deficiency caused a 24.4% increase in corneal optical path differences. Further, corneas with limbal stem cell deficiency were less dichroic than controls. Conclusions: These results suggest that rabbit models of limbal stem cell deficiency must be rigorously screened for use in preclinical studies to ensure experimental homogeneity because protocols used to create limbal stem cell deficiency could be not associated with good intra-laboratory reproducibility of clinical features. Limbal stem cell deficiency, as induced herein, altered the optical anisotropic properties of the corneal stroma. Such alterations are indicative of changes in collagen packing and the spatial orientation of glycosaminoglycan chains from proteoglycans. Knowledge of these changes is important to potentiate strategies aimed at restoring the morphofunctional integrity of the corneal stroma affected by limbal stem cell deficiency.

摘要
研究目的：探究角膜缘干细胞缺乏症（limbal stem cell deficiency）家兔模型的临床特征（clinical features）实验室内重复性，并评估其角膜光学各向异性（corneal optical anisotropies）。
方法：采用高侵袭性造模方案，对23只成年新西兰白兔的右眼实施角膜缘损伤，具体操作包括360°角膜缘周切术（limbal peritomy）、角膜缘切除术（keratolimbectomy）、碱性化学灼伤（alkaline chemical burn）及上皮机械刮除术（mechanical removal of the epithelium）。对损伤眼进行为期28天的临床评估，其中包含角膜印迹细胞学（corneal impression cytology）检查。收集呈现典型角膜缘干细胞缺乏症临床表型的病变角膜，进行组织病理学检查（histopathological examination），并检测其光学各向异性。以健康家兔眼球角膜作为对照。借助定量偏振光显微镜（quantitative polarized light microscope）对角膜切片进行检测，测量由基质胶原双折射（stromal collagen birefringence）引起的光程差，以及与蛋白聚糖（proteoglycans）糖胺聚糖链（glycosaminoglycan chains）的表达及空间取向相关的线性二色性（linear dichroism）。
结果：1只家兔右眼出现前房积脓（hypopyon）征象，予以排除。剩余22只家兔的术眼均观察到眼部炎症征象。角膜印迹细胞学检查未检出杯状细胞。22例角膜中，12例呈现典型的角膜缘干细胞缺乏症临床表型，表现为上皮缺损、炎性细胞浸润、中至重度角膜混浊及新生血管形成（neovascularization）。偏振光显微镜下的显微研究显示，与对照组相比，角膜缘干细胞缺乏症可使角膜光程差增加24.4%；此外，角膜缘干细胞缺乏症模型的角膜线性二色性弱于对照组。
结论：本研究结果提示，若将角膜缘干细胞缺乏症家兔模型用于临床前研究，需进行严格筛选以确保实验同质性，因为当前造模方案可能无法保证临床特征的良好实验室内重复性。本研究诱导的角膜缘干细胞缺乏症可改变角膜基质的光学各向异性特性，该改变提示基质胶原排列及蛋白聚糖糖胺聚糖链的空间取向发生变化。明确此类变化，有助于优化旨在恢复受角膜缘干细胞缺乏症影响的角膜基质形态与功能完整性的治疗策略。