Brookes N H, Loh I-P, Clover G M, Poole C A, Sherwin T
Department of Ophthalmology, Faculty of Medicine and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand.
Exp Eye Res. 2003 Oct;77(4):515-24. doi: 10.1016/s0014-4835(03)00148-9.
Keratoconus is a debilitating corneal thinning disease that principally develops in the second and third decades of life. Our group previously developed a novel approach to studying keratoconus, based on the observation that there is a gradient of damage across the keratoconic cone. We identified a number of cellular characteristics of keratoconus such as discrete incursions of fine cellular processes from the anterior keratocytes in association with localised indentation of the basal epithelium, and increased levels of the lysosomal enzymes Cathepsin B and G in aberrant keratocytes, located beneath compromised regions of Bowman's layer, but also deeper in the stroma. Enzyme activity by these cells seemed to be causing localised structural degradation of the anterior stroma, leading to near-complete destruction of both Bowman's layer and the stroma, often necessitating a full-thickness corneal graft for sight restoration. This current study extends our initial findings by investigating the role of corneal nerves passing between the stroma and epithelium at the sites of early degradative change observed previously, and may be facilitating the keratocyte-epithelial interactions in this disease. Cells in sections of normal and keratoconic human corneas were labelled with the fixable fluorescent viability dye 5-chloromethylfluorescein diacetate, antibodies to alpha-tubulin (nerves), alpha3beta1 integrin, Cathepsin B and G, and the nuclear dye DAPI, and then examined with a confocal microscope. Anterior keratocyte nuclei were seen wrapping around the nerves as they passed through the otherwise acellular Bowman's layer, and as the disease progressed and Bowman's layer degraded, these keratocytes were seen to express higher levels of Cathepsin B and G, and become displaced anteriorly into to the epithelium. Localised nerve thickenings also developed within the epithelium in association with Cathepsin B and G expression, and appeared to be very destructive to the cornea. Insight into the molecular mechanisms of keratoconic disease pathogenesis and progression can be gained from the process of extracellular matrix remodelling known from studies of connective tissues other than the cornea, and wound healing studies in the cornea. Further studies are required to determine how well this model fits the actual molecular basis of the pathogenesis of keratoconus.
圆锥角膜是一种使人衰弱的角膜变薄疾病，主要在人生的第二个和第三个十年发展。我们的研究小组先前基于圆锥角膜圆锥体上存在损伤梯度这一观察结果，开发了一种研究圆锥角膜的新方法。我们确定了圆锥角膜的一些细胞特征，比如前角膜细胞的精细细胞突起离散侵入，伴有基底上皮的局部凹陷，以及位于Bowman层受损区域下方但也在基质更深层的异常角膜细胞中溶酶体酶组织蛋白酶B和G的水平升高。这些细胞的酶活性似乎导致前基质的局部结构降解，导致Bowman层和基质几乎完全破坏，通常需要进行全层角膜移植来恢复视力。当前这项研究通过调查先前观察到的早期降解变化部位基质与上皮之间通过的角膜神经的作用，扩展了我们最初的发现，并且可能在促进这种疾病中角膜细胞与上皮的相互作用。正常和圆锥角膜人角膜切片中的细胞用可固定的荧光活力染料5-氯甲基荧光素二乙酸酯、抗α-微管蛋白（神经）抗体、α3β1整合素、组织蛋白酶B和G以及核染料DAPI进行标记，然后用共聚焦显微镜检查。在前角膜细胞核穿过原本无细胞的Bowman层时，可以看到它们围绕着神经，并且随着疾病进展和Bowman层降解，可以看到这些角膜细胞表达更高水平的组织蛋白酶B和G，并向前移位到上皮中。上皮内还出现了与组织蛋白酶B和G表达相关的局部神经增粗，并且似乎对角膜具有很强的破坏性。从对角膜以外的结缔组织的研究以及角膜伤口愈合研究中已知的细胞外基质重塑过程，可以深入了解圆锥角膜疾病发病机制和进展的分子机制。需要进一步研究来确定这个模型与圆锥角膜发病机制的实际分子基础的契合程度。
