Müller Linda J, Pels Elizabeth, Schurmans Lucas R H M, Vrensen Gijs F J M
The Netherlands Ophthalmic Research Institute, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands.
Exp Eye Res. 2004 Mar;78(3):493-501. doi: 10.1016/s0014-4835(03)00206-9.
The purpose of the present study was to re-evaluate the three-dimensional organization of collagen fibrils and proteoglycans (PGs) in the human corneal stroma using an improved ultrastructural approach. After a short aldehyde prefixation, one half of seven fresh corneal buttons was stained for PGs with Quinolinic Phtalocyanin (QP) or Cupromeronic Blue (CB). Strips of 1 mm width were cut, subsequently treated with aqueous phosphotungstic acid (PTA) and further processed for light and electron microscopy. The other half of the corneas served as control and was routinely processed with OsO4. Embedding was as such that ultrathin sections could be cut precisely parallel (frontal sections) or perpendicular (cross sections) to the corneal surface. The mutual connections between collagen fibrils and PGs were studied and the length of PGs and their mutual distance were measured manually at a calibrated final magnification of 70,000 x. Prefixed fresh corneal tissue treated with QP and CB shows no signs of swelling and exhibits well contrasted PGs. In cross sections PGs form a repeating network of ring-like structures (approximately 45 nm) around the collagen fibrils. In frontal sections PGs are aligned orthogonal to the collagen fibrils, are equidistant (approximately 42 nm) attached to the collagen fibrils along their full length and have a thickness of approximately 11 nm and a length of approximately 54 nm. The observed maximal length of the PGs and the occurrence of ring-like structures enwrapping the collagen fibrils urged us to revisit the prevailing model of maurice (1962) on the organization of the corneal stroma. In the new model hexagonal arranged collagen fibrils are interconnected at regular distances with their next-nearest neighbours by groups of six PGs, attached orthogonal to the circumference of the fibrils. In this way a regular meshwork of ring-like structures enwrapping the collagen fibrils is formed. It is discussed that this new model more convincingly explains corneal resistance to compression and stretching and further rationalizes corneal transparency because of the low refractive index difference between the regularly arranged collagen fibrils and their inter-space filled with PGs.
本研究的目的是使用改进的超微结构方法重新评估人角膜基质中胶原纤维和蛋白聚糖（PGs）的三维组织结构。在进行短时间醛类预固定后，将七个新鲜角膜片的一半用喹啉酞菁（QP）或铜铬蓝（CB）对PGs进行染色。切成1毫米宽的条带，随后用磷钨酸水溶液（PTA）处理，并进一步进行光镜和电镜处理。另一半角膜用作对照，常规用四氧化锇处理。包埋方式使得超薄切片可以精确地平行（正面切片）或垂直（横截面）于角膜表面切割。研究了胶原纤维和PGs之间的相互连接，并在校准的最终放大倍数70000倍下手动测量PGs的长度及其相互距离。用QP和CB处理的预固定新鲜角膜组织没有肿胀迹象，并且PGs的对比度良好。在横截面中，PGs在胶原纤维周围形成重复的环状结构网络（约45纳米）。在正面切片中，PGs与胶原纤维正交排列，沿其全长等距（约42纳米）附着于胶原纤维，厚度约为11纳米，长度约为54纳米。观察到的PGs的最大长度以及围绕胶原纤维的环状结构的出现促使我们重新审视莫里斯（1962年）关于角膜基质组织结构的主流模型。在新模型中，六边形排列的胶原纤维通过六组PGs与其最近邻在规则距离处相互连接，这些PGs垂直于纤维的圆周附着。通过这种方式，形成了围绕胶原纤维的规则环状结构网络。讨论了这个新模型更令人信服地解释了角膜对压缩和拉伸的抵抗力，并进一步使角膜透明度合理化，因为规则排列的胶原纤维与其充满PGs的间隙之间的折射率差异很小。
