ELECTRON MICROSCOPIC STUDIES OF THE CRYSTALLINE LENS

我们研究计划的长期目标一直是，并将继续阐明与晶状体功能相关的正常人类晶状体形态，以及晶状体中与年龄相关的和/或病理变化如何表现为最终导致特定白内障的晶状体光学受损。我们对实验性视网膜色素变性(RP)动物模型的后囊下白内障(PSC)以及羟甲基戊二醛(HMG)-CoA还原酶抑制剂对PSC形成的影响进行了初步研究，结果表明，这些晶状体的PSC混浊是后部缝线解剖学逐渐妥协的结果，而不是由于发育不良的韦德尔细胞向后极部迁移的结果。在我们之前的研究中，我们已经开发并成功地应用了相关技术，允许在混浊之前和导致混浊之前准确量化晶状体缝线解剖的进行性、负性变化。因此，在这笔赠款的五年中建议进行的研究旨在实现以下具体目标：第一，确定在PSC形成之前、期间和之后，由于不同的病因，晶状体结构(特别是缝合解剖学)和功能(球差，即焦距变化)之间是否存在可比较的、渐进的和可量化的负相关关系，这些原因包括：1)HMG-CoA还原酶抑制剂的长期治疗；2)RP；3)类固醇治疗；4)糖尿病；和5)艾滋病。第二，进一步阐明灵长类晶状体细胞(中央、萌发前、萌发和移行带上皮细胞，以及伸长、成熟和老化的纤维)的超微结构与发育、生长、年龄和病理的关系。关于第一组具体目标：实验动物晶状体的光学质量(焦距可变性；即球差)将通过使用我们自己设计的低功率氦-霓激光扫描装置进行分析来评估，特别是参考晶状体缝合。然后，激光扫描的晶状体将被我们开发的一种方法精确解剖，该方法允许从前极部和后极部逐渐恢复完整的缝线图案，用于扫描电子显微镜(SEM)的结构分析。通过这种方式，晶状体缝线解剖作为发育、生长、年龄和白内障发生的函数可以被准确地表征。所有如上解剖的晶状体的原位3D缝合解剖将通过3D-CAD重建分析确定，以确定激光扫描分析发现的对晶状体功能产生不利影响的结构相关性是否与PSC混浊相对应。还将通过生物显微镜裂隙灯分析来检查可比组的实验动物晶状体，以确定表现为异常缝合的晶状体生长受损是否与这些非灵长类晶状体通常不具有的间断区的产生有关。这些结果将与从激光扫描晶状体的3D-CAD重建中获得的模拟裂隙灯图像以及由上述病因导致的人类PSC的裂隙灯图像进行比较。随着对我们实验室开发和应用的技术所提供的晶状体结构/功能之间的相互关系有了更深入的了解，本建议中描述的研究结果可能会因为不同的病因而导致更早地发现和改善临床治疗。

The long term objective of our research program has been, and continues to be, the elucidation of normal human lens morphology as it relates to lens function, and how age-related and/or pathological changes in the lens are manifested as compromised lens optics leading ultimately to specific cataracts. Our preliminary studies of posterior subcapsular cataracts (PSC) from experimental animal models for Retinitis Pigmentosa (RP) and for the effects of Hydroxymethlyglutaryl (HMG)-CoA reductase inhibitors on PSC formation, suggest that the PSC opacities of these lenses result from a progressive compromise in posterior sutural anatomy rather than from a proliferation of dysplastic 'Wedl' cell migrating to the posterior pole. In our previous studies we have developed, and successfully applied, correlative techniques that allow for the accurate quantification of progressive, negative changes in lens sutural anatomy, prior to and resulting in opacification. Thus, the studies proposed in the five years of this grant are designed to accomplish the following specific aims: First, to determine if a comparable, progressive, and quantifiable, negative relationship exists between lens structure (particularly sutural anatomy) and function (spherical aberration, i.e. focal length variability) before, during and after PSC formation, as a consequence of different etiologies including: 1) long term therapeutic treatment with HMG-CoA reductase inhibitors; 2) RP; 3) therapeutic treatment with steroids; 4) diabetes; and 5) AIDS. Second, to further elucidate the ultrastructure of primate lens cells (central, pre-germinative, germinative, and transitional zone epithelial cells, as well as elongating, mature and aged fibers) as a function of development, growth, age and pathology. As regards the first set of specific aims: The optical quality (focal length variability; i.e spherical aberration) of the experimental animal lenses will be assessed by analysis with a low power helium-neon laser scan unit of our own design with particular reference to lens sutures. The laser scanned lenses will then be precisely dissected by a method that we have developed permitting the retrieval of complete intact suture patterns at progressive depths from the anterior and posterior poles for structural analysis by scanning electron microscopy (SEM). In this manner, lens sutural anatomy as a function of development, growth, age and cataractogenesis can be accurately characterized. The in situ 3D sutural anatomy of all lenses dissected as above, will then be ascertained by 3D-CAD reconstructional analysis to determine if structural correlates found to adversely effect lens function by laser scan analysis, correspond to the PSC opacities. Comparable groups of experimental animal lenses will also be examined by biomicroscopic slit-lamp analysis to determine if compromised lens growth manifested as abnormal sutures, correlate with the production of zones of discontinuity that are not normally characteristic of these non primate lenses. These results will be compared to simulated slit-lamp images taken from the 3D-CAD reconstructions of laser scanned lenses and with slit-lamp images taken of human PSCs resulting from the above described etiologies. With a greater understanding of the inter-relationship between lens structure/function afforded by techniques that we have developed and applied in our laboratories, then the results of studies described in this proposal could lead to the earlier detection and improved clinical management of human PSCs as a consequence of different etiologies.