ELECTRON MICROSCOPIC STUDIES OF THE CRYSTALLINE LENS

我们研究项目的长期目标一直是，并将继续是，阐明正常人类晶状体形态，因为它与晶状体功能有关，以及晶状体中与年龄相关和/或病理变化如何表现为晶状体光学受损，最终导致特定的白内障。我们通过实验动物模型对色素性视网膜炎（RP）的后囊膜下白内障（PSC）进行了初步研究，并对羟甲基戊二酰(HMG)-辅酶a还原酶抑制剂对PSC形成的影响进行了研究，结果表明，这些晶状体的PSC混色是由于后针孔解剖结构的进行性损害，而不是由于发育不良的“Wedl”细胞向后极迁移的增殖。在我们之前的研究中，我们已经开发并成功应用了相关技术，可以准确量化在混浊之前和导致混浊的晶状体缝合线解剖结构的进行性、负性变化。因此，在该资助的五年内提出的研究旨在实现以下具体目标：首先，确定在PSC形成之前，期间和之后，晶状体结构（特别是缝合线解剖）和功能（球差，即焦距变异性）之间是否存在可比较的，渐进的和可量化的负相关关系，这是不同病因的结果，包括：1)长期使用HMG-CoA还原酶抑制剂进行治疗；2) RP;3)类固醇治疗；4)糖尿病;5)艾滋病。其次，进一步阐明灵长类晶状体细胞的超微结构（中央、萌发前、萌发和过渡带上皮细胞，以及伸长、成熟和老化纤维）在发育、生长、年龄和病理中的作用。关于第一组具体目标：实验动物透镜的光学质量（焦距变化率，即球差）将通过我们自己设计的低功率氦氖激光扫描单元进行分析，特别是参考透镜缝合线。然后，我们开发的一种方法可以精确地解剖激光扫描的透镜，这种方法允许从前后两极的渐进深度检索完整的完整缝合模式，以便通过扫描电子显微镜（SEM）进行结构分析。通过这种方式，晶状体缝合线解剖学作为发育、生长、年龄和白内障发生的功能可以准确地表征。然后通过3D- cad重建分析确定所有如上解剖的晶状体的原位3D缝合解剖，以确定激光扫描分析发现的对晶状体功能不利的结构相关性是否与PSC不透明相对应。类似的实验动物晶状体组也将通过生物显微裂隙灯分析来检查，以确定受损的晶状体生长是否表现为异常缝合线，是否与这些非灵长类晶状体通常不具有的不连续性区域的产生有关。这些结果将与从激光扫描透镜的3D-CAD重建中获得的模拟裂隙灯图像以及由上述病因引起的人类PSCs的裂隙灯图像进行比较。通过我们在实验室开发和应用的技术，我们对晶状体结构/功能之间的相互关系有了更深入的了解，那么本提案中描述的研究结果可能会导致早期检测和改进人类psc的临床管理，这是不同病因的结果。

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.