Bioengineering of Transport Across Bruch's Membrance

描述(由申请人提供)：更好地了解老年性黄斑病变(ARM)早期阶段的病理生理学可能会指导开发更好的治疗和/或预防老年性黄斑变性的方法。早期手臂的显著临床和组织病理损害涉及Bruch膜，这是一种位于脉络膜毛细血管和RPE之间的薄层结缔组织。与ARM晚期的情况不同，ARM的几个危险因素已经被确定，而早期ARM唯一一致被确定的危险因素是高龄。因此，重要的是要了解Bruch膜中与年龄相关的变化如何使一些人容易患上视网膜功能障碍。 Bruch膜转运能力下降，可能是由脂质堆积引起的，已被认为是ARM发病机制中的关键早期事件。然而，形态计量学研究还没有成功地确定那些部分负责限制布鲁赫膜的运输能力。QFDE是一种形态技术，它以精细的细节保存细胞外基质，并允许可视化使用传统的透射电子显微镜(TEM)技术看不到的结构。QFDE提供了一种技术，在其他结缔组织中，它为这些组织的运输特性提供了新的见解。我们的目标是将快速冷冻/深度蚀刻(QFDE)形态学技术与生物工程分析方法相结合，使用保存完好的正常人类捐赠者的眼睛来评估这一假说。 我们的具体目标是(I)使用快速冷冻/深蚀刻(QFDE)形态和脂肪组织化学方法，结合传统的电子显微镜方法，定量表征20-70岁供体眼Bruch膜与脂质沉积相关的早期超微结构变化；(Ii)将这些结果与分析生物工程工具相结合，评估与年龄相关的变化对Bruch膜运输特性的影响；以及(Iii)使用通过细胞外基质的液体和脂肪运输的体外模型来验证分析研究的结果，并表征脂质积累对运输的影响。

DESCRIPTION (provided by applicant): Better understanding of the pathophysiology of the early stages of age-related maculopathy (ARM) may guide development of better treatments and/or preventions of age-related macular degeneration. Prominent clinical and histopathologic lesions of early ARM involve Bruch's membrane, a thin connective tissue between the choriocapillaris and the RPE. Unlike the case for late ARM, for which several risk factors have been identified, the only consistently identified risk factor for early ARM is advanced age. It is, therefore, important to understand how age-related changes in Bruch's membrane predispose some individuals for retinal dysfunction. A decreased transport capacity of Bruch's membrane, perhaps caused by lipid accumulation, has been postulated to be a critical early event in the pathogenesis of ARM. However, morphometric studies have not yet been successful in identifying those moieties responsible for limiting the transport capacity of Bruch's membrane. QFDE is a morphological technique that preserves extracellular matrix in exquisite detail and allows structures to be visualized that are not seen using conventional transmission electron microscopy (TEM) techniques. QFDE offers a technology that, in other connective tissues, has provided new insights into the transport characteristics of these tissues. Our goal is to combine the technique of quick-freeze/deep-etch (QFDE) morphology with bioengineering analytic methods to evaluate this hypothesis, using well-preserved eyes from normal human donors. Our specific aims are (i) to use the methods of quick-freeze/deep-etch (QFDE) morphology and lipid histochemistry in conjunction with conventional electron microscopy methods to characterize quantitatively the earliest ultrastructural changes in Bruch's membrane associated with lipid deposition in 20-70 year old donor eyes; (ii) to combine these results with analytical bioengineering tools to evaluate the effects of age-related changes on transport properties of Bruch's membrane; and (iii) to use an in vitro model of fluid and lipid transport through extracellular matrix to validate the results of the analytical studies and characterize the impact of lipid accumulation on transport.