ASSESSMENT OF RETINAL FUNCTION IN HEALTH AND DISEASE FROM MOUSE TO HUMAN

 描述(申请人提供)：在发达国家，导致失明的主要原因包括黄斑变性、青光眼和糖尿病视网膜病变，这些疾病都表现为视网膜中特定细胞类型的功能障碍和退化。这些疾病的分子机制和病理生理学还不是很清楚。视网膜疾病的病理生理学研究广泛使用的技术包括在体视网膜电信号(ERG)和单细胞电生理学。在活体实验中，ERG可以评估视网膜的整体健康状态，但在提供有关疾病视网膜功能缺陷的细胞和分子来源的定量信息方面存在局限性。另一方面，单细胞记录提供了定量数据 来自单个细胞但具有挑战性的，只提供有限的记录时间，并且不容易放大来评估整个视网膜的功能和信号。这项建议的目的是推进技术和方法论，以剖析完整的小鼠、灵长类动物、猪以及最终的人类视网膜中光感受器、双极细胞和Müler神经胶质细胞的功能。我将首先开发一种方法，使用来自分离的野生型小鼠视网膜的Ex Vivo ERG来定量评估这些细胞的内在功能“状态”(K99)。EX Vivo ERG提供了横跨视网膜侧轴的所有细胞的平均功能状态的信息。然而，一些视网膜疾病只影响局部视网膜(如DR)或主要针对神经节细胞(如青光眼)，这在Ex Vivo ERG信号中不一定可见。该方案的一个目标是开发一种结合了Ex Vivo ERG和多电极阵列(MEA)方法的新型设备。该装置将被用来评估整个野生型小鼠视网膜(K99)的光感受器、双极细胞、Müler神经胶质细胞和神经节细胞的局部功能。EX Vivo ERG(K99)将被用于确定视杆和视锥双极细胞、Müler胶质细胞和神经节细胞在视网膜色素变性(RP，P23H视紫红质突变)和糖尿病视网膜病变(DR，链脲佐菌素诱导的糖尿病)模型中的功能如何受到影响，MEA-ERG设备将被用于评估激光(K99和R00)局部损伤的视网膜的局部功能。这些实验将促进对这些疾病的病理生理学的理解，这些疾病分别主要影响视网膜外部和内部。为小鼠视网膜开发的方法学将被用于建立方案，以剖析灵长类动物、猪以及最终人类供体视网膜中光感受器、双极细胞和Müler神经胶质细胞的功能。我将首先开发记录协议以从灵长类动物那里获得可行的反应 安乐死后立即摘除眼球的视网膜(K99)。然后，与汉尼肯博士合作，我们将使用猪眼(R00)来确定死亡和摘除之间的可接受时间范围。最后，基于灵长类动物和猪的实验，我们将设计和进行记录来评估人类视网膜黄斑和周边区域(R00)的光感受器、双极和Müler胶质细胞的功能。

 DESCRIPTION (provided by applicant): Leading causes of blindness in developed countries include macular degeneration, glaucoma and diabetic retinopathy which are all manifested as dysfunction and degeneration of specific cell types in the retina. The molecular mechanisms and pathophysiology of these diseases are not well understood. Widely used techniques to study the pathophysiology of retinal diseases include In Vivo electroretinogram (ERG) and single cell electrophysiology. In Vivo ERG can assess the overall health state of the retina but is limited in providing quantitative information about the cellular and molecular origin of the functional deficits in a diseased retina. Single cell recordings, on the other hand, provide quantitative data from individual cells but are challenging, offer only limited recording time and are not easily scaled up to assess the function and signaling across the whole retina. The objective of this proposal is to advance techniques and methodology to dissect the function of photoreceptor, bipolar and Müller-glial cells in the intact mouse, primate, pig and ultimately human retinas. I wll first develop methodology to quantitatively assess the intrinsic functional "state" of these cells y using Ex Vivo ERG from isolated wild-type mouse retinas (K99). Ex Vivo ERG provides information about the average functional state of all cells across the lateral axis of the retina. However, some of the retinal diseases affect retina only locally (e.g. DR) or target primarily ganglion cells (e.g. glaucoma) that will not necessarily be observable in the Ex Vivo ERG signal. One objective of this proposal is to develop a novel device combining Ex Vivo ERG and multi-electrode array (MEA) methods. This device will be used to assess the local function of photoreceptors, bipolar, Müller-glial and ganglion cells across the whole wild-type mouse retinas (K99). Ex Vivo ERG (K99) will be applied to determine how the function of rod and cone bipolar, Müller-glial and ganglion cells are affected in the mouse models of retinitis pigmentosa (RP, P23H rhodopsin mutation) and diabetic retinopathy (DR, Streptozotocin-induced diabetes) and MEA-ERG device will be used to assess the local function of the retina that has been focally injured by laser (K99 and R00). These experiments will advance the understanding of pathophysiology of these diseases known to affect primarily outer and inner retina, respectively. The methodology developed for mouse retinas will be used to establish protocols to dissect the function of photoreceptors, bipolar and Müller-glial cells in primate, pig, and ultimately human donor retinas. I will first develop the recording protocols to obtain viable responses from primate retinas dissected from eye balls enucleated immediately following the euthanasia (K99). Then, in collaboration with Dr. Hanneken, we will determine the acceptable time frame between death and enucleation by using pig eyes (R00). Finally, based on the primate and pig experiments we will design and conduct recordings to assess function of photoreceptor, bipolar and Müller glia cells in a macula and peripheral regions of the human retina (R00).