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

从小鼠到人类的健康和疾病中的视网膜功能评估

基本信息

批准号：
9249586
负责人：
Frans Vinberg
金额：
$ 9.48万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-01 至 2017-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9249586
关键词：
Affect Age related macular degeneration Animal Model Animal Testing Area Argon Blindness Burn injury Cell physiology Cells Cessation of life Clinical Trials Collaborations Cone Data Developed Countries Developing Countries Devices Diabetes Mellitus Diabetic Retinopathy Disease Disease model Dissection Electrophysiology (science)Electroretinography Environment Euthanasia Eye Eye diseases Family suidae Functional disorder Genetic Glaucoma Health Human Individual Lasers Lateral Light Link Macular degeneration Measures Mediating Methodology Methods Molecular Muller&apos s cell Mus Mutation Neuroglia Neurons Noise Opsin Peripheral Pharmaceutical Preparations Pharmacology Photoreceptors Positioning Attribute Primates Protocols documentation Retina Retinal Retinal Diseases Retinitis Pigmentosa Rhodopsin Rodent Safety Shapes Signal Transduction Streptozocin Techniques Time Vertebrate Photoreceptors Vision Wild Type Mouse base cell type design experimental study fovea centralis ganglion cell in vivo injured macula mouse model multi-electrode arrays novel patch clamp public health relevance response scale up screening

项目摘要

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).

描述（由适用提供）：发达国家失明的主要原因包括黄斑变性，青光眼和糖尿病性视网膜病，它们都表现为视网膜中特定细胞类型的功能障碍和变性。这些疾病的分子机制和病理生理学尚不清楚。广泛研究视网膜疾病的病理生理学的技术包括体内电图（ERG）和单细胞电生理学。体内ERG可以评估视网膜的整体健康状况，但在提供有关视网膜功能性缺陷的细胞和分子起源的定量信息方面受到限制。另一方面，单细胞记录提供了定量数据从单个单元格中，但受到挑战，仅提供有限的记录时间，并且不容易扩展以评估整个视网膜的功能和信号。该提案的目的是推进技术和方法，以剖析完整小鼠，灵长类动物，猪和最终人类视网膜中光感受器，双极和müller-glial细胞的功能。我首先要使用分离的野生型小鼠视网膜（K99）的ex vivo ERG定量评估这些细胞y的固有功能“状态”的方法。 Ex Vivo ERG提供了有关视网膜侧轴所有细胞的平均功能状态的信息。但是，某些残留疾病仅影响视网膜（例如DR）或靶向原代神经节细胞（例如青光眼），而这些细胞不一定在离体ERG信号中可观察到。该提案的一个目的是开发一种新型设备，结合了离体ERG和多电极阵列（MEA）方法。该设备将用于评估整个野生型小鼠视网膜中光感受器，双极，müller-glial和神经节细胞的局部功能（K99）。在体内ERG（K99）将用于确定杆和双极性，müller-glial和神经节细胞的功能如何在色素性视网膜炎的小鼠模型（RP，p23h rhotopsin突变）的小鼠模型中受到影响，并评估糖尿病性的糖尿病和糖尿病诱导的型型糖尿病，并将其置于型型糖尿病诱导的手段。激光受伤（K99和R00）。这些实验将分别对这些已知疾病的病理生理学的理解分别影响原发性外部和内部视网膜。为小鼠视网膜开发的方法将用于建立方案，以在灵长类动物，猪和最终的人类供体视网膜中在灵长类动物，猪和最终的人类供体视网膜中剖析光感受器，双极和müller-glial细胞的功能。我将首先制定记录协议，以从私有在安乐死之后立即从眼球中解剖视网膜（K99）。然后，通过使用Pig Eyes（R00），我们将与Hanneken博士合作确定死亡和摘除之间可接受的时间范围。最后，基于灵长类动物和猪实验，我们将设计和进行记录，以评估人类视网膜（R00）的黄斑和外围区域中光感受器，双极和müller胶质细胞的功能。