Imaging retinal astrocytes, ganglion cells and axonal transport in vivo

描述（由申请人提供）：星形胶质细胞是脊椎动物视网膜中的一类主要胶质细胞。它们主要位于视网膜最内层；其过程围绕视网膜神经节细胞（RGC）轴突和轴突束以及所有血管。由于这种解剖关系和各种生理证据，星形胶质细胞被认为在视网膜血流自动调节机制中起主要作用，即在眼灌注压变化的反应中维持近乎恒定的血流。星形胶质细胞也被认为在许多眼部疾病的病理生理中发挥重要作用，通过响应各种损伤，如缺血、眼压升高和神经变性，以一种被描述为胶质瘤的方式。因此，在体内对星形胶质细胞成像的能力可以帮助阐明疾病病理生理的各个方面。同样，有证据表明，RGC轴突细胞骨架成分，特别是微管，在对实验性损伤（如轴截术和实验性青光眼）反应的早期阶段被破坏。这种破坏是重要的，因为微管是驱动轴突运输的“轨道”。因此，如果损伤反应早期出现微管异常，由此产生的轴突运输中断可能会加剧损伤，并抑制保护或救援反应发挥全部潜力。本R21项目的总体目标是开发视网膜星形胶质细胞、RGCs及其轴突和轴突运输在体内的成像方法。具体目的1：建立大鼠眼内视网膜星形胶质细胞、RGCs及其轴突和活跃轴突运输的体内可视化方法。评估体内标记物的最佳浓度、随访时间和持续时间，并进行组织病理学研究以证实体内观察结果。具体目标2：通过视网膜功能（视网膜电图，ERG）和视网膜结构（光谱域光学相干断层扫描，SDOCT）的敏感测量来评估体内星形胶质细胞标记物和轴突运输示踪剂的潜在毒性，从而评估其在灵长类动物实验模型中的应用潜力。具体目的3：通过比较四种单侧实验性损伤模型（玻璃体内注射诺可达唑/秋水珠碱破坏轴突微管并抑制轴突主动运输、急性眼压升高、慢性眼压升高和视神经挤压）与双侧对照眼的影响，评估我们新开发的方法的敏感性。本文提出的新方法将为今后的研究提供可能，研究星形胶质细胞异常和RGC轴突运输异常的发生，并将这些现象与RGC损伤实验模型中RGC和轴突变性的过程进行比较。

DESCRIPTION (provided by applicant): Astrocytes are a major class of glia in the vertebrate retina. They are located primarily within the innermost retinal layers; their processes surround retinal ganglion cell (RGC) axons and axon bundles as well as all blood vessels. Because of this anatomical relationship, and a variety of physiological evidence, astrocytes are thought to have a major role in the mechanisms of retinal blood flow autoregulation, i.e. the maintenance of nearly constant blood flow in response to variations of ocular perfusion pressure. Astrocytes are also thought to play an important role in the pathophysiology of many ocular diseases by responding to a variety of insults such as ischemia, increased intraocular pressure and neuronal degeneration in a manner that has been characterized as gliosis. Hence, the ability to image astrocytes in vivo could help to elucidate aspects of disease pathophysiology. Similarly, there is evidence to suggest that RGC axonal cytoskeletal components, specifically microtubules, are disrupted during the earliest stages of response to experimental injuries such as axotomy and experimental glaucoma. This disruption is significant because microtubules are the "tracks" upon which axonal transport is driven. Thus, if microtubule abnormalities develop early in response to injury, the resultant axonal transport disruption could exacerbate the injury and inhibit protective or rescue responses from achieving full potential. The overall goal of this R21 project is to develop the methods for imaging retinal astrocytes, RGCs, their axons and axonal transport in vivo. The specific objectives are as follows: Specific Aim 1: To establish methodologies for in vivo visualization of retinal astrocytes, RGCs, their axons and active axonal transport in the rat eye. To evaluate the optimal concentration, follow-up duration and persistence of in vivo markers as well as perform histopathological studies to corroborate in vivo observations. Specific Aim 2: To evaluate potential toxicity of in vivo astrocyte markers and axonal transport tracers using sensitive measures of retinal function (electroretinography, ERG) and retinal structure (spectral domain optical coherence tomography, SDOCT), so as to assess potential for use in primate experimental models. Specific Aim 3: To evaluate the sensitivity of our newly developed methods by comparing the impact of four unilateral experimental injury models (intravitreal injection of nocodazole/colchicine to disrupt axonal microtubules and inhibit active axonal transport; acute elevation of intraocular pressure; chronic elevation of intraocular pressure; and optic nerve crush) with results obtained in bilateral control eyes. The novel methods developed in this proposal will make possible in future proposals, studies about the onset of astrocyte abnormalities and RGC axonal transport abnormalities and comparisons of those phenomena to the course of RGC and axonal degeneration in experimental models of RGC injury. PUBLIC HEALTH RELEVANCE: Glaucoma is one of the most common causes of blindness in the United States and around the world. It is a chronic disease with no known cure. Though prospective longitudinal trials have found that treatment to lower intraocular pressure decreases the rate of progressive vision loss, some individuals continue to lose vision despite successful therapy to lower their intraocular pressure. Thus, a more thorough understanding of the events leading to damage and vision loss in glaucoma is required. The goal of this project is to develop methods for evaluating two groups of cells and aspects of their function in the living eye using specialized imaging techniques.