Imaging retinal astrocytes, ganglion cells and axonal transport in vivo

描述(申请人提供)：星形胶质细胞是脊椎动物视网膜中的一种主要的神经胶质细胞。它们主要位于视网膜最内层；它们的突起围绕着视网膜神经节细胞(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学证据，星形胶质细胞被认为在视网膜血流自动调节机制中发挥重要作用，即对眼灌注压的变化保持近乎恒定的血流量。星形胶质细胞也被认为在许多眼部疾病的病理生理学中起着重要作用，它通过对各种损伤的反应，如缺血、眼压升高和神经元变性，以胶质增生为特征。因此，在体内成像星形胶质细胞的能力可以帮助阐明疾病病理生理学的各个方面。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(如轴突切断和实验性青光眼)反应的最早阶段被破坏。这种破坏是显著的，因为微管是轴突运输的“轨道”。因此，如果损伤早期出现微管异常，由此产生的轴突运输中断可能会加剧损伤，抑制保护性或救助性反应的充分发挥。R21项目的总体目标是发展体内视网膜星形胶质细胞、RGC及其轴突和轴突运输的成像方法。具体目标如下：具体目标1：建立大鼠视网膜星形胶质细胞、RGC及其轴突和主动轴突运输的体内可视化方法。评估体内标记物的最佳浓度、随访时间和持久性，并进行组织病理学研究以证实体内观察。具体目的2：使用视网膜功能(视网膜电信号，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志物和轴突运输示踪剂的潜在毒性，以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(玻璃体内注射诺康唑/秋水仙碱破坏轴突微管并抑制主动轴突运输；急性眼压升高；慢性眼压升高；视神经挤压)的影响，评价我们新开发的方法的敏感性。这项建议中发展的新方法将使未来的建议成为可能，关于星形胶质细胞异常和RGC轴突运输异常的研究，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。 与公共健康相关：青光眼是美国和世界各地最常见的致盲原因之一。这是一种慢性疾病，目前尚无治愈方法。虽然前瞻性纵向试验发现，降低眼压的治疗可以降低进展性视力丧失的比率，但一些人尽管成功地进行了降低眼压的治疗，但仍继续失明。因此，需要对青光眼中导致损害和视力丧失的事件有更全面的了解。该项目的目标是开发使用专门的成像技术来评估两组细胞及其在活眼中的功能的方法。

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.