Axonal cytoskeletal changes in experimental glaucoma

描述（申请人提供）：该项目提出，在青光眼视网膜神经节细胞（RGC）死亡之前，以及永久性视力丧失之前，存在一个以轴突微管（MT）降解为特征的RGC功能障碍阶段。新出现的证据表明，MT降解最初可能发生在轴突口径没有实质性变化。因此，有人提出，早期阶段的RGC功能障碍，涉及MT降解应优先通过扫描激光偏振（SLP）的视网膜神经纤维层（RNFL）的RNFL厚度的变化之前检测。这是因为SLP的基本光学原理是基于检测偏振光的相位延迟，这是由于长而薄的圆柱形MT在RNFL中产生的光学性质双折射。初步研究表明，在几种不同的RGC损伤实验模型（包括实验性青光眼（EG））中，RNFL延迟先于RNFL厚度下降，且下降速度快于RNFL厚度。在没有RNFL厚度变化的情况下，通过SLP对轴突MT破坏的临床检测可能代表了神经性昏迷损伤的早期和潜在可逆阶段，并为治疗调整提供了临床可检测的标志物。因此，该建议的中心假设是，乳头周围RNFL轴突内MT的破坏是神经性昏迷损伤的早期指标，先于轴突口径的变化和这些轴突的物理损失。使用EG的非人灵长类动物（NHP）模型，在三个特定目的中对从该假设产生的预测进行了测试。具体目标1：在具有EG的NHP眼中，测试在通过谱域光学相干断层扫描（sd-OCT）测量的RNFL厚度变化之前和在通过共焦扫描激光断层扫描（CSLT）测量的视神经乳头（ONH）表面变化之前，视乳头周围RNFL延迟将下降的预测;具体目标2：为了检验乳头周围RNFL MT破坏的组织学证据将比组织学上更明显的预测，明确的RNFL厚度变化和球后视神经轴突缺失;具体目标3：检验RGC功能异常与以异常轴突MT为特征的RGC变性中间阶段相关的预测。为了实现这些目标，将通过激光光凝小梁网来诱导EG，以在24名NHP中引起中度、单侧慢性IOP升高。在4周激光前基线期内和EG发作后长达8个月（目标1），将分别使用SLP、sd-OCT和CSLT每周测量每只NHP的双眼视乳头周围RNFL延迟、RNFL厚度和ONH表面形貌。对于每个参数，统计学显著变化定义为超过每只眼睛的基线期间变异性的任何变化，两次确认。一旦每只动物进展到其终点，将其处死以进行组织学数据收集和分析（目的2）。在针对目标1的每周体内结构测试期间，还将使用三种经证实的视网膜电图形式在双眼中评估RGC功能（目标3）。青光眼是美国和世界上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，但前瞻性纵向试验发现，降低眼内压的治疗可降低进行性视力丧失的发生率。因此，早期诊断能够及时进行治疗干预，并减少青光眼的总体影响。然而，实现及时诊断需要临床检测视神经乳头（ONH）和视网膜神经纤维层（RNFL）的青光眼损伤的发作和进展，这仍然是每个青光眼患者临床护理中的核心挑战。该项目评估和发展用于检测青光眼早期损伤和进展的临床工具。

DESCRIPTION (provided by applicant): This project proposes that prior to retinal ganglion cell (RGC) death in glaucoma, and before permanent loss of vision, there exists a stage of RGC dysfunction characterized by degradation of axonal microtubules (MTs). Emerging evidence suggests that MT degradation can occur initially without substantial changes in axonal caliber. Therefore, it is proposed that early stage RGC dysfunction involving MT degradation should be preferentially detectable by scanning laser polarimetry (SLP) of the retinal nerve fiber layer (RNFL) prior to changes in RNFL thickness. This is because the fundamental optical principle of SLP is based on detecting phase retardance of polarized light, which is due to the optical property birefringence produced in the RNFL by the long, thin cylindrical MTs. Preliminary studies demonstrate that RNFL retardance declines prior to, and faster than RNFL thickness in several different experimental models of RGC injury, including experimental glaucoma (EG). Clinical detection of axonal MT disruption by SLP, in the absence of RNFL thickness changes, might represent an early and potentially reversible phase of glaucomatous damage and provide a clinically detectable marker for therapeutic adjustment. Thus the central hypothesis of this proposal is that disruption of MTs within the axons of the peripapillary RNFL is an early indicator of glaucomatous damage, preceding both changes in axonal caliber and physical loss of those axons. Predictions arising from this hypothesis are tested in three Specific Aims using a non-human primate (NHP) model of EG. Specific Aim 1: To test the prediction that peripapillary RNFL retardance will decline prior to RNFL thickness changes measured by spectral domain optical coherence tomography (sd-OCT) and prior to optic nerve head (ONH) surface changes measured by confocal scanning laser tomography (CSLT) in NHP eyes with EG; Specific Aim 2: To test the predictions that histological evidence of peripapillary RNFL MT disruption will be more pronounced than histologically-defined RNFL thickness changes and retrobulbar optic nerve axon loss; Specific Aim 3: To test the prediction that RGC functional abnormalities are associated with the intermediate stage of RGC degeneration characterized by abnormal axonal MTs. To achieve these Aims, EG will be induced via laser photocoagulation of the trabecular meshwork to cause moderate, unilateral chronic IOP elevation in 24 NHPs. Weekly measurements of peripapillary RNFL retardance, RNFL thickness and ONH surface topography will be made in both eyes of each NHP using SLP, sd-OCT and CSLT, respectively, during a 4-week pre-laser baseline period and for up to 8 months after onset of EG (Aim 1). For each parameter, statistically significant change is defined as any change exceeding the baseline intersession variability for each individual eye, twice confirmed. Once each animal progresses to its endpoint, it is sacrificed for histological data collection and analysis (Aim 2). During each week of in vivo structural testing for Aim 1, RGC function will also be assessed in both eyes using three proven forms of electroretinography (Aim 3). 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, but prospective longitudinal trials have found that treatment to lower intraocular pressure decreases the rate of progressive vision loss. Thus, early diagnosis enables timely therapeutic intervention and reduces the overall impact of glaucoma. However, achieving a timely diagnosis requires clinical detection of the onset and progression of glaucomatous damage to the optic nerve head (ONH) and retinal nerve fiber layer (RNFL), which remain a central challenge in the clinical care of every glaucoma patient. This project evaluates and advances clinical tools for detecting early damage and progression of glaucoma.