Downregulation of Rod Metabolism in Retinopathy of Prematurity

描述（由申请人提供）：本提案概述了广泛挑战领域（05）比较有效性研究下的一组实验，该研究将解决最高优先级挑战05- ey -102儿童眼病和疾病的治疗。具体来说，它将比较两项早产儿视网膜病变（ROP）新干预措施的临床前试验结果。从某种意义上说，持续的ROP流行病是一个好问题：新生儿学的进步使最早产的婴儿得以生存。不幸的是，这些患者患ROP的风险最高。因此，中等收入国家的ROP发病率和严重程度接近历史最高水平。在世界范围内，至少有5万名儿童双目失明，是单眼失明人数的许多倍，而且许多儿童因ROP而终生遭受视力损害。在美国，每年大约有15,000例新诊断病例，尽管有最好的医疗管理，但仍有400-600例进展为失明。这将ROP列为儿童失明的主要眼部原因，并强烈推动对干预措施有效性的研究。血管病变的治疗一直是许多良好工作的焦点，但对神经视网膜的损害却很少受到关注。最近的发现表明，在杆光感受器氧化应激在ROP中起因果作用。这启发了另一种治疗ROP的方法：调节杆代谢。我们将在大鼠ROP模型中进行临床前试验，通过下调杆状体中两个能量需求过程：暗电流和视觉周期。我们预计这种方法将比目前最好的医疗管理更有利。为了在大鼠身上产生与人类ROP相似的血管异常，幼崽在出生后的头几天暴露在高氧和低氧环境中；引产在出生后第14天结束。大鼠按两种方式分组：按氧暴露史分组和按治疗分组。“ROP大鼠”和对照组分别接受dorzolamide暗电流调制（DCM）治疗、视黄胺视觉周期调制（VCM）治疗或载药治疗。在出生后19岁左右，当血管异常接近峰值时（在未经治疗的动物中），通过视网膜电图（ERG）客观地评估神经视网膜（光感受器和后感受器）的功能。通过将图像分析软件（RISA）应用于眼底的数字照片，获得了浅表血管异常的无偏数值测量。ERG和视网膜成像都是非侵入性技术，允许在同一只眼睛中进行后续分析。根据ERG分析的结果选出一小部分接近组中值的大鼠，对其视力进行严格的心理物理（行为）测试；在每只大鼠中进行5次阈值测量（绝对值加4个增量值），并将数据对角线或垂直方向相对于对照进行调换，将提供任何功能障碍部位的线索。对剩下的动物进行了详细的解剖和分子研究。在甲苯胺蓝染色视网膜横截面的光学显微照片中评估视网膜层，可获得有关视网膜细胞损伤的信息。采用酶联免疫吸附法和免疫印迹法检测氧化应激标志物。qPCR用于补充这些措施，并评估神经源性和血管生成生长因子如VEGF、PEDF和信号素的表达。在DCM和VCM治疗的ROP大鼠中，视网膜结构、功能和脉管系统的改善，氧化应激分子标志物的减少和生长因子表达的正常化都是假设的。

DESCRIPTION (provided by applicant): This proposal outlines a set of experiments under broad challenge area (05) Comparative Effectiveness Research which will address the highest-priority challenge 05-EY-102 Treatment of Pediatric Eye Diseases and Disorders. Specifically, it will compare results from two preclinical trials of novel interventions in retinopathy of prematurity (ROP). In one sense, the continuing ROP epidemic is a good problem to have: Advances in neonatology are permitting the most prematurely born infants to survive. Unfortunately, these are the very patients at highest risk for ROP. Thus, the incidence and severity of ROP in middle income countries is near all-time highs. Worldwide, at least 50,000 children are binocularly blind, many times that number monocularly blind, and many fold again suffering from lifelong visual impairment as a consequence of ROP. In the United States, approximately 15,000 cases are newly diagnosed each year and, despite the best available medical management, 400-600 progress to blindness. This ranks ROP as the leading ocular cause of childhood blindness and strongly motivates research into the effectiveness of interventions. Treatment of the vasculopathy has been the focus of much good work, but the insult to the neural retina has received less attention. Recent discoveries indicate that oxidative stress in the rod photoreceptors plays a causal role in ROP. This inspires an alternative way to treat ROP: modulating rod metabolism. We will conduct a preclinical trial in rat models of ROP by downregulating two energy-demanding processes in the rods: the dark current and the visual cycle. We anticipate that this approach will compare favorably with the best currently available medical management. To produce in rats vascular abnormalities similar to those that characterize human ROP, pups are exposed to high and low oxygen during the first days after birth; the induction ends at postnatal day 14. Rats are grouped two ways: by oxygen exposure history and by treatment. "ROP rats" and controls are assigned to either dark current modulation (DCM) treatment with dorzolamide, visual cycle modulation (VCM) treatment with retinylamine, or vehicle treatment. At around postnatal age 19, when the vascular abnormalities are near their peak (in untreated animals), function of the neural retina (both photoreceptor and postreceptor) is objectively assessed by electroretinography (ERG). Unbiased, numeric measures of abnormality of the superficial vessels are obtained by application of image analysis software (RISA) to digital photographs of the ocular fundus. Both the ERG and retinal imaging are noninvasive techniques that permit subsequent analyses to be conducted in the same eye. A small subset of rats, selected based on the results of their ERG analyses to be near the medians for their group, are assigned to rigorous psychophysical (behavioral) testing of their vision; five threshold measurements (absolute plus four increment) are made in each rat and a transposition of the data, diagonally or vertically relative to controls, will provide clues as to the site of any dysfunction. Detailed anatomic and molecular studies are undertaken in the remaining animals. Assessment of the retinal layers in light micrographs of toluidine blue-stained retinal cross-sections yields information about damage to retinal cells. Markers of oxidative stress are assessed by ELISA and western blotting. qPCR is used to supplement these measures and to evaluate the expression of neurogenic and angiogenic growth factors such as VEGF, PEDF and semaphorins. Improved retinal structure, function and vasculature, as well as reduction in molecular markers of oxidative stress and normalization of growth factor expression, are all hypothesized in both DCM and VCM treated ROP rats. PUBLIC HEALTH RELEVANCE: Retinopathy of prematurity (ROP) is clinically diagnosed by abnormal retinal vasculature, but damage to retinal neurons underpins functional sequelae and may also underpin the vasculopathy. At the same age as the onset of acute vasculopathy, an escalation in the metabolic demands of the rod photoreceptors occurs that simultaneously renders them vulnerable to blood oxygen swings and powerful provocateurs of pathological angiogenesis. Thus, suppression of metabolically demanding processes in the rods promises preservation of visual function and prevention of vascular disease.