Downregulation of Rod Metabolism in Retinopathy of Prematurity

描述(由申请人提供)：本提案概述了在广泛的挑战领域(05)比较有效性研究下的一系列实验，这些实验将解决最优先的挑战05-EY-102儿童眼病和疾病的治疗。具体地说，它将比较两个治疗早产儿视网膜病变(ROP)的新干预措施的临床前试验的结果。在某种意义上，持续的ROP疫情是一个很好的问题：新生儿学的进步让最早产的婴儿得以存活。不幸的是，这些正是ROP风险最高的患者。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50,000名儿童双目失明，是这个数字的许多倍，许多儿童因ROP而再次遭受终身视力障碍。在美国，每年约有15000例新诊断病例，尽管有最好的医疗管理，但仍有400-600人失明。这将ROP列为导致儿童失明的主要眼病原因，并强烈推动了对干预措施有效性的研究。血管病变的治疗一直是许多好工作的重点，但对神经视网膜的侮辱却没有得到太多关注。最近的发现表明，视杆感光细胞中的氧化应激在ROP中起着因果作用。这启发了另一种治疗ROP的方法：调节杆状代谢。我们将在ROP的大鼠模型上进行临床前试验，下调视杆细胞中两个需要能量的过程：暗电流和视觉周期。我们预计，这种方法将与目前最好的医疗管理相媲美。为了在大鼠身上产生类似于人类ROP特征的血管异常，幼鼠在出生后的头几天暴露于高氧和低氧环境中；诱导过程在出生后14天结束。大鼠被分成两组：根据氧气暴露历史和通过治疗。ROP大鼠和对照组被分配到多唑胺的暗电流调制(DCM)治疗、视黄胺的视觉周期调制(VCM)治疗或赋形剂治疗。在出生后19岁左右，当血管异常接近高峰时(在未经治疗的动物中)，神经视网膜(包括光感受器和感受器后)的功能被客观地用视网膜电图仪(ERG)评估。通过将图像分析软件(RISA)应用于眼底的数字照片来获得浅血管异常的无偏的数字测量。ERG和视网膜成像都是非侵入性技术，可以在同一只眼睛中进行后续分析。根据ERG分析结果选出的一小部分大鼠被分配到严格的视觉心理物理(行为)测试中；每只大鼠都进行了五个阈值测量(绝对加四个增量)，数据相对于对照组的对角或垂直方向的移位将提供任何功能障碍位置的线索。对其余的动物进行了详细的解剖学和分子研究。对甲苯胺蓝染色的视网膜切片的光镜下视网膜层进行评估，可获得视网膜细胞受损的信息。氧化应激的标志物用ELISA法和Western blotting法进行评估。定量聚合酶链式反应被用来补充这些措施，并评估神经源性和血管生成生长因子的表达，如血管内皮生长因子、PEDF和信号素。在DCM和VCM治疗的ROP大鼠中，视网膜结构、功能和血管的改善，以及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。 公共卫生相关性：早产儿视网膜病变(ROP)临床诊断为视网膜血管异常，但视网膜神经元的损伤是功能性后遗症的基础，也可能是血管病变的基础。在急性血管病变发生的同一年龄，杆状感光细胞的代谢需求升级，同时使它们容易受到血氧波动和强大的病理性血管生成激动者的影响。因此，抑制视杆的代谢过程保证了视觉功能的保存和血管疾病的预防。

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.