Paranthan Riya R, Bargagna-Mohan Paola, Lau Daniel L, Mohan Royce
Ophthalmology & Visual Science, University of Kentucky, Lexington, KY, USA.
Mol Vis. 2011;17:1901-8. Epub 2011 Jul 14.
To develop an animal model for simultaneously eliciting corneal angiogenesis and retinal gliosis that will enable the assessment of inhibitor efficacy on these two pathological processes in separate anatomic sites of the ocular globe.
Four to six week-old mice in a C57BL/6J background were anesthetized and 0.15 N NaOH was applied to the cornea, followed by mechanical scraping of the epithelium from limbus and central cornea. After this injury, mice were treated with vehicle or with an inhibitor (withaferin A [WFA]), which were delivered by intraperitoneal injection, to assess the pharmacological effects on angiogenesis and/or gliosis. Mice were sacrificed after 14 days and tissues (corneas and retinas) were prepared for analysis of corneal neovascularization and retinal gliosis by immunohistochemistry and western blotting, respectively. This protocol was also suited for studying earlier disease end points, for assessment of drug dose efficacy or genetic influences and the entire procedure and this analysis was completed in 16-17 days.
Both corneal angiogenesis and retinal gliosis were maximally sustained at fourteen days following chemical and mechanical injury of the cornea. 1) Injured corneas showed abundant CD31+ staining, with new blood vessels branching out from the limbus to the central cornea. WFA treatment potently inhibited corneal neovascularization. 2) Retinal gliosis in injured mice was associated with upregulated expression of glial fibrillary acidic protein (GFAP) that appeared as polymeric filaments and soluble forms expressed in reactive Müller glial cells. WFA treatment potently downregulated the expression of soluble and filamentous GFAP; the latter protein was fragmented.
We have developed a mouse model for investigating retinal gliosis and corneal neovascularization. We used this model to demonstrate the simultaneous inhibitory effects of WFA on both of these disease processes. Retinal gliosis occurs in several major degenerative conditions of the eye, including age-related macular degeneration, where angiogenesis is also a prevailing pathological feature. Thus, inhibitors of both gliosis and angiogensis used as combination therapy are currently being explored for treatment of such complex diseases. The model presented here affords a very simple preclinical assay for screening combination of drugs or polypharmacological agents and reduces the numbers of animals because of the different anatomic sites of these pathologies. Finally, given that endogenous mediators elicit angiogenesis and gliosis in this model, the combination of genetics and pharmacology can be exploited to study drug mechanisms and for target validation in vivo.
建立一种能同时引发角膜血管生成和视网膜胶质增生的动物模型，以便在眼球的不同解剖部位评估抑制剂对这两种病理过程的疗效。
对4至6周龄的C57BL/6J背景小鼠进行麻醉，将0.15 N氢氧化钠应用于角膜，随后从角膜缘和中央角膜机械刮除上皮。损伤后，小鼠接受载体或抑制剂（Withaferin A [WFA]）治疗，通过腹腔注射给药，以评估对血管生成和/或胶质增生的药理作用。14天后处死小鼠，分别制备组织（角膜和视网膜），通过免疫组织化学和蛋白质印迹法分析角膜新生血管形成和视网膜胶质增生。该方案也适用于研究疾病的早期终点、评估药物剂量疗效或遗传影响，整个过程和分析在16至17天内完成。
角膜化学和机械损伤后14天，角膜血管生成和视网膜胶质增生均达到最大程度的持续状态。1）损伤的角膜显示大量CD31+染色，新血管从角膜缘向中央角膜分支。WFA治疗有效抑制角膜新生血管形成。2）损伤小鼠的视网膜胶质增生与胶质纤维酸性蛋白（GFAP）表达上调有关，GFAP以聚合物丝和反应性Müller胶质细胞中表达的可溶性形式出现。WFA治疗有效下调可溶性和丝状GFAP的表达；后者蛋白发生片段化。
我们建立了一种用于研究视网膜胶质增生和角膜新生血管形成的小鼠模型。我们使用该模型证明了WFA对这两种疾病过程的同时抑制作用。视网膜胶质增生发生在眼睛的几种主要退行性疾病中，包括年龄相关性黄斑变性，其中血管生成也是一个主要的病理特征。因此，目前正在探索将胶质增生和血管生成抑制剂联合治疗此类复杂疾病。本文介绍的模型为筛选药物组合或多药理学药物提供了一种非常简单的临床前试验，并由于这些病理的不同解剖部位而减少了动物数量。最后，鉴于内源性介质在该模型中引发血管生成和胶质增生，可以利用遗传学和药理学的结合来研究药物机制并在体内进行靶点验证。
