AQUEOUS HUMOR DYNAMICS STUDIES IN VIVO AND IN VITRO

体内和体外房水动力学研究

• 批准号: 8173153
• 负责人: PAUL L KAUFMAN
• 金额: $ 3.1万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8173153
• 关键词: Adrenergic Agents; Anterior; Aqueous Humor; Binding; Caliber; Cell Adhesion; Cells; Ciliary Muscle; Computer Retrieval of Information on Scientific Projects Database; Custom; Cytoskeleton; Denervation; Detection; Dose; Drainage procedure; Environment; Enzyme Immunoassay; Enzymes; Eye; Fluorescence; Fluorophotometry; Funding; Gene Delivery; Gene Expression; Genes; Glaucoma; Goals; Grant; Human; Immunohistochemistry; In Vitro; Institution; Isotopes; Lentivirus Vector; Life; Matrix Metalloproteinases; Measures; Mediating; Microscope; Monkeys; Muscle Cells; NG-Nitroarginine Methyl Ester; Neurotransmitters; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Organ; Organ Culture Techniques; PGF receptor; Pathway interactions; Perfusion; Pharmaceutical Preparations; Pharmacotherapy; Physiologic Intraocular Pressure; Presbyopia; Production; Prostaglandin-Endoperoxide Synthase; Prostaglandins; Proteins; Pupil; Refractometry; Research; Research Design; Research Personnel; Resources; Risk Factors; Role; Sclera; Simulate; Source; Stromelysin 1; System; Techniques; Trabecular meshwork structure; United States National Institutes of Health; Western Blotting; adrenergic; base; cholinergic; extracellular; gene therapy; in vivo; inhibitor/antagonist; insight; kinase inhibitor; nonhuman primate; novel; novel strategies; pressure; prostaglandin-F synthase; public health relevance; response; small molecule; src-Family Kinases; tonometry

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Note: Full grant title is "Aqueous humor dynamics studies in vivo and in vitro, extra-lenticular aspects of accommodation and presbyopia" Objective: To investigate new approaches to lower intraocular pressure, a significant risk factor for the progression of glaucoma. Outflow through the trabecular meshwork will potentially be enhanced by ILK, PI3K and Src kinase inhibitors, which have been shown to alter the cytoskeleton and cellular adhesions of trabecular meshwork (TM) cells in culture. The effects of these compounds on intraocular pressure (IOP) and outflow facility will be studied in organ-cultured anterior segments and in monkey eyes in vivo. Gene therapy approaches will be utilized to lower IOP by enhancing outflow via the uveoscleral pathway. One of the current most effective pharmacotherapies for glaucoma is based on derivatives of prostaglandin (PG)F2a which binds to the FP receptor leading, in part, to enhanced matrix metalloproteinase (MMP) synthesis. This, in turn, alters the composition of the extracellular environment of the ciliary muscle (CM) and sclera leading to an enhancement of uveoscleral outflow. Lentiviral vectors carrying genes for PGF synthase and for stromelysin (MMP-3) will be injected into the anterior segment of monkey eyes in vivo and the effects on IOP and uveoscleral outflow will be assessed. PGF2a and MMP-3 production will also be assessed in the aqueous humor and in the media from human TM and CM cells transduced in vitro. A final objective will be to investigate in vivo the role of the neurotransmitter, nitric oxide, in regulating IOP, aqueous humor inflow and outflow. To simulate the dramatically reduced levels of nitric oxide synthase (NOS) in the anterior segment in glaucoma, a short-term equivalent of nitrergic denervation will be created in monkeys in vivo following topical or intracameral dosing with the non-selective NOS inhibitor L-NAME and/or the relatively selective neuronal nitric oxide synthase inhibitors 7-NI and/or NPLA. The effects on basal pupil diameter, refraction, aqueous humor formation and/or outflow facility will be determined. Conversely, these parameters will be measured in response to the nitric oxide donor, SNP, in the presence and absence of cholinergic and/or adrenergic blockade. These results will provide important insights for developing new therapies to lower IOP. Studies will be conducted in living nonhuman primates, in vitro in organ-cultured anterior segments and in trabecular meshwork cells and ciliary muscle cells in culture. The following techniques will be employed: aqueous humor formation by fluorophotometry; outflow facility by two-level constant pressure perfusion; uveoscleral outflow calculated from isotope dilution and accumulation measures; IOP by Goldmann applanation tonometry; refraction by Hartinger coincidence refractometry; pupil diameter via vernier calipers; gene expression in vivo by coexpression of GFP fluorescence detected by a custom designed research microscope system; gene product detection by Western blots and enzyme immunoassays and immunohistochemistry. PUBLIC HEALTH RELEVANCE: The proposed studies will investigate new approaches to lower intraocular pressure that may be further developed for glaucoma therapy. The first two objectives are to enhance aqueous humor outflow through the two known outflow pathways using novel small molecules (PI3K, ILK and Src kinase inhibitors) to enhance trabecular outflow, and gene therapy (lentiviral vector mediated delivery of genes that increase production of enzymes (prostaglandin synthase and stromelysin)) to increase uveoscleral outflow. A third goal is to better understand the role of an important neurotransmitter (nitric oxide) in regulating normal and drug induced changes in aqueous humor formation and drainage in order to provide the basis for novel glaucoma therapies.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 注：完整的资助标题是“体内和体外房水动力学研究，调节和老花眼的晶状体外方面” 目的：研究降低眼内压的新方法，眼内压是青光眼进展的重要危险因素。 ILK、PI3K 和 Src 激酶抑制剂可能会增强通过小梁网的流出，这些抑制剂已被证明可以改变培养物中小梁网 (TM) 细胞的细胞骨架和细胞粘附。这些化合物对眼压（IOP）和流出能力的影响将在器官培养的眼前段和猴眼体内进行研究。基因治疗方法将用于通过增强葡萄膜巩膜途径的流出来降低眼压。目前治疗青光眼最有效的药物疗法之一是基于前列腺素 (PG)F2a 衍生物，它与 FP 受体结合，在一定程度上增强基质金属蛋白酶 (MMP) 的合成。这反过来又改变了睫状肌（CM）和巩膜的细胞外环境的组成，导致葡萄膜巩膜流出增强。将携带 PGF 合酶和溶基质素 (MMP-3) 基因的慢病毒载体注射到体内猴眼前节，并评估对 IOP 和葡萄膜巩膜流出的影响。还将评估体外转导的人 TM 和 CM 细胞的房水和培养基中 PGF2a 和 MMP-3 的产生。最终目标是在体内研究神经递质一氧化氮在调节眼压、房水流入和流出中的作用。为了模拟青光眼眼前段一氧化氮合酶 (NOS) 水平的显着降低，在局部或前房内给药非选择性 NOS 抑制剂 L-NAME 和/或相对选择性神经元一氧化氮合酶抑制剂 7-NI 和/或 国家人民解放军。将确定对基础瞳孔直径、屈光、房水形成和/或流出设施的影响。相反，在存在和不存在胆碱能和/或肾上腺素能阻断的情况下，将响应一氧化氮供体SNP来测量这些参数。这些结果将为开发降低眼压的新疗法提供重要见解。研究将在活的非人类灵长类动物、体外器官培养的前段以及培养的小梁网细胞和睫状肌细胞中进行。将采用以下技术：通过荧光光度法形成房水；两级恒压灌注流出设施；根据同位素稀释和累积测量计算葡萄膜巩膜流出量；采用 Goldmann 压平眼压计测量 IOP；通过 Hartinger 符合折射仪进行折射；通过游标卡尺测量瞳孔直径；通过定制设计的研究显微镜系统检测 GFP 荧光的共表达来进行体内基因表达；通过蛋白质印迹、酶免疫分析和免疫组织化学检测基因产物。 公共健康相关性：拟议的研究将调查降低眼内压的新方法，这些方法可能会进一步开发用于青光眼治疗。前两个目标是通过两种已知的流出途径增强房水流出，使用新型小分子（PI3K、ILK 和 Src 激酶抑制剂）来增强小梁流出，以及基因治疗（慢病毒载体介导的基因递送，增加酶（前列腺素合酶和溶基质素）的产生）以增加葡萄膜巩膜流出。第三个目标是更好地了解重要的神经递质（一氧化氮）在调节正常和药物诱导的房水形成和排出变化中的作用，以便为新型青光眼治疗提供基础。