AQUEOUS HUMOR DYNAMICS STUDIES IN VIVO AND IN VITRO

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 注：基金的完整标题是“体内和体外的水幽默动力学研究，调节的晶状体外方面和老花眼”。 目的：探索降低眼压的新方法，眼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加小梁网的流出，它们已被证明可以改变培养中的小梁网细胞的细胞骨架和细胞黏附。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节和在体猴眼中进行研究。基因治疗方法将被用来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之一是基于前列腺素(PG)F2a的衍生物，它与FP受体结合，部分地导致基质金属蛋白酶(MMP)的合成增强。这反过来改变了睫状肌(CM)和巩膜的细胞外环境的组成，导致葡萄膜巩膜流出增加。携带前列腺素F合成酶和基质分解酶基因的慢病毒载体将被注射到猴眼的眼前段，并将评估其对眼压和葡萄膜巩膜流出的影响。在体外转导的人TM和CM细胞的房水和培养液中也将评估PGF2a和MMP-3的产生。最后一个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节一氧化氮合酶水平的显著降低，在体猴体内局部或皮下注射非选择性一氧化氮合酶抑制剂L-NAME和/或相对选择性的神经元型一氧化氮合酶抑制剂7-NI和/或NplA后，将产生短期相当于氮能失神经的现象。将确定对基础瞳孔直径、屈光度、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/或肾上腺素能阻滞剂的情况下，这些参数将根据一氧化氮供体SNP的反应进行测量。这些结果将为开发降低眼压的新疗法提供重要的见解。研究将在活体非人类灵长类动物、体外器官培养的前节以及培养的小梁网络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；通过两级恒压灌流实现流出；通过同位素稀释和聚集测量计算葡萄膜巩膜流出量；通过Goldmann压平眼压计计算眼压；通过Hartinger符合屈光度计测量屈光度；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荧光的共表达在体内的基因表达；通过Western blotts和酶免疫分析以及免疫组织化学方法检测基因产物。 与公共健康相关：拟议的研究将探索降低眼压的新方法，这些方法可能会进一步开发用于青光眼治疗。前两个目标是使用新的小分子(PI3K、ILK和Src激酶抑制剂)来促进小梁流出，以及使用基因治疗(慢病毒载体介导的增加酶(前列腺素合成酶和基质分解素)产生的基因)来增加葡萄膜巩膜流出，从而通过两种已知的流出途径促进房水流出。第三个目标是更好地了解一种重要的神经递质(一氧化氮)在调节正常和药物诱导的房水形成和引流中的作用，以便为青光眼的新疗法提供基础。

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.