AQUEOUS HUMOR DYNAMICS STUDIES IN VIVO AND IN VITRO

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 研究中心，而研究中心不一定是研究者所在的机构。 注：完整的授权题目是"眼内和眼外调节和老花眼的眼内和眼外晶状体动态研究" 目的：探讨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激酶抑制剂将潜在地增强通过小梁网的流出，这些抑制剂已显示出改变培养物中小梁网（TM）细胞的细胞骨架和细胞粘附。这些化合物对眼内压（IOP）和外流设施的影响将在器官培养的前段和猴眼体内进行研究。基因治疗方法将用于通过增强经由葡萄膜巩膜途径的流出来降低IOP。目前最有效的青光眼药物疗法之一是基于前列腺素（PG）F2a的衍生物，其结合FP受体，部分导致基质金属蛋白酶（MMP）合成增强。这又改变了睫状肌（CM）和巩膜的细胞外环境的组成，导致葡萄膜巩膜流出的增强。将携带PGF合酶和基质溶解素（MMP-3）基因的慢病毒载体在体内注射到猴眼睛的前段中，并评估对IOP和葡萄膜巩膜流出的影响。还将在来自体外转导的人TM和CM细胞的房水和培养基中评估PGF 2a和MMP-3的产生。最后一个目标将是在体内研究神经递质，一氧化氮，在调节眼内压，房水流入和流出的作用。为了模拟青光眼中前段中一氧化氮合酶（NOS）水平的显著降低，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型一氧化氮合酶抑制剂7-NI和/或NPLA后，将在猴体内产生短期等效的氮能去神经支配。将确定对基础瞳孔直径、屈光、房水形成和/或流出功能的影响。相反，这些参数将在存在和不存在胆碱能和/或肾上腺素能阻断的情况下响应于一氧化氮供体SNP来测量。这些结果将为开发新的治疗方法以降低IOP提供重要的见解。研究将在活体非人灵长类动物、体外器官培养的眼前节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;通过两级恒压灌注的流出设施;从同位素稀释和累积测量计算的葡萄膜巩膜流出;通过Goldmann压平眼压计的IOP;通过Hartinger符合折射计的折射;通过游标卡尺的瞳孔直径;通过由定制设计的研究显微镜系统检测的GFP荧光的共表达的体内基因表达;通过蛋白质印迹和酶免疫测定和免疫组织化学检测基因产物。 公共卫生相关性：拟议的研究将探讨新的方法来降低眼内压，可能会进一步发展青光眼治疗。前两个目的是通过两个已知的流出途径，使用新的小分子（PI3K、ILK和Src激酶抑制剂）来增强房水流出，以增强小梁流出，以及基因治疗（慢病毒载体介导的增加酶（前列腺素合酶和基质溶解素）产生的基因的递送）来增加葡萄膜巩膜流出。第三个目标是更好地理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变化中的作用，以便为新型青光眼治疗提供基础。

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.