Mechanotransduction in the Ciliary Muscle

睫状肌的机械传导

• 批准号: 8737265
• 负责人: ANDREW D PUCKER
• 金额: $ 13.77万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-30 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8737265
• 关键词: Adolescent; Animal Experiments; Animals; Applications Grants; Architecture; Atomic Force Microscopy; Biochemical Pathway; Biomechanics; Bladder; Cavia; Cell Size; Cell Volumes; Cells; Cellular Structures; Chickens; Child; Ciliary Muscle; Clinical; Crystalline Lens; Cues; Data; Development; Electrons; Enzyme-Linked Immunosorbent Assay; Equilibrium; Evidence based treatment; Exercise; Eye; Eyeglasses; Functional disorder; Future; Gene Expression; Glaucoma; Goals; Growth; Growth and Development function; Human; Hypertrophy; Image; Immunologist; In Vitro; Intervention; Kidney Diseases; Laboratories; Lead; Learning; Mammals; Measures; Mechanics; Mentors; Microscopy; Modeling; Molecular; Molecular Biology; Mus; Muscle Cells; Myopia; Myosin Light Chain Kinase; Optics; Organ; Pathway interactions; Peripheral; Physiology; Presbyopia; Primates; Prolate; RNA; Recruitment Activity; Research; Research Personnel; Research Support; Retina; Risk; Scanning; Shapes; Smooth Muscle; Smooth Muscle Actin Staining Method; Stretching; Techniques; Testing; Tissues; Training; Transmission Electron Microscopy; Up-Regulation; Visual; Work; base; cell growth; cell immortalization; effective therapy; emmetropization; genetic analysis; improved; insight; lens; multi-photon; multidisciplinary; prevent; public health relevance; research study; response; skills; tissue culture; transcriptome sequencing

DESCRIPTION (provided by applicant): The goal of this K08 application is to provide the candidate with the skills needed to become an independent translational researcher in the field of ciliary muscle physiology and juvenile myopia. The applicant will gain an understanding of how to induce myopia in the guinea pig, and he will receive advanced training in microscopy and genetic analysis in order to characterize smooth muscle growth and development. The applicant will also learn tissue culture immortalization and cell stretching techniques. The full mechanism of emmetropization, eye growth that produces a clear image on the retina, is unknown. Visually guided eye growth using foveal defocus cues has been the main paradigm, but treatments based on this hypothesis have resulted in little clinical benefit. Primate and human studies suggest that peripheral hyperopic defocus from a relatively prolate ocular shape may be a stronger stimulant to ocular growth than foveal defocus. An alternative hypothesis proposed by this laboratory is that the enlarged eye at risk for myopia places mechanical stretch on the ciliary muscle, which triggers specific biochemical pathways (mechanotransduction) leading to hypertrophy, altered ocular shape, and accelerated axial elongation. Increased expression of ¿-smooth muscle actin (¿ -SMA) along with myosin light chain kinase (MLCK) and increased cell size are known markers of hypertrophy. Experiments in Aim 1 will determine alterations in in vitro ciliary muscle cell structure, gene expression, and cell modulus after mechanical stretch of primary guinea pig and human ciliary muscle cells. Human immortal cells will then be developed and stretched, and results will be compared to the primary cells. Experiments in Aim 2 will determine the ex vivo consequences of experimentally induced myopia and mechanical stretch on guinea pig ciliary muscle. We will induce minus spectacle lens myopia monocularly, and we will biaxially stretch segments of ciliary muscle; results from both experiments will be compared to untreated ciliary muscle from the fellow eye. In Aims 1 and 2, we will measure cytoskeletal architecture and cell volume with Multi-Photon Confocal, Scanning Electron, and Transmission Electron Microscopy. Using Atomic Force Microscopy, we will evaluate mechanical changes in cells (modulus). We will measure alterations in ¿ -SMA and MLCK expression with ELISA. ¿-SMA and MLCK expression will be confirmed with RNA-Seq. RNA-Seq will also indicate if other pathways are altered in response to ocular growth and/or ciliary muscle stretch. We chose the guinea pig model because the guinea pig can accommodate, and because spectacle lenses can induce a significant amount of myopia. To achieve these Aims, the applicant has recruited a strong, multidisciplinary mentoring team, which comprises a primary mentor, Dr. Donald Mutti, who conducts human juvenile myopia research, and three co-mentors: Dr. Kirk McHugh, a cell and molecular biologist who is an expert in the study of smooth muscle in bladder and obstructive (stretch) nephropathy in the mouse; Dr. Andy Fischer, a neuroscientist who is also an expert in chicken myopia development and molecular biology, and Dr. Sudha Agarwal, an immunologist who is an expert in exercise mechanotransduction. This K08-supported research will form the basis for a future R01 grant application for independent research aimed at creating targeted interventions for treating and preventing ciliary muscle related myopic growth.

描述（由应用程序提供）：该K08应用程序的目标是为候选人提供成为睫状肌肉生理和少年近视领域的独立翻译研究人员所需的技能。该应用将了解如何在豚鼠中诱导近视，他将接受显微镜和遗传分析的高级培训，以表征平滑的肌肉生长和发育。该应用还将学习组织培养的免疫化和细胞拉伸技术。未知的透气化的完整机制，即在视网膜上产生清晰图像的眼睛生长。使用中央凹形提示的目视引导的眼睛生长一直是主要的范式，但是基于此假设的治疗方法几乎没有临床益处。灵长类动物和人类的研究表明，来自相对pr酸眼形的外围触角脱焦，可能比中央凹形散热器具有强烈的刺激性。该实验室提出的另一种假设是，近视肌肉的机械伸展的风险增加，这会触发特定的生化途径（机械转导），从而导致肥大，眼部形状改变和加速轴向伸长。麦片肌肉肌动蛋白（� -SMA）以及肌球蛋白轻链激酶（MLCK）的表达增加和细胞大小增加是已知的肥大标志物。 AIM 1中的实验将确定在原发性豚鼠和人纤毛肌肉细胞机械延伸后体外纤毛肌肉细胞结构，基因表达和细胞模量的改变。然后将开发和拉伸人类不朽的细胞，并将结果与​​原代细胞进行比较。 AIM 2中的实验将确定实验诱导的近视和机械伸展的豚鼠纤毛肌肉的后果。我们将单眼诱导减去镜面近视，并将双轴伸展睫状肌肉。两种实验的结果将与其他眼睛的未处理睫状肌肉进行比较。在目标1和2中，我们将用多光子共聚焦，扫描电子和透射电子显微镜测量细胞骨架结构和细胞体积。使用原子力显微镜，我们将评估细胞的机械变化（模量）。我们将用ELISA测量� -SMA和MLCK表达的变化。 RNA -Seq将确认 - SMA和MLCK表达。 RNA-seq还将指示其他途径是否会因眼部生长和/或睫状肌肉拉伸而改变。我们之所以选择豚鼠模型，是因为豚鼠可以容纳，并且因为眼镜镜可以诱导大量的近视。为了实现这些目标，适用的招募了一个强大的多学科指导团队，该团队包括主要的导师Donald Mutti博士，他进行了人类青少年近视研究和三个同事：Kirk McHugh博士，一个细胞和分子生物学家，他是Bladder and theStractive nephrop的肌肉研究中的专家（神经科学家安迪·菲舍尔（Andy Fischer）博士也是鸡肉近视开发和分子生物学专家，以及免疫学家Sudha Agarwal博士，他是运动机械转导的专家。这项由K08支持的研究将成为未来R01赠款申请的独立研究申请的基础，旨在创建针对性的干预措施，以治疗和预防睫状肌肉相关的近视增长。