Mechanotransduction in the Ciliary Muscle

睫状肌的机械传导

基本信息

批准号：
8487047
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
项目类别：
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/8487047
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

项目摘要

ABSTRACT: 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.

抽象的：该K08应用程序的目标是为候选人提供成为独立的技能睫状肌肉生理学和青少年近视领域的转化研究员。申请人将获得了解如何在豚鼠中诱导近视，他将接受显微镜的高级培训和遗传分析以表征平滑肌的生长和发育。申请人也将学习组织培养的免疫化和细胞拉伸技术。润气化的完整机制，眼睛在视网膜上产生清晰图像的生长尚不清楚。视觉引导的眼睛使用凹形散焦提示是主要的范式，但基于此假设的治疗几乎没有临床好处。灵长类动物和人类研究表明，来自相对prate眼的外围性触角散异焦与中央凹形散焦相比，形状可能是眼部生长的刺激性。提出的另一种假设该实验室是，近视的风险扩大，将机械肌肉机械伸展到这会触发特定的生化途径（机械转移）导致肥大，改变眼形状和加速轴向伸长。增加表达�-光滑肌肉肌动蛋白（�-SMA）以及肌球蛋白轻链激酶（MLCK）和增加的细胞大小是已知的肥大标志物。实验 AIM 1将确定体外睫状肌肉细胞结构，基因表达和细胞模量的改变原发性豚鼠和人类睫状肌细胞的机械拉伸。然后，人类不朽的细胞将是开发和拉伸，结果将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体内后果睫状肌肉。我们将单眼诱导近视镜头近视，我们将双轴拉伸段纤毛肌肉；将两个实验的结果与研究员的未处理睫状肌肉进行比较眼睛。在AIMS 1和2中，我们将用多光子共焦测量细胞骨架结构和细胞体积，扫描电子和透射电子显微镜。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。 RNA -Seq将确认 - SMA和MLCK表达。 RNA-seq也将指示其他响应眼部生长和/或睫状肌肉拉伸的途径改变了途径。我们选择豚鼠型号是因为豚鼠可以容纳，并且奇观镜头可以引起显着的近视的数量。为了实现这些目标，适用的招募了强大的多学科指导由主要心理组成的团队唐纳德·穆蒂（Donald Mutti）博士进行了人类少年近视研究和三个共同发源地：柯克·麦克休（Kirk McHugh）博士，一位细胞和分子生物学家，他是平滑研究的专家膀胱中的肌肉和阻塞性（拉伸）肾病；神经科学家安迪·菲舍尔（Andy Fischer）博士还是鸡肉近视开发和分子生物学的专家，以及Sudha Agarwal博士免疫学家是运动机械转导专家。这项由K08支持的研究将构成未来R01授予申请的独立研究申请的基础，旨在创建针对性的干预措施治疗和预防睫状肌肉相关的近视生长。