In vivo 2-photon imaging of retinal biochemistry before and after retinal organoid transplantation

项目摘要 求职者和职业发展计划：布朗博士是一名从事玻璃体视网膜手术的助理教授 并在加州大学欧文分校担任工程师和内科科学家。布朗博士 作为一名博士生，他建立了工程和生物科学的基本技能。他有平衡的临床职责 他的实验室研究活动和。他的长期职业目标是设计功能成像工具 这将促进对早期和晚期眼病的了解，并促进所需的治疗 治疗人类。到目前为止，他的训练一直只使用体外成像。为了实现他的职业目标，他 请求对开发体内功能成像工具和正确开发分子工具的培训提供支持 解释从动物模型观察到的双光子成像(2pi)。这一K08奖项将使布朗博士能够 培养他在高级成像、荧光显微镜和视网膜细胞方面的科学和专业技能 生物学应用于活体。布朗博士和他的共同导师帕尔切夫斯基博士、塞勒博士和库普曼博士 制定了实践策略，通过相关的课程作业、教学、 实验室技术和加州大学欧文分校的合作。培训计划将为布朗博士提交 R01级独立研究和优化功能性视网膜成像作为治疗工具的建议 发现号。 研究计划：许多致盲的视网膜疾病，如老年性黄斑变性，都是其后果 生化功能障碍以及炎症和细胞死亡的次要影响。常规临床工具 提供有关视网膜的有价值的结构信息，但无法可视化视网膜功能。非线性 光学成像现在可以在体外揭示亚细胞生物化学，并已成为研究的可靠工具 视网膜疾病。2PI在小鼠模型中表现出能量和光周期代谢的亚细胞变化。 细胞替代疗法已经成为晚期视力丧失和临床前试验的候选疗法。 在大鼠身上已经显示出视觉功能的恢复。了解视网膜类器官的代谢功能 (RTOG)在盲鼠体内移植前后，我们将按照概述的那样在体外和体内使用功能性2PI 在以下具体目标中。1)用2pi研究RTOG的成熟度，并将功能成像数据与 体外细胞特异性报道和活体后分子标记，2)确定IN的功能成像生物标记物 用2pi研究正常大鼠和视网膜变性大鼠的体内代谢，3)体内功能2pi的研究 大鼠移植rtOgs以确定功能成像生物标志物和相关成像结果的变化 进行视功能测试。完成这些目标将产生特定细胞的时间分辨代谢细节 发展中的RtOgs人群，利用关于健康视网膜的视网膜生物化学的体内新信息， 病变的视网膜，以及病变的视网膜接受视网膜薄片移植。这些发现将指导我们 功能成像和组织替代疗法在人类中的未来翻译。

Project Summary Candidate and Career Development Plan: Dr. Browne is an assistant professor practicing vitreoretinal surgery and functioning as an engineer and physician-scientist at the University of California, Irvine. Dr. Browne established his engineering and basic biological sciences skills as a PhD student. He has balanced clinical duties with his laboratory research activities and. His long-term career goals are to engineer functional imaging tools that will advance the understanding of early and advanced eye diseases and facilitate the therapies needed to treat humans. His training thus far has been using in vitro imaging alone. To achieve his career goals, he is requesting support for training to develop functional imaging tools in vivo and the molecular tools to correctly interpret Two-photon imaging (2PI) observations from animal models. This K08 award will enable Dr. Browne to develop his scientific and professional skills in advanced imaging, fluorescence microscopy, and retinal cellular biology applied in vivo. Dr. Browne and his co-mentors, Drs. Palczewski, Seiler and Kuppermann, have developed a hands-on strategy to fulfill the training requirements through relevant course work, didactics, laboratory techniques, and collaborations at UC Irvine. The training program will prepare Dr. Browne to submit R01-level proposals to independently investigate and optimize functional retinal imaging as a tool for therapeutic discovery. Research Plan: Many blinding retinal conditions, like age-related macular degeneration, are the consequence of biochemical dysfunction and the secondary effects of inflammation and cell death. Conventional clinical tools provide valuable structural information about the retina but are unable to visualize retinal function. Non-linear optical imaging can now reveal subcellular biochemistry in vitro and has emerged as a reliable tool to study retinal disease. 2PI in mouse models demonstrates subcellular changes in energy and light cycle metabolism. Cell replacement therapy has emerged as a therapeutic candidate for advanced vision loss, and preclinical trials in rats have demonstrated visual function restoration. To understand metabolic function in retinal organoids (RtOg) before and after transplantation into blind rats, we will employ functional 2PI in vitro and in vivo as outlined in the following specific aims. 1) Investigate RtOg maturation with 2PI and correlate functional imaging data with cell-specific reporters in vitro and molecular signatures post vivo, 2) Identify functional imaging biomarkers for in vivo metabolism using 2PI of normal rats and rat with retinal degeneration, 3) Study in vivo functional 2PI of transplanted RtOgs in rats to identify alterations in functional imaging biomarkers and correlate imaging findings with visual function testing. Completion of these aims will yield time-resolved metabolic detail for specific cell populations in developing RtOgs, avail novel in vivo information about retinal biochemistry in healthy retina, diseased retina, and diseased retinas treated with retinal sheet transplantation. These discoveries will guide future translation of both functional imaging and tissue replacement therapy in humans.