Imaging immune cell type and behavior in the living retina using adaptive optics

摘要： 免疫细胞对眼部炎症的反应参与了各种常见的视网膜疾病，这些疾病导致 失明，如青光眼、老年性黄斑变性、葡萄膜炎和糖尿病视网膜病变。然而， 循环和常驻免疫细胞的微观和半透明性质使此类细胞的研究具有挑战性. 活生生的眼睛。更复杂的是，免疫细胞在体内循环时移动速度很快(厘米/秒)。 在组织中，大血管和冰川的速度为微米/分钟。该项目将结合多项创新， 克服其中的许多挑战，研究活着的眼睛中单个免疫细胞对炎症的反应。这个 使用自适应光学扫描光检眼镜(AOSLO)对小鼠视网膜进行成像将在体内提供一种无创的 一种成像方法，可以分解单个免疫细胞，并在整个炎症过程中跟踪它们的行为。 通过我们的AOSLO，我们最近展示了快速和延时相结合的相位对比度 现在，摄像技术使我们能够单独使用和不使用近红外光来可视化这些微小的免疫细胞 荧光对比。因此，该项目将寻求在两种炎症模型中研究免疫细胞动力学。 活着的小鼠观察并跟踪细胞对启动、升级和解析的反应。虽然方法是 无创性和不需要使用荧光，我们将结合荧光确认特定的白细胞 参与炎症反应的人群。我们的项目针对跟踪动态的三个协同目标 使用两个已建立的模型研究炎症的性质：模型1)内毒素诱导的葡萄膜炎模型 眼玻璃体内注射脂多糖。这会导致一种急性的、短期的炎症。我们会 通过监测组织驻留的小胶质细胞和系统性炎症的行为来跟踪视网膜炎症的初始升级 早期应答者中性粒细胞。和模型2)在健康宿主小鼠中诱导的自身免疫性葡萄膜炎 注射一个亚群的对光感受器间视黄酸结合蛋白有反应的荧光CD4+T细胞 不同的供体小鼠。在这里，我们将描述这些外来反应性T细胞在健康视网膜中的行为，以及它们的 与宿主免疫系统的相互作用。在这两种炎症模型中，我们将描述炎症的启动、升级、 通过纵向跟踪免疫细胞的行为，从几小时到几天到 月份。最后，在第三个目标中，我们还将研究响应这些炎症模型的血液流动的变化。为 无论是内毒素介导的炎症模型还是自身免疫性炎症模型，我们都将监测结构和功能 视网膜血管系统从最大的动脉和静脉到单根毛细血管的变化以了解相互作用 在血液流动和免疫细胞进入视网膜之间。我们已经观察到血管反应不是 同质的，小动脉、小静脉和毛细血管对炎性损伤的反应方式不同，很可能 调节免疫细胞通道的位置、到达和类型。总而言之，炎症的纵向监测和 血流将利用高分辨率眼底镜的创新为视网膜免疫反应提供新的见解。

Abstract: Immune cell response to ocular inflammation is involved in various prevalent retinal diseases that lead to blindness such as glaucoma, age related macular degeneration, uveitis and diabetic retinopathy. However, the microscopic and translucent nature of circulating and resident immune cells has made study of such cells challenging in the living eye. Further complicating matters, immune cells move at fast speeds (centimeters/second) when circulating in big vessels and glacial pace at micrometers/minute when in tissue. This project will combine a number of innovations that overcome many of these challenges to study single immune cells in the living eye in their response to inflammation. The use of adaptive optics scanning light ophthalmoscope (AOSLO) to image mouse retina will provide a noninvasive in vivo imaging method that can resolve single immune cells and track their behavior through the entire course of inflammation. With our AOSLO, we have recently demonstrated that phase contrast combined with both fast and time-lapse videography now enables us to visualize these microscopic immune cells using near infrared light alone and without fluorescence contrast. Therefore, this project will seek to study immune cell dynamics in two models of inflammation in the living mouse eye and track the cellular responses to initiation, escalation and resolution. And while the approach is non-invasive and does not require use of fluorescence, we will combine fluorescence confirmation of specific leukocyte populations that contribute to the inflammatory response. Our project tackles three synergistic aims to track the dynamic nature of inflammation using two established models: Model 1) An endotoxin induced uveitis model using lipopolysaccharide (LPS) injection in vitreous of the eye. This results in an acute, short-term form of inflammation. We will track the initial escalation of inflammation in the retina by monitoring behavior of tissue resident microglia and systemic early responder neutrophils. And Model 2) An autoimmune uveitis condition that is induced in a healthy host mouse by injecting a subpopulation of fluorescent CD4+ T cells that are reactive to interphotoreceptor retinoid binding protein from a different donor mouse. Here, we will characterize the behavior of these foreign reactive T cells in healthy retina, and their interaction with the host immune system. In both inflammatory models, we will characterize the initiation, escalation, infiltration and resolution of immune cells in the retina by longitudinally tracking their behavior from hours to days to months. Finally, in a third aim, we will also study the changes in blood flow in response to these inflammatory models. For both endotoxin mediated and autoimmune inflammatory models described above, we will monitor structural and functional changes in retinal vasculature from the largest arteries and veins to single file flow capillaries to understand the interplay between blood flow and immune cell arrival into the retina. Already, we have observed that the vascular response is not homogeneous, and arterioles, venules and capillaries respond in different ways to the inflammatory insult and likely mediate the location, arrival and type of immune cell passage. Together, the longitudinal monitoring of inflammation and blood flow will provide new insights to the retinal immune response using innovations in high-resolution ophthalmoscopy.