In vivo tools for analyzing interstitial fluid flow

间质液（ISF）流具有许多功能，包括维持离子流。 平衡、冲洗废物并为细胞信号和 细胞最近，使用多光子显微镜，这使得在体内研究， 细胞分辨率，导致了新的发现，特别是在大脑中，关于动力学 和解剖结构参与ISF流。值得注意的是，ISF流在处理 阿尔茨海默病中的蛋白质积累，并受睡眠调节。虽然大部分 虽然取得了一些进展，但对一些基本问题仍存在争议。 关于ISF的流程这在很大程度上可能是由于实验中的并发症。 方法.迄今为止的研究需要注射示踪剂，示踪剂可以通过 多光子显微术或其他成像方法如MRI。但其工艺 示踪剂的注入本身可能会影响流动， 脑内压力。此外，注入的示踪剂不能模拟 蛋白质废物和细胞因子。研究仅限于表面部位 可以通过当代成像技术来访问。该建议产生光学 以及利用体内多光子来解决这些短时间内的问题的生物学工具 显微镜首先，在原位产生示踪剂的新方法中， 感兴趣的细胞将被转导，以便它们将荧光蛋白分泌到细胞中。 细胞外间隙这将被用来解决现有的争议的路线， ISF在大脑中流动。第二，新发现的大脑神经元被认为 连接到作为ISF导管的前房或房旁空间。这项工作将使用 新的分泌示踪剂来回答这些淋巴通道是否以及如何连接到 这些空间。这种流体流动在不同的条件下可能会改变，因此将对此进行研究 在模拟睡眠和清醒的正常功能中，以及在中风模型中。第三、 三光子显微镜现在能够比传统的双光子显微镜更深入地成像 显微镜这使得能够对以前无法访问的解剖结构进行成像。这项工作将 研究海马中的ISF流动，海马是记忆和认知的关键大脑结构， 似乎特别容易受到ISF的干扰。这项工作将建立 新的工具，可以使新的实验，以解决在许多系统中的ISF。

Interstitial fluid (ISF) flow has many functions including the maintenance of ionic balances, flushing of wastes and providing a route for migration of both cell signals and cells. Recently, the use of multiphoton microscopy, which enables in vivo studies with cellular resolution, has resulted in novel findings, especially in the brain, about dynamics and anatomy involved in ISF flow. Notably, ISF flow may be critical in dealing with protein accumulation in Alzheimer's disease and is regulated by sleep. Although much progress has been made, there remains controversy about some of the fundamentals regarding ISF flow. Much of this may be due to complications in the experimental methods. Studies to date require the injection of tracers which can be imaged by multiphoton microscopy or other imaging methods such as MRI. However, the process of injection of the tracers may itself affect the flow due to the delicate balance of pressures within the brain. In addition, injected tracers do not mimic the origin of proteins, wastes and cytokines made by the brain. Studies are limited to superficial sites accessible by current generation imaging technologies. This proposal generates optical and biological tools that address these short comings using in vivo multiphoton microscopy. First, in a new way to generate tracers in situ, cells within the tissue of interests will be transduced so that they secrete fluorescent proteins into the extracellular space. This will be used to resolve existing controversies about the route of ISF flow within the brain. Second, the newly discovered brain lymphatics are thought to link to the peri or paravacular spaces that serve as conduits for ISF. This work will use the new secrete tracers to answer whether and how these lymphatic channels link to the these spaces. This fluid flow may be altered in different conditions, so this will be studied in normal function mimicking sleep and waking, as well as with a stroke model. Third, three-photon microscopy now enables much deeper imaging than traditional two-photon microscopy. This enables imaging of anatomy previously not accessible. This work will study ISF flow in the hippocampus, a critical brain structure in memory and cognition, that seems to be particularly vulnerable to disruptions to ISF. This work will establish novel tools that can enable new experiments to address ISF in many systems.