Novel tracers for in vivo studies of waste transport by fluid flows in the brain

总结 假设间质流体以对流的形式运动有助于β淀粉样蛋白（Aβ）和废物 通过将细胞外蛋白带出大脑来清除。这个系统的故障可能会导致 阿尔茨海默病（AD），但这一理论的某些方面仍然是推测性的.例如，虽然流入 虽然有大量文献证明脑实质中有液体流出，但流出路径不太清楚。外源性示踪剂和 脑蛋白质从组织进入神经系统仍然是一个悬而未决的问题。至少有两个想法。的 胶质淋巴/淋巴转运机制预测来自小动脉血管周围空间的液体流入 通过小静脉血管周围空间的流出来平衡。壁间动脉周围引流 （iPad）camp已经确定，细胞外染料和蛋白质聚集在基底膜附近， 小动脉血管，并假设该室提供了废物清除的管道。iPad理念 预测废物沿小动脉沿着排出，与胶质淋巴预测的方向相反，尽管两者都 都被认为是进入神经系统的在研究这种运输方面存在实验挑战， 流体运动通常通过注入改变压力平衡的外源指示剂来跟踪， 用于急性测量。为了克服这些障碍，新的基因工程分泌示踪剂 模拟内源性蛋白质产生且不因注射产生的伪影而复杂化的GEST具有以下优点： 被原型化了。显微注射腺相关病毒（AAV）载体驱动神经元中GEST的表达 在大脑的一个小体积内。利用荧光成像技术研究分泌型荧光蛋白在大肠杆菌中的分布 组织切片和活体成像都可以用于绘制运输图。因为这种示踪剂是由 在注射病毒载体后很长时间，脑中的压力和流体流动不受干扰。在 此外，蛋白质如Aβ也由神经元分泌，因此GEST分布将复制天然的 蛋白质运输，而不是运动的外源注入示踪剂。虽然这一战略显示出希望， 需要新的能力来解决关于废物蛋白质的流体运输的具体问题。第一、 脑中的间质流体流速需要更快的分钟级时间分辨率。一种可光活化的 一个版本的GEST被提出，这样一个体积的示踪剂可以被“突出显示”，并跟踪它移动通过 脑组织运动的方向和速度可以支持或反驳不同的流动介导的理论 废物清理其次，在流速较快或浓度较低的区域，如 那些人为了检测分泌蛋白所采取的路径，开发了一种新的分泌型、细胞渗透型Cre。 这将被用于停止呼吸的报告动物，以打开遇到液体的细胞中的报告基因 带着克雷因为基因激活是永久性的，这提供了暴露于 对低浓度具有良好的灵敏度。最后，这些新的检测方法将用于研究 在AD小鼠模型中转运改变。

SUMMARY Interstitial fluid motion in the form of convective flow is hypothesized to contribute to amyloid beta (Aβ) and waste clearance by carrying extracellular proteins out of the brain. Failures of this system could contribute to Alzheimer’s disease (AD), but are some aspects of this theory are still speculative. For examples, while the inflow of fluid to the brain parenchyma is well documented, the outflow path is less clear. How exogenous tracers and brain proteins get from the tissue into the lymphatics is still an open question. There are at least two ideas. The glymphatic/lymphatic transport mechanism predicts that the inflow of fluid from the arteriole perivascular spaces would be balanced by an outflow through the venular perivascular spaces. The Intermural Peri-Arterial Drainage (IPAD) camp has identified that extracellular dyes and proteins aggregate near the basement membrane of the arteriole vessels and hypothesizes that this compartment provides a conduit for waste clearance. The IPAD idea predicts that waste exits along the arterioles, in the opposite direction of the glymphatic prediction, although both are thought to feed into the lymphatics. There are experimental challenges in studying this transport because fluid motion is usually tracked by injection of exogenous indicators that alter pressure balances and can only be used for acute measurements. To overcome these barriers, new genetically engineered secreted tracers (GESTs) which mimic endogenous protein production and are not complicated by artifacts from injection have been prototyped. Microinjected adeno-associated virus (AAV) vectors drive the expression of GESTs in neurons within a small volume of brain. The distribution of the secreted fluorescent protein using fluorescence imaging in both tissue sections and intravital imaging can be used to map the transport. Because this tracer is produced by neurons long after the injection of the viral vectors, pressures and fluid flow in the brain are not disturbed. In addition, proteins such as Aβ are also secreted by neurons, so that the GEST distribution will replicate native protein transport rather than the motion of exogenously injected tracers. While this strategy has shown promise, new capabilities are needed to address the specific questions about fluid transport of waste proteins. First, interstitial fluid flow rates in the brain require faster time resolution on the scale of minutes. A photoactivable version of the GEST is proposed so that a volume of tracer can be “highlighted” and tracked as it moves through the brain tissue. The direction and speed of motion could support or refute the different theories of flow-mediated waste clearance. Second, it is not possible to detect GESTs in areas of faster flow or low concentration such as the lymphatics. To detect the paths taken by secreted proteins, a new secreted, cell-permeant Cre is developed. This will be used in floxed-stopped reporter animals to turn on reporter genes in cells that encounter fluids carrying the Cre. Because the gene activation is permanent, this provides a cumulative assay of exposure to Cre with good sensitivity to low concentrations. Finally, these new assays will be used to investigate whether transport is altered in AD mouse models.