Microscale Oxygenation Mapping During Stroke

项目摘要 缺血性脑卒中后脑损伤的程度是一个时间和空间依赖性的功能， 缺氧的症状虽然已经研究了缺血性损伤的氧阈值， 多年来，局部氧水平和神经元结构改变之间的详细关系 在体内研究其功能是极具挑战性的。树突棘特别令人感兴趣 因为它们可能在脑损伤后皮质可塑性中起关键作用， 康复训练策略。然而，氧水平在单枝晶水平上的作用是 了解甚少，在很大程度上是由于缺乏能够用微米量化氧合的方法。 在三个维度上缩放分辨率。虽然氧敏电极能够精确地测量氧气， 在单一位置测量，其侵入性使其无法进行慢性研究， 绘制氧气分布图氧敏感磷光染料的最新进展已经开启了 当与多光子结合时，为啮齿动物模型的非侵入性氧标测提供了新的可能性 激发尽管这种方法有可能对微血管氧合产生新的见解，但低 信号电平限制氧合测量的速度和穿透深度。因此 本项目的目标是开发一种改进的成像方法，用于三维测定 血管内和血管外氧合水平，同时进行树突状形态成像， 用这个系统来量化局部氧水平和树突重塑之间的关系 脑缺血后。氧气测绘的技术进步将涉及两个方面， 件.首先，我们将通过以下方式提高氧敏感探针的磷光信号水平： 开发空间复用再生放大器激发源， 空间偏移激发斑点。空间分辨率的最小折衷将被 在时间分辨率和成像深度上有相当大的增益。第二，我们将改进一种方法， 使用微泡辅助聚焦超声短暂破坏血脑屏障， 允许将染料输送到血管外空间。这些进步将使长期 以微米级分辨率测量氧气水平。然后，我们将使用这些工具来量化 纵向氧梯度从表面和穿透微动脉通过毛细血管和排水 在基线条件下和在闭塞单个小动脉后的小静脉。最后，我们将量化 个别树突周围的氧水平和这些树突中的形态变化之间的关系 树突在急性和慢性缺血。

PROJECT SUMMARY The extent of brain injury following ischemic stroke is a time and space dependent function of the degree of oxygen deprivation. Although oxygen thresholds for ischemic damage have been investigated for many years, the detailed relationship between local oxygen levels and alterations in neuronal structure and function has been extremely challenging to study in vivo. Dendritic spines are of particular interest since they likely play a key role in cortical plasticity following brain injury and are a potential target for rehabilitative training strategies. However, the role of oxygen levels at the level of single dendrites is poorly understood, in large part due to a lack of methods capable of quantifying oxygenation with micron scale resolution in three dimensions. Although oxygen sensitive electrodes enable precise oxygen measurement at a single location, their invasive nature precludes them from chronic studies and for mapping oxygen distributions. Recent advances in oxygen sensitive phosphorescent dyes have opened up new possibilities for noninvasive oxygen mapping in rodent models when combined with multiphoton excitation. Despite the potential for this approach to yield new insight into microvascular oxygenation, low signals levels limit the speed and penetration depth of the oxygenation measurements. Therefore, the goal of this project is to develop an improved imaging method for three-dimensional determination of intra- and extra-vascular oxygenation levels simultaneously with imaging of dendritic morphology and to use this system to quantify the relationship between local oxygen levels and dendritic remodeling following cerebral ischemia. The technological advances in oxygen mapping will involve two components. First, we will improve the phosphorescence signal levels of the oxygen sensitive probe by developing a spatially multiplexed regenerative amplifier excitation source that will produce several spatially offset excitation spots. The minimal tradeoff in spatial resolution will be offset by the considerable gain in temporal resolution and imaging depth. Second, we will refine a method to transiently disrupt the blood brain barrier using microbubble-assisted focused ultrasound, which will permit delivery of the dye to the extravascular space. Together these advances will enable chronic measurement of oxygen levels with micron scale resolution. We will then use these tools to quantify the longitudinal oxygen gradients from surface and penetrating arterioles through capillaries and draining venules under baseline conditions and after occlusion of a single arteriole. Finally, we will quantify the relationship between oxygen levels surrounding individual dendrites and morphological changes in these dendrites during both acute and chronic ischemia.