Microscale Oxygenation Mapping During Stroke

描述（由申请人提供）：缺血性卒中后脑损伤的程度是缺氧程度的时间和空间依赖性函数。虽然缺血性损伤的氧阈值已研究多年，但局部氧水平与神经元结构和功能改变之间的详细关系在体内研究中极具挑战性。树突棘特别令人感兴趣，因为它们 可能在脑损伤后的皮质可塑性中起关键作用，并且是 康复训练策略。然而，氧水平在单树突水平上的作用知之甚少，这在很大程度上是由于缺乏能够在三维中以微米级分辨率量化氧合的方法。尽管氧敏电极能够在单个位置进行精确的氧气测量，但其侵入性使其无法进行慢性研究和绘制氧气分布图。氧敏感磷光染料的最新进展为结合多光子激发在啮齿动物模型中进行非侵入性氧映射开辟了新的可能性。尽管这种方法有可能对微血管氧合产生新的见解，但低信号水平 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，并使用该系统量化局部氧水平和脑缺血后树突状重塑之间的关系。氧气测绘的技术进步将涉及两个组成部分。首先，我们将通过开发空间复用再生放大器激发源来提高氧敏感探针的磷光信号水平，该空间复用再生放大器激发源将产生几个空间偏移的激发点。最小的权衡 在空间分辨率上的增加将被在时间分辨率和成像深度上的相当大的增益所抵消。其次，我们将改进一种方法，使用微泡辅助聚焦超声瞬时破坏血脑屏障，这将允许将染料输送到血管外空间。这些进展将使长期测量氧气水平与微米级分辨率。然后，我们将使用这些工具来量化在基线条件下和单个小动脉闭塞后从表面和穿透小动脉通过毛细血管和引流小静脉的纵向氧梯度。最后，我们将量化在急性和慢性缺血过程中单个树突周围的氧水平和这些树突的形态变化之间的关系。

DESCRIPTION (provided by applicant): 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.