Integrating TPM and PAM to examine the metabolic underpinning of neurovascular repair after stroke

项目摘要 每年，美国有超过80万人患有中风。虽然绝大多数幸存下来， 在急性事件中，超过一半的幸存者在运动、感觉或认知功能方面遭受中度至重度损伤。 因此，中风仍然是长期残疾的主要原因，每年花费超过340亿美元， 直接医疗成本和间接成本（生产力损失）。面对这种巨大的疾病 然而，由于负担过重，几乎没有改善中风恢复的疗法。大脑有一些内在的修复能力，但 我们对潜在机制的了解仍然非常有限。最近的研究表明，成功的 从中风损伤中恢复需要神经血管重塑以重组受损的脑网络。的确， 电路修复和由此产生的重新映射对于中风恢复是必要的。此外，脑血管重塑 脑氧代谢的变化在动物和中风后患者中观察到， 改善结果。可能需要神经修复和脑血管重塑的紧密协调， 满足大脑修复的能量需求。然而，神经血管修复的时空协调和 中风后伴随的氧代谢变化仍然不完全清楚。我们寻求答案 通过开发一种新的双模式活体成像技术， 荧光显微镜（TPM）和多参数光声显微镜（PAM），用于高分辨率，时间- 脑卒中后神经血管修复和代谢变化的延时和综合成像。为此我们 已经开发了原型TPM-PAM系统和具有双透明性的新颅窗（即，光和 超声波）、长寿命以及与清醒脑成像的兼容性。在坚实的科学基础上， 拟议的项目将侧重于开发用于纵向成像的高灵敏度TPM-PAM系统 中风后神经修复和脑血管重塑的时空相互作用，以及动态 神经元活动、血流和血氧供应之间的耦合成像，在单神经元单 毛细血管水平。本研究有三个具体目标：（1）开发一种光学 透明且声敏的微谐振器，用于高灵敏度地集成TPM和PAM，（2） 开发并验证用于GCaMP小鼠神经血管成像的基于微谐振器的TPM-PAM，以及（3） 确定功能性血管修复和神经回路修复之间的时空关系， 中风通过TPM-PAM的开发和应用， 揭示了有前途的新的治疗靶点，以促进功能恢复。

PROJECT SUMMARY Each year, over 800,000 people in the United States suffer from a stroke. Although the vast majority survive the acute event, over half of survivors suffer moderate to severe impairment in motor, sensory, or cognitive function. As a consequence, stroke remains the leading cause of long-term disability, costing over $34 billion annually in direct medical costs and indirect costs (lost productivity) in the United States. In the face of this enormous disease burden, there are few therapies to improve stroke recovery. The brain has some intrinsic capacity for repair, but our understanding of the underlying mechanisms remains very limited. Recent studies suggest that a successful recovery from stroke injury requires neurovascular remodeling to reorganize the damaged brain network. Indeed, circuit repair and the resultant remapping is essential for stroke recovery. Moreover, cerebrovascular remodeling and changes in cerebral oxygen metabolism are observed in animals and patients after stroke and are associated with improved outcomes. Tight coordination of neural repair and cerebrovascular remodeling is likely required to meet energy requirements of brain repair. However, the spatiotemporal coordination of neurovascular repair and the attendant changes in oxygen metabolism after stroke remain incompletely understood. We seek to answer these important questions by developing a new dual-modal intravital imaging technique that integrates 2-photon fluorescence microscopy (TPM) and multi-parametric photoacoustic microscopy (PAM) for high-resolution, time- lapse and comprehensive imaging of neurovascular repair and metabolic changes after stroke. To this end, we have developed a prototype TPM-PAM system and a new cranial window with dual transparency (i.e., light and ultrasound), long lifetime, and compatibility for awake-brain imaging. Building on the strong scientific basis, this proposed project will focus on the development of a high-sensitivity TPM-PAM system for longitudinal imaging of the spatiotemporal interplay of post-stroke neural repair and cerebrovascular remodeling, as well as dynamic imaging of the coupling between neuronal activity, blood flow, and blood oxygen supply, at single-neuron single- capillary level in the awake mouse brain. The proposed research has three specific aims: (1) develop an optically transparent and acoustically sensitive microresonator for integration of TPM and PAM with high sensitivity, (2) develop and validate the microresonator-based TPM-PAM for neurovascular imaging in GCaMP mice, and (3) determine the spatiotemporal relationship between functional vascular repair and neuronal circuit repair after stroke. Advancing our understanding of stroke repair through the development and application of TPM-PAM may reveal promising new therapeutic targets to enhance functional recovery.