Modeling cerebral reperfusion injury using photoacoustic microscopy and oxygen-sensing nanoparticles

This project explores a novel non-invasive, dynamic imaging approach capable of quantifying functional changes in the brain following reperfusion after stroke.

Stroke is the third leading cause of death and the leading cause of long-term morbidity in the United States (1). In 2008, the cost of care for patients amounted to $18.8 billion, and this population had an additional $15.5 billion in loss of productivity. Virginia has an annual age-adjusted stroke rate of 3%, placing it amongst the states most affected by stroke. This means that this year, 199,319 Virginians have had a stroke and are living with the disability caused by this stroke; and, that in the past year nearly 3,300 Virginians have died due to stroke (2). For patients suffering from ischemic stroke (85% of all strokes), the only proven treatments are rapid revascularization using clot-busting agents (3) or mechanical thrombectomy (4).  Revascularization or reperfusion can itself initiate a cascade of events that lead to further injury. Understanding mechanisms of reperfusion injury can allow for development of therapies that minimize this injury, thereby improving stroke care. Uncovering this new understanding requires a novel combination of non-invasive, dynamic imaging approaches that are capable of simultaneously quantifying the numerous functional changes in the brain vasculature—including changes in hemodynamics, microvascular wall structure and composition, and tissue oxygen levels—minutes following reperfusion (hyperacute changes) and hours following reperfusion (acute changes). 

Photoacoustic microscopy (PAM), which is uniquely capable of simultaneous label-free imaging of blood perfusion, oxygenation and flow in the mouse brain microvasculature, is a novel tool that can be used to address early changes in oxygen metabolism after reperfusion in the setting of ischemia. Integrating this technique with multiphoton microscopy (MPM), which is able to image genetically encoded lineage-specific indicators, allows for studying the contribution of various cellular components in the pathophysiology of reperfusion injury. Incorporating the use of ultraviolet light activated boron-based oxygen-sensing nanoparticles (BNPs) provides quantitative information about oxygen levels and gradients in the brain tissue. Here, we propose to combine, for the first time ever, PAM, MPM, and BNPs to quantify, correlate, and better understand dynamic changes in blood flow, oxygenation, and vascular structure during the time course of reperfusion injury in a widely accepted murine stroke model, with the following specific aims:

Aim 1. To use the integrated PAM, MPM, and BNP imaging system to study the acute changes associated with reperfusion injury within 4 hours of reperfusion following ischemia.

Hypothesis: Reperfusion results in rapid alterations in blood and tissue oxygenation in the ischemic brain, which may result in generation of oxygen free radicals, vasospasm of capillary beds, and accumulation of metabolites that result in further injury.

Aim 2. To use the integrated PAM, MPM, and BNP imaging system to study the hyperacute changes associated with reperfusion injury.

Hypothesis: Reperfusion of an ischemic territory will result in vasospasm of capillary beds, which may result in further ischemia.

Desired outcomes

Given the impact of ischemic stroke on the population of the United States, in particular Virginia, and the lack of availability of agents that can assist with post-stroke recovery, a therapeutic regimen targeting this patient population and minimizing reperfusion injury is greatly needed. Therapies that can improve patient outcomes after stroke have the potential to improve patient quality of life and decrease costs to the health-care system and the families of patients afflicted by stroke. This project will advance therapeutic development by establishing a new tri-modal approach for high-resolution brain imaging, and by offering new insights about the coordination between microvascular adaptations and tissue responses to reperfusion injury.