Combined Nanodrops Imaging and Ultrasound Localization Microscopy for Detecting Intracerebral Hemorrhage

Objective: Advanced imaging methods are crucial for understanding stroke mechanisms and discovering effective treatments to reduce bleeding and enhance recovery. In pre-clinical in vivo stroke imaging, MRI, CT and optical imaging are commonly used to evaluate stroke outcomes in rodent models. However, MRI and CT have limited spatial resolution for rodent brains, and optical imaging is hindered by limited imaging depth of penetration. Here we introduce a novel contrast-enhanced ultrasound imaging method to overcome these challenges and characterize intracerebral hemorrhage with unique insights. Methods: We combined microbubble-based ultrasound localization microscopy (ULM) and nanodrop (ND)-based vessel leakage imaging to achieve simultaneous microvascular imaging and hemorrhage detection. ULM maps brain-wide cerebral vasculature with high spatial resolution and identifies microvascular impairments around hemorrhagic areas. NDs are sub-micron liquid-core particles that can extravasate due to blood-brain barrier breakdown, serving as positive contrast agents to detect hemorrhage sites. Results: Our findings demonstrate that NDs could effectively accumulate in the hemorrhagic site and reveal the location of the bleeding areas upon activation by focused ultrasound beams. ULM further reveals the microvascular damage manifested in the form of reduced vascularity and decreased blood flow velocity across areas affected by the hemorrhagic stroke. Conclusion: The results demonstrate that sequential ULM combined with ND imaging is a useful imaging tool for basic in vivo research in stroke with rodent models where brain-wide detection of active bleeding and microvascular impairment are essential.