Visualization of Mouse Cerebral Thrombi Using Near-Infrared Fluorescence Imaging

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Ex Vivo Near-infrared fluorescent (NIRF) Thrombus Imaging of the Brain Tissue

1. After decapitation, remove the scalp and cut through the skull with scissors from the foramen magnum up toward the sagittal suture. Remove the cranial vault by carefully elevating the edges of the incised skull using scissors while avoiding injury to the underlying brain, thus laying bare the hemispheres.
2. Cut out the optic nerves in the brain base as close as possible to the brain surface because they could overlap with the circle of Willis (COW) arteries that should be visualized on near-infrared fluorescent (NIRF) imaging. Then, to visualize the Y-shape 'middle cerebral artery (MCA) / anterior cerebral artery (ACA) / distal internal carotid artery (ICA)' for the NIRF thrombus imaging, cut out the distal ICAs as far as possible to the brain surface after gently compressing the cerebellar base to expose the cutting points (Figure 1A).
3. Perform NIRF imaging (excitation/emission, 675 / 690 nm; 1-sec exposure) of the removed brain tissue with its base pointing up (Figure 1B, C), which visualizes the fluorescently labeled thromboemboli in the arteries of the COW (Figure 1D). Then, perform additional imaging with the vertex of the brain pointing up, which visualizes cortical thromboemboli. Avoid drying the brain by putting a few drops of saline on the tissue.

Figure 1: Overview of Ex Vivo Near-Infrared Fluorescent (NIRF) Imaging of Cerebral Thromboemboli. (A and B) A representative brain tissue was removed from a mouse with a thromboembolic stroke. Take precautions not to lose thromboemboli (red arrow) in the middle cerebral artery (MCA) – anterior cerebral artery (ACA) bifurcation area of the distal internal carotid artery (ICA, blue arrows) when removing the brain. (C and D) Ex vivo NIRF thrombus imaging. The brain tissue is placed inside the light-tight chamber of an NIRF imager (C) for the visualization of the thrombus (red arrow in D) pre-labeled with the Cyanine 5.5 (Cy5.5) fluorescent marker C15 before embolic occlusion of the MCA–ACA bifurcation area.