Microvascular Embolism Mouse Model for <em>In Vivo</em> Two-photon Microscopy Using Fluorescent Polystyrene Microspheres

Microvascular occlusions in the brain are relatively common but remain largely understudied. The event rate, long-term consequences, and potential clearing mechanisms are largely unknown. Current models used to study these events, such as photothrombosis and micro-emboli injection, each have their own advantages and limitations. This study developed a detailed protocol that combines the preparation of a chronic cranial window with the induction of cerebral microvascular embolisms via intra-arterial injection of microspheres in mice. This setup allows for long-term in vivo imaging of the micro-vasculature and micro-occlusions using two-photon microscopy. Microspheres are delivered through a catheter placed in the external carotid artery (ECA), which is permanently ligated. Importantly, the common carotid artery (CCA) and internal carotid artery (ICA) remain intact throughout and after the procedure, thereby minimizing disturbance to cerebral blood flow. To facilitate immediate in vivo imaging and prevent microsphere clustering or adhesion to pipette tips and the catheter, microspheres are suspended in a mixture of FITC-Dextran and 0.1% Tween 20. The injection technique was validated using post mortem in situ 3D imaging to determine the microspheres' distribution. This study further demonstrates its utility with an in vivo two-photon microscopy example. This approach provides a consistent and robust method for inducing and studying microvascular embolisms, enabling investigation of their impact and clearance dynamics using high-resolution in vivo imaging.