Microvascular Embolism Mouse Model for In Vivo Two-photon Microscopy Using Fluorescent Polystyrene Microspheres

Using this microsphere injection method, we aim to unravel the short and long-term consequences of capillary plugging to investigate potential mechanisms of clearing of embolus in cerebral capillary network. It allows for real-time in vivo imaging and tracking of fluorescent particles in the cortical capillary network. The microspheres and the local consequences can be followed for weeks after injection.

Our studies help to understand local tissue damage caused by microvascular occlusions. These results may hopefully lead to new treatment strategies. We can now study day by day and for weeks how capillary plugging alters vessels, activates cells, changes blood flow, and damages the blood-brain barrier.

To begin, pinch the toe of an anesthetized mouse to verify the depth of anesthesia. Make a midline incision of approximately 0.5 centimeters over the trachea below the mandible using surgical scissors and dissecting forceps. Dissect the connective tissue superficial to the cervical fascia using micro-suture-tying forceps to expose the underlying sternal hyoid muscles.

Now, separate the left and right sternal hyoid muscles by gently tearing the connective tissue between them. Retract the right omohyoid muscle caudal-laterally with a tissue hook. Ensure that the right carotid triangle, including the common carotid artery, internal carotid artery, and external carotid artery, are clearly visible.

Next, remove any remaining fascia and adipose tissue surrounding the common carotid artery. Carefully separate the common carotid artery from the vagus nerve. Place two 4/0 1.5 threads around the common carotid artery and tie them loosely, ensuring the knots do not impede blood flow.

Remove fascia and adipose tissue from the internal carotid artery and posterior parietal artery. Then temporarily ligate the posterior parietal artery and all internal carotid side branches, including the occipital artery, by tying a knot with a 4/0 1.5 thread. Remove fascia and adipose tissue from the external carotid artery and the temporal artery.

Place two loose knots around the external carotid artery and temporal artery using 4/0 size 1.5 threads. Position the most distal thread as far away from the Y-shaped bifurcation as possible, and use it to permanently ligate the external carotid artery and temporal artery. Leave the proximal knot untied.

Homogenize and sonicate the 10 micrometer microspheres to obtain a uniform suspension of individual particles. Add 20 microliters of homogenized microspheres to 140 microliters of FITC-Dextran to obtain a final volume of 160 microliters of mixture containing 1.44 times 10 to the power of five microspheres. Briefly retract the syringe connected to the catheter to create an airlock.

Then immerse the catheter tip into the prepared FITC-Dextran/Tween20/microsphere mixture and slowly pull back the syringe until the mixture is in the catheter. Ensure no air bubbles are present in the catheter. Place the syringe with the loaded catheter in the syringe pump.

Set the pump to 10 microliters per minute and run until a small drop of microsphere mixture appears at the catheter tip. Now, place a vessel clip on the internal carotid artery and tighten one of the previously prepared proximal knots around the common carotid artery. Using micro-scissors, make a small diagonal incision in the external carotid artery approximately 0.5 millimeters distal to the ligation thread, ensuring the incision size is slightly smaller than the catheter diameter.

Absorb any blood with a sterile cloth. Briefly start the syringe pump to form a drop of microsphere mixture at the catheter tip, confirming that no air remains in the catheter tip. Open the external carotid artery incision and insert the catheter using fine micro-forceps.

Then secure the catheter in the external carotid artery by tightening the proximal suture around the external carotid artery. Gradually pump at 10 microliters per minute until the artery visibly enlarges and the green FITC dye fills the external carotid artery, confirming successful injection. Next, remove the vessel clip from the internal carotid artery.

Then increase the syringe pump flow rate to 20 microliters per minute. Confirm successful microsphere injection by verifying that the internal carotid artery distal to the posterior parietal artery bifurcation is filled with FITC dye. Once injection is successful, remove the thread that ligated the common carotid artery to enable the microsphere mixture to flow into the internal carotid artery.

During the injection, the FITC dye can still be observed in the external carotid artery. Switch off the syringe pump once the mixture in the catheter reaches the 80 microliter mark to stop injection. Then ligate the common carotid artery, clip the internal carotid, and gently remove the catheter, while keeping the securing thread in place.

Permanently ligate the external carotid artery by tightening the thread that had secured the catheter. Remove the vessel clip and threads around the common carotid artery, internal carotid artery, and posterior parietal artery to restore blood flow. The success of the microvascular embolism surgery was confirmed by full body and full brain post mortem in situ imaging four days after surgery.

A sagittal brain slice showed lodging of microspheres in the brain. Extracted mouse brains imaged one day post-surgery showed that microspheres were predominantly lodged in the ipsilateral hemisphere within the flow territory of the middle and anterior cerebral arteries. Microsphere counts in brain slices were significantly increased when Tween20 was added to the microsphere mixture.

Mice lost less weight and recovered faster after surgery when injected with a microsphere mixture that contained Tween20. Fluorescence microscopy images of the brain cortex taken 0.5 hours after surgery showed individual white dots, representing microspheres occluding capillaries. Two-photon z-stack projection images showed microspheres causing cerebral microvascular embolisms with impaired perfusion.

Embolisms also cause disruption of the blood-brain barrier with visible dye leakage from the vessels.