Microvascular Embolism Mouse Model for In Vivo Two-photon Microscopy Using Fluorescent Polystyrene Microspheres
November 21st, 2025
This protocol contains a thorough description of a step-by-step approach for a chronic cranial window and microvascular embolism mouse model optimized for in vivo two-photon microscopy imaging 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.
Overview
This protocol describes a detailed step-by-step approach for creating a chronic cranial window and microvascular embolism mouse model, optimized for in vivo two-photon microscopy imaging. The method utilizes fluorescent polystyrene microspheres to investigate the effects of capillary plugging.
Key Study Components
Area of Science
- Neuroscience
- Microvascular research
- In vivo imaging techniques
Background
- Understanding the consequences of microvascular occlusions is crucial for developing new treatment strategies.
- Real-time imaging allows for tracking of fluorescent particles in the cerebral capillary network.
- Previous studies have shown that capillary plugging can lead to local tissue damage.
- This research aims to elucidate the mechanisms of embolus clearance in the brain.
Purpose of Study
- To investigate the short and long-term effects of capillary plugging.
- To understand how microvascular occlusions affect blood flow and the blood-brain barrier.
- To explore potential treatment strategies for conditions related to microvascular damage.
Methods Used
- Creation of a chronic cranial window in mice for imaging.
- Injection of fluorescent microspheres to induce microvascular embolism.
- Utilization of two-photon microscopy for real-time imaging.
- Post-surgery imaging to confirm microsphere lodging in the brain.
Main Results
- Microspheres were predominantly lodged in the ipsilateral hemisphere.
- Increased microsphere counts were observed with the addition of Tween20.
- Mice injected with Tween20 showed faster recovery post-surgery.
- Fluorescence microscopy revealed occlusion of capillaries by microspheres.
Conclusions
- This method allows for detailed study of microvascular occlusions in vivo.
- Findings may lead to new insights into treatment strategies for cerebral microvascular conditions.
- The study enhances understanding of the dynamics of blood flow and tissue damage in the brain.
Frequently Asked Questions
Executive Industry Relevance
Microvascular embolism models using fluorescent microspheres and in vivo two-photon microscopy provide a robust platform for dissecting the mechanisms and consequences of cerebral micro-occlusions. This approach enables high-resolution, longitudinal tracking of vascular events and tissue responses, supporting predictive confidence in early-stage neurovascular target validation. The model's reproducibility and quantitative imaging outputs position it as a valuable asset for portfolio triage and mechanistic de-risking in neurovascular drug discovery.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of neurovascular injury hypotheses through controlled induction of micro-occlusions.
- Supports mechanistic de-risking by visualizing vessel, cellular, and blood-brain barrier responses over time.
- Facilitates functional target validation by linking embolism events to downstream tissue damage and clearance dynamics.
Screening & Assay Development
- Provides a validated in vivo system for quantitative assessment of microvascular occlusion and reperfusion.
- Delivers reproducible, high-content imaging outputs suitable for assay standardization and cross-study comparisons.
- Enables screening of candidate interventions targeting embolism clearance or neurovascular protection.
Translational & Preclinical Research
- Aligns with disease-relevant models of cerebral microvascular injury for translational biomarker exploration.
- Supports continuity from discovery through preclinical validation by enabling longitudinal outcome tracking.
- Informs risk-adjusted advancement decisions by quantifying tissue and vascular responses to micro-occlusions.
Pipeline & Workflow Integration
This model integrates into the discovery-to-preclinical continuum, bridging mechanistic studies and translational research in neurovascular biology.
- Discovery Biology: Facilitates hypothesis testing on microvascular injury and clearance mechanisms using real-time imaging.
- Screening: Provides quantitative, reproducible readouts for evaluating candidate therapeutics in vivo.
- Analytics: Enables statistical comparison of embolism burden, tissue damage, and recovery across experimental groups.
- Translational Research: Connects mechanistic findings to disease-relevant endpoints and biomarker development.
- Enterprise Reuse: Offers a standardized, reusable platform for neurovascular research and intervention testing.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in neurovascular target validation and mechanistic de-risking.
- Operational Value: Enhances reproducibility and scalability through standardized imaging and injection protocols.
- Strategic Value: Improves go/no-go decisions by providing quantitative, longitudinal data on vascular and tissue outcomes.
- Portfolio Impact: Supports risk-adjusted prioritization of neurovascular programs based on robust in vivo evidence.
Implementation Considerations
- Requires expertise in microsurgery, in vivo imaging, and quantitative analysis of vascular events.
- Demands access to two-photon microscopy and post-mortem 3D imaging infrastructure.
- Necessitates cross-team standardization of injection mixtures and imaging protocols for reproducibility.
- Adaptable to various mouse models but may require optimization for different vascular territories.
- Potential limitations include technical complexity and the need for longitudinal animal monitoring.
