The Stroke Preclinical Assessment Network Multi-Laboratory Model of Thromboembolic Stroke with Thrombolysis: TE-MCAo
December 19th, 2025
To simulate thromboembolic stroke, emboli prepared from heterologous rat blood were injected into the middle cerebral artery, followed by administration of systemic thrombolysis for recanalization. This version of the model is optimized for use in a multi-laboratory network and supports testing multiple candidate therapeutics.
The scope of our research is to develop a thromboembolic stroke model with controlled thrombolysis for use in rigorous multi-laboratory preclinical therapeutic testing. Our protocol addresses the absence of a simple reproducible thromboembolic stroke model that mimics clinical thrombolysis and supports multicenter preclinical trials. To begin render a PE-50 tubing blunt by cutting the end at a beveled angle.
Trim off the very tip of the bevel point using micro scissors. After preparing the anesthetized rat for surgery, make a vertical incision on the inner thigh, approximately 1.5 to two centimeters long along the natural fold of the inguinal intersection with the leg. The incision should be orthogonal to the expected anatomic course of the femoral artery.
Using blunt dissection, carefully dissect down to the femoral vessels. Then separate the femoral artery from the vein and nerve that run in the same bundle. Skeletonize the femoral artery as far proximal as possible, tracing the artery to its retroperitoneal origin.
Loosely place a loop of 5-0 silk around the femoral artery, and use the tails to provide gentle tension on the proximal end of the artery. Then distally occlude the femoral artery with a 5-0 silk ligature, and use the tails of the suture to provide slight tension distally with hemostatic clamps. About two thirds of the distance from the proximal suture loop, place a piece of silk suture under the artery and make a small arteriotomy distal to the loose suture using micro scissors.
Using sharp forceps, lift the top opening of the arteriotomy and introduce the blunt beveled PE-50 catheter into the opening. Advance the catheter proximally as far as possible, such that the loose loop when tightened, encompasses the catheter. Tighten the suture gently, ensuring it does not occlude the catheter or sit on the bevel.
Release the rubber shod clamp. The catheter should immediately fill with blood. Fill three 1.5 milliliter centrifuge tubes, and store them at four degrees Celsius.
Once the tubes are filled, remove the PE-50 catheter from the femoral artery, ligate the proximal end, and close the incision with interrupted 5-0 Prolene sutures. To prepare the thrombus, transfer 500 microliters of freshly drawn or stored donor blood into a 1.5-milliliter microcentrifuge tube, and add one milliliter of calcium chloride for anticoagulation reversal. Then immediately aspirate the blood from the microcentrifuge tube into a 100-centimeter PE-50 tubing, using gentle suction, and incubate the coiled tubing in prewarmed PBS at 37 degrees Celsius for two hours in a tabletop oven.
After expelling the thrombus into a Petri dish containing PBS, cut it into sections of approximately six centimeters, using a razor blade. Draw each thrombus gently into a PE-50 catheter and fully expel it five times to wash it. Wash the thrombus again by drawing it gently into a PE-10 catheter and fully expelling it 15 times.
Add 200 microliters of 4%Evans blue into a Petri dish containing 10 milliliters of PBS to create Dilution number 1. Then pipette 400 microliters of Dilution number 1 into a separate Petri dish containing 20 milliliters of PBS to create Dilution number 2. Place the clot in the more concentrated Evans blue Petri dish solution for one second, then transfer it to the diluted Evans blue Petri dish.
Load one washed thrombus into prefabricated microcatheters without air emboli, using the wet-to-wet method. Once the thrombus is fully loaded, extrude enough thrombus so that exactly five centimeters remains in the microcatheter. Trim the excess with a razor blade.
Mark the microcatheter 16 millimeters from the tip to demarcate the extent of catheter that should be advanced intraluminally. The preloaded thromboemboli were injected into the internal carotid artery of 135 subjects across six research laboratories to test the feasibility of the thromboembolic middle cerebral artery occlusion model. Magnetic resonance imaging was performed after three days in 102 animals, indicating a 75%scan completion rate.
Animal loss before imaging was consistent across all six laboratories. The mean lesion volume across all sites was 13%of the ipsilateral hemisphere, with some variation across the six laboratories. Our protocol mimics human thrombus and thrombolysis, reduces animal use, it standardizes surgery and achieves reliable multicenter reproducibility.
As a significant outcome of our research, we created a scalable reproducible TE-MCAo model that's usable across multiple labs with consistent outcomes and structured quality control. This model can be consistently used in both single and multi-laboratory stroke studies, improving therapeutic screening and accelerating translation of candidate cerebral protective treatments.
Overview
This protocol demonstrates a reproducible thromboembolic stroke model that mimics clinical thrombolysis, supporting multicenter preclinical trials. It aims to improve therapeutic screening and accelerate the translation of candidate treatments.
Key Study Components
Area of Science
- Neuroscience
- Stroke Research
- Preclinical Testing
Background
- Thromboembolic strokes are a major health concern.
- Existing models lack reproducibility and clinical relevance.
- Multi-laboratory studies require standardized protocols.
- This model addresses these gaps in stroke research.
Purpose of Study
- To develop a standardized thromboembolic stroke model.
- To facilitate rigorous therapeutic testing across multiple laboratories.
- To mimic human thrombus and thrombolysis conditions.
Methods Used
- Preparation of emboli from rat blood.
- Injection into the middle cerebral artery.
- Administration of systemic thrombolysis.
- Magnetic resonance imaging for outcome assessment.
Main Results
- The model achieved a 75% scan completion rate in imaging.
- Mean lesion volume was 13% of the ipsilateral hemisphere.
- Consistent outcomes were observed across six laboratories.
- The protocol supports multicenter reproducibility.
Conclusions
- The TE-MCAo model is scalable and reproducible.
- It enhances the reliability of preclinical stroke studies.
- This model can accelerate the development of therapeutic interventions.
Frequently Asked Questions
Executive Industry Relevance
The TE-MCAo multi-laboratory thromboembolic stroke model with controlled thrombolysis addresses a critical gap in preclinical stroke research by enabling reproducible, clinically relevant studies across sites. This model enhances predictive confidence for therapeutic screening and supports robust translational continuity from discovery to preclinical validation. Its standardized approach reduces biological variability and operational barriers, directly impacting portfolio advancement decisions in neurovascular drug development.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables rigorous interrogation of neurovascular therapeutic hypotheses in a disease-relevant system.
- Supports functional target validation by modeling human-like thromboembolic and thrombolytic events.
- Facilitates mechanistic de-risking through controlled recanalization and standardized lesion assessment.
- Improves predictive confidence for candidate selection and portfolio triage in stroke research.
Screening & Assay Development
- Prepares validated rodent models for high-fidelity compound screening in multicenter settings.
- Standardizes surgical and thrombus preparation protocols to ensure reproducibility and quantitative outputs.
- Enables scalable, cross-laboratory assay deployment for reliable efficacy evaluation.
- Supports robust comparison of therapeutic interventions using consistent imaging and histological endpoints.
Translational & Preclinical Research
- Aligns preclinical models with clinical thrombolysis scenarios, enhancing translational biomarker relevance.
- Provides continuity from early discovery through preclinical validation with multicenter reproducibility.
- Reduces translational risk by mimicking human neuroinflammatory and recanalization processes.
- Enables risk-adjusted advancement of neuroprotective and thrombolytic candidates.
Pipeline & Workflow Integration
This model integrates from early discovery through lead identification to preclinical validation, supporting multicenter therapeutic screening and translational research in stroke.
- Discovery Biology: Facilitates hypothesis testing and pathway clarification in a clinically relevant stroke context.
- Screening: Delivers reproducible, quantitative lesion and imaging outputs for compound evaluation.
- Analytics: Provides standardized MRI and histological readouts for cross-condition comparison.
- Translational Research: Bridges preclinical and clinical workflows by modeling systemic thrombolysis and recanalization.
- Enterprise Reuse: Offers a scalable, standardized platform for repeated use across single and multi-laboratory studies.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in neurovascular target validation.
- Operational Value: Promotes standardization, reproducibility, and scalability across research sites.
- Strategic Value: Enables informed go/no-go decisions and capital-efficient advancement of stroke therapeutics.
- Portfolio Impact: Supports risk-adjusted prioritization and cross-functional advancement of candidate therapies.
Implementation Considerations
- Requires expertise in rodent microsurgery and vascular catheterization.
- Needs access to imaging infrastructure and standardized surgical tools.
- Demands cross-team adherence to SOPs and training for reproducibility.
- Adaptable to various rodent strains and laboratory environments with protocol fidelity.
- Dependent on consistent blood handling and thrombus preparation to minimize variability.
