Transient Middle Cerebral Artery Occlusion Model of Stroke
August 11th, 2023
This protocol describes the model of transient focal cerebral ischemia in mice through intraluminal occlusion of the middle cerebral artery. Additionally, examples of outcome assessment are shown using magnetic resonance imaging and behavioral tests.
The middle cerebral artery occlusion model is widely used to simulate ischemic stroke in mice. This model allows to investigate a temporal progression of ischemic damage, evaluate effectiveness of neuroprotective agents, and explore the role of a specific genes and pathways in stroke pathology with a final aim of finding new potential treatments. The implementation of neuroimaging techniques, such as MRI, allows to make better diagnosis, improve the assessment of outcomes, and reduce the number of animals needed in each study.
This protocol first a clear and detailed explanation of the most commonly used technique for inducing transient middle cerebral artery occlusion. Moreover, it implements the technique by introducing the recession modification of anesthesia with during occlusion time, reducing the detrimental effect of prolonged anesthesia time. To begin, position the anesthetized mouse on the operating table.
Maintain body temperature using a rectal probe connected to a heating pad. Make an incision on the skin of the head in the direction of the sagittal suture, starting from the ears to the area between the eyes. Retract the skin and remove the periosteum on the right side of the skull.
Then find the coordinates, 2.5 millimeters lateral from bregma. Apply cyanoacrylate to the doppler holder and attach it for laser doppler flowmetry. After the glue has dried, connect the doppler probe.
Then check for the correct signal readout for assessing the cerebral blood flow. To perform artery occlusion, turn the mouse over to the supine position and fix it to the surgical table with medical tape. Then, make a midline incision on the neck.
Pull the skin laterally, followed by pulling away the salivary glands using retractors. This exposes the carotid territory. Identify the vascular anatomy of the common carotid artery, or CCA, the ICA and the ECA as well as the different arteries derived from them.
For easier handling, detach CCA, followed by ECA from the connective tissue. Then, cauterize the superior thyroid artery and detach ICA from the connective tissue. Check again to make sure the main arteries are fully detached from the connective tissue.
Next, wrap 6-0 silk sutures around the ECA at the maxillary lingual bifurcation. Then suture around the CCA. Place a vascular clip interrupting blood circulation from the ICA.
Make a small incision in the ECA close to the area where the tight knot is located. Insert the monofilament until the thick coating has completely entered the arterial lumen. Tighten the second knot to hold the monofilament inside the artery and to prevent the pressure exerted by the blood from pushing it out.
Cut the ECA below the first knot and rotate the stump to orient it in the direction of the ICA. Advance the monofilament via the ICA until the point where the MCA branches out. The occlusion is reflected in an abrupt blood flow drop in the LDF readout.
After 40 minutes, anesthetize the mouse again and place it back on the surgical table. Remove the stitches from the neck. After 45 minutes of occlusion, loosen the knot holding the monofilament in place.
Pull slowly and gently on the filament and check that tissue recanalization occurs. Then, pull out the filament and tighten the knot to prevent blood loss. Clean the blood and untie the CCA knot.
Ensure that there is no arterial wall damage. Remove the retractors and reposition the muscles, glands and skin. Suture the skin using a 6-0 suture and apply disinfectant.
Disconnect the doppler probe and detach the holder. Finally suture and disinfect the skin of the head. The outcome of the transient middle cerebral artery occlusion procedure was evaluated using in vivo MRI neuroimaging, Lesion evolution assessed at different time points after reperfusion showed that progression of the lesion volume takes approximately two days to complete.
Behavioral tests, such as grip strength test, showed a loss of strength in mice after 24 hours of transient middle cerebral artery occlusion Also, upon stimulation in the corner test, the mice showed a preference to turn to the right side, which was ipsilateral to the lesion indicating a successful middle cerebral artery occlusion on the right side.
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Overview
This study presents a protocol for inducing transient focal cerebral ischemia in mice using middle cerebral artery occlusion. The approach is evaluated through magnetic resonance imaging and behavioral assessments to understand the progression of ischemic damage and the effectiveness of potential neuroprotective agents.
Key Study Components
Area of Science
- Neuroscience
- Stroke research
- Neuroimaging
Background
- The middle cerebral artery occlusion model simulates ischemic stroke in mice.
- This model analyzes the temporal progression of ischemic damage.
- Behavioral tests assess motor deficits following ischemic injury.
- Neuroimaging techniques aid in outcome assessment and reduce animal use.
Purpose of Study
- To provide a detailed protocol for inducing transient middle cerebral artery occlusion.
- To assess the effectiveness of neuroprotective agents in ischemic models.
- To explore specific genes and pathways involved in stroke pathology.
Methods Used
- The study utilizes a mouse model for transient focal cerebral ischemia.
- Involves surgical procedures for middle cerebral artery occlusion while monitoring cerebral blood flow.
- Outcomes are evaluated using MRI and behavioral tests at various time points post-occlusion.
- Details include anesthesia management and surgical coordination to minimize stress and complications.
Main Results
- MRI measurements revealed lesion progression over approximately two days after occlusion.
- Behavioral assessments indicated reduced grip strength and a directional turning bias towards the injury site.
- The approach successfully demonstrates ischemic mechanics and potential therapeutic interventions.
Conclusions
- This protocol enables comprehensive evaluation of ischemic injury and the effectiveness of treatments.
- The findings enhance understanding of cerebral ischemia mechanisms and support future therapeutic development.
- Overall, this work contributes to improved models of stroke and neuroprotective strategies.
Frequently Asked Questions
Executive Industry Relevance
The transient middle cerebral artery occlusion (MCAO) model in mice provides a robust, reproducible platform for evaluating ischemic stroke mechanisms and candidate neuroprotective agents. This model enables quantitative assessment of lesion progression and functional outcomes, supporting predictive confidence in early-stage CNS therapeutic discovery. Its integration of neuroimaging and behavioral endpoints enhances translational continuity from preclinical research to clinical hypothesis generation.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of gene and pathway involvement in stroke pathology using genetically modified mice.
- Supports biological de-risking by clarifying mechanistic links between molecular targets and ischemic outcomes.
- Facilitates predictive confidence in target selection for neuroprotective strategies.
Screening & Assay Development
- Provides a validated in vivo system for screening neuroprotective compounds under controlled ischemic conditions.
- Standardizes outcome measurement through MRI and behavioral assays, ensuring reproducibility across studies.
- Generates quantitative data on lesion volume and functional deficits for reliable compound evaluation.
Translational & Preclinical Research
- Aligns preclinical endpoints with clinical imaging and behavioral assessments, supporting translational biomarker development.
- Enables longitudinal tracking of lesion evolution and therapeutic response post-reperfusion.
- Reduces animal use through advanced neuroimaging, optimizing resource allocation in preclinical programs.
Pipeline & Workflow Integration
The MCAO model bridges early discovery, lead identification, and preclinical validation phases in CNS drug development pipelines.
- Discovery Biology: Supports hypothesis testing on gene function and pathway relevance in ischemic injury.
- Screening: Delivers reproducible, quantitative readouts for compound efficacy in a disease-relevant system.
- Analytics: Integrates MRI and behavioral data for robust statistical comparison of experimental groups.
- Translational Research: Provides continuity between preclinical findings and clinical imaging endpoints.
- Enterprise Reuse: Offers a standardized, scalable platform adaptable to diverse genetic and pharmacological studies.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in CNS target validation.
- Operational Value: Enhances reproducibility and standardization through integrated imaging and behavioral protocols.
- Strategic Value: Informs go/no-go decisions and prioritizes assets with translational potential.
- Portfolio Impact: Supports risk-adjusted advancement and resource allocation across neurotherapeutic programs.
Implementation Considerations
- Requires expertise in microsurgical techniques and neuroimaging analysis.
- Demands access to MRI infrastructure and behavioral testing platforms.
- Necessitates rigorous cross-team standardization of surgical and analytical procedures.
- Adaptable to various genetic backgrounds and pharmacological interventions in mice.
- Potential variability in surgical outcomes requires careful quality control and statistical planning.
