Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis
May 6th, 2021
Described here is the photothrombotic stroke model, where a stroke is produced through the intact skull by inducing permanent microvascular occlusion using laser illumination after administration of a photosensitive dye.
The photothrombosis stroke model approach may by suitable for cellular and molecular studies of cortical plasticity because of the limited borders and flexibility to induce injury in different areas of the cortex. Three principle advantages distinguish the photothrombosis model from other stroke models, the possibility to direct the lesion to the desired region, it's high reproducibility, and low mortality. Proper rose bengal dosing is critical for the success and for the stability of the model.
Results depend on technical specification like laser power, cross application timing, and optical shilling of the unaffected cortex. Begin by dissolving rose bengal in 0.9%saline solution for a final concentration of 10 milligrams per milliliter. Meticulously sterilize all instruments using a hot lead sterilizer and disinfect all surfaces before and after surgery with a microbial disinfectant spray.
Prepare a syringe filled with saline solution to maintain the operation area hydrated and prepare the anesthesia gas. Measure the body weight of the mouse to adjust the dose of rose bengal to be injected. Set the associated feedback controlled heating pad to maintain the mouse body temperature.
Once the mouse is completely anesthetized and fixed in the stereotactic frame, gently insert the rectal probe to monitor the body temperature of the mouse throughout the surgical procedure. Apply the Dex panthenol eye ointment to both eyes and clean the skin and the surrounding fur with disinfecting agent. Use scissors to make a single two to two and a half centimeter longitudinal incision and retract to expose the skull.
Use cotton to gently remove the periosteum and locate the coronal sutures. Wear protective glasses while switching on the 561 nanometer laser and mark the plus three millimeters to the left, then switch off the laser and hook the sticker with four millimeter diameter hole in the marked coordinates. Inject the mouse intraparietal with rose bengal.
Place the laser beam at a four to five centimeter distance from the skull. Switch on the laser and illuminate the skull for 20 minutes. Rehydrate the skull by applying two drops of 0.9%saline.
After suturing the wound, place the mouse in a recovery chamber at 37 degrees Celsius to recover from anesthesia. After one hour, place the mouse back in the cage in a temperature controlled room. The crystal violet stain to serial coronal brain sections were used for the infarct volume entry analysis after 24 hours of the stroke induction.
The variability of this stroke model was low, and the mean infarct volume was 29.3 cubic millimeters, representing 23%of the one brain hemisphere. The phototrombosis lesion caused a moderate and longterm sensory motor impairment indicated by the composite neuro score. Stroke animals had a significant change in the neuro score 24 hours after the surgery.
Even though the differences persisted, the stroke mice improved over time. In the mice subjected to the rose bengal plus illumination, the body weight and body temperature decreased 24 hours after the surgery. However, within three days after the surgery, recovery was observed.
Ischemic changes were confirmed using laser imaging. The results indicated that the rose bengal or laser illumination alone did not produce a lesion while the simultaneous application of both generated a round hypoperfused area surrounded by the narrow oligomeric zone. The crystal violet and tunnel staining for the assessment of infarct volume 24 hours after surgery revealed no tissue damage either in the rose bengal or laser illumination surgeries.
On the other hand, the rose bengal plus laser illumination generated a well demarcated lesion. It's important to measure the body weight of the animal to adjust the rose bengal injection. The correct condition of coroner switchers is important to induce a consistent lesion between animals.
Since this protocol leaves the skull intact and can even be performed through cranial windows, it can be combined with wide field or multi photon in vivo imaging.
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Overview
This article demonstrates a standardized photothrombotic mouse model for inducing focal cortical stroke using intraperitoneal injection of Rose Bengal and targeted 561 nm laser illumination. The protocol enables precise, reproducible induction of cortical lesions without invasive surgery, facilitating studies of post-stroke mechanisms and therapeutic interventions. Detailed surgical steps and histological analysis methods are provided to support reproducibility and address the replication crisis in preclinical stroke research.
Key Study Components
Area of Science
- Neuroscience
- Stroke research
- Experimental models
Background
- Stroke is a major cause of death and disability worldwide.
- Current therapeutic options for stroke are limited, necessitating further research into pathophysiological mechanisms such as inflammation, angiogenesis, and neuronal plasticity.
- In vitro models cannot fully replicate brain complexity, making in vivo models essential for studying stroke and evaluating new drug targets.
- Standardized, reproducible animal models are needed to improve research reliability and address the replication crisis.
Purpose of Study
- To present a detailed, standardized protocol for photothrombotic stroke induction in mice.
- To enable targeted induction of cortical lesions with high reproducibility and low mortality.
- To facilitate the study of post-stroke mechanisms and evaluation of therapeutic strategies.
Methods Used
- Preparation of Rose Bengal solution (10 mg/mL in 0.9% saline).
- Intraperitoneal injection of Rose Bengal, with dose adjusted to mouse body weight.
- Targeted illumination of the intact skull with a 561 nm laser for 20 minutes at specified cortical coordinates.
- Post-surgical care including wound suturing and recovery monitoring.
- Histological analysis using crystal violet and TUNEL staining to assess infarct volume and tissue damage.
- Behavioral assessment using composite neuro score.
Main Results
- The model produced well-demarcated, reproducible cortical lesions with low variability (mean infarct volume: 29.3 mm³, ~23% of one hemisphere).
- Stroke induction resulted in moderate, long-term sensorimotor impairment, with significant neuro score changes at 24 hours and gradual improvement over time.
- Body weight and temperature decreased 24 hours post-surgery but recovered within three days.
- Lesions were only observed when both Rose Bengal and laser illumination were applied simultaneously; controls showed no tissue damage.
- The protocol is compatible with in vivo imaging techniques due to the intact skull.
Conclusions
- The photothrombotic stroke model allows precise, reproducible induction of cortical lesions in mice without invasive surgery.
- It is suitable for cellular and molecular studies of cortical plasticity and post-stroke mechanisms.
- Standardization of this protocol supports reproducibility and reliability in preclinical stroke research.
