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.
