Modeling Photothrombotic Stroke in Mouse Model: A Laser Illumination Technique Through Mouse Skull Following Photosensitive Dye Administration to Induce Photothrombosis

To begin, stabilize an anesthetized mouse in a stereotactic unit. Attach a probe to monitor the mouse’s temperature. Regulate its body temperature using a heating pad.

Next, sterilize the surgical site and make a longitudinal incision to expose the skull.

Scrape off the underlying periosteum - a connective tissue layer from the skull surface.

Identify the coronal suture and mark the position of interest from the bregma - the point of intersection of the coronal and sagittal sutures.

Fix a tape with a small aperture on the marked region to restrict the exposed area during laser illumination. Now, inject the desired photosensitive dye into the peritoneal cavity of the mouse.

The injected dye molecules travel through the circulatory system and reach the brain.

Next, position the appropriate laser source above the marked region and illuminate the area for the desired duration. Following illumination, the circulating dye is activated and produces highly reactive oxygen species. These act by damaging endothelial cell membranes in the blood vessels in the illuminated area.

The endothelial cell damage promotes local activation and aggregation of platelets and subsequent thrombi formation, interrupting local cerebral blood flow. Thereafter, rehydrate the skull using saline. Close the surgical incision and return the mouse to its cage.

The interruption of cerebral blood flow damages the brain tissue, leading to dysfunction of the brain in the mouse.

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's 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 dexpanthenol eye ointment to both eyes, and clean the skin and the surrounding fur with a disinfecting agent.

Use scissors to make a single 2.0 to 2.5-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 bregma +3 millimeter to the left. Then, switch off the laser, and hook the sticker with a 4-millimeter diameter hole in the marked coordinates.

Inject the mouse intraperitoneally with Rose Bengal. Place the laser beam at a 4 to 5-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.