This article describes a method for monitoring blood flow in the brain of an anesthetized mouse using photothrombosis. The technique involves injecting a photosensitive dye and using laser excitation to induce clot formation.
Take an anesthetized mouse having a cranial window with a thin skull layer to enable brain visualization with minimal invasiveness.
Under a microscope, identify an artery based on the blood flow from larger to smaller vessels.
Inject a photosensitive dye into the tail vein that reaches the brain via circulation.
Excite the dye with light to emit fluorescence, enabling blood flow monitoring.
Increase the light intensity to induce clot formation, known as photothrombosis.
The dye absorbs high-intensity light, reacts with oxygen, and generates reactive oxygen species, which damage endothelial cells.
The damaged cells release mediators that cause platelets to adhere, initiating clot formation. Fibrin in the blood helps entrap additional blood cells, creating a clot.
The clot, detected by the accumulation of dye preceding it, obstructs blood flow to the brain.
Close the incision, apply antibiotics to prevent infection, and administer analgesics to reduce pain and allow recovery.
After the mouse is set up at the microscope, prepare fresh rose Bengal in artificial CSF at 20 milligrams per milliliter. Filter sterilize the mixture and dispose of it after the experiment is completed. It must always be used fresh.
Now, while scanning the cranial window with a 561-nanometer laser, inject 0.1 of the mixture via the tail vein. Within 5 seconds, the dye should easily be seen in the cranial vasculature. After sufficient dye has been injected, select a 40 to 80-micron vessel for thrombosis. Arteries can be differentiated from veins, based on the flow of larger to smaller vessels, and vice versa.
Now, prepare to laser the selected vessel. Increase the dwell time to whatever is appropriate for the system in use. Increase the laser power to 100%, and set the image collection to one frame per second with maximum scan speed. Scan the mouse until a staple clot is formed. Usually this takes about five minutes. Ideally, only one field of view should be scanned to create the clot. Choose the objective accordingly. If no clot forms, try using more dye.
Once the clot has formed, remove the mouse from the imaging area and prepare to complete the surgery under the dissecting microscope. First, carefully remove the ring while checking for bleeding. If any bleeding occurs, the experiment must be terminated.
Next, using a 6-0 suture, close the incision. Then, apply an antibiotic to the incision line. For pain relief, provide subcutaneous injections of buprenex for three days. After administering the first injection of buprenex, place the mouse in a recovery chamber and monitor it until it is ambulatory. Then, return it to its home cage, which should be cleaned. Longitudinal imaging is detailed in the text protocol.
繁體中文
