Assessing Blood-Brain Barrier Integrity in a Laser-Induced Brain Injury Rat Model

Take an anesthetized control rat and a laser-induced brain injury rat model.

The laser application on the right hemisphere damaged cells and the blood-brain barrier, or BBB, which separates blood from brain tissue.

Inject a tracking dye into the cannulated tail vein that travels through the systemic circulation to reach the brain.

The dye binds to plasma proteins and leaks into brain tissue through the damaged BBB.

Euthanize the rats.

Perfuse the rats through the heart to clear the dye from the circulation.

Extract and slice the brain, isolate the slices from the injured hemisphere, and homogenize them.

Treat with an acidic solution to denature proteins, releasing the protein-bound dye.

Centrifuge and isolate the dye-containing supernatant. Add alcohol to enhance dye fluorescence.

Transfer the solution to a microplate. Upon excitation, a higher fluorescence intensity in the injured brain indicates laser-induced BBB damage.

To evaluate the incidence of blood-brain barrier breakage, 24 hours after the laser-induced injury, load a syringe with 2% Evans blue dye, diluted in 4 milliliters per kilogram of saline solution, and deliver the solution intravenously to the injured and control rats via