This study investigates the effects of laser-induced brain injury on the blood-brain barrier (BBB) in a rat model. The methodology includes the use of a tracking dye to assess BBB integrity following injury.
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 the cannulated tail vein.
Allow the solution to circulate for one hour, before using surgical pin sets and scissors to open the chest of the first animal. Perfuse the exposed heart, with cooled 0.9% saline via the left ventricle at 110 millimeters of mercury of pressure, until a colorless perfusion liquid is observed from the right atrium.
Next, harvest the brain, and obtain 2-millimeter rostrocaudal slices. Separate the left brain slices from the right brain slices to allow evaluation of the injured and non-injured hemispheres separately, and weigh the samples. Homogenize the samples, using a mortar and pestle, and incubate the tissue samples in 50% trichloroacetic acid for 24 hours.
The next day, centrifuged the homogenized brain tissue samples, for 20 minutes at 10,000 times g and room temperature. Mix 1 milliliter of the supernatant with 1.5 milliliters of 96% ethanol at 1 to 3 ratio. Then, use a fluorescence detector at a 620 nanometer excitation and a 680 nanometer emission wavelength to assess the blood-brain barrier breakage.
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