We seek to understand the intricate mechanisms underlying mild traumatic brain injury caused by exposure to blast-induced shockwaves. We are also investigating methods for alleviating these symptoms. It has been a comprehensive description of the initial phase of blood-brain barrier breakdown due to low intensity blast-induced shockwaves.
Specifically, the breakdown starts abruptly approximately three hours after exposure, and the affected lesions undergo remodeling for approximately one day following the onset. This method allows us to examine the breakdown of the BBB in more detail, providing an additional metric to evaluate the effectiveness of various treatments that can be employed in the event of an explosion. To begin, prepare a shock tube for exposing the animal to the blast-induced shockwaves.
Position the anesthetized mouse five centimeters away from the exit end of the shock tube, ensuring its body axis is parallel to, but not aligned with the tube's axis. Then deliver a single blast shockwave with a peak overpressure of 25 kilopascals to the animal's head. For the Evans blue injection, prepare a 4%weight by volume Evans blue solution in saline.
Vortex the solution and store it in the dark until use. Inject the Evans blue solution into the tail vein at a dosage of 2.5 microliters per gram. After perfusing the anesthetized mouse with the FITC-dextran dye and paraformaldehyde, use surgical scissors and tweezers to carefully remove the brain.
Postfix the brain overnight in the same fixative at four degrees Celsius. On the following day, replace the fixative with PBS. To begin, obtain the fixed brain from a blast shockwave-treated mouse injected with Evans blue and FITC-dextran dyes.
Prepare a 24-well plate with 500 microliters of 20%glycerol in phosphate buffer adjusted to pH 7.4 in each well. Using a brain slicer, cut the brain coronally into 12 slices, each one millimeter thick. Transfer each slice to the corresponding well of the 24-well plate, and store the plate at four degrees Celsius for at least two hours.
Then replace the solution with 50%glycerol phosphate buffer and store it again at four degrees Celsius for at least two hours. Finally, replace the solution with 100%glycerol. For immediate microscopic observation, allow the slices to stand for at least two hours at room temperature.
Add 500 microliters of glycerol to the bottom of a 35-millimeter glass-bottom dish. Transfer the brain slice to the glycerol solution and cover the surface with a cover glass. Remove excess glycerol from the edge of the cover glass.
Measure the fluorescence intensity of the slice under a fluorescence microscope. After the microscopic measurement, return the slice to the plate's well. Add 500 microliters of phosphate buffer to each well and store the 24-well plate at four degrees Celsius overnight.
Replace the solution with 30%glycerol phosphate buffer and incubate the plate at four degrees Celsius for at least two hours before performing immunohistochemistry Evans blue labeling demonstrated that blood-brain barrier or BBB breakdown began within six hours after blast shockwave exposure and lasted up to seven days. Fluorescence hotspots varied in size and intensity, with Evans blue-only hotspots indicating BBB breakdown during Evans blue injection and repair after FITC-dextran injection. Clusters of reactive astrocytes were closely associated with BBB breakdown sites confirmed by immunohistochemistry.
Activated microglia and ameboid microglia were observed alongside reactive astrocytes three days after the blast shockwave exposure.
