Pulsed ultrasound expands the extracellular and perivascular spaces of the brain

Abstract Diffusion within the extracellular and perivascular spaces of the brain plays an important role in biological processes, therapeutic delivery, and clearance mechanisms within the central nervous system. Recently, ultrasound has been used to enhance the dispersion of locally administered molecules and particles within the brain, but ultrasound-mediated effects on the brain parenchyma remain poorly understood. We combined an electron microscopy-based ultrastructural analysis with high-resolution tracking of non-adhesive nanoparticles in order to probe changes in the extracellular and perivascular spaces of the brain following a non-destructive pulsed ultrasound regimen known to alter diffusivity in other tissues. Freshly obtained rat brain neocortical slices underwent sham treatment or pulsed, low intensity ultrasound for 5 minutes at 1 MHz. Transmission electron microscopy revealed intact cells and blood vessels and evidence of enlarged spaces, particularly adjacent to blood vessels, in ultrasound-treated brain slices. Additionally, ultrasound significantly increased the diffusion rate of 100 nm, 200 nm, and 500 nm nanoparticles that were injected into the brain slices, while 2000 nm particles were unaffected. In ultrasound-treated slices, 91.6% of the 100 nm particles, 20.7% of the 200 nm particles, 13.8% of the 500 nm particles, and 0% of the 2000 nm particles exhibited diffusive motion. Thus, pulsed ultrasound can have meaningful structural effects on the brain extracellular and perivascular spaces without evidence of tissue disruption.