Blood-brain Barrier Disruption Induced By Diagnostic Ultrasound Combined With Microbubbles In Mice

ObjectiveTo investigate the effects of the microbubble(MB)dose,mechanism index(MI)and sonication duration on blood-brain barrier(BBB)disruption induced by diagnostic ultrasound combined with MBs as well as optimizing parameters to increase BBB permeability and reduce brain tissue damage.Meanwhile the changes of BBB permeability after BBB disruption under the optimal parameters were observed,and the potential mechanism was preliminarily studied.Methods1.MBs were prepared by the thin-film hydration method and mechanical oscillation method.Morphology and concentration of MBs were detected under microscopy.Size distribution was measured by partical size analyzer.Stability of MBs was evaluated.Effect of contrast enhanced imaging of MBs within cardiac chamers was observed.2.Effects of various MB doses,MIs and sonication durations on the degree of BBB disruption at right striatum were preformed under the flash mode of diagnostic ultrasound.The extent of BBB disruption was qualitatively assessed by Evans blue(EB)staining and quantitatively analyzed by an EB extravasation measurement.HE staining was performed to evaluate tissue damage.Based on these findings,optimal parameters were screened out.Expression of tight junction(TJ)related proteins ZO-1,occludin and claudin-5 before and after BBB disruption was determined by western blotting analysis and immunohistofluorescence staining.Transmission electron microscopy(TEM)was performed to observe ultrastructure changes of TJs after BBB disruption.3.Changes of BBB permeability after BBB disruption under the optimal parameters was qualitatively assessed by EB staining and quantitatively analyzed by an EB extravasation measurement.Expression of TJ related proteins ZO-1,occludin and claudin-5 at different time points after BBB disruption was determined by western blotting analysis and immunohistofluorescence staining.HE staining was performed to evaluate adverse effects of BBB disruption on major organs of mice.Results1.Homemade MBs presented as spheres with translucent center.Mean concentration of MBs was(3.63±0.11)×10~9 MBs/ml.Mean size of MBs was 1546.8±114.2 nm.Size and concentration of MBs remained relatively stability within the first 6 h after preparation.Long-lasting contrast enhanced imaging of MBs was observed within cardiac chamers.Half-time of MBs was 2.97±0.54 min.2.With increases in MB doses,MIs and sonication durations,the amount of EB extravasation inceased,degree of EB staining was also enhanced.There was no damage was observed when groups sonicated with a MI less than or equal to 0.4.Increasing the MB dose to 1.0×10~7 MBs,MI to 0.8,or sonication duration to 3 min,BBB disruption was only associated with a few scattered erythrocytes to small groups of erythrocyte extravasation.At a MB dose of 2.0×10~7 MBs or a sonication duration of 4 min,erythrocyte extravasation increased,associated with individual dark-stained ischemic or apoptotic neurons and slight vacuolization of the neuropil surrounding impaired vessels.The most extensive erythrocyte extravasation along with distinct neuron loss and acute degeneration of the neuropil was detected at a MB dose of 3.0×10~7 MBs.Expression of all three TJ related proteins significantly decreased after BBB disruption,at the same time,the opening of TJs were observed under TEM.3.The highest BBB permeablity was achieved immediately after sonication in both the cortex and striatum,and then returned to normal approximately 6 h and 4 h after sonication,respectively.Expression of all three TJ related proteins significantly decreased immediately after sonication,and gradually increased to the same level as the control group at 6 h after sonication.Apart from a small amount of erythrocyte extravasation in brain tissues at 6 h after sonication,there was no damage was observed in slices of major organs of mice at the rest of time points.Conclusions1.Lipid MBs were prepared successfully,which were characterized by higher concentration,uniform size distribution,good stability,longer half-time and long-lasting contrast enhanced imaging in vivo.2.Extent of BBB disruption increased with MB dose,MI and sonication duration.Concurrently,the risk of tissue damage additionally increased.3.Guided by GE Vivid E9 diagnostic ultrasound system,ultrasonic frequency at1.5/3.0MHz,a MB dose of 1.0×10~7 MBs,a MI of 0.8 and a sonication duration of 3 min,a relatively larger extent of BBB disruption in mice can be achieved transcranially,noninvasively and temporarily.4.Under optimal parameters,duration of BBB disruption in the cortex and striatum were 6h and 4 h,respectively.5.Changes of BBB permeability in line with the trends of expression of all three TJ related proteins after BBB disruption induced by diagnostic ultrasound combined with MBs.Decreased expression of TJ related proteins ZO-1,occludin and claudin-5 caused the opening of TJs may be one of the mechanisms of BBB disruption induced by diagnostic ultrasound combined with MBs.