Mechanisms Of The Blood-brain Barrier Disruption Induced By Focused Ultrasound Combined With Microbubbles

Background and ObjectiveThe blood-brain barrier (BBB) is composed of brain microvascularendothelial cells (BMECs), basal lamina, pericytes, and astrocyte processes.This specialized vascular system marks the interface between the peripheralcirculation and the central nervous system (CNS). Within the CNS, the BBBexcludes large (>400Da) molecules from entering the brain parenchyma andprotects it from toxic foreign substances. However, the presence of thisbarrier represents a substantial obstacle to the delivery of mostneurotherapeutic drugs to the brain. Although some methods focusing onovercoming this problem were carried out to show some promise for drugdelivery to the brain, they still cannot fully target and deliver therapeuticmaterials across the BBB both safely and efficiently. Recent experimentshave shown that focused ultrasound (FUS), at suitable parameters andcombined with an appropriate dose of microbubbles, can induce a targeted,transient, noninvasive, and reversible BBB disruption. Although the exactmechanisms behind this method remain unclear and the potential drawbacksfor its use in clinical care are unknown, it is, in light of these results, possible that targeted drug delivery to treat CNS diseases may become realistic in thefuture. It’s assumed that, in order to transduce external forces to intracellularbiochemical signals, mechanosensitive ion channels in the cell membranesof endothelial cells can respond to the mechanical forces of the combinationof FUS and microbubbles. These effects can increase the BBB permeatilitythrough two main routes exist: the paracellular (between cells) and thetranscellular (across cells) pathways. This research was designed todetermine whether disrupting the BBB in rats by applying focusedultrasound (FUS) combined with microbubbles induced changes in thedensity of caveolae and the expression of the structural proteincaveolin-1and determine the role of transcellular pathway in the BBBdisruption; and to determine the effects of caveolin-1on claudin-5and therole of the paracellular and transcellular pathways in the BBB disruption.MethodsThe rats were devided to three sets randomly.The rats in the first set were all treated with FUS combined withmicrobubbles and randomly assigned to six groups (group1, control; group2, rats immediately after treatment; group3, rats at1h after treatment; group4, rats at2h after treatment; group5, rats at4h after treatment; and group6,rats at8h after treatment). Two methods, fluorospectro photometer analysisof the EB extravasation and MRI enhancement analysis of the signalintensity were both employed to evaluate the BBB disruption quantitatively at different times after sonication. Then the change rules of BBBpermeability that corresponded with the elapsed time after treatment wereobserved.The rats in the second set were assigned randomly to four groups (group1, control; group2, rats treated with microbubbles only; group3, rats treatedwith FUS only; and group4, rats treated with a combination of FUS andmicrobubbles). Changes in caveolae density and caveolin-1expressionlevels were detected in each group.1. SP immunohistochemistry were used to examine the distribution ofcaveolin-1and the changes in its expression under different treatmentconditions.2. qRT-PCR and western blot analysis was used to determine the geneand protein expression levels of caveolin-1.3. Transmission electron microscopy (TEM) was used to observe theultrastructure of BMECs and to determine the number changes of caveolae.The rats in the third set were assigned randomly to three groups (group1, control; group2, rats treated with a combination of FUS and microbubbles;group3， rats treated with filipin before a combination of FUS andmicrobubbles). The effects of filipin on caveolin-1and claudin-5underlyingthe BBB disruption induced by FUS and microbubbles were researched inthe rats.1. Fluorospectro photometer analysis of the EB extravasation was used to the effects of filipin on the BBB disruption.2.Western blot analysis was used to detect the expression changes ofcaveolin-1and claudin-5disturbed by filipin.Results1. The results of MRI enhancement of the signal intensity revealed that,compared with the normal control rats, the signal intensity increasedimmediately after the administration of FUS and microbubbles. The signalintensity was most significantly increased1h after combined FUS andmicrobubble treatment. In groups that were imaged2,4, and8hpost-treatment, the signal intensity was gradually restored to normal.2. The results of fluorospectro photometer analysis of the EBextravasation revealed that, compared with the normal control rats, the EBextravasation increased immediately after the combination of FUS andmicrobubbles. The EB extravasation was highest1hours after thecombination treatment.2,4and8h later, the EB extravasation was graduallyrestored to normal.3. The results of the SP immunohistochemical analysis revealed that thecaveolae structural protein caveolin-1was expressed in BMECs. Theexpression of caveolin-1was not significantly different between control andmicrobubbletreated rats, FUS-treated rats showed a slight increase inexpression levels, and rats treated with a combination of FUS andmicrobubbles showed the most significant increase on caveolin-1 expression.4. The results of western blot analysis revealed that, compared withcontrol rats, treatment with microbubbles alone had no effect on caveolin-1expression, but both FUS alone and FUS combined with microbubblesincreased the expression levels of caveolin-1with the combination oftreatments showing the most significant increase.5. The results of qRT-PCR analysis revealed that, compared withcontrol rats, treatment with microbubbles alone had no effect on caveolin-1gene expression, but both FUS alone and FUS combined with microbubblesincreased the gene expression levels of caveolin-1with the combination oftreatments showing the most significant increase.6. The results of transmission electron microscopy experimentsrevealed that, a few pinocytotic vesicles (caveolae) were observed in thecontrol rats and those treated with microbubbles alone. The number ofcaveolae increased in rats treated with FUS alone with the highest numberfound in rats treated with a combination of FUS and microbubbles.7. The BBB disruption induced by FUS and microbubbles wasdisturbed by Using of filipin.8.Filipin could decrease the expression of caveolin-1to increase theexpression of claudin-5to regulate the BBB disruption.Conclusions1. The results of MRI enhancement of the signal intensity consisted with the results of fluorospectro photometer analysis of the EB extravasation.The combination of FUS and microbubbles could induce a targeted, transient,noninvasive, and reversible BBB disruption.2.1h after treatment with FUS and microbubbles was the time whenthe permeability of the BBB was the most significantly altered.3. The effects of BBB disruption induced by combination of FUS andmicrobubbles was stronger than the effects induced by microbubbles aloneor FUS alone.4. The results of SP immunohistochemical, qRT-PCR and western blotanalysis revealed that the combination of FUS and microbubbles couldsignificantly increase the gene and protein expression of caveolin-1.5. The results of transmission electron microscopy experimentsrevealed that the combination of FUS and microbubbles could significantlyincrease the density of caveolae.6. Caveolin-1-mediated transcellular pathways played an important rolein the BBB disruption induced by FUS combined with microbubbles.7.The paracellular and transcellular pathways both had an importantrole in the BBB disruption induced by FUS combined with microbubbles,and caveolin-1in the transcellular pathway most likely could regulate thetight junction complex.