Abstract
BackgroundThe Blood Brain Barrier (BBB) maintains the homeostasis of central nervous system by preventing the free passage of macromolecules from the systemic circulation into the brain. This normal physiological function of the BBB presents a challenge for delivery of therapeutic compounds into the brain. Recent studies have shown that the application of focused ultrasound together with ultrasound contrast agent (microbubbles) temporarily increases the permeability of the BBB. This effect is associated with breakdown of tight junctions, the structures that regulate the paracellular permeability of the endothelial cell layer. The influence of this ultrasound effect on the activation of intracellular signaling proteins is currently not well understood. Therefore, the aim of this study was to investigate the activation of cell survival signaling molecules in response to ultrasound-mediated BBB opening;MethodsThe BBB was disrupted in two four-spot lines (1-1.5 mm spacing) along the right hemisphere of rat brain with ultrasound beams (0.3 MPa, 120 s, 10 ms bursts, repetition frequency = 1 Hz) in the presence Definity microbubbles. Contrast-enhanced MRI images were acquired to assess the extent of BBB opening upon which the animals were sacrificed and the brains removed and processed for biochemical and immunohistochemical analyses;ResultsImmunoblotting of sonicated brain lysates resolved by SDS-PAGE demonstrated an increase in phosphorylation of Akt and its downstream signaling molecule, GSK3β, while the phosphorylation of MAPK remained unchanged. The elevated levels of pAkt and pGSK3β are still evident after 24 hours post-sonication, a time point where the integrity of the BBB is known to be re-established. Furthermore, immunofluoresence staining localized this increase in pAkt and pGSK3β levels to neuronal cells flanking the region of the disrupted BBB;ConclusionsOur data demonstrates that ultrasound-mediated BBB disruption causes an activation of the Akt signaling pathway in neuronal cells surrounding the disrupted BBB.
Highlights
The Blood Brain Barrier (BBB) maintains the homeostasis of central nervous system by preventing the free passage of macromolecules from the systemic circulation into the brain
In the following study we show that the application of focused ultrasound in combination with ultrasound contrast agent disrupts the BBB integrity, and that this disruption is associated with reduced interaction of the tight junction protein occludin with ZO-1
Extravasation of IgG and enhancement of MRI contrast agent Application of focused ultrasound (Figure 1) in the presence of ultrasound contrast agent increases the permeability of the BBB, the effect of which is shown by enhancement of MRI contrast agent (Figure 2A, arrow)
Summary
The Blood Brain Barrier (BBB) maintains the homeostasis of central nervous system by preventing the free passage of macromolecules from the systemic circulation into the brain. Recent studies have shown that the application of focused ultrasound together with ultrasound contrast agent (microbubbles) temporarily increases the permeability of the BBB This effect is associated with breakdown of tight junctions, the structures that regulate the paracellular permeability of the endothelial cell layer. The homeostasis of the neuroparanchymal milieu is controlled by the presence of specialized tight junction structures existing between endothelial cells of brain microvessels. It has been shown that the nasal cavity provides a special anatomical feature so that drugs can pass the olfactory epithelium and enter the central nervous system [10] These modes of delivery into the brain are effective, they do not result in a focal disruption of the BBB for the treatment of localized diseases such as brain cancer
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