Effects on P-Glycoprotein Expression after Blood-Brain Barrier Disruption Using Focused Ultrasound and Microbubbles

Abstract

Many blood-borne substances attempting to pass through the luminal membrane of brain endothelial cells are acted upon by a variety of metabolizing enzymes or are actively expelled back into the capillary lumen by embedded efflux transporters, such as Permeability-glycoprotein (Pgp). Overexpression of this protein has also been linked to multidrug resistance in cancer cells. Previous studies have shown that focused ultrasound (FUS), when combined with a microbubble agent, has ability to temporarily disrupt blood-brain barrier (BBBD). In this work, we investigated whether modulation of Pgp expression is part of the FUS-induced effects. We found that ultrasound can temporarily suppress Pgp expression. When BBBD was produced at 0.55 MPa, Pgp was suppressed up to 48 hours and restored by 72 hours. At 0.81 MPa, suppression can last 72 hours or longer. These findings support the idea that microbubble-enhanced FUS disrupts the functional components of the BBB through suppression of drug efflux.

Fig 1. Schematic of the MRI-guided FUS system used in this work.

The function generator, amplifier, and power meter were located outside the MRI room. A passive cavitation detector (PCD) was used to monitor the acoustic emissions.

Fig 2. Sonication planning, monitoring, and assessment of FUS-induced BBBD after microbubble-enhanced sonication at 0.55 (left) and 0.81 (right) MPa.

A. Axial MRI acquired during FUS-BBBD. T2-weighted imaging (T2WI) was used for treatment planning, contrast-enhanced T1-weighted imaging (CE-T1WI) to verify BBB permeabilization, and T2*-weighted imaging (T2*WI) to detect tissue damage. The targeted spots are indicated by circles; the yellow curves outline the extent of MRI contrast enhancement due to BBB permeabilization. Hypointense areas were evident in the focal plane at 0.81 MPa in T2*WI (arrow), indicating vessel damage and regions with extravasated erythrocytes; hyperintense areas in these images were due to MRI contrast agent extravasation. B. Appearance in H&E stained sections after BBBD at 0.55 MPa (left, 24 hours) and at 0.81 MPa (right, one hour). At 0.55 MPa, most of the sonicated region appeared unaffected, but in a few areas with tiny clusters of extravasated erythrocytes were seen (inset). Larger regions of erythrocytes were evident at 0.81 MPa. C. Examples of acoustic emissions recordings. These power spectra were normalized to recordings obtained without microbubbles. In each case, strong harmonic emissions were observed. Ultraharmonic and subharmonic emission were observed occasionally (arrows).

Fig 3. Method of scoring Pgp intensity after immunohistochemical staining.

The microphotograph shows a section from the anterior right caudate putamen at a depth of 3 mm relative to the dural surface, the depth of the focal plane. The monoclonal antibody C219 was used for Pgp staining. Prominent immunoreactivity is evident in endothelial cells of capillaries. The intensity of the P-gp reaction was scored for each vessel in the section on a scale from 0–2, with 0 = not stained, 1 = partially-stained, 2 = fully-stained. Scale bar: 20 μm.

Fig 4

A. Representative microphotographs from regions in the sonicated and control hemispheres at different time points after BBBD induced by microbubble-enhanced sonication at 0.55MPa. Staining intensity was clearly reduced at one, 24, and 48 hours, but not at 72 hours. Fig 4. B. Pgp staining intensity scores at different time points after FUS-induced BBBD at 0.55MPa. A comparison of Pgp immunostaining in sonicated and control hemispheres revealed statistically significant differences in animals that were sacrificed one, 24 and 48 hours after BBBD (p<0.05, p<0.05, p<0.01, respectively), but not at 72 hours (p = 0.43). Scale bar: 40 μm.

Fig 5

A. Representative microphotographs from regions in the sonicated and control hemispheres at different time points after BBBD induced by microbubble-enhanced sonication at 0.81 MPa. Except at 72 hours, the center of the sonicated region was diffusely stained. At the periphery, the staining intensity was reduced at all time points. Fig 5. B. Pgp staining intensity scores at different time points after BBBD induced by microbubble-enhanced sonication at 0.81MPa. A comparison of Pgp immunostaining in sonicated and control hemispheres found statistically significant differences between animals that were sacrificed 48, 72 hours after BBBD (p<0.05) but not at one (p = 0.25) or 24 hours (p = 0.094). Scale bar: 40 μm.

Fig 6. Relative Pgp staining intensity scores for the two exposure levels as a function of time.

The scores were normalized to that measured in the control hemisphere. The difference in the relative intensity scores were not significant (p = 0.25, 0.3, 0.24 and 0.058, for one, 24, 48 and 72 hours, respectively).

Fig 7. Strength of harmonics recorded during ultrasound mediated BBBD for the two exposure levels tested.

The difference in the strength of the fourth harmonic was statistically significant (P<0.05), but not at the second or third harmonics (P = 0.054, 0.06, respectively).