Jour nal of Biosci enc e s an d M e dic ine s, 2013, 1, 10-13 JBM Published Online October 2013 (
The effects of ultrasound on blood-brain barrier
Feng-Yi Yang, Wei-Ting Lin
Department of Biomedical Imaging and R adi ological Sciences , National Yang-Ming University, Taipei, Taiwan
Received August 2013
The brain is protected from the entry of foreign sub-
stances by blood-brain barrier (BBB), but becomes a
barrier while chemotherapy is needed for the brain
diseases. Ultrasound with microbubbles (MBs) has
been shown to noninvasively increase the permeabili-
ty of the BBB in the normal tissue and brain tumor.
The real mechanism for disruption is still unknown.
Hemorrhage was usually found in the sonicated re-
gion of the brain. Thus, treatment safety is the pri-
mary concern when considering clinical application
of BBB disruption induced by ultrasound in the pre-
sence of MBs. Here we investigate the effects of ul-
trasound on the permeability of BBB whether the
MBs were administered. The data reveals that Evans
blue (EB) a ccu mulation w as highest in t he brain after
sonication with MBs. However, the permeability of
BBB also can be significantly increased by ultrasound
alone. These results demonstrated that noninvasive
disruption of BBB by ultrasound alone with no dam-
age i s p ossible.
Keywords: Ultrasound; Blood-Brain Barrier;
Permeability; Drug Delivery; Brain Disease
Most brain diseases are hard to treat with chemotherapeu-
tics due to the inability of molecules to pass the bloo d-brain
barrier (BBB). The endothelial cells of the brain are
tightl y fused to each othe r known as tight j unctions [1].
Several methods have been developed to disrupt the
BBB to facilitate drug delivery. Recently, it has been
shown that BBB can be locally and noninvasively dis-
rupted by focused ultrasound (FUS) in the presence of
microbubbles (MBs) [2-4]. However, small areas of
erythrocyte extravasation were found in the sonicated
site [5,6]. Interactions between the ultrasound and
MBs—which include oscillatory forces, acoustic cavi-
tation, and shear stress related to streaming of fluid
around the bubblesare likely to trigger various physi-
ological responses [7]. The side effect of hemorrhage
may b e induced by the widening of the tight junc tion or
vessel damage after FUS sonication. In this study, we
investigate if BBB can be disrupted noninvasively by
FUS without MBs administration in order to avoid the
brain damage from cavitation effects.
2.1. Animal Prepara tion
A total of t welv e male Spr ague-Dawley rats weighing
from 280 to 350 g were used in these experiments. All
the procedures of the animal experiment adhered to the
Guidelines for Care and Use of Experimental Animals
by our institutional animal committee.
2.2. Ultrasound System
FUS was produced by a 1 MHz single-element focused
transducer (A392S, Panametrics, Waltham, MA, USA)
with a diameter of 38 mm and a radius of curvature of
63.5 mm. The half-maximum of the pressure amplitude
of the focal zone had a diamete r and length o f 3 and 26
mm, respectively. The transducer was mounted on a
removable cone filled with deionized and degassed wa-
ter whose tip was capped by a polyurethane memb r ane ,
and the center of the focal spot was at approximately
5.7 mm below the cone tip. FUS beam was precisely
targeted using a stereotaxic apparatus (Stoelting, Wood
Dale, IL, USA). A function generator (33220A, Agilent
Inc., Palo Alto, USA) was connected to a power am-
plifier (500-009, Advanced Surgical Systems, Tucson,
AZ) to drive the FUS transducer and a power meter/
sensor module (Bird 4421, Ohio, USA) was used to
measure the input electrical power. The animal posi-
tioning for the sonication arrangement was the same as
our previous works [8,9]. The rat was laid prone be-
neath the cone tip and ultrasound transmission gel
(Pharmaceutical Innovations, Newark, NJ, USA) was
used to maximize the transmission of ultrasound be-
tween the transducer and the rats b r ai n.
2.3. Soncation
Ultrasound contrast agent (UCA, SonoVue, Bracco In-
F.-Y. Yang, W.-T. Lin / Journal of Biosci ences and Medicines 1 (2013) 10-13
Copyright © 2013 SciRes. OPEN ACCESS
ternational, Amsterdam, The Netherlands) was injected
into the tail vein of the rats about 15 s before each soni-
cation. This agent contains phospholipid-coated micro-
bubbles at a concentration of 1 - 5 × 108 bubbles/ml, with
the bubbles having a mean diameter of 2.5 μm. The so-
nication was precisely targeted using a stereotaxic appa-
ratus. The ultrasound beam was delivered to one location
in one brain hemisphere. The following sonication para-
meters were used: an acoustic power of 2.86 W with or
without an inj ec tio n o f 300 μl/k g UC A, an acoustic power
of 1.01 W without UCA injection, a pulse repetition fre-
quency of 1 Hz, and a duty cycle of 5%. The schematic
diagram of the FUS system for sonication is shown in
Figure 1.
2.4. Evans Blue Extravasation
When measuring vascular permeability, it has been
shown that BBB disruption can be quantified based on
the extravasation of EB. The animals were sacrificed
approximately 4 hours after the EB injection. These rats
were then perfused with saline via the left ventricle until
colorless perfusion fluid appeared from the right atrium.
After perfusion and brain removal, the brain was sec-
tioned into fo ur slices (6 mm posterior to the bregma)
and mounted on glass slides. The four sections were then
divided into r ight and left hemispher es before measuring
the amount of EB extravasation. The unsonicated he-
mispheres formed the control groups. Samples were
weighed and then soaked in 50% trichloroacetic acid
solution. After homogenization and centrifugation, the
extracted dye was diluted with ethanol (1:3), and the
amount of dye present measured using a spectrophoto-
meter (PowerWave 340, BioTek, USA) at 620 nm. The
EB present in the tissue samples was quantified using a
linear regression standard curve derived from seven con-
centrations of the dye; the amount of dye was denoted in
absorbance per gram of tissue. All data are typically ex-
pressed as means ± SEM. Statistical analysis was per-
Figure 1. Diagrams of the experi-
mental FUS setup for BBB disrup-
formed with a standard t-test. Statistical sig nificance was
defined as p values 0.05.
Two experimental protocols were performed to assess
firstly the degree of the BBB disruption that occurs at
an acoustic power of 2.86 W with an injection of 300
μl/kg MBs and secondly the effect of various sonication
powers on the BBB per meability.
3.1. BBB Disruption by FUS with MBs
In the first protocol, three rats were sonicated with FUS
in the presence of MBs for the sonication time of 1 min.
Figure 2 shows that a sonication with MBs is able to
significantly increase the accumulation of EB in the
sonicated brain compared to the unsonicated brain.
3.2. Effect of FUS on BBB Permeability
Another experiment was performed to investigate the
effect of FUS alone on the permeability of BBB at an
acoustic power of 2.86 W or 1.01 W for the sonication
time of 5 min.
In F i g u re 3 , we s h owed compar iso ns of EB extravasa-
tion in the right or left hemisphere brain after FUS ex-
posure alone at an acoustic power of 2.86 W. No matter
right or left hemisphere brain, EB extravasation in the
contralateral brain was significantly higher than that in
the sonicated brain, especially for the left contralateral
brain. In addition, the EB extravasation was similar be-
twee n the right bra i n and le ft brai n after FUS expos ure.
Oppositely, Figure 4 shows that EB extravasation in
the sonicated brain was significantly higher than that in
the contralateral brain while a lower acoustic power of
1.01 W was applied.
Figure 2. EB extr avasation for the brain sonicat ed at an acou s-
tic power of 2.86 W with 300 μl/kg UCA. *Denote significant
difference compared with the contralateral normal brain.
F.-Y. Yang, W.-T. Lin / Journal of Biosci ences and Medicines 1 (2013) 10-13
Copyright © 2013 SciRes. OPEN ACCESS
Figure 3. EB extravasation for the brain sonicated at an
acoustic power of 2.86 W in the right “a” or left “b” hemis-
phere. *Denote significant difference compared with the con-
tralateral normal brain.
This stud y demon strated tha t the various acoustic powers
of FUS exposure alone that affecting BBB permeability
as evaluated by EB accumulation. BBB permeability can
be enhanced by FUS exposure alone and the effic ie ncy is
dependent on the acoustic power of sonication.
Many methods have been created to overcome BBB
while chemotherapy is needed, such as chemical modifi-
cation of drugs, and direct injection through a catheter
[10,11]. Recently, FUS with MBs has been developed as
a promising tool for target drug delivery in the brain be-
cause BBB can be locally and noninvasively disrupted
by this technology. One major shortcoming of FUS with
MBs is that hemorrhage or brain damage is usually pro-
duced by inertial cavitation when the ultrasound para-
meters and the dose of MBs are not optimized. So far,
the behaviors of MBs after sonication are still unable to
approximately predict by mathematical model. Therefore,
safety concern is the key fa ct or fo r this tec hno log y in the
clinical applica tion.
Figure 4. EB extravasation for the brain sonicated with an
acoustic power of 1.01 W in the right hemisphere brain. *De-
note significant difference compared with the contralateral
normal brain.
One study demonstrated BBB disruption was pro-
duce d in the thermal lesio ns after so nication alone, b ut it
has always been associated with tissue damage [12].
Here, we proposed that BBB disruption can be induced
by pul sed FUS in the absence of MBs. Figure 3 showed
that EB extravasation did not increase in the sonicated
brain when sonication was applied at a higher acoustic
power of 2.86 W. On the contrary, the permeability of
BBB in the contralateral brain significantly increased due
to the reflecting ultrasound wave from the bottom skull.
Figure 4 showed that EB significantly increased in the
sonicated brain when a lower acoustic power of 1.01 W
was applied. The results suggest that the degree of BBB
disruption induced by sonication alone was not propor-
tional to the acoustic power. Thus, the degree of locali-
zation of BBB disruption can be enhanced by FUS ex-
posure alone at an appropriate acoustic power. The in-
creases in the BBB permeability reported here are im-
portant for the safe de live r y o f d rugs t o the br a i n. F urt he r
investigations are necessary for optimal ultrasound pa-
rameters and histological examinations.
This study was supported by grants from the National Science Council
of Taiwan (no. NSC 101-2320-B-010-036-MY3, NSC 102-2221-E-
010-005-MY3), Cheng Hsin General Hospital Foundation (no. 102-
F218C1 1 an d 1 0 1F19 5CY18).
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