The mechanical index threshold for bloodbrain barrier disruption induced by ultrasound

and an ultrasound contrast agent appears to be

independent of ultrasonic frequency
Nathan McDannold, Natalia Vykhodtseva, and Kullervo Hynynen
Department of Radiology, Brigham & Womens Hospital, Harvard Medical School,
75 Francis St., Boston, MA 02115, USA
Abstract. This work investigated the effect of ultrasonic frequency on the
threshold for blood-brain barrier disruption induced by ultrasound pulses
combined with an ultrasound contrast agent. We found that the threshold
expressed in terms of the peak negative pressure amplitude increased as a
function of frequency. It appeared to be constant, however, when the exposures
were expressed as a function of mechanical index (frequency divided by square
root of frequency). Histological examination of representative samples with
similar amounts of blood-brain barrier disruption found that the number of
regions containing extravasated red blood cells per unit area was substantially
lower on average for lower ultrasound frequencies.
Keywords: Ultrasound, blood-brain barrier, drug delivery.

INTRODUCTION
The blood-brain barrier (BBB) is a major limitation to the delivery of drugs to the
central nervous system (CNS). This barrier, which is formed by the anatomical and
biochemical properties of brain endothelium, prevents all large-molecule agents and
even most small-molecule agents from being delivered to the brain [1]. It is arguably
the principle impediment to the use of therapeutics for CNS disease.
In the past few years, several studies in animals have demonstrated that low-power
ultrasound pulses combined with an ultrasound contrast agent can temporarily disrupt
the BBB with only negligible effects to the brain [2-5]. This technique is promising in
that it can be applied non-invasively and can be targeted.
While the exact mechanisms for the disruption are not known, it is presumably
related to the interaction between the ultrasound field, the microbubbles that make up
the ultrasound contrast agent, and the microvasculature. The interaction between
microbubbles and the ultrasound field is strongly affected by the ultrasound frequency
[6]. The purpose of this work was thus to examine the relationship between the
ultrasound frequency, the threshold for BBB disruption and the resulting tissue effects.

METHODS
The experiments were approved by our institutional animal committee. Sonications
were targeted one cm deep at two non-overlapping locations in each hemisphere of the
brains of male New Zealand white rabbits (weight: approximately 4 kg). The animals
were anesthetized using IM injections of a mixture of 12 mg of sodium xylazine
(Xyla-ject; Phoenix Pharmaceuticals, St Joseph, MO, USA) and 48 mg of ketamine
hydrochloride (Abbott Laboratories, North Chicago, IL, USA) given per kg of body
weight per hour. IA craniotomy (approximately 22 cm) performed at least two weeks
before the experiments provided an acoustic path into the brain. The fur on the skin
overlying the craniotomy was removed before the experiments. A total of 44 locations
were sonicated over the course of the experiments in 11 rabbits. Four locations were
excluded from the analysis due to operator errors.
An air-backed spherically curved transducer (center frequency: 2.04 MHz;
diameter/radius of curvature: 10/8 cm) generated the ultrasound beam. The transducer
was driven by a function generator (Model 395, Wavetek, San Diego, CA, USA) and
RF amplifier (model 240L, ENI Inc, Rochester, NY, USA). The electrical power was
measured with a power meter (model 438A, Hewlett Packard, Palo Alto, CA, USA)
and dual directional coupler (model C173, Werlatone, Brewster, NY, USA). The
transducers electrical impedance was matched to the output impedance of the
amplifier (50) by an external matching network. Measurements of the peak negative
pressure amplitude were performed with a calibrated membrane hydrophone (spot
diameter 0.5 mm, GEC-Marconi Research Center, Chelmsford, England) for the entire
pressure amplitude range used in degassed and deionized water. The values reported
here are in the brain after taking into account ultrasound attenuation through 10 mm of
brain, assuming an attenuation coefficient of 5 Np/m/MHz (0.43 dB/cm/MHz) [7].
Ten seconds before each sonication (parameters: 20 s duration; burst length: 10 ms;
pulse repetition frequency: 1 Hz), a bolus of ultrasound contrast agent (Optison, GE
Healthcare, Milwaukee, WI) was injected intravenously through the ear vein at a
dosage of 50 l per kg of body weight. This dosage was selected as it in the range
recommended for human use (0.55.0 ml; i.e., 7.1-71 m/kg for a 70 kg adult).
The transducer was attached to a three axis manual positioning system and
submerged in a tank of degassed, deionized water. The rabbit was positioned supine
on a plastic plate that was placed above this tank. The ultrasound beam propagated
vertically out of the tank through a hole in this plate. This hole was the opening of a
thin plastic bag filled with degassed water that provided acoustic coupling between the
water tank and the animal. The experiments were performed in a clinical 1.5T MRI
scanner (GE Healthcare, Milwaukee, WI, USA). A receive-only surface coil (7.6 mm
diameter, GE Healthcare, Milwaukee, WI, USA) was placed below the head to
provide imaging with a high signal-to-noise ratio.
BBB disruption was detected in contrast-enhanced T1-weighted fast spin echo
images acquired immediately after the last sonication in each brain (parameters:
TR/TE: 500/15-23 ms; ETL: 4; BW: 16 kHz; matrix size: 256256; NEX: 4; FOV: 10
cm; slice thickness: 1.5 mm; interslice spacing: 1.5 mm; contrast agent: Magnevist,
Berlex Laboratories, Inc., Wayne, NJ, USA administered IV at a dose of 0.125 mmol
per kg of body weight as a bolus injection).

The animals were sacrificed 4h after the last sonication. Representative examples
from were selected for histological examination. The samples selected had a signal
enhancement in contrast MRI that was within one standard deviation of the mean
enhancement for sonications applied at a peak pressure amplitude of 0.5 MPa using an
ultrasound frequency of 0.69 MHz. This value was selected because a previous study
found that these parameters produced negligible effects to the brain [3]. The brains
were fixed via transcardial perfusion (0.9%NaCl-250 ml, 10% buffered formalin
phosphate 500 ml) followed by immersion fixation (10% buffered formalin
phosphate). These brains were then embedded in paraffin and serially sectioned at 5
micrometers. Every 50th section (250 m between sections) was stained with
hematoxylin and eosin (H&E) for light microscopy evaluation. For each targeted
location, the section that showed the largest tissue effects was identified. The number
of regions containing extravasated erythrocytes was counted for these samples and
compared to counts from previous studies (see table 1). One author who was blind to
the acoustic parameters used performed the histology examination. This author did
know the locations of the sonications.

Data Analysis
The signal intensity enhancement was found by calculating the percent increase in
image intensity in a 33 voxel region of interest (ROI) at the sonication target in the
contrast-enhanced T1-weighted imaging. The percent increase in a 55 voxel ROI in a
nearby non-sonicated region (control) was subtracted from this value to exclude
enhancement due to contrast in the vasculature. BBB disruption was determined to
have occurred if the mean percent change in the target ROI was greater than the
standard deviation of the larger control ROI. The probability for BBB disruption, with
95% confidence intervals, was estimated as a function of peak negative pressure
amplitude using probit analysis, a regression model for the analysis of categorical data
often used for bioassay work [8]. The threshold was defined as the value where the
probability for BBB disruption was estimated to be 50%.
The BBB disruption threshold for sonications at 2.04 MHz was compared with
results at other frequencies obtained previously in rabbits [2,3,5,9-11] and rats [4] (see
table 1 for details). Except where noted, these experiments were performed under
identical conditions. This data was used to investigate the functional form of the
threshold (T) as a function of frequency (f). The equation:
T = afb
(1)
was fit using weighted non-linear least squares regression to find values of the
constants a and b. The weights were the one divided by the square of the 95%
confidence intervals found in the probit regression. Based on the value of b found in
this regression (see results), we combined the data at the different frequencies and
found a single BBB disruption threshold expressed in terms of the mechanical index
(MI), which is defined as the peak negative pressure amplitude divided by the square
root of the frequency. The number and density of the number of extravasations were
compared using an unpaired students t-test.

TABLE 1. Data for the different frequencies tested.

Frequency
(MHz)
0.26
0.69
1.50
1.63
2.04

Pressure
amplitude (MPa)*
0.1 - 0.6
0.4 - 1.5
0.4 - 2.6
0.7 - 4.7
0.3 - 2.3

N locations
(threshold)
54
60
119**
139***
40

Reference(s)
[2,10]
[3,11]
[4]
[5,9]
N/A

N locations
(histology)
7
8
-13****
8

Reference
[10]
[11]
-[9]
N/A

*Estimates in the brain; **30s sonications in rats, 3T MRI; all other data acquired in rabbits with 20s sonications (1.5T
MRI); ***100 ms bursts; all other data used 10 ms bursts; ****Animals sacrificed 72h after sonication; all other cases,
animals were sacrificed 4h after sonication

RESULTS

25

100

20

BBBD probability

Signal Intensity Change (%)

The MRI signal intensity enhancement is plotted as a function of peak negative

pressure amplitude for the 2.04 MHz sonications in fig. 1. The probability for BBB
disruption as a function of this pressure amplitude and the probit regression used to
estimate the value at which the probability was 50% are also shown. For this
frequency, this threshold was estimated to be 0.69 MPa (CI: 0.55 - 0.87).
To investigate the frequency dependence on this threshold, this data was compared
to previous studies performed with different ultrasound frequencies in rabbits
[2,3,5,9,10] and rats [4] (see table 1). Representative examples of contrast-enhanced
MRI from these different experiments are shown in fig. 2. The threshold for BBB
disruption increased as a function of ultrasound frequency (fig. 3). Regression of this
data to equation (1) yielded a value for b of 0.47 (95% confidence intervals: 0.430.51). Since the mechanical index (MI) is defined as the peak negative pressure
amplitude divided by the square root of the ultrasound frequency (i.e., b=0.5), this
result suggests that the threshold for BBB disruption for each frequency occurred at
the same MI. Plotting the threshold in MI as a function of frequency also suggests this
finding (fig 3, right). Based on these results, the data from all ultrasound frequencies
was combined and a single threshold for BBB disruption was found in MI. The
threshold where the probability for BBB disruption was 50% occurred at a MI of 0.47
(95% confidence intervals: 0.43-0.51).

15
10
5
0

80
60
40
20
0

MPa

Mpa

FIGURE 1: Left: Signal enhancement in contrast-enhanced T1-weighted MR images

as a function of peak negative pressure amplitude for sonications applied at an
ultrasound frequency of 2.07 MHz. Right: Probability for BBB disruption (BBBD) as
a function of peak negative pressure amplitude. The solid line indicates the probit
regression of this data; dotted lines indicate 95% confidence intervals.

0.26 MHz

0.69 MHz

1.63 MHz

2.04 MHz

cm

FIGURE 2: Examples of contrast-enhanced T1-weighted MR images of rabbit

brains showing targeted BBB disruption, indicated by focal contrast enhancement, for
the different frequencies tested in these studies. Four sonications were targeted in each
brain. Note that two locations sonicated at 0.26 MHz did not result in BBB disruption
in these examples.

0.8
0.7
0.6
0.5
0.4
0.3
Rabbit
Rat

0.2
0.1

0.25

0.75

1.25

1.75

US Frequency (MHz)

BBBD Threshold (MI)

BBBD Threshold (MPa)

Histological changes at 2.04 MHz were small and generally limited to minor
vascular damage, as indicated by tiny clumps of extravasated erythrocytes or
individual extravasated red blood cells that were scattered about the sonicated regions
(fig. 4). The total number of extravasations produced per sonication decreased slightly
as the ultrasound frequency increased for the representative samples examined,
although substantial variation was observed and the difference between the different
frequencies was not significant (fig. 5A). However, the density of extravasations
(number of extravasations divided by r, where r is the beam radius measured in
water) was often much higher for the 1.63 and 2.04 MHz sonications than any of the
lower frequency sonications (fig. 5B). On average, significantly more extravasations
per unit area (P<0.05) were found with 1.63 MHz than at 0.26 or 0.69 MHz. These
differences were evident even though the amount of contrast enhancement was similar
for these samples (fig. 5C).
0.8
0.7
0.6
0.5
0.4
0.3
Rabbit
Rat

0.2
0.1

0.25

0.75

1.25

1.75

US Frequency (MHz)

FIGURE 3: Left: BBB disruption (BBBD) threshold as a function of ultrasound

frequency. Right: BBB disruption threshold as a function of the mechanical index
(MI), which appeared to be the same for the different frequencies tested. Solid line:
regression of data to eq. (1); dotted lines: 95% confidence intervals.

(a)

# Extravasations

20
15
10
5
0
-5

0.26

0.69

1.63

US Frequency (MHz)

2.04

40

MRI Signal Enhancement (%)

25

Density of Extravasations (#/mm)

FIGURE 4: Typical tissue effects as seen in histology. Histological changes were

limited generally to minor vascular damage, as indicated by the presence of individual
extravasated red blood cells or tiny clumps of extravasated erythrocytes. Bar: 100 m
(b)

30
20
10
0
0.26

0.69

1.63

2.04

US Frequency (MHz)

25

(c)
20
15
10
5
0
-5

0.26

0.69

1.63

2.04

US Frequency (MHz)

FIGURE 5: A: Number of regions containing extravasations as a function of

ultrasound frequency. B: The density of extravasations vs. frequency. This density was
defined as the number of areas with extravasation divided by r, where r is the half
width of beam plots measured in water with a needle hydrophone. Lower ultrasound
frequencies had fewer cells per unit area on average, but substantial variation was
present. C: MRI contrast enhancement. Mean standard deviation shown.

DISCUSSION
These results suggest that the BBB disruption threshold is independent of
ultrasound frequency when expressed in MI. This result is in agreement with some
prior work that found that thresholds for lung damage in mice [12], killing of fruit fly
larvae [12], membrane damage in plants [13], and inertial cavitation of ultrasound
contrast agents in solution [14] are consistent with the frequency dependence that
defines the MI. However, several other studies have clearly shown that this is not
always the case [15-18]. Indeed, other work suggests that the threshold and magnitude
of the BBB disruption depends on the burst length [19]. Nevertheless, this work
supports the use of MI as a meaningful index for ultrasound-induced vascular
bioeffects in the presence of microbubbles provided that other sonication parameters
such as burst length are kept constant.
The MI was developed based on theoretical formulation of inertial cavitation
thresholds in water and blood and is used as a standard for setting limits on nonthermal bioeffects produced by ultrasound. This formulation was based on analytical
models of microbubble growth as a function of ultrasound frequency. While
ultrasound-induced BBB disruption is presumably related to the interaction of the
microbubbles and the brain vasculature, inertial cavitation does not appear necessary
to produce the disruption [10]. Other mechanisms that could be responsible include
radiation force, stretching of the vessel walls during bubble oscillation, and acoustic

streaming of the fluid surrounding the bubbles. Each of these effects should be related
to bubble growth, so perhaps our results are not surprising.
We also found that lower ultrasound frequencies produced fewer extravasations per
unit area. This result agrees with our earlier qualitative observations at 0.26 MHz
[2,10] These extravasations are likely the result of micro-damage to the blood vessels
resulting from inertial cavitation. The larger amount at higher frequencies may be due
to the resonant frequency of the microbubbles that make up Optison, which is close to
2 MHz. Perhaps inertial cavitation was more likely to occur at frequencies near the
resonant size. However, the bubbles behavior inside blood vessels may be different
than in solution [20]
This data is potentially limited by the resolution of the MRI. It is possible that small
regions with BBB disruption existed below our detection limit for the higher
frequency sonications. Such opening may also have resulted in fewer regions with
extravasated red blood cells. Future work using higher resolution imaging methods or
testing overlapping sonications are necessary to confirm that this did not occur.
In conclusion, the results from this study suggest that the BBB disruption threshold
is independent of ultrasound frequency when expressed in MI. They also suggest that
lower frequency sonication is associated with less micro-damage to the vasculature.
This latter result is favorable, as lower frequency devices are less affected by the skull.

ACKNOWLEDGMENTS
Support: NIH (R01EB003268, R33EB000705, U41RR019703). The authors thank
Yongzhi Zhang for his help with these experiments.

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