Perspectives in Medicine (2012) 1, 510

Bartels E, Bartels S, Poppert H (Editors):

New Trends in Neurosonology and Cerebral Hemodynamics an Update.
Perspectives in Medicine (2012) 1, 510

journal homepage: www.elsevier.com/locate/permed

Advances in neurosonology Brain perfusion,

sonothrombolysis and CNS drug delivery
Stephen Meairs

Department of Neurology, Universittsklinikum Mannheim, University of Heidelberg, Germany

KEYWORDS Summary Advances in microbubble pharmacology together with novel ultrasound technolo-
gies are leading to new emerging applications for both assessment and treatment of stroke
Microbubbles;
patients. Ultrasound perfusion imaging, for example, has added new perspectives for diagnosis
Ultrasound;
and monitoring of both ischemic and hemorrhagic stroke. Real-time brain perfusion imaging
Stroke;
of cerebral infarctions is now possible and quantitative algorithms for evaluation of regional
Therapy;
cerebral blood ow are being applied for the rst time in humans. There is growing interest
Sonothrombolysis;
in therapeutic applications of ultrasound, particularly in the eld of sonothrombolysis. Recent
Perfusion;
results indicate that ultrasound and microbubbles may be effective in clot lysis of ischemic
Drug delivery;
stroke even without additional thrombolytic drugs. Moreover, this combination may be effective
BBB
in treating the microcirculation, which will give new dimensions to the application of sonothrom-
bolysis in stroke patients. Further therapeutic avenues include opening of the bloodbrain
barrier (BBB) with ultrasound and microbubbles to enable novel drug delivery to the brain.
2012 Elsevier GmbH. Open access under CC BY-NC-ND license.

Brain perfusion imaging towards new mechanical index imaging. Because of the high acoustic
clinical applications intensities that are emitted by bursting bubbles, bubbles
that are behind the emitting bubbles (further away from
the ultrasound transducer) are shadowed by this effect
The most important advance in brain perfusion imaging dur-
and thus obscured from data analysis. Thus, areas of tissue
ing the last several years has been low-mechanical index
that are shadowed may not be available for analysis of tissue
(MI) real time perfusion scanning. This technique allows the
perfusion. The problem of shadowing is basically eliminated
detection of ultrasound contrast agent (UCA) in the cerebral
with low mechanical index imaging, since bubbles are not
microcirculation with little or no bubble destruction as com-
destroyed with such low acoustic pressures. Moreover, the
pared to the high MI-imaging. Because of minimal contrast
technique can obtain multi-planar real-time images of brain
agent bubble destruction, a high frame rate can be applied,
perfusion [1]. This is a signicant breakthrough for ultra-
which leads to a better time resolution of bolus kinetics
sound perfusion imaging, since previous approaches were
(Fig. 1). Low-MI imaging of contrast agent also avoids the
conned to a single image plane and therefore limited in
shadowing effect, a signicant problem associated with high
their assessment of the extents of brain infarction and low
perfusion states. The disadvantage of this new low-MI tech-
Correspondence address: Department of Neurology, Universitt-
nique, however, is the limited investigation depth due to the
low MI used. Recent technological advances suggest, how-
sklinikum Mannheim, University of Heidelberg, 68167 Mannheim,
ever, that perfusion imaging of the contralateral hemisphere
Germany. Tel.: +49 621 383 3550/3953; fax: +49 621 383 3807.
E-mail address: meairs@neuro.ma.uni-heidelberg.de through the temporal bone window will be possible.

2211-968X 2012 Elsevier GmbH. Open access under CC BY-NC-ND license.

doi:10.1016/j.permed.2012.02.038
6 S. Meairs

Figure 1 Real-time acoustic intensity curve of the contrast agent SonoVueTM after bolus injection on a Philips IU22 platform using
a 15 MHz dynamic pulsed array transducer. A very low mechanical index of 0.017 is used, which is then attenuated about 90% by
the skull bone before entering the brain. A region of interest has been drawn in the upper image to determine where mean acoustic
intensity will be measured. The smaller images in the middle of the gure are the individual image frames at 14 Hz. The red curve
displays the mean acoustic intensity of contrast bolus arrival from the region of interest for a total of 264 frames during 18.81 s.

If a constant concentration of contrast agent is deliv-
ered to brain tissue using a constant UCA infusion rate, then
after destruction with high MI ultrasound, new microbub-
bles will enter this eld with a certain velocity, will travel a
determined distance and ll a certain tissue volume depend-
ing on blood velocity. The intensity of the echo response
signal is directly related to the contrast agent concentra-
tion in the tissue; therefore, the blood ow assessment is
based on monitoring the intensity of the echo response signal
of the insonated volume after bubbles destruction. Low-
MI ultrasound imaging can be used to monitor microbubble
replenishment in real time (Fig. 2) following the applica-
tion of destruction pulses at high MI. The behavior of the
rell kinetics can be assessed with an exponential curve t
[2]. The parameters of this exponential curve are related to
cerebral blood ow: blood ow velocity is directly related to
the rate constant , the fractional vascular volume is related
to the plateau echo enhancement (A), and the product of
both (A ) is associated with blood ow [3]. More sophisti-
cated algorithms for characterization of microbubble rell
have been recently introduced [4,5], which should increase
reproducibility and improve the quantication of cerebral
blood ow with ultrasound perfusion imaging.
Since individual microbubbles can be depicted owing
through small vessels in the brain with low MI imaging, it
is possible to track these bubbles and map perfusion over
time. Dynamic microvascular microbubble maps provide
excellent demarcation of MCA infarctions (Fig. 3) and pro-
vide impressive displays of low velocity tissue microbubble
rell following destruction with high mechanical index imag-
ing. In brain regions showing delayed contrast bolus arrival
on perfusion-weighted MRI, ultrasound shows decreased
or absent microbubble rell kinetics. This new technique
has been applied for diagnosis of acute ischemic stroke.
Recent results demonstrate that real-time ultrasound per-
fusion imaging with analysis of microbubble replenishment
correctly identies ischemic brain tissue in acute MCA stroke
[6]. Pulse compression methods are being combined with
the nonlinear bubble imaging techniques discussed above for
highly sensitive contrast imaging with very low noise. These
advances will lead to further improvements of microbubble
imaging of the brain microcirculation, making ultrasound
perfusion imaging a viable application for bedside assess-
ment of acute stroke patients.
Therapeutic applications of ultrasound
sonothrombolysis
Ultrasound applied as an adjunct to thrombolytic therapy
improves recanalization of occluded intracerebral vessels
and microbubbles can amplify this effect. New data sug-
gest that ultrasound and microbubbles alone may be as
Advances in neurosonology Brain perfusion, sonothrombolysis and CNS drug delivery 7

Figure 2 Real-time acoustic intensity curve of the contrast agent SonoVueTM demonstrating rell following bubble destruction
with transient high mechanical index imaging. Mean acoustic intensity is measured in the region of interest in the upper image. The
smaller images in the middle of the gure are the individual image frames at 14 Hz. The yellow arrow shows the rst of a series of
high mechanical index images for microbubble destruction.

Figure 3 Microvascular map of left MCA infarction (arrows) characterized by absent or diminished contrast agent. The excellent
infarction demarcation compares well with MRI diffusion-weighted imaging (bottom images).
8 S. Meairs
effective as t-PA thrombolysis and also safer with less risk of
hemorrhage [7,8].
Ultrasound-sensitive thrombolytic drug delivery com-
bined with specic targeting is highly attractive. Targeting
of clot-dissolving therapeutics can potentially decrease the
frequency of complications while simultaneously increasing
treatment effectiveness by concentrating the available drug
at the desired site and permitting a lower systemic dose [9].
Clinical studies support the use of ultrasound for therapy
of ischemic stroke, and rst trials of enhancing sonothrom-
bolysis with microbubbles have been encouraging. A recent
meta-analysis of all published clinical sonothrombolysis
studies conrmed that ultrasound and tPA (with or with-
out microbubbles) increases recanalization compared to
tPA alone [10]. These observations have led to design
of CLOTBUSTER, a phase III controlled clinical trial of
sonothrombolysis.
Sonothrombolysis of spontaneous intracranial
hemorrhages
One emerging clinical application is sonothrombolysis of
intracranial hemorrhages for clot evacuation using catheter-
mounted transducers. As compared with MISTIE (Minimally
Invasive Surgery plus T-PA for Intracerebral Hemorrhage
Evacuation) and CLEAR (Clot Lysis Evaluating Accelerated
Resolution of Intraventricular Hemorrhage II) studies data,
the rate of lysis during treatment for IVH and ICH was faster
in patients treated with sonothrombolysis plus rt-PA versus
rt-PA alone [11]. Thus, lysis and drainage of spontaneous ICH
and IVH with a reduction in mass effect can be accomplished
rapidly and safely through sonothrombolysis using stereotac-
tically delivered drainage and ultrasound catheters via a bur
hole.
MRI-guided focused ultrasound for clot lysis
Histotripsy is a process which fractionates soft tissue through
controlled cavitation using focused, short, high-intensity
ultrasound pulses. Histotripsy can be used to achieve effec-
tive thrombolysis with ultrasound energy alone at peak
negative acoustic pressures >6 MPa, breaking down blood
clots in about 1.55 min into small fragments less than 5 m
diameter [12]. Recent developments in using MR-guided
focused ultrasound therapy through the intact skull suggest
that this technology could be useful for clot lysis in humans.
Experimental studies are currently being undertaken to test
this possibility, both in ischemic and hemorrhagic stroke.
Ultrasound and microbubbles for treatment of the
microcirculation
Ultrasound and microbubbles may improve ow to the
microcirculation irrespective of recanalization, thus open-
ing new opportunities for application of sonothrombolysis
in acute ischemic stroke. This was suggested by results of
a study on possible adverse bioeffects [13] of 2 MHz ultra-
sound and microbubbles (SonovueTM ) in a middle cerebral
artery permanent occlusion model in rats at different steps
in the cascade of tissue destruction after ischemic stroke
[14]. While deleterious effects were not observed, infarc-
tions were unexpectedly smaller in the treatment group,
despite the fact that in all animals recanalization of the MCA
did not occur. This suggested a benecial effect of ultra-
sound and microbubbles in the microcirculation. A similar
tissue protective effect has been found in an in vivo ani-
mal study using intravenous microbubbles and transthoracic
ultrasound to treat acute coronary thromboses. Pigs treated
with ultrasound and intravenous peruorocarbon microbub-
bles (PESDA) had signicantly greater improvements in ST
segments over a 30-minute treatment period when com-
pared with pigs treated with ultrasound alone or with control
animals. Moreover, there was a signicantly smaller myocar-
dial contrast defect size after treatment with ultrasound
and PESDA [15]. Recently, nano-CT was used to demon-
strate complete reversal of microcirculatory impairment in
a rodent reperfusion model following treatment with rt-PA,
ultrasound and microbubbles [16]. The mechanism of the
microcirculatory effect of ultrasound and microbubbles may
involve improvement of blood ow to risk tissue via col-
laterals and changes in the microenvironment of damaged
tissue, like decreased cell damaging factors, e.g. glutamate
or enhanced enzyme activity of endothelial nitric oxide [17].
Further work is necessary to elucidate the exact mecha-
nisms of salvaging of tissue-at-risk by ultrasound-mediated
microbubble thrombolysis.
Opening the bloodbrain barrier with focused
ultrasound and microbubbles
The bloodbrain barrier is a signicant obstacle for delivery
of both small molecules and macromolecular agents. Indeed,
potential therapeutic substances, which cannot be applied
in the presence of an intact BBB are neuropeptides, proteins
and chemotherapeutic agents. Likewise, large-molecules
such as monoclonal antibodies, recombinant proteins, anti-
sense, or gene therapeutics do not cross the BBB.
There is a good deal of evidence showing that ultra-
sound can be used to permeate blood-tissue barriers. Large
molecules and genes can cross the plasma membrane of cul-
tured cells after application of acoustic energy [18]. Indeed,
electron microscopy has revealed ultrasound-induced mem-
brane porosity in both in vitro and in vivo experiments [19].
High-intensity focused ultrasound has been shown to allow
selective and non-destructive disruption of the BBB in rats
[20]. If microbubbles are introduced to the blood stream
prior to focused US exposure, the BBB can be transiently
opened at the ultrasound focus without acute neuronal dam-
age [21]. Thus, the introduction of cavitation nuclei into the
blood stream can conne the ultrasound effects to the vas-
culature and reduce the intensity needed to produce BBB
opening (Fig. 4). This can diminish the risk of tissue damage
and make the technique more easily applied through the
intact skull. In most studies, the conrmation of BBB disrup-
tion has been obtained with MR contrast imaging at targeted
locations [2123] or with post mortem histology [20,24].
Targeted delivery of antibodies to the brain has been
accomplished with focused ultrasound. Dopamine D(4)
receptor-targeting antibody was injected intravenously and
shown to recognize antigen in the murine brain follow-
ing disruption of the BBB with ultrasound [22]. Likewise,
Advances in neurosonology Brain perfusion, sonothrombolysis and CNS drug delivery
Figure 4 Example of bloodbrain disruption by ultrasound
and microbubbles in a rat brain. Extravasation of Evans blue
marks BBB disruption. Exposure time was 20 s with a 1 MHz sec-
tor transducer at a pressure amplitude of 2.5 MPa through the
rat skull with about 50% attenuation.
doxorubicin, a chemotherapeutic drug that does not cross
the BBB, has been administered to the brain using ultra-
sound and microbubbles [25]. Different levels of doxorubicin
in the brain were accomplished through alteration of the
microbubble concentration. These results are encouraging
and provide an important framework for future studies
aimed at local disruption of the BBB for delivery of macro-
molecular agents to the brain.
Several avenues of transcapillary passage after ultra-
sound sonication have been identied. These include
transcytosis, passage through endothelial cell cytoplas-
mic openings, opening of tight junctions and free passage
through injured endothelium [26]. One study investigated
the integrity of the tight junctions (TJs) in rat brain
microvessels after BBB disruption by ultrasound bursts (1.5-
MHz) in combination with Optison [27]. BBB disruption, as
evidenced by leakage of i.v. administered horseradish perox-
idase (HRP) and lanthanum chloride, was paralleled by the
apparent disintegration of the TJ complexes, the redistri-
bution and loss of the immunosignals for occludin, claudin-5
and ZO-1. At 6 and 24 h after sonication, no HRP or lan-
thanum leakage was observed and the barrier function of
the TJs, as indicated by the localization and density of
immunosignals, appeared to be completely restored. The
results of these studies demonstrate that the effect of ultra-
sound upon TJs is very transient, lasting less than 4 h.
Although much effort has been undertaken to demon-
strate the safety of BBB opening with ultrasound and
microbubbles, further work is needed to elucidate the
molecular effects of this application. Recent data demon-
strate that at the upper thresholds of acoustic pressure
for safe BBB opening a reorganization of gap-junctional
plaques in both neurons and astrocytes may occur [28].
This is important because gap junctions allow transfer of
information between adjacent cells and are responsible for
tissue homeostasis. Likewise, there is evidence that focused
ultrasound-induced opening of the BBB in the presence of
ultrasound contrast agents can lead to increased ubiqui-
tinylation of proteins in neuronal cells [29], indicating that
9
brain molecular stress pathways are affected by this treat-
ment. Nevertheless, this new technology for delivering drugs
across the BBB will offer exciting opportunities for treat-
ment of a variety of brain diseases in the future.
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