A Clinical System for Non-invasive

Blood–Brain Barrier Opening Using a

Neuronavigation-Guided
Single-Element Focused Ultrasound
Transducer Antonios N. Pouliopoulos &
Shih-Ying Wu & Mark T. Burgess &
Maria Eleni Karakatsani & Hermes A.S.
Kamimura & Elisa E. Konofagou
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 ARTICLE IN PRESS
Ultrasound in Med. & Biol., Vol. 00, No. 00, pp. 117, 2019
Copyright © 2019 World Federation for Ultrasound in Medicine & Biology. All rights reserved.
Printed in the USA. All rights reserved.
0301-5629/$ - see front matter

https://doi.org/10.1016/j.ultrasmedbio.2019.09.010

Original Contribution

A CLINICAL SYSTEM FOR NON-INVASIVE BLOODBRAIN BARRIER OPENING

USING A NEURONAVIGATION-GUIDED SINGLE-ELEMENT FOCUSED
ULTRASOUND TRANSDUCER

TAGEDPANTONIOS N. POULIOPOULOS,* SHIH-YING WU,* MARK T. BURGESS,* MARIA ELENI KARAKATSANI,*

HERMES A.S. KAMIMURA,* and ELISA E. KONOFAGOU*,yAGEDNTE
* Department of Biomedical Engineering, Columbia University, New York City, New York, USA; and y Department of Radiology,
Columbia University, New York City, New York, USA
(Received 29 July 2019; revised 16 September 2019; in final from 16 September 2019)

Abstract—Focused ultrasound (FUS)-mediated blood-brain barrier (BBB) opening is currently being investigated in
clinical trials. Here, we describe a portable clinical system with a therapeutic transducer suitable for humans, which
eliminates the need for in-line magnetic resonance imaging (MRI) guidance. A neuronavigation-guided 0.25-MHz
single-element FUS transducer was developed for non-invasive clinical BBB opening. Numerical simulations and
experiments were performed to determine the characteristics of the FUS beam within a human skull. We also validated
the feasibility of BBB opening obtained with this system in two non-human primates using U.S. Food and Drug Admin-
istration (FDA)-approved treatment parameters. Ultrasound propagation through a human skull fragment caused 44.4
§ 1% pressure attenuation at a normal incidence angle, while the focal size decreased by 3.3 § 1.4% and
3.9 § 1.8% along the lateral and axial dimension, respectively. Measured lateral and axial shifts were 0.5 § 0.4 mm
and 2.1 § 1.1 mm, while simulated shifts were 0.1 § 0.2 mm and 6.1 § 2.4 mm, respectively. A 1.5-MHz passive cavita-
tion detector transcranially detected cavitation signals of Definity microbubbles flowing through a vessel-mimicking
phantom. T1-weighted MRI confirmed a 153 § 5.5 mm3 BBB opening in two non-human primates at a mechanical
index of 0.4, using Definity microbubbles at the FDA-approved dose for imaging applications, without edema or hemor-
rhage. In conclusion, we developed a portable system for non-invasive BBB opening in humans, which can be achieved
at clinically relevant ultrasound exposures without the need for in-line MRI guidance. The proposed FUS system may
accelerate the adoption of non-invasive FUS-mediated therapies due to its fast application, low cost and portability. (E-
mail: ek2191@columbia.edu) © 2019 World Federation for Ultrasound in Medicine & Biology. All rights reserved.

Key Words: Bloodbrain barrier, Focused ultrasound, Clinical system, Drug delivery.

INTRODUCTION circulating microbubbles, FUS can perform targeted, non-

invasive and reversible bloodbrain barrier (BBB) opening
Focused ultrasound (FUS) serves as a non-invasive and
(Hynynen et al. 2001; Konofagou 2012). FUS-mediated
non-ionizing therapeutic modality, with applications in lith-
BBB opening has been successfully and safely tested for
otripsy (Miller and Thomas 1996), tumor ablation (Xia
well over 15 years in a variety of animal models, from
et al. 2012), neuromodulation (Kamimura et al. 2016; Tyler
rodents (Choi et al. 2007; Sheikov et al. 2008) to non-
et al. 2018) and essential tremor treatment (Lipsman et al.
human primates (NHPs) (Marquet et al. 2011; Arvanitis
2013; Elias et al. 2016). Microbubbles are routinely used as
et al. 2012; Wu et al. 2018). The success of these pre-
contrast agents in ultrasound imaging (Cosgrove and Har-
clinical studies has paved the way toward clinical imple-
vey 2009) and as stress mediators in ultrasound therapy
mentation of this technology.
(Coussios and Roy 2008) to deliver drugs into cells (Fan
The first published clinical study regarding the appli-
et al. 2012; Shamout et al. 2015), tumors (Graham et al.
cation of ultrasound and microbubbles to increase BBB
2014; Sun et al. 2017; Arvanitis et al. 2018) or tissues
permeability was reported by Carpentier et al. (2016). The
(Kotopoulis et al. 2013). In conjunction with systemically
authors used an implantable 11.5-mm unfocused single-ele-
ment 1.05-MHz transducer called SonoCloud (Goldwirt
Address correspondence to: Elisa E. Konofagou, 351 Engineer- et al. 2016; Horodyckid et al. 2017), which was fixed
ing Terrace, 1210 Amsterdam Avenue, Mail Code: 8904, New York,
NY 10027, USA. E-mail: ek2191@columbia.edu within the skull bone and connected to an external power

1
 ARTICLE IN PRESS
2 Ultrasound in Medicine & Biology
supply via a transdermal needle. Glioblastoma multiforme
(GBM) patients were enrolled in this study and were
exposed to repeated monthly FUS treatments before receiv-
ing systemic chemotherapy with carboplatin. The results
indicated that the BBB was disrupted at acoustic pressures
up to 1.1 MPa (i.e., mechanical index [MI] = 1.07) without
detectable adverse effects on magnetic resonance imaging
(MRI) or clinical examination. Results from a larger cohort
of 19 subjects revealed an increase in median progression-
free survival for patients with clear BBB disruption (Idbaih
et al. 2019). The same group has developed a quantification
method for FUS treatment assessment (Asquier et al. 2019)
and is conducting a clinical trial with Alzheimer’s disease
(AD) patients (NCT03119961). In addition, GBM patients
are currently treated with a new-generation SonoCloud,
which enlarges the treatment volume by nine times (SC9;
NCT03744026); this device has recently received U.S.
Food and Drug Administration (FDA) approval for a phase
1/2a trial in the United States.
Another non-invasive approach involves the gener-
ation of FUS through a 1024-element 0.22-MHz hemi-
spherical array embedded within the MRI bore.
Real-time acoustic emission monitoring is used to deter-
mine the pressure levels during the FUS treatment
(O’Reilly and Hynynen 2012). The first study revealing
BBB opening in 5 AD patients using the MR-guided
ExAblate system developed by Insightec (Insightec Inc.,
Tirat Carmel, Israel) was published by Lipsman et al.
(2018). AD patients received FUS treatments aimed at
the dorsolateral prefrontal cortex. BBB opening was
fully reversible, with no contrast enhancement detected
1d after treatment. The same group recently published
results from a trial with a cohort of five GBM patients,
who have been treated with FUS in combination with
temozolomide or doxorubicin (Mainprize et al. 2019).
BBB opening was observed in all patients and an
increase of the delivered chemotherapy was measured in
the 2 patients for whom data were available. Currently,
there are multiple clinical trials using the ExAblate
Neuro system throughout the world, for targeted BBB
opening in patients with GBM (NCT03322813,
NCT03712293, NCT03616860, NCT03551249); AD
(NCT02986932, NCT03671889, NCT03739905), Her2-
positive breast cancer brain metastases (NCT03714243);
amyotrophic lateral sclerosis (NCT03321487); and
Parkinson’s disease (PD) dementia (NCT03608553).
Among the advantages of multi-element arrays are the
ability to correct for skull-induced aberrations based on
computed tomography (CT) scans of the treated subject
(Clement and Hynynen 2002; Aubry et al. 2003), simul-
taneous treatment monitoring via passive cavitation
mapping (Jones et al. 2013), standing wave reduction
and flexibility in the positioning of the focal volume
through electronic steering.
Volume 00, Number 00, 2019
An alternative approach is to employ neuronavi-
gation systems instead of MRI for FUS guidance.
Neuronavigation-assisted BBB opening using a
0.4-MHz single-element transducer was proposed by
Wei et al. (2013). It was found that the precision of
this technique was comparable to that of stereotactic
procedures in a swine model, with a targeting error of
2.3 § 0.9 mm. BBB opening was observed above a
derated pressure threshold of 0.43 MPa at 0.4 MHz
(i.e., MI = 0.68), using a constant infusion of 0.3 mL/
kg/min SonoVue microbubbles. Clinical trials with
this system, called NaviFUS, are currently in progress
in Taiwan, recruiting GBM (NCT03626896) and
drug-resistant epilepsy (NCT03860298) patients.
Our group has reported on successful BBB opening
through neuronavigation targeting in an NHP model with
simultaneous real-time passive cavitation detection (PCD)
and passive acoustic mapping (PAM) (Wu et al. 2018). In
this study, the average precision between planned and actual
targeting was 3.1 mm, compared with 4.3 mm for the
frame-based stereotaxis. The BBB opening threshold was
350 kPa at 0.5 MHz (MI = 0.5), using 4- to 5-mm monodis-
perse microbubbles at a dose of 2.5 £ 108 microbubbles/kg.
We found that 2-D PAM can be used to predict and verify
the BBB opening location. The total FUS procedure dura-
tion was less than 30 min, which is equivalent to the dura-
tion of standard radiation therapy.
Here, we sought to establish the clinical relevance
of the previously described approach and develop a neu-
ronavigation-guided focused ultrasound (NgFUS) sys-
tem suitable for use in humans. Our objectives were to
(i) perform BBB opening at a lower frequency of
0.25 MHz, which would allow for lower attenuation/dis-
tortion of the ultrasound beam and would thus be more
suitable for humans; (ii) determine the transducer char-
acteristics (e.g., radius of curvature and aperture size)
required to expand the treatment envelope and enable
coverage of the human brain using a single-element FUS
transducer; (iii) confirm the ability of low-frequency
PCD transducers to detect cavitation signals through the
human skull; and (iv) investigate whether BBB opening
with real-time PCD monitoring is possible in an NHP
model using Definity microbubbles at the FDA-approved
dose for ultrasound imaging applications and clinically
relevant ultrasound parameters. The same system is cur-
rently being designated for testing in a small cohort of
AD subjects.
METHODS
Numerical simulations
Numerical simulations of ultrasound propagation
through the human skull were performed in two dimen-
sions using the k-Wave acoustics toolbox (Treeby and
 ARTICLE IN PRESS
A Portable System for FUS-mediated BBB Opening
A. N. POULIOPOULOS et al.
Table 1. Transducer parameters used in numerical simulations
Transducer Center Outer Inner Radius of
frequency diameter diameter curvature
(MHz) (mm) (mm) (mm)
1 0.2 110 44 70
2 0.35 60 44 76
3 0.25 110 44 110
Cox 2010; Treeby et al. 2012), to test different trans-
ducer characteristics. We first investigated the trade-off
between the focal depth and aperture size, that is, the
f-number, within the human skull. Our aim was to deter-
mine the center frequency, outer diameter and radius of
curvature, to be able to target both cortical and subcorti-
cal regions of the human brain, thus enlarging the treat-
ment envelope. We tested three different transducer
configurations (Table 1) based on commercially avail-
able low-frequency models (transducer 1: Sonic Con-
cepts H-149, transducer 2: Sonic Concepts H-209) and a
custom-designed transducer (transducer 3). H-149 and
H-209 were commercially available models that we
identified as potentially appropriate for BBB opening
applications in humans. These transducers were chosen
as examples of small and large f-number, respectively
(0.64 vs. 1.27). The custom-designed transducer (outer
diameter: 110 mm, radius of curvature: 110 mm,
f-number: 1) was optimized after multiple iterations of dif-
ferent designs, with emphasis on the outer diameter (search
space: 60140 mm) and radius of curvature (search space:
70120 mm). To allow for insertion of a PCD transducer
or a receiving ultrasound array, an inner gap 44 mm in
diameter was applied in all transducer designs.
A human CT skull DICOM file from the Cancer
Imaging Archive (2017; HeadNeck Cetuximab demo)
was used as input in our simulations. Hounsfield CT units
were converted to sound speed and medium density, as
described previously (Aubry et al. 2003; Wu et al. 2018).
Sound speed, medium density and attenuation coefficient
within the brain were set to be equal to those of water at
37˚C (i.e., 1524 m/s, 1000 kg/m3 and 3.5 £ 104 dB/
MHz¢cm, respectively). The transducers were positioned
close to the skull in an effort to place the focal volume as
close to the brain median plane as possible, while maintain-
ing a reasonable radius of curvature and realistic housing
dimensions (Table 1). A number of axial offsets were tested
(range: 30 to +30 mm, step 10 mm), to determine the
evolution of focal shifts across different depths. In the case
of an axial offset of 0 mm, the transducer’s nominal focus
was positioned at the human brain midline. The primary
aim of these simulations was to evaluate the effect of dif-
ferent focusing depths on the focal volume distortion;
therefore, we tested only axial offsets and not lateral off-
sets. Introducing lateral offsets would produce a large
3
variation in the incidence angle, deviating significantly
from the desirable 90o incidence. Therefore, the lateral
position of the FUS transducer center was fixed at
y = 0 mm for all simulations. We have also tested pulses of
different lengths (i.e., 1, 5, 25 and 2500 cycles) to investi-
gate the effects of interference and standing waves within
the human skull. To calculate the theoretical ultrasound
transmission coefficient through the human skull, we
repeated the simulations with different pulse lengths in free
field by replacing the human skull with water. The simula-
tion grid was equal to 300 £ 300 mm, at 1-mm spatial reso-
lution, while the temporal resolution was 143 ns with a
total of 7000 time steps or exposure time of 1 ms. For the
pulse length of 2500 cycles, the simulation consisted of
70,000 time steps or 10 ms, to enable comparison with the
treatment scheme typically used for in vivo BBB opening.
Shear waves were not taken into account in these simula-
tions. Axial (i.e., x) and lateral (i.e., y) axes were defined
with respect to the FUS transducer, and had left to right
and anterior to posterior directions, respectively.
Clinical system description
In the proposed clinical system (Fig. 1), we chose a
low center frequency (i.e., 0.25 MHz) to reduce the
attenuation caused by the human skull and decrease the
pressure threshold for cavitation induction (Apfel and
Holland 1991). The first step was to refine the dimen-
sions and characteristics of the single-element spherical-
segment transducer based on numerical simulations. We
then constructed the chosen single-element FUS trans-
ducer (Part No. H-231, center frequency: 0.25 MHz;
Sonic Concepts, Bothell, WA, USA) and attached it onto
a robotic arm (Kinova Jaco2, Kinova, Boisbriand, QC,
Canada). The robotic arm had 4 degrees of freedom and
a maximum midrange loading capacity of 4.4 kg, and
was controlled via a joystick. The whole construct was
fixed onto a wheeled cart, making the system portable to
any location.
The clinical FUS transducer was driven by a func-
tion generator (33500B Series, Agilent Technologies,
Santa Clara, CA, USA) through a 55-dB radiofrequency
power amplifier (A150, E&I, Rochester, NY, USA)
using clinically relevant parameters (Table 2). A water
degassing system (WDS105+, Sonic Concepts) was used
to fill the transducer cone with degassed water and inflate
or deflate the cone according to the sonicated location.
Reflective beads were attached to the transducer to
enable real-time tracking of its location through an infra-
red camera acting as a position sensor and neuronaviga-
tion guidance (BrainSight; Rogue Research, Montreal,
QC, Canada). Using the bull’s eye view function, we
have previously shown high targeting accuracy with spa-
tial error lower than 2 mm (Wu et al. 2018).
 ARTICLE IN PRESS
4 Ultrasound in Medicine & Biology Volume 00, Number 00, 2019

Fig. 1. Clinical setup with a single-element transducer and neuronavigation guidance. FUS = focused ultrasound;
MRI = magnetic resonance imaging; RF = radiofrequency.

Table 2. Clinically relevant ultrasound parameters for blood (Pouliopoulos et al. 2017). Here, we used PCD to define the
brain barrier opening in vivo using the neuronavigation-guided cavitation mode in vitro and in vivo by calculating the sta-
focused ultrasound system ble cavitation dose (SCD) and inertial cavitation dose
Parameter Value (ICD), as described before (Tung et al. 2010). Briefly, the
recorded time-domain signals were transformed into the
Center frequency 0.25 MHz frequency domain through a fast Fourier transform (seg-
Derated peak-negative pressure 0.2 MPapk-neg
Mechanical index 0.4 ment size: 524,288 data points), performed in MATLAB
Definity microbubble dose 10 mL/kg (1 £ clinical dose) (The MathWorks, Natick, MA, USA). Three spectral areas
Pulse length 10 ms or 2500 cycles were filtered to derive the relevant cavitation levels or cavi-
Pulse repetition frequency 2 Hz
Sonication duration 2 min tation dose per pulse as follows:

1. Harmonic peaks, fh,n = nfc

Microbubble acoustic emissions were recorded (sam-
pling frequency: 50 MHz, capture length: 10 ms) with a
1.5-MHz passive cavitation detector (PCD; diameter:
32 mm, focal depth: 114 mm, ndtXducer, Northborough,
MA, USA). PCD provides information on the cavitation
magnitude, duration and mode within the focal volume,
using either separate transducers (Tung et al. 2010) or a
therapeutic transducer alone (Heymans et al. 2017). Cavita-
tion signals also provide indirect information about the
microbubble velocity through the Doppler effect,
which can be captured either with a single-element PCD
(Pouliopoulos and Choi 2016) or using an array of receivers
2. Ultraharmonic peaks, fu,n = (n  1/2) fc
3. Broadband emissions fb with fh,n + 10 kHz < fb <
fu,n  10 kHz and fu,n + 10 kHz < fb < fh,n + 1  10 kHz
Here, fc is the center frequency (i.e., 0.25 MHz) and n the
harmonic number (n ¼ 3; 4; 5; :::; 10). Fundamental and
second harmonics were excluded from the calculations
because of the strong skull reflections at these frequen-
cies in control experiments.
Stable harmonic (dSCDh), stable ultraharmonic
(dSDCu) and inertial cavitation (dICD) levels were then
calculated as the mean root-mean-square (RMS) of the
 ARTICLE IN PRESS
A Portable System for FUS-mediated BBB Opening
A. N. POULIOPOULOS et al. 5

maximum absolute Fast Fourier Trransform (FFT) the same setup, replacing the human skull fragment with
amplitude of the detected signal within each frequency a NHP skull fragment. The human and NHP skull frag-
region for each acoustic pulse as follows: ments were positioned right on top of the water cone and
vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ffi
u *  2+ at a perpendicular incidence angle, to imitate the clinical
u   scenario. Pressure profiles and transcranial loss were
dSCDh ¼ t FFT
f h;n
expected to be extremely sensitive to the incidence angle
n
and distance from the transducer surface. Here, we tested
vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi only one incidence angle (i.e., »90o) and transducer surfa-
u *  2+
u   ceskull distance (i.e., 62 mm), which are clinically rele-
dSCDu ¼ t  
FFT vant for treatment of dorsolateral prefrontal cortex.
f u;n n Pressure profiles and losses were estimated at skull loca-
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
v* tions of variable thickness (n = 10, thickness range:
u   2+ 37.5 mm, measured with a caliper), as attenuation
u  
dICD ¼ t FFT : depends on the skull thickness (Gerstenmayer et al.
fb 2018). All reported pressure values refer to the derated
peak-negative pressure.
The total cavitation dose in vivo was calculated as the sum Cavitation detection through the human skull was
of all the cavitation levels throughout the FUS treatment: also conducted within a water tank. A 0.8-mm silicon
X
T elastomer tube was submerged and fixed at a horizontal
SCDh ¼ dSCDh;t position within the focal volume of the clinical trans-
t¼0 ducer (120 mm from transducer surface). The tube was
filled with either water, which served as a control, or
X
T
Definity microbubbles (0.2 mL microbubbles/L of solu-
SCDu ¼ dSCDu;t
tion) flowing at a rate of 1.8 mL/min. Measurements
t¼0
were conducted both in free field and with the human
skull fragment in the beam path, positioned 62 mm away
X
T
ICD ¼ dICDt from the transducer surface. We tested three derated
t¼0 acoustic pressures, 200, 300 and 400 kPa, corresponding
to MIs of 0.4, 0.6 and 0.8, respectively. Cavitation levels
The total sonication duration was T = 2 min. were calculated across the experimental conditions
In vitro characterization (n = 10 consecutive pulses per condition) to establish the
Skull-induced aberrations were characterized in a ability of the PCD transducer to detect cavitation signals
water tank. A capsule hydrophone (HGL-0200, §3-dB through the human skull at each acoustic pressure.
frequency range: 0.2540 MHz, electrode aperture: 200 In separate experiments, a tissue-implantable type-
mm; Onda Corp., Sunnyvale, CA, USA) was used to mea- T thermocouple (Physitemp instruments, Clifton, NJ,
sure the emitted pressure profiles in free field and with a USA) was attached to the skull surface to measure the
human skull fragment in the beam path. The skull frag- heating profile during clinically relevant FUS exposure
ment was submerged in water and degassed before the (MI: 0.40.8, duty cycle: 2%; Table 2). A positive con-
experiment using a vacuum pump, to reduce the gas con- trol sonication at a higher duty cycle (20% at an MI of
tent within the bone. Raster scans around the focal point 0.8) was conducted to compare with the low-duty-cycle
were performed at a spatial resolution of 0.1 mm laterally BBB opening scheme. Temperature data were recorded
and 1 mm axially. The scans had lateral/elevational and at a sampling rate of 100 samples/s. Temperature
axial ranges of 10 and 60 mm, respectively, and were cen- increase on the skull surface was calculated by subtract-
tered at the geometric focus of the FUS transducer ing the temperature before FUS exposure from the value
(110 mm from transducer surface). Shifts along the lateral measured during FUS exposure (n = 3).
and elevational dimensions were averaged, assuming an
axisymmetric distortion of the beam. Ultrasound pressure In vivo feasibility
transmission coefficient through the human skull was cal- All animal experiments were reviewed and
culated (in %) by dividing the maximum pressure of the approved by the local Institutional Animal Care and Use
focal volume after the skull placement by the maximum Committee prior to all performed studies and were in
pressure of the focal volume in free field, for both simula- accordance with the National Institutes of Health guide-
tions and experiments. Transcranial pressure loss was cal- lines for animal welfare. Two male adult Rhesus maca-
culated as 100%  transmission coefficient. To determine ques (weight: 811 kg, age: 1220 y) were treated with
the ultrasound attenuation through an NHP skull, we used the clinical FUS transducer, targeting the thalamus
 ARTICLE IN PRESS
6 Ultrasound in Medicine & Biology
(NHP 1) and the dorsolateral prefrontal cortex (NHP 2),
to examine the performance of the system at both corti-
cal and subcortical regions. To accommodate the NHP
experiment, the patient chair (Fig. 1) was replaced with
a surgical table equipped with a stereotactic apparatus
for head fixation. NHPs were initially sedated with a
mixture of ketamine (10 mg/kg) and atropine (0.02 mg/
kg) through intramuscular injection. Once sedated, the
animals were intubated and catheterized via the saphe-
nous vein. Anesthesia was induced and maintained
throughout the experiment using inhalable isoflurane
mixed with oxygen (1%2%).
The ultrasound parameters used here (Table 2) were
identical to those approved by the FDA for use in
Alzheimer’s patients using our system (derated peak-
negative pressure: 0.2 MPa, pulse length: 10 ms, pulse
repetition frequency: 2 Hz, total sonication duration:
2 min). We maintained the MI below the FDA-approved
limit for ultrasound imaging applications with Definity
microbubbles to avoid compromising safety. BBB open-
ing in the NHP model was attempted at a peak-negative
pressure of 0.2 MPa or an MI of 0.4. This MI is approxi-
mately five times lower than the maximum MI approved
by the FDA for imaging applications (i.e., MI of 1.9),
twice lower than the BBB opening threshold found by
Carpentier et al. (2016) in humans and similar to the
threshold found in our previous NHP studies (Wu et al.
2018). In contrast to our previous studies that used 4- to
5-mm size-isolated microbubbles at a dose of 2.5 £ 108
microbubbles/kg (Karakatsani et al. 2017; Wu et al.
2018), here we used commercially available Definity
microbubbles at the FDA-approved clinical dose for
ultrasound imaging applications (i.e., 10 mL/kg). Defi-
nity microbubbles were infused as a bolus via a single
injection, on treatment initiation.
Bloodbrain barrier opening was assessed approxi-
mately 60 min post-sonication with T1-weighted MRI (3-D
spoiled gradient-echo, TR/TE: 20/1.4 ms, flip angle: 30˚,
number of excitations [NEX]: 2, spatial resolution:
500 £ 500 mm2, slice thickness: 1 mm with no inter-slice
gap). T1-weighted scans were acquired before and after
intravenous administration of 0.2 mL/kg gadodiamide MRI
contrast agent (Omniscan; GE Healthcare, Bronx, NY,
USA), which is normally impermeable to the BBB (molecu-
lar weight: 591.7 Da). BBB opening was quantified by com-
paring pre- and post-contrast administration T1 scans. Safety
outcomes were assessed with axial T2-weighted MRI (TR/
TE: 3000/80 ms, flip angle: 90˚, NEX: 3, spatial resolution:
400 £ 400 mm2, slice thickness: 2 mm with no inter-slice
gap) and susceptibility-weighted imaging (SWI; TR/TE:
19/27 ms; flip angle: 15˚, NEX: 1, spatial resolution:
400 £ 400 mm2; slice thickness: 1 mm with no inter-slice
gap). All scans were performed in a 3-T clinical MRI
scanner.
Volume 00, Number 00, 2019
BBB opening quantification
We developed a graphics user interface (GUI) in
MATLAB for BBB opening quantification and analysis.
To calculate the BBB opening volume, the pre-contrast
T1 scan was subtracted from the post-contrast T1 scan.
An intensity threshold was set to isolate the BBB open-
ing area in the difference image, and a contour plot was
applied to the pixels above the threshold within the
selected region of interest. The area of the BBB opening
contour was calculated for each coronal MRI slice, and
the total BBB opening volume (in mm3) was found by
summing the BBB opening areas in all slices.
Statistical analysis
All measurements presented here are expressed as
the mean § standard deviation. Simulations were per-
formed for n = 4 pulse lengths and n = 6 transducer axial
positions. Cavitation detection was established by com-
paring control and microbubble-seeded cavitation levels
in free field and through the human skull, using a two-
sample t-test in MATLAB (n = 10 pulses). Statistically
significant differences were assumed at p < 0.05.
RESULTS
Numerical simulations
Numerical simulations revealed that transducer 3
was able to target the brain median plane while maintain-
ing a tightly focused beam, without multiple sidelobes
(Fig. 2). Transducer 1 did not have sufficient radius of
curvature to produce a long enough focal depth for the
human skull, because of its low f-number. Transducer 2
produced multiple sidelobes similar in amplitude to the
main lobe because of the large f-number and the low
outer-to-inner diameter ratio. Furthermore, the focal
volume was subject to greater distortion because of the
higher center frequency compared with transducers 1
and 3 (0.35 MHz vs. 0.2 MHz and 0.25 MHz). We found
that in the case of a single-element transducer, an
f-number of 1 (transducer 3) was more suitable for appli-
cations in the human brain, compared with lower or
larger f-numbers within the subset we have tested.
Such a transducer design allows targeting of both
superficial cortical areas and deeper subcortical areas
(Fig. 3). By physically moving the FUS transducer
toward/away from the skull surface, one can achieve a
treatment envelope up to 80 mm in depth. Simulations
revealed that the focal dimensions, pressure profile and
skull-induced focal shift depend on the transducer axial
offset and the pulse length (Fig. 4). The transducer axial
offset was defined as the distance of the free-field focus
from the simulation center (x = 0 mm). Intracranial
acoustic pressures only moderately changed throughout
the axial offsets. Highest pressures were observed near
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A Portable System for FUS-mediated BBB Opening
A. N. POULIOPOULOS et al. 7

Fig. 2. Numerical simulations of ultrasound propagation with different single-element transducers (top to bottom: 1, 2,
3) emitting pulses of variable length (left to right: 1, 5, 25, 2500 cycles). Transducer 3 was the only configuration that
was able to treat deep structures without presenting multiple sidelobes within the human skull. Color bar: Normalized
focal pressure. Each pressure profile was self-normalized to the maximum acoustic pressure within the skull to illustrate
the 3-dB focal volume. Pressure values refer to the maximum instantaneous pressure at each location.

the skull center, while there was a decrease of up to
7% toward the proximal and distal skull. The amplitude
of lateral sidelobes increased with pulse length, from
49% of the main lobe at 1 cycle to 76% of the main lobe
at 2500 cycles. All pressure profiles shown in Figures 2
and 3 were normalized to the maximum pressure within
the skull and plotted in the range [0.5, 1], to visualize the
3-dB focal volume following transcranial ultrasound
propagation.
Pulse lengths longer than 1 cycle produced con-
structive and destructive interference at the distal part of
skull, with nodes and antinodes appearing at a spacing of
half-wavelength (i.e., 3 mm). The interference spatial
extent was equal to half the spatial length of the acoustic
pulse (e.g., 2.5 cycles or 15 mm for a pulse length of
5 cycles or 30 mm). For the clinically relevant pulse length
of 2500 cycles, the interference profile reached equilibrium
and extended throughout the interior of the human skull.
The theoretical limit for standing wave generation at
0.25 MHz and a skull size of 130 mm is 43 cycles.
The presence of the human skull led to the distor-
tion and spatial shift of the simulated focal volume
(Fig. 5). In water medium without the human skull, the
axial and lateral full widths at half-maximum (FWHM)
were simulated to be 65.5 £ 5.6 mm. The focal width
and length were reduced by 2.7 § 2.4% and by 8.4 §
4.8% along the lateral and axial dimensions, respec-
tively, because of skull-induced aberrations (n = 4 pulse
lengths and n = 6 transducer positions). The focus was
also negatively shifted toward the transducer (Fig. 5b).
Axial shifts depended on the transducer position. Inter-
estingly, shifts were smaller for larger offsets. In other
words, the farther the focus from the brain midline, the
smaller the axial shift. On average, the axial and lateral
focal shifts were 6.1 § 2.4 and 0.1 § 0.2 mm, respec-
tively (Fig. 5c). Pressure attenuation caused by the
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8 Ultrasound in Medicine & Biology Volume 00, Number 00, 2019

Fig. 3. Numerical simulations of ultrasound propagation with the clinical focused ultrasound transducer targeting struc-
tures of variable depth within a human skull. Examples are shown for transducer axial offset of 30 to 20 mm
(offset = 0 mm when the focus in free-field coincides with the midline). Center frequency: 0.25 MHz, pulse length: 2500
cycles. Color bar: normalized focal pressure. Each pressure profile was self-normalized to the maximum acoustic pres-
sure within the skull to illustrate the 3-dB focal volume. Pressure values refer to the maximum instantaneous pressure
at each location.

Fig. 4. Lateral (top) and axial (bottom) profiles of the simulated pressure field within a human skull. Lateral sidelobes
and interference patterns emerge for pulse lengths larger than one cycle. The spatial length of interference away from
the distal skull bone increases linearly with the pulse length.
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A Portable System for FUS-mediated BBB Opening
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Fig. 5. Simulated human skull-induced focal distortion. (a) Full width at half maximum (FHWM) change caused by the
presence of the human skull. FWHM changes were first averaged across the pulse lengths for each axial offset (n = 4
pulse lengths), and then averaged across all depths (n = 6 axial offsets). (b) Simulated focal shifts along the axial (red
crosses) and lateral (blue boxes) dimensions. Diagonal dotted-dashed line and parallel dotted line denote axial and lateral
shifts equal to zero, respectively (n = 4 pulse lengths). (c) Average focal shifts across the lateral and axial dimensions
(n = 6 axial offsets). Data are expressed as the mean § standard deviation.

human skull was simulated to be 36.1 § 3.4% (n = 10
different CT slices).
In vitro characterization
To confirm the simulation findings, we performed a
detailed estimation of the 2-D beam profiles along the
lateral/elevational and lateral/axial dimensions, with and
without the presence of a human skull fragment (Fig. 6).
Using the capsule hydrophone and the 3-D positioning sys-
tem (Fig. 6a), we measured the pressure profiles along the
axial, lateral and elevational dimensions. The free-field focal
length and width were 47.6 £ 5.6 mm (Fig. 6b, 6c: left side).
These values were close to the nominal focal dimensions of
49 £ 6 mm provided by the manufacturer. Ultrasound propa-
gation through the human skull was expected to attenuate
and shift the acoustic focus. Inserting the skull fragment
within the beam path attenuated the pressure amplitude by
44.4 § 1.3% and distorted the focal region (Fig. 6b, 6c: right
side). The lateral and axial FWHMs decreased by 3.3 §
1.5% and 3.9 § 1.8%, respectively (Fig. 6d). Experimental
focal shifts along the lateral and axial dimensions were 0.5
§ 0.4 and 2.1 § 1.1 mm, respectively (Fig. 6e).
Passive cavitation detection measurements con-
firmed that the 1.5-MHz PCD transducer can detect cavi-
tation signals through the human skull (Fig. 7). Using
the in vitro setup described earlier (Fig. 7a), we detected
stationary reflections at the fundamental and the second
harmonic for the control experiment, both from the tube
and from the human skull (Fig. 7b). When Definity
microbubbles were flowing through the vessel phantom,
we observed a rise in the higher harmonics (up to the
fifth harmonic or 1.25 MHz) and ultraharmonics (up to
the third ultraharmonic or 0.825 MHz).
Higher acoustic pressures led in general to higher har-
monic and ultraharmonic peaks. In Figure 7d, light-color
bars represent control sonications, while dark-color bars rep-
resent sonications with Definity microbubbles. The two left-
most bars in each cavitation dose represent free-field
sonications, while the two rightmost bars represent sonica-
tions through the human skull fragment. Ten distinct thera-
peutic pulses were emitted for each condition. Harmonic
stable cavitation levels were significantly higher for micro-
bubbles than the control, for MIs of 0.4 and 0.6 both in free-
field and through the human skull (Fig. 7d). Ultraharmonic
stable cavitation levels with microbubbles were significantly
higher than those of the control at MIs of 0.4 and 0.6 only in
free-field. There was a significant difference through the
human skull at an MI of 0.4, but a non-significant increase
at an MI of 0.6. At the highest acoustic pressure, stable har-
monic and inertial cavitation levels were significantly higher
for the control than for microbubbles. This was likely due to
inadequate degassing of the human skull fragment, which
resulted in intracranial cavitation nuclei in the control exper-
iment. Inertial cavitation levels rose considerably above
the noise level at all MIs in free-field, and also during the
control experiments in the presence of skull for MIs of 0.6
and 0.8.
Next, we measured the ultrasound-induced heating
during clinically relevant ultrasound exposure. A wire
thermocouple was attached below the human skull frag-
ment and within the ultrasound beam path. To simulate
the clinical scenario, 2-min sonications were performed
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10 Ultrasound in Medicine & Biology Volume 00, Number 00, 2019

Fig. 6. Experimental human skull-induced focal distortion. (a) Experimental setup for measuring focal distortion using a
hydrophone. A raster scan was performed to measure the focal volume in (b, cleft side) free field and (b, cright side)
with a human skull fragment. Pressure maximum was 10 mm closer to the transducer compared with the geometric
focus. White crosses denote the position of the free-field focus. Green crosses denote the position of the focus following
transcranial propagation. (d) Full width at half maximum change and (e) focal shifts along the lateral and axial dimen-
sions. Data are expressed as the mean § standard deviation (n = 10 scans with ultrasound propagating through skull seg-
ments of different thickness). FUS = focused ultrasound; FWHM = full width at half-maximum.

using the parameters intended for the clinic (Table 2).
The maximum temperature increase was between 0.11 §
0.05˚C and 0.16 § 0.03˚C (n = 3) during sonication at
MIs of 0.40.8 (Fig. 8). This negligible heating was
expected, given the low duty cycle of ultrasonic pulse
sequences used in BBB opening (i.e., 2%). A control
sonication at 10 £ higher duty cycle (i.e., 20%) and an
MI of 0.8 did increase the temperature by 0.59 § 0.23˚C.
In vivo feasibility
Finally, we tested the proposed clinical system in an
NHP model to perform non-invasive and targeted BBB
opening at a peak-negative pressure of 200 kPa or an MI
of 0.4, using the clinically recommended Definity dose
(10 mL/kg). Two NHPs were treated targeting the
thalamus (NHP 1) and the dorsolateral prefrontal cortex
(NHP 2). The two targets were selected as examples of
deep and superficial structures, respectively. Despite the
low pressure and microbubble dose, we achieved BBB
opening in both targeted structures (Fig. 9). BBB opening
was more pronounced in the gray matter rather than in the
white matter tracts, as reported before (Karakatsani et al.
2017). The total BBB opening volume was 153 mm3 for
NHP 1 and 164 mm3 for NHP 2. Safety was evaluated
with T2-weighted MRI and SWI (Fig. 9). There was nei-
ther a hyper-intense region in T2 scans nor a hypo-intense
region in SWI an hour post-sonication, indicating lack of
hemorrhage or edema in the sonicated region. Although
this was an acute safety evaluation, we did not expect any
long-term effects at this low-pressure regime, based on
our previous studies (Downs et al. 2015).
Safety outcomes were corroborated by the captured
PCD data which confirmed in real time the absence of vio-
lent cavitation events within the focal volume (Fig. 10).
Before microbubble administration, the spectral content of
the received signals included the fundamental frequency
(i.e., 0.25 MHz) and the first two or three harmonics
(Fig. 10a, 10d). Following microbubble bolus injection, there
was an increase in higher harmonics and, for NHP 2, ultra-
harmonics (Fig. 10b, 10e). However, there was no consider-
able increase in the broadband signal floor following
microbubble administration (white dashed line), as illustrated
in the spectrograms of both FUS treatments (Fig. 10c, 10f).
These qualitative traits were quantified with SCD and ICD
(Fig. 10gi). SCDh increased by 5.44 § 1.16-fold on micro-
bubble infusion (t > 15 s), while SCDu and ICD increased
by 1.46 § 0.01- and 1.48 § 0.21-fold, respectively. We can
thus infer that microbubbles underwent stable and recurrent
oscillations, with stable cavitation dominating over transient
and inertial cavitation throughout treatment. On average, the
total cavitation dose was 1.37 § 0.17 £ 104 mV.
DISCUSSION
A clinical system using a single-element transducer
and neuronavigation guidance for BBB opening offers
distinct advantages compared with alternative
approaches. First, BBB opening can be achieved in a
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A Portable System for FUS-mediated BBB Opening
A. N. POULIOPOULOS et al. 11

Fig. 7. Passive cavitation detection through the human skull. (a) In vitro setup for passive cavitation detection. A 0.8-mm tube
filled with Definity microbubbles was used as a vessel-mimicking phantom. (b) Spectra of control (transparent orange line) and
microbubble (black line) acoustic emissions for mechanical indexes (MIs) of 0.4 (left), 0.6 (middle) and 0.8 (right) in free-field.
(c) Spectra of control and microbubble acoustic emissions through the human skull. (d) Cavitation levels in free-field (circles,
plus signs) and through the human skull (crosses, diamonds), for control (light bars) and microbubbles (dark bars), at MIs of
0.4 (left), 0.6 (middle) and 0.8 (right). Data are expressed as the mean § standard deviation (n = 10 pulses). *p < 0.05.
FFT = fast Fourier transform; FUS = focused ultrasound; PCD = passive cavitation detector.

non-invasive manner, which is preferable especially for regions (Figs. 35), although at the expense of a large
long-term repeated treatments required in AD or PD. axial-to-lateral focal size ratio and variable focal distor-
Second, such a system can provide access to both shal- tion in different depths (Fig. 5). Also, there is no need
low (i.e., cortical) and deep (i.e., subcortical) brain for an MRI system during BBB opening which may be a
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12 Ultrasound in Medicine & Biology Volume 00, Number 00, 2019

Fig. 8. Skull heating using the clinical focused ultrasound transducer at mechanical indexes (MIs) of 0.4 (red line),
0.6 (green line) and 0.8 (blue line) and clinically relevant ultrasound parameters (center frequency: 0.25 MHz,
pulse length: 2500 cycles or 10 ms, pulse repetition frequency: 2 Hz, duty cycle: 2%, total duration: 2 min). A
higher duty cycle (i.e., DC: 20%) was used as a positive control for heating (black line). Data are expressed as the
mean § standard deviation (n = 3).

costly and formidable hurdle for widespread use of FUS-

mediated treatments, especially given that temperature
elevation is not incurred. Neuronavigation systems are
typically available for neurosurgical procedures (Grunert
et al. 2003), so the additional cost for hospitals is the sin-
gle-element transducer, the driving electronics and the
robotic arm. The targeting and sonication procedure is
efficient and simple (<30 min) as opposed to MR-guided
FUS treatment (34 h). Moreover, the NgFUS is porta-
ble so treatment can take place at any location without
the need of an MRI unit. Finally, low-frequency and
low-duty-cycle treatment leads to limited skull-induced
aberrations (Figs. 5 and 6) and FUS-induced skull heat-
ing (Fig. 8), respectively.
Lower frequencies favor cavitation-mediated bio-
effects at low acoustic pressures (Apfel and Holland
1991; Ilovitsh et al. 2018). We have shown here that the
BBB can be opened in an NHP model at an MI of 0.4
(Fig. 9), which is twice lower than the minimum MI
required using the unfocused implanted 1.05-MHz trans-
ducer in humans (Carpentier et al. 2016) and similar to
other NHP studies (McDannold et al. 2012; Downs et al.
2015; Karakatsani et al. 2017; Wu et al. 2018). Low-
pressure treatments not only ensure safety (Fig. 9),
but also facilitate regulatory approval because they are Fig. 9. In vivo feasibility in a non-human primate (NHP)
compatible with routinely used ultrasound imaging pro- model. Coronal T1-weighted, T2-weighted and susceptibility-
weighted imaging (SWI) for NHPs 1 (left) and 2 (right). T1-
tocols. Such acoustic pressure instigates cavitation activ- Weighted magnetic resonance imaging-confirmed bloodbrain
ity that is detectable in real time with the co-aligned barrier opening in the thalamus (NHP 1) and dorsolateral pre-
PCD transducer (Fig. 7), with stable cavitation emissions frontal cortex (NHP 2), using the clinical focused ultrasound
dominating the spectra during FUS treatment in an NHP (FUS) transducer with clinically relevant parameters (MI: 0.4)
model (Fig. 10). Clinically relevant parameters (Table 2) and U.S. Food and Drug Administration-approved Definity
microbubble dose (10 mL/kg). T2-Weighted imaging and SWI
are thus not expected to lead to violent inertial cavita- revealed that there is no acute hemorrhage or edema after the
tion, which was detected in higher-MI sonication FUS treatment. Color bar: Normalized contrast enhancement.
(Fig. 7). Bar = 1 cm.
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A Portable System for FUS-mediated BBB Opening
A. N. POULIOPOULOS et al. 13

Fig. 10. In vivo passive cavitation detection measurements confirmed that stable cavitation dominated throughout ultra-
sound treatment at clinically relevant conditions. Spectral amplitude (a, d) before and (b, e) after microbubble injection,
for non-human primate (NHP) 1 (a, b) and NHP 2 (d, e). Spectrogram of the entire treatment session for NHP 1 (c) and
NHP 2 (f). Higher harmonic emissions were detected, with no substantial increase in the broadband floor after microbub-
ble entrance into the focal volume (white dashed lines). (g, h) Stable harmonic cavitation levels (black line) rose right
after microbubble administration (dashed line) and remained relatively constant throughout the sonication, for both NHP
1 (g) and NHP 2 (h). Stable ultraharmonic (blue line) and inertial cavitation levels (red line) had a moderate increase,
indicating absence of violent cavitation events at an MI of 0.4. Arrows indicate the time points shown in (b) and (e). (i)
Average stable harmonic (black), stable ultraharmonic (blue) and inertial (red) cavitation dose during focused ultrasound
treatment for NHP 1 (filled bars) and NHP 2 (patterned bars), following microbubble administration (t > 15 s). Data are
expressed as the mean § standard deviation (n = 210 pulses). FFT = fast Fourier transform.

Here, we reported successful BBB opening using
10-ms-long pulses. However, such pulse lengths produce
interference and standing waves within the human skull
(O’Reilly et al. 2010), as illustrated here (Figs. 24),
and promote primary (Dayton et al. 2002; Koruk et al.
2015) and secondary (Dayton et al. 1997; Lazarus et al.
2017) acoustic radiation forces, which may indirectly
compromise safety. In our future work, we aim to use
either coded excitation (Kamimura et al. 2015) or shorter
pulses on the order of microseconds (<50 cycles) to
avoid standing wave formation (O’Reilly et al. 2011).
Such pulses have been reported to produce more uniform
cavitation activity within the focal area by extending the
microbubble lifetime (Pouliopoulos et al. 2014), avoid-
ing cluster formation and spreading the microbubble
activity in space and time (Pouliopoulos et al. 2016; Pou-
liopoulos 2017). In vivo, rapid short-pulse sequences
produce uniform BBB openings that last less than
10 min and do not allow extravasation of inflammation-
inducing proteins, such as albumin, into the brain paren-
chyma (Morse et al. 2019). On the other hand, it is more
difficult to deliver large therapeutic molecules using
short pulses (Choi et al. 2011).
Short pulses also allow for improved passive map-
ping of cavitation signals, through the synchronization
of the therapeutic and imaging processes (i.e., using
absolute time-of-flight information) (Gateau et al.
2011; Burgess et al. 2018). PAM in either the time
(Gy€ongy and Coussios 2010; Coviello et al. 2015) or
frequency (Salgaonkar et al. 2009; Haworth et al.
2012; Arvanitis et al. 2015a, 2017; Haworth et al.
2017; Wu et al. 2018; Burgess et al. 2018) domain can
be achieved by replacing the single-element PCD
transducer with a multi-element linear array operating
in receive mode. Using a PAM array, one can account
for skull-induced aberrations in receive and localize
acoustic cavitation activity in a more precise manner
(Jones et al. 2013, 2015; O’Reilly et al. 2014; Arvanitis
 ARTICLE IN PRESS
14 Ultrasound in Medicine & Biology
et al. 2015b). Furthermore, we plan to use pulse inver-
sion in the short therapeutic pulses, to be able to detect
weak cavitation emissions through the thick human
skull (Pouliopoulos et al. 2018). Future efforts will
finally focus on using short pulses and PAM in closed-
loop (Sun et al. 2017; Jones et al. 2018; Kamimura
et al. 2018; Patel et al. 2019) to improve the spatiotem-
poral control of acoustic cavitation activity within the
brain.
The proposed system is limited by a number of fac-
tors. First, the axial focal length is eight times larger
than the lateral focal width. The elongated focus was
necessary to increase the targetable brain coverage or
treatment envelope; however, this comes at the expense
of potentially asymmetric BBB opening. Hemispherical
arrays provide lower axial-to-lateral focal beam width
even at large steering angles, in both emission and recep-
tion modes. Second, real-time single-element PCD mon-
itoring does not allow for either subfocal volume
localization of cavitation activity or detection of cavita-
tion signals over a large bandwidth. The clinical trans-
ducer was designed with an inner diameter of 44 mm to
allow for insertion of not only single-element trans-
ducers but also multi-element arrays for passive mapping
of cavitation activity. Third, the proposed system is cur-
rently capable of compensating for skull-induced aberra-
tions by predicing, and accounting for, the shift using a
simulation framework similar to the one presented herein
but not by phasing the elements in a way similar to
multi-element hemispherical arrays. Another possible
solution is to use 3-D printed holographic phase plates
tailored to each skull contour and targeted structure
(Melde et al. 2016; Ferri et al. 2018; Maimbourg et al.
2018). Accurate knowledge of the intracranial pressure
is impossible; therefore, one needs to simulate the pres-
sure field within the targeted location on a patient-by-
patient basis using head CT scans. However, this
remains an approximation, and emitted pressures should
remain below the safety limits assuming the lowest
attenuation coefficient possible. Finally, the robotic sys-
tem used to hold and position the transducer in place for
treatment has only 4 degrees of freedom, which limits
the achievable range of incidence angles. Subsequent
improvements will include a robotic arm with 6 degrees
of freedom.
In this study, numerical simulations were performed
in 2-D space, assuming axisymmetric beam profiles
along the axial dimension. However, the human skull is
asymmetric and highly inhomogeneous in 3-D space;
therefore the simulated profiles are a first-order approxi-
mation. The single-element transducer was simulated in
k-Wave as a collection of 1-mm point sources firing
simultaneously. This may be the cause of the overesti-
mation of the axial beam width in the simulations
Volume 00, Number 00, 2019
compared with the experiment (65.53 mm vs. 47.57 mm
in free field). Also, we used a human skull fragment for
our experiments as opposed to a complete human skull.
In previous studies from our group, we investigated the
effect of varying incidence angles at the BBB opening
volume (Karakatsani et al. 2017; Wu et al. 2018). Here,
our primary interest was to study the effects of focusing
the therapeutic beam at different depths (Figs. 35);
thus, only one incidence angle (approximately 90o) was
set for both simulations and experiments. For this reason,
the lateral position of the FUS transducer remained con-
stant in the numerical simulations. Yet, there was a dis-
crepancy between the simulated and experimental
pressure losses following transcranial propagation (36%
vs. 44.4%), which can be reduced by using 3-D simula-
tions, finer grids and time steps and identical skull
shapes/dimensions. In upcoming clinical trials, 3-D sim-
ulations will be performed for each patient, using a grid
with isotropic resolution of 0.5 mm, a specific beam tra-
jectory and a well-defined target within the prefrontal
cortex.
On average, axial shifts were of similar magnitude
to those predicted in simulations than in the experiments
(Figs. 5 and 6). Averaging in the simulations was con-
ducted over different pulse lengths and focusing depths
(Fig. 5), whereas experimental measurements (Fig. 6)
were conducted with a single pulse length (i.e., 25
cycles) and fixed transducerskull distance (i.e., 62
mm). The axial shift in the simulation which resembled
the experimental skulltransducer distance (i.e., axial
offset of 30 mm) was 2.25 § 1.92 mm (Fig. 5), similar
to the experimentally derived shift of 2.1 § 1.1 mm
(Fig. 6). The in vitro cavitation detection experiment
was conducted using a single 0.8-mm vessel-mimicking
tube, which does not capture the complexity and vari-
ability of the in vivo vasculature. Finally, although all
simulations and bench-top experiments focused on the
human skull, the initial in vivo feasibility testing of the
NgFUS system was conducted using two NHPs.
Although this model is the closest resemblance to
humans, the safety and performance of this approach
remains to be tested in the clinic.
CONCLUSIONS
We developed a clinical setup for BBB opening based
on a single-element transducer with neuronavigation guid-
ance and real-time cavitation monitoring. Using this system,
one can achieve non-invasive and targeted BBB opening
with limited focal distortion and induced skull heating. Lat-
eral and axial shifts were experimentally measured to be 0.5
§ 0.4 and 2.1 § 1.1 mm, and were simulated as 0.1 § 0.2
and 6.1 § 2.4 mm. We found that the focal volume
decreased by 3.3 § 1.4% and 3.9 § 1.8% along the lateral
 ARTICLE IN PRESS
A Portable System for FUS-mediated BBB Opening
A. N. POULIOPOULOS et al.
and axial dimensions, respectively, following transmission
through a human skull fragment. Maximum temperature
increase on the skull surface was 0.16 § 0.03˚C. Using this
clinical system, we produced a 153 § 5.5 mm3 BBB open-
ing in an NHP model with clinically relevant parameters
and without any detectable damage. Ongoing work is
focused on the short- and long-term safety profile of the cur-
rent clinical system, including histopathology and behavioral
studies in multiple NHPs, and progressing onto human
applications. In our future work, we plan to use the NgFUS
system, which was recently granted an investigational
device exemption (IDE G180140) by the FDA, to achieve
non-invasive and targeted BBB opening in AD patients.
Acknowledgments—This work was supported by National Institutes of
Health Grants 5R01EB009041 and 5R01AG038961. The authors
acknowledge the members of the Ultrasound Elasticity and Imaging
Laboratory (UEIL) at Columbia University for their contribution in the
development this system. ANP thanks Javier Cudeiro for advice on the
numerical simulations, Cindy Han for guidance on regulatory issues
and Rogue Research Inc. for assistance with the BrainSight platform.
Conflict of interest disclosure—EEK and HASK are inventors and own
intellectual property on some aspects of the technology presented
herein. All other authors have no conflict of interest.
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Another random document with
no related content on Scribd:
 "He says Enra lives as though all were well with the world; but all is not
well!"

"All is not well?" Maki repeated, frowning with the childish effort to
understand.

"Do you remember Yuma's death?" Rita went on. Yuma was a little
black slave who died of a tarantula sting.

"She turned greyish-white all over, just like an autumn fly covered with
mildew, and a smell of corruption came from her before she died. And the
day before yesterday someone had attacked a little girl of five by the very
walls of Aton's temple, strangled her and thrown the body to the pigs. Is
that well? And Enra lives as though none of these things happened. Perhaps
all is well for God, but Enra is not God; people say he is, but he himself
knows he isn't."

She pondered for a while and began again:

"He does not know how to cry, but one cannot live without tears;
nothing is sweeter than tears..."

"Does Shiha say that?" Maki asked.

"No, I say it .... or perhaps Shiha, I don't remember.... What is Aton, the
Sun? A spark in darkness: death will blow and the sun will be extinguished.
Darkness is more than light; first there was darkness and then light. Maybe
God dwells in darkness."

She laughed suddenly.

"You, too, are a sensible girl; you are afraid of light and love darkness.
The daughter does not take after the father."

Maki listened greedily; sometimes she strove to say something, but

words froze on her lips; she merely looked at her sister with wide open eyes
and seemed like one bound hand and foot waiting for a blow.
 "Well, that's enough moping, let us go! You must walk about, it is good
for you," Rita said, lifting her with apparent roughness, but in reality with
tender care, and led her into the garden.

It was dusk. The sky was clear, but mist was creeping over the ground.
The water in the river had only just returned to its normal level; there were
still pools of water about. Drops fell from the wet leaves. Frogs croaked
ecstatically. The smell of the flowers was intoxicating. All at once the mist
turned rosy from the moon that was rising invisibly.

They walked to the big pond where Maki's birch tree grew. It had never
recovered after the scorching Sheheb. Everything round it was green and in
flower, but it was dead and only on some of the bare branches a withered
leaf showed black.

Maki put her arms round the pale, slender stem and pressed her cheek
against it.

"Poor, poor darling!" she whispered tenderly as though saying good-bye

to a friend who had died.

"A-ah, you remember the superstition!" Rita said, with a laugh. "If one
plants a tree and it dies, the person who planted it will die also. Well, even
if you do die, that's nothing very dreadful—you will have had a child
anyway. To think of God sending such happiness to one who doesn't want
it! Why, I would die ten times if I could only have a child."

After walking round the big pond, they came to the Lotos pond, with
Aton's chapel on a little island and a small bridge leading to it. A huge
lotos, not yet fully opened, showed white on the water. A boat was tied near
by. Rita jumped into it, and seeing a garden knife at the bottom, took it to
cut the lotos; she gave the flower to Maki and hid the knife in the bosom of
her dress.

They went back to the Water-House and sat down in the old place.

"You haven't been to see Shiha for some time, have you?" Rita asked.
 "No, I haven't."

"And I often go to him. It is interesting. A regular abode of love. All our

dignitaries' wives keep going there. These eunuchs are excellent
matchmakers. And they are themselves fond of women—and the women
like it, of course. Shiha tempts me, too, with the god's marriage-bed: 'the
god will come down to you in the night like a bridegroom to his bride,' he
says. But I am not a fool to buy a pig in a poke. Instead of a god a slave or a
dirty Jew may come and disgrace one..."

She paused and then spoke again, looking straight into Maki's eyes:

"Extraordinary! How is it possible not to find out who the father of your
child is? Why, I would get the wretch from the bottom of the sea! But Shiha
knows who came to you then. Would you like me to threaten him so that he
should tell? ... Well, why are you silent? Do you want me to?"

"Do what you like, but don't torment, don't torment me so! Better make
an end of it." Maki moaned, pale and trembling as though she were on the
rack.

Rita drew back, and she trembled, too.

"Make an end of it? Do you think I know everything and merely tease
you, play cat and mouse with you? Well, perhaps I do know.... What's the
matter, why are you so frightened? Perhaps you know, too? A-ah, I've
caught you! Speak, tell me, who is it? He?"

"Yes, he, Saakera," Maki answered, with apparent calm, looking straight
into her eyes. "Well, kill me, I don't care..."

Rita brought the knife out of her bosom and flung it far away. She
buried her face in her hands and sat for a few minutes without moving; then
she drew her hands away from her face and put them on Maki's shoulders.

"There, it's a good thing you told me or very likely I would have killed
you, really. Do you remember Ankhi's doll?"
 When Rita and Ankhi were little they had once a fight over a clay doll,
a hideous thing that they both loved passionately. Rita took it from her
sister, who pulled it out of her hands, and broke it into bits against the wall.
Then Rita fell upon Ankhi like a fury and bit her throat; the nurses had
difficulty in dragging her off. And in the night she stole away into the
garden and ate some poison berries, 'spiders' eggs'; she very nearly died.

"The devil entered into me then, and now, too. We have all taken after
father—we are possessed.... Yes, it is a good thing you told me. All is well
now—it's over! But I do wonder at myself: I thought I would kill you if you
told me; and now I don't feel anything. Silly girls had a fight over a doll, but
perhaps it was not worth while, after all. You know what a number of wives
Saakera has. Sheep are in the stalls, fish in the hatchery and we in his
palace. You and I are no better than the others. You gave me your betrothed,
I gave you my husband, so we are quits and that's an end of it. We'll be
friends as before, better than before. When the baby is born—it must be a
boy, we don't want a girl—we'll look after it together.... What's the matter,
why are you silent again? Don't you believe me?"

"I do, but I am afraid...."

"What of?"

"I don't know.... You may forgive me, but I will torment myself to
death.... Oh, Rita, darling, why didn't you kill me straight away? It would
have been better to make an end of it!"

"Nonsense! All will be forgiven and forgotten if only one lives and
loves. And you do love me, don't you—more than before?"

"More, much more! I love you dreadfully, that's why I will die—
because I love you so. You know, Rita, if one loves very much, one cannot
live, it's too great a joy...."

"It was after that you got to love me so?" Rita asked, with slight
mockery.
 Instead of answering Maki hid her face on Rita's breast and burst into
tears.

"There, that will do," Rita said, drily. "It is time to go home—see what
heavy dew there is."

She took her by the arms and again led her along carefully like a nurse.

They went indoors. Rita put her sister to bed and sat down beside her,
waiting for her to go to sleep.

"Don't go," Maki begged.

"I won't, don't be afraid, I'll sleep here beside you."

"You do love me, yes?" Maki whispered in her ear.

"No, I don't love you a bit. Why, you silly girl, if I didn't love you, I
wouldn't torment you so.... there, that's enough talking, go to sleep."

"No, wait a minute, what was I going to say? ... Oh, yes! You know I do
not know for certain who came to me then. I told you it was he, Saakera,
but I don't really know—perhaps it wasn't he."

"Who was it, then?"

"He Whom I was expecting. I doubted, I did not believe—and this is

why I suffer now. I shall die in misery, but when I am dead, perhaps He will
come again."

"There, don't let us talk of it, sleep. Shall I tell you a story?"

"Do," Maki answered in a sleepy, childish voice.

"Once upon a time there lived a king and queen," Rita began the tale of
the Bewitched Prince, in a sing-song voice like the old nurse, Asa. "One
day they prayed to the gods and the gods gave them a son. And when he
was born the seven Hathor came to decree his destiny and said, 'this man
will meet with death from a crocodile, a snake, or a dog.' And the king was
very, very sorry when he heard of this. And he caused a tower to be built in
the mountains and settled the prince there. And the prince was very, very
happy there...."

She stopped, listened to her sister's even breathing and kissing her on
the eyes, that she might have good dreams, went out of the room.

Old Asa, the princesses' nurse, could not go to sleep in her stuffy room
and went out into the garden; seeing something white flit among the trees
she was frightened and wondered if it were Tiy—she knew that the dead
queen walked about at night. But, recognizing Princess Meritatona, she
called to her. The girl stopped, looked round without answering and ran
along, disappearing among the thick bushes.

Used as Asa was to the princess's whims, she was surprised and then
frightened—in a different way than at first: she felt there was something
really alarming about the white phantom.

She ran after Rita, but her old legs did not obey her very well. She went
on and on, calling her name, but there was no trace of her.

She met the gardener.

"Have you seen the princess?"

"Yes, I have."

"Where?"

"On the Lotos pond, in the chapel."

"What was she doing there?"

"I can't say."

"Let us go and look."

They walked to the chapel. The gardener did not dare to go in; Asa went
in, but ran out immediately, screaming wildly, and fell to the ground, almost
knocking the gardener off his feet.

He went into the chapel and saw the princess hanging on the brass rod
of the curtain before the altar. She had made the noose out of the curtain
drawstring but so badly that it slipped off. Hanging unevenly, her body
rested with the toes of the left foot on the corner of the bench she had
knocked down after climbing on to it to throw the string over the rod.

When the gardener cut the string and took the noose off Rita's neck she
did not breathe and her face was so blue and fixed that he thought her dead.

Maki dreamt that she was lying on the marriage-bed in a high tower in
the starry sky, waiting for Him as she had done then, in the temple of Attis;
she knew that He would come and that His face would be like the moon, the
sun of the night, not burning, not terrible, like the face of the god whose
name is Quiet Heart.

She woke up and called:

"Rita!"

She looked round—there was no one in the room; only the moon looked
in at the window, bright as the sun of the night.

Suddenly far away in the garden cries were heard. Maki jumped up, ran
into the garden and listened. The cries came nearer and nearer. Men with
torches ran about shouting.

Maki ran towards the torches. The men were carrying something long
and white. Maki rushed forward with a shriek. The men made way for her.
Moonlight fell upon Rita's face, and Maki fell fainting upon the ground.

Rita was in a deep swoon. She was saved in the end, but for several
weeks she was at death's door as in childhood when she had eaten 'spiders'
eggs.'
 The same night Maki's labour pains began and by the morning she was
safely delivered of a son.

VI
 here was brilliant sunshine outside, but it was dark as
night in the bedchamber of the Maru-Aton palace, with the
shutters closed and the windows curtained; only the gilded
columns glimmered faintly in the dim lamplight.

A bed of carved ebony and ivory, painted and gilded,

stood in the middle of the room on a platform with four steps. It was shaped
like a fantastic monster, a mixture of crocodile and hippopotamus, with
lion's feet and open jaws: it guarded the sleeper; the more fearful the bed,
the sweeter the sleep. Strangely convex, round and hard, with a wooden
crescent for a pillow, it seemed uncomfortable, but was in truth better than
any other bed, for it was cool to sleep on in the hot nights when feather beds
and pillows were unendurable.

Princess Makitatona lay on the bed. On the fourth day after her delivery
she had been attacked with child-bed fever.

The dark, stuffy room smelt of drugs. Pentu, the physician, was
pounding in a mortar of stone a complicated remedy, composed of forty-six
ingredients, corresponding to the same number of blood vessels in the
human body. In addition to medicinal herbs it contained lizard's blood,
sulphur from pigs' ears, powder from the head and wings of the sacred
beetle, Kheper, a pregnant woman's milk, a hippopotamus's tooth and flies'
dirt.

In another corner of the room a Babylonian sorcerer, Assursharatta, was

boiling in a cauldron the blood of a freshly slain lamb with magical herbs
and muttering a spell against the seven demons of fever:
Sibiti shunu, sibiti shunu,
Sibit adi shina shunu.
They are seven, they are seven,
They are seven twice over.
There are seven of them in heaven,
There are seven of them in hell.
They are neither male nor female,
They are childless and unmarried.
Whirlwinds that bring destruction
 Do not know what mercy is,
Do not bend their ear to prayer.
They are evil, they are mighty,
They are seven, they are seven!

But nothing helped the patient—neither the medicines nor the spells,
nor even the healing water from the well of the Sun in Heliopolis, where the
god Ra washed his face when he lived on earth.

In vain old Asa whispered the incantation:

Mother Isis cries
From the top of the hill:
"Horus my son,
The hill is on fire,
Bring me water,
Quench the fire."

The fire of the fever would not be quenched.

In vain the queen read over her daughter the prayer of Mother Isis.
When a scorpion stung baby Horus she cried to the sun and the sun was
darkened, night was upon the earth until the god Tot healed the baby and
gave it back to its mother. Since then the magical prayer of Isis had always
been read over sick children.

"Stand still, O Sun, stand still until the child is restored to its mother!"
the queen repeated with frenzied entreaty, but she knew the miracle would
not happen, the sun would not stop.

She recalled the hymn to Aton:

Thou conquerest all through love,

Thou soothest the babe in the womb
Before its mother can soothe it.
 But now He failed to soothe her.

Thou hast mercy upon a worm

And upon the midges of the air.

But now He had no mercy upon her.

The king and queen never left the invalid's side, but she was delirious
and did not recognize them. If a ray of sunlight penetrated between the
curtains or through a crack in the door, she grew restless and cried:

"It is coming, it is coming again! There it is stretching out its leg....

Abby darling, do drive it away, quick! It will seize me and suck me dry like
a fly ... whoever could have let a spider into the sky?"

The king understood: Aton the Sun was the spider, the hand shaped rays
were the spider's legs.

But most often she talked in her delirium about Shiha, the eunuch.

"Shiha, what does it mean 'light is greater than darkness'? Who has
blasphemed against divine darkness? Do you say King Uaenra is godless?
.... How dare you, you old monkey? .... Drink, drink! Isn't there something
cooler? You gave me boiling water last time, it scalded my mouth...."

They gave her the freshest water out of porous Tyntyrian vessels, but
she pushed the cup away:

"Hot water again!"

The baby boy had been born prematurely; it had no nails, no hair and
was weak and pale like a blade of grass grown in darkness. It hardly cried at
all and only wrinkled its face painfully at the lamplight.
 "It isn't right for a baby to be in darkness: it may go blind; it must be
taken into the sunshine," the midwives decided.

But as soon as they took it out into the light of day it screamed and
struggled as in a fit; they had to take it back into the dark. It was born an
enemy of Aton the Sun.

"Is it night outside?" Maki asked in one of her lucid intervals.

"No, it is day," the king answered.

"The day of life is short, the night of death is long," she said, with a
quiet smile, looking into his eyes, as it were into his very heart. "Shiha says
'darkness was before light; sunshine is a veil over darkness.' Shall the dead
see the sun? What do you think, Enra?"

He was about to answer, but she began to wander again.

"A hen, a white hen with a red wig on like Ty's.... It is running after
me.... Oh, it has stuck its teeth into me!"

The king remembered that the white hen was the mate of the cock with
which he and the princesses had played once. Old Asa wept bitterly: she
thought the hen with teeth was a bad omen.

"Do explain, Shiha," Maki wandered, "King Uaenra is wiser than all the
sons of men: how is it he does not know death? He lives and sings to the
sun as though there were no death and all were well... What will he sing
when he does know death?"

Sometimes the king fancied it was not mere delirium: it was as though
she knew that he was there without seeing him and spoke for his benefit,
passed dreadful judgment upon him, laughed at him with a terrible laugh.

"Enra, Enra, why don't you pray?" the queen repeated like one insane,
looking at him with dry, tearless eyes. "Pray! Your prayer is strong: the
Father will hear His son. Save her, Enra!"
 The king was silent. He felt so ashamed that he could have screamed
with shame, as with pain, but worse than shame, pain and death was the
mockery "what will you sing when you do know death?"

At the same time princess Meritatona was lying ill in the apartments of
Saakera, the heir-apparent.

Maki kept talking of her as of one dead.

"All through me, through me!" she repeated in anguish.

"But, darling, Rita is alive," her mother said, trying to comfort her. But
she would not believe it.

"No, mother, don't deceive me, I saw how they carried her, dead."

"She might be saved if only she believed me," the queen thought. "But
how can I convince her? And what has happened between them? A fine
mother I am—I don't know why one daughter strangled herself and by
whom the other has had a child.... Perhaps Enra knows? He spoke with her
then—he must know."

She questioned the king when they were alone together.

"Enra, do you know who the baby's father is?"

"No, I don't know."

"Haven't you asked her?"

"I have, but she did not say."

"How is it you didn't find out, how could you have left her to face such
torture alone?"

"I pitied her."

 "And don't you pity her now? Enra, Enra, what have you done!"

The baby did not live long: it died by the evening of the fifth day as
quietly as though it had gone to sleep.

"Where is the boy?" Maki asked, coming to herself.

"It is asleep," the queen answered.

"Never mind, I'll be very careful, let me have him!"

No one moved or spoke.

"Give him to me, do!" Maki repeated, looking round at them all.
"Mother, where is he? Tell me the truth.... What has happened? Is he dead?"

The queen covered her face with her hands.

"Well, perhaps it is better so," Maki said quietly. "We shall soon be
together...."

That same night before dawn the death struggle began. She no longer
tossed about or wandered; she lay quite naked: the lightest covering
oppressed her; the slender, childish body seemed flat and crushed as though
it had been trodden on like a blade of grass; the head with the elongated
skull was thrown back, the eyes closed, the face immovable and the
breathing so faint that at times it was not noticeable.

Pentu, the physician, brought to her lips a round brass mirror and when
it grew slightly clouded, he said:

"She breathes."

Suddenly she opened her eyes and called:

 "Enra, where is Enra?"

"Here," the king answered bending over her, and she whispered in his
ear, like a blade of grass rustling:

"Open the shutters."

He knew she was afraid of light and did not venture to open all the
shutters at once, but ordered them to draw the curtains from one window
only.

"All, all," she whispered.

All the windows were flung open. The morning sun flooded the room—
the rays of the god Aton like a child's hands embraced her naked body.

"Lift me up," the blade of grass rustled, and the king lifted her as easily
as though she were a blade of grass. The sun lighted her face.

"Akhnaton, Sun's joy, Sun's only Son!" she said, looking into his eyes so
that he understood this was not delirium, "I know that you are...."

She did not finish, but he understood: "I know that you are He."

Suddenly she trembled in his arms, like a leaf in a storm. He laid her
down on the bed.

Pentu put the mirror to her lips, but this time its brass surface remained
clear. The rays of the Sun—a child's hands—embraced the body of the
dead.

There was the sound of weeping in the chamber. The women cried,
wailed frantically, beat their breasts, tore their hair, scratched their own
faces till they bled, with a kind of rapture of despair. But all was decorous
like a holy rite: this was how they had wailed thousands of years before and
how they would wail thousands of years hence.
 The king heard the wailing, but there was bitter laughter in his heart:
"you are He!"

VII

want to have Maki buried according to the ancient

custom," the queen said.

"It shall be as you wish," the king answered. He

understood what 'according to the ancient custom' meant.
The hieroglyphics and mural paintings of the new tombs
in the province of Aton contained no name or image of the
ancient gods, no prayers, no incantations from the ancient scrolls of Going
out into the World, Unsealing of lips and eyes, from the Book of the Gates,
the Book of what is beyond the Tomb; no name or image of Osiris himself,
the bringer of life to the dead.

It was only now that the king grasped the meaning of the inscription in
Merira's tomb: 'may Aton and Unnofer revive the flesh on my bones.'
Unnofer was the Good Spirit, Osiris, the King Akhnaton himself. There was
a challenge and a temptation in it: "if you are He, conquer death!"

"I am not He! I am not! I am not!" he wanted to cry out in terror.

The mild fanatic, "the holy fool," Panehesy, looked straight into his
eyes, as though asking: "Will you renounce the work of your whole life,
will you lie, You-Who-live-in-truth, Ankh-em-maat?" And he read in his
eyes the answer, "Yes, I will lie."

The best embalmers of Thebes, Memphis and Heliopolis, herhebs and

mesras, "divine sealers, cutters and cleaners," were working at the dead
body of princess Makitatona.
 Cauldrons were boiling night and day in the House of Life, the
embalming chamber: ointments and unguents were being cooked—balm,
styrax, cinnamon, myrrh and cassia; piles of wood were heaped up—sandal,
terebinth, cedar, currant wood, mastic and the fragrant wood of the shuu
tree; heaps of incense from Punt in lumps as big as a fist lay about. Clouds
of dust rose over the copper mortars in which powders were made. Anyone
unaccustomed to these pungent smells would have fainted coming into the
chamber.

For thirty days and thirty nights they were cleaning, soaking, drying,
salting, embalming, smoking and pickling the body.

The king watched everything. He saw the entrails being taken out
through a slanting slit in the stomach, and the lapis lazuli sun beetle,
Kheper, being put in the place of the heart. He heard the cracking of the
bones when the nose was broken and the brain scooped out with a long flint
knife.

Eyes of glass were set into the empty sockets. The hair of the wig,
eyebrows and eyelashes was smoothed carefully. The nails, finger and toe,
were gilded. A narrow plaited Osiris's beard in a wooden box was tied to
the chin, for, in the resurrection of the dead, woman becomes man, the god
Osiris. The bandage of the god Ra was put round the forehead, of the god
Horus round the neck, of the god Tot on the ears, of the goddess Hathor on
the mouth. And the mummy spun round and round like a spindle in the
clever hands that bound it in endless bandages like a chrysalis in a cocoon.

A wooden, crescent-shaped support for the head was prepared and a

prayer to the Sun—not the new god, Aton, but to the ancient god Ra—was
written on a new sheet of papyrus: "Give warmth under his head. Do not
forget his name. Come to the Osiris Makitaton. His name is the Radiant, the
Ever-Living, the Ancient of Days. He is Thee."

Thus a new great and terrible god Makitaton grew out of little Maki.

Ancient wisdom went hand in hand with ancient crudeness and

childishness: the hieroglyphic of the serpent in the tomb inscription was cut
into two, so that the snake should not sting the dead, and fledgelings on the
paintings had their feet cut so that they should not run away. A silver boat
was placed in the tomb for the dead to sail the Sunset Sea, also a mirror,
rouge, powder, a book of fairy tales and draughts: the dead could play a
game with her soul; toys were also put in, among them Ankhi's broken doll,
carefully pieced together.

A tomb effigy was made for the mummy: the bird Ba with a human face
and hands, the soul of the dead girl, placing its hands upon her heart and
looking lovingly upon her face was saying:

"The heart of my birth, my mother's heart, my earthly heart, do not

forsake me. Thou art in me; thou art my Ka, my Double within my body;
thou art Khnum, the Potter who hath made my limbs."

The Germinating Osiris was prepared, too: linen was stretched on a

wooden frame, the likeness of Osiris's mummy was drawn upon it, a thin
layer of black earth was placed over it and thickly sown with wheat. The
frame was watered until the seeds germinated; then the crop was cut down
smoothly like grass on the lawn. This green, spring-like resurrected body of
Osiris was placed in the tomb by the side of the corpse. The living seemed
to be teaching the dead: "Look, the seed has come to life—you do the
same!"

The ancient custom was not observed in one respect only: the head of
queen Nefertiti, the earthly mother, was sculptured in the four corners of the
granite tomb instead of the head of Isis, the heavenly mother. When the
queen heard of this she was indignant and rebelled against the king for the
first time in her life. But it was too late to prepare a new tomb.

On the fortieth day after Maki's death the funeral procession started out.
The coffin was put into a boat, the boat into a sledge—the carriage of the
ancient times when there were no wheels; two pairs of oxen drew it and the
runners slowly creaked on the white sand of the desert as it were on snow.

Mourners dressed in blue—the colour of the sky, the colour of death—

walked in front, throwing dust over their heads with a wail, monotonous
like the howling of jackals.
 "Weep, weep, weep, O sisters! Shed tears, shed endless tears! Draw
your mistress to the West, oxen, draw her to the West! Poor darling, you
were so fond of talking to me, why are you silent now, why don't you speak
a word? So many friends you had, and now you are alone, alone, alone! The
little feet that walked so fast, the little hands that held so tight are bound,
confined, tied down. Weep, weep, weep, O sisters! shed tears, shed endless
tears!"

The sun was setting when they entered the Princesses' Valley, with the
yawning openings of the tombs cut in the rocks. Close by an old fig tree
was an unfading patch of green against the dead sands and a sweetbriar
flowered fragrant with the scent of honey and roses: the secret waters of an
underground spring kept them fresh. The drowsy humming of bees sounded
like faraway cymbals.

The mummy was placed at the entrance of the tomb and stood against
the yawning blackness of the cave, bathed in the last radiance of the setting
sun. Two priests, one wearing the mask of the jackal-headed Anubis and the
other of the falcon-headed Horus, stood on either side of the mummy, while
the officiating priest, herheb, performing the sacrament Apra, the opening
of lips and eyes, read the magical words from a papyrus:

"Get up, get up, get up, Osiris Makitaton! I, thy son Horus, have come
to give thee back thy life, to join thy bones, to bind thy flesh, to put thy
limbs together. I am Horus, thy son, who gives birth to his father. Horus
opens thy eyes that they may see, thy lips that they may Speak, thy ears that
they may hear; he strengthens thy legs that they may walk and thy arms that
they may work!"

The priest embraced the mummy, brought his face near its face and
breathed into its mouth.

"Thy flesh increases, thy blood flows and all thy limbs are whole."

"I am, I am! I live, I live! I shall not know corruption," another priest,
hidden behind the mummy, answered as though it were itself speaking.
 "Thou art a god among gods, transfigured, indestructible, ruling over
other gods;" the officiating priest declared.

"I am one. My being is the being of all the gods throughout eternity,"
the mummy answered and the dead eyes glittered more brightly than the
living. "He is—I am; I am—He is."

The king fell on his face: he understood that this new terrible god,
Lightgiving, Everlasting, Ancient of days, Makitaton, had overthrown Aton.

He breathed with relief when the body was put back in the coffin and
Makitaton became little Maki once more.

He bent over her, kissed her on the mouth and put upon her heart a
branch of mimosa: the tender, feathery leaves were to respond with their
tremour to the first stir of the heart at resurrection.

The king spent the night in a tent in the desert, waiting for sunrise, to
say the morning prayers at the tomb.

He could not go to sleep for hours. At last he got up, lifted the side of
the tent and looked out. The Milky Way stretched like a cloud rent in two
from one end of the desert to the other, the Pleiades glowed, and the seven
stars of Tuart the Hippopotamus glittered with a cold brilliance. Dead
stillness was all round; only the jackals' howling and the hooting of owls
came from the gorge below.

He lay down again and went to sleep.

He dreamt he was standing on a square platform at the top of Cheops'

great pyramid. The desert below was thronged with a countless multitude of
men: there seemed to be as many heads as there were grains of sand in the
desert; it was as though all tribes and peoples had gathered together for the
last judgment on him, Uaenra. They were looking at him and waiting with
bated breath.