In Vivo Monitoring of Blood-Brain Barrier Leakage
by Using Photoacoustic Microscopy
Weitao Li, Lidong Xing, Ling Tao, Zhiyu Qian
Department of Biomedical Engineering, Nanjing University
of Aeronautics and Astronautics
Nanjing, 210016, China
liweitao@nuaa.edu.cn
Liming Nie
Center for Molecular Imaging and Translational Medicine,
State Key Laboratory of Molecular Vaccinology and
Molecular Diagnostics, School of Public Health, Xiamen
University, Xiamen 361102, China.
limingnie@gmail.com
Abstract—Monitoring and evaluation of vascular leakage in
brain is important in the research area of brain injuries and
edema. A high-resolution photoacoustic microscopy (PAM) was
developed to monitor the blood-brain barrier (BBB) permeability
using Evans blue (EB) dye. We used a mouse traumatic brain
injury (TBI) to validate the usefulness of our system. The uptake
of EB in brain vessels was continuously studied for 2 hours after
injection in a normal mouse. The TBI mice with and without
injection of EB were both scanned by PAM for 2 hours,
respectively. The PAM results showed that EB was a high
contrast agent for PAM images and the degree of BBB leakage
was accessed by EB leakage. Our studies demonstrate that PAM
has potential in monitoring BBB leakage in vivo.
Keywords—blood-brain barrier; photoacoustic microscopy;
traumatic brain injury; evans blue; optical imaging
I. INTRODUCTION
Mild to severe traumatic brain injury (TBI) causes brain
damage, intellectual and cognitive deficits and is a serious
problem globally [1]. Traumatic brain injury is the main
leading cause of death and disability in youth [2]. There are
~1.5 million people who die, and overall about 10 million
people who are either killed or hospitalized because of TBI
annually [3]. The disruption of the blood-brain barrier (BBB)
and secondary neurodegeneration is caused by many
neurological conditions, such as brain tumors [4] and traumatic
brain injury [5]. So an in vivo method for monitoring BBB
leakage is needed. Traditionally, Evans Blue (EB) dye
extravasation has been used to measure BBB permeability [6].
The amount of EB was measured at 625 nm by optical density
(OD), which was not an in vivo method. An optical imaging
method based on fluorescence was used to map BBB in order
to study BBB leakage [7]. However, the detection depth is less
than 1 mm. Therefore, an in vivo monitoring method is needed
to advance studies of BBB leakage detection.
More recently, photoacoustic methods have been applied
mainly to in vivo evaluation of materials distribution in
biological tissues. The principle of Photoacoustics (PA)
combines optical imaging with ultrasound, so it can achieve
both deep and high resolution images. Thus, PA has been used
in many biological imaging areas, such as mouse brain
metabolism [8], functional brain imaging [9,10], mouse brain
vasculature [11], and traumatic brain injury of a mouse [12]. If
the materials have strong absorption in visible or/and near-
infrared light, they will be suitable as contrast agents for PA
imaging. Due to the high absorption coefficient at 620 nm, the
EB dye could be used for this purpose. At 532 nm, the
absorption coefficient of blood corresponds to an EB solution
with a concentration of 3.6 mg/mL [11]. EB is nontoxic and
soluble in water. Moreover, PAM imaging based on EB is used
for the evaluation of leakage of blood vessels in the brain.
Here, for the first time, we have developed a high
resolution photoacoustic microscopy system based on EB dye
that can monitor BBB leakage caused by TBI. The PA system
works on the principle of an absorption peak of EB at 620 nm.
The transport kinetics of EB in blood circulation in the brain
was monitored. Furthermore, we can obtain the extent of EB
distribution in the brain, in response to BBB disruption. The
mouse models were used to demonstrate the usefulness of our
method for monitoring vascular leakage in vivo. This method is
simple to use and requires no tissues processing.
II. M
ATERIALS AND METHODS
A. Experimaental Details
The experimental set up of homemade AR-PAM is shown
schematically in Fig. 1. This system consists of a pulse laser
light source with a 7 ns pulse duration and 20 Hz pulse
repetition rates. The light source is a tunable dye laser (Surelite
OPO Plus, Continuum) pumped by a Nd:YAG laser. The laser
beam was reflected by some mirrors (Thorlab Inc.) and passed
through one conical lens and one condenser to focus on the
surface of the mouse brain. The maximum energy at 532 nm at
the skin of the mouse was ~7 mJ/cm
2
, which is less than the
American National Standards Institute (ANSI) limit (20
mJ/cm
2
). A two-dimensional (x-y) axis motorized stage
(Edmud, Barrington, NJ) was controlled by a computer via a
programmable motion controller (Dover DMM 0200, MA) and
the minimum step used was 10 um. The focus regions of light
and ultrasonic transducer were adjusted by this motor stage.
The PA signal was received by an ultrasonic transducer with
2.54 cm focal length (center frequency: 10 MHz, Olympus
NDT V-315). Then the signal was amplified by an amplifier
(Olympus CSE 1422). After that it was digitized by a 14-bit
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