Noninvasive Optical Measurement of Cerebral Blood
Flow in Mice Using Molecular Dynamics Analysis of
Indocyanine Green
Taeyun Ku1, Chulhee Choi1,2,3*
1 Graduate School of Medical Science and Engineering, KAIST, Daejeon, Republic of Korea, 2 Department of Bio and Brain Engineering, KAIST, Daejeon, Republic of Korea,
3 KI for the BioCentury, KAIST, Daejeon, Republic of Korea

Abstract
In preclinical studies of ischemic brain disorders, it is crucial to measure cerebral blood flow (CBF); however, this requires
radiological techniques with heavy instrumentation or invasive procedures. Here, we propose a noninvasive and easy-to-use
optical imaging technique for measuring CBF in experimental small animals. Mice were injected with indocyanine green(ICG) via tail-vein catheterization. Time-series near-infrared fluorescence signals excited by 760 nm light-emitting diodes
were imaged overhead by a charge-coupled device coupled with an 830 nm bandpass-filter. We calculated four CBF
parameters including arrival time, rising time and mean transit time of a bolus and blood flow index based on time andintensity information of ICG fluorescence dynamics. CBF maps were generated using the parameters to estimate the statusof CBF, and they dominantly represented intracerebral blood flows in mice even in the presence of an intact skull and scalp.
We demonstrated that this noninvasive optical imaging technique successfully detected reduced local CBF during middle
cerebral artery occlusion. We further showed that the proposed method is sufficiently sensitive to detect the differences
between CBF status in mice anesthetized with either isoflurane or ketamine–xylazine, and monitor the dynamic changes in
CBF after reperfusion during transient middle cerebral artery occlusion. The near-infrared optical imaging of ICG
fluorescence combined with a time-series analysis of the molecular dynamics can be a useful noninvasive tool for preclinicalstudies of brain ischemia.
Citation: Ku T, Choi C (2012) Noninvasive Optical Measurement of Cerebral Blood Flow in Mice Using Molecular Dynamics Analysis of Indocyanine Green. PLoS
ONE 7(10): e48383. doi:10.1371/journal.pone.0048383
Editor: Jean-Claude Baron, University of Cambridge, United Kingdom
Received April 27, 2012; Accepted September 24, 2012; Published October 31, 2012
Copyright: ß 2012 Ku, Choi. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by a grant (2011K000286) from the Brain Research Center of the 21st Century Frontier Research Program, funded by the
Ministry of Education, Science and Technology, the Republic of Korea (to C.C.). The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: cchoi@kaist.ac.kr

preclinical setting are limited. Laser Doppler flowmetry is notsensitive enough to estimate regional CBF and detects only the
relative changes [8]; whereas optical micro-angiography has acritical limitation in that the scalp of small animals must be
removed to obtain sufficient resolution to identify red blood cell
movement in small vessels.
In contrast to label-free techniques, optical techniques using
fluorescent tracers can significantly enhance the signal-to-noise
ratio (SNR) using intravascular contrast agents. In addition, thesetechniques have the advantage of extracting kinetic information by
time-series analysis of fluorescence signals after the bolus injectionof the tracers. Indocyanine green (ICG) is a fluorescent probe
clinically used for liver function tests [9] and angiography inophthalmology [10]. ICG is excited in the near-infrared (NIR)
wavelength range [11], and deeper penetration of NIR wavelength
light in tissues is enabled due to a lesser degree of absorption andscattering [12]. Intravenously injected ICG rapidly binds to serum
albumin and remains confined in the vessels until it is removed via
clearance through the liver [11]. Kinetic information of the ICG
bolus enhances detection of deep organs by enriching of organspecific information including differences in filling and wash-out
patterns [13,14] and detection of tissue blood flow in experimental
animals [15,16] and humans [17–20]. Hillman et al. described

Introduction
Measurement of cerebral blood flow (CBF) is essential for the
diagnosis and monitoring of stroke patients. Nuclear imaging
methods that detect radioactive tracers by positron emission
tomography [1] and radiological imaging methods, such as
computed tomography (CT) [2,3] and magnetic resonanceimaging (MRI) [4,5], are widely used for noninvasive measurement of CBF. In basic research involving small animals, theapplication of these methods is restricted especially for longitudinalstudies due to limitations that include the high cost of equipment,risk from radiation, and the low availability of portable devices. Asan alternative, optical approaches have gained increasing attention
for detection of CBF.
Optical methods for measuring CBF include the use offluorescent tracers and label-free techniques. Laser Doppler
flowmetry, a label-free optical technique, measures the velocity
of circulating red blood cells in the bloodstream by the Doppler
effect, and is commonly used to monitor the relative change in
CBF in experimental animals [6]. Optical micro-angiography,
based on optical coherence tomography, was recently developed
for the label-free measurement of blood velocity in small vessels inmice [7]; however, practical applications of these techniques in the
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Noninvasive Optical Imaging of Brain Perfusion

such approaches in detail and termed them dynamic contrast
enhancement (DyCE) technique [21]. We demonstrated recently
that analysis of ICG molecular dynamics can be used to estimate
peripheral tissue perfusion semi-quantitatively in mice [16] andhumans [17,22]. However, this approach has not been applied forthe detection of CBF due to the different hemodynamic
characteristics of the brain, which has a rapidly moving andabundant blood supply. Near-infrared spectroscopy (NIRS) has
been used primarily to detect ICG absorption signals in the brain[23,24]. Measurement of CBF using NIRS and ICG was first
demonstrated to be effective in animals [15,25], and was thenrapidly applied to humans because of its noninvasive characteristics [18,26–28]. However, detection of NIRS signals above thehuman scalp is highly limited by the presence of a large amount ofnoise. This limitation is mainly due to the thick extracerebral
tissues, including the scalp, skull, and dura mater; therefore, there
have been significant efforts to separate intracerebral fromextracerebral signals [27,29,30]. While methods such as CT or
MRI provide two- (2D) or three-dimensional (3D) images thatprovide valuable information about CBF, NIRS can detect bloodflow information only at several points upon the head due to its
limited spatial resolution. It is reported that the detection of ICG
fluorescent signal is more sensitive than the detection of absorption
signals by NIRS when used as a point detection approach [31,32].
Woitzik and his colleagues obtained 2D blood flow maps using
ICG fluorescent imaging, although the method was applicable
only during an invasive surgical procedure where the scalp andskull of the patient were totally removed [33].
While optical penetration is restricted by the thick extracerebral
tissues in humans, sufficiently high SNR can be achieved inexperimental small animals–such as mice–because of theirrelatively thin extracerebral tissues. We propose an optical imaging
technique that measures CBF in mice noninvasively through a
combined ICG molecular dynamics analysis. To the best of ourknowledge, this is the first report to examine the feasibility ofnoninvasive fluorescence imaging for measurement of CBF insmall animals.

Intracranial CBF was Dominantly Detected by Time-series
Optical Imaging of ICG Fluorescence
We next investigated whether the blood flow information
observed in the area of the shape of the mouse cerebrum on the
CBF map was originated from intracranial brain tissue. Above thecerebral cortex of the mouse are multiple layers of extracerebral
tissues, including the scalp, skull, and dura mater. Because these
extracerebral tissues also have blood vessels and blood supply, we
compared fluorescence dynamics over the cranium to those
directly over the cerebrum. When we removed the extracerebral
tissues above the hemisphere of a scalp-removed mouse by
craniotomy with complete excision of the dura mater (Figure 2A),
we found no difference in the pattern in dynamics between thecontralateral region with the skull and dura mater and theipsilateral region lacking these extracerebral tissues (Figure 2B).
We further evaluated whether ICG fluorescence imaging with
time-series analysis of molecular dynamics could detect CBF
noninvasively. We shaved only the scalp to minimize theinvasiveness of the procedure. The CBF maps successfully
displayed the shape of the underlying brain both in white ICR
mice and black C57Bl/6 mice (Figure 2C and D). Even though
the dynamics of the extracerebral flow were not extractable fromthe dynamics of the intracerebral flow, the degree of influence by
the extracerebral signals warranted further evaluation. Therefore,
we selectively blocked either the extracerebral or the intracerebral
blood flow by selective vascular occlusion, and subsequently
examined the CBF maps. To selectively reduce intracerebral
blood flow, we ligated the ICA and occluded the MCA byinserting a suture filament that preserved the intact ECA(Figure 2E). To selectively reduce the extracerebral flow, weligated the ECA (Figure 2F). Selective blockade of the intracerebral blood flow prominently increased the Trising parameters of the
pixels in the ipsilateral hemisphere, both in a scalp-removed
mouse and in an intact-scalp mouse (Figure 2G and H). In
contrast, although ligation of the ECA effectively reduced thedelivery of ICG to the extracerebral tissues (Figure S1), it did not
significantly alter the Trising map regardless of scalp removal(Figure 2I and J). These results collectively indicate that the timeseries optical imaging of ICG fluorescence can successfully detectblood flow, dominantly representing intracerebral blood flow evenin the presence of extracerebral tissues in mice.

Results
Analysis of the Molecular Dynamics of ICG for Twodimensional Measurement of CBF in MiceTo obtain the molecular dynamics of ICG in mouse brain, we
acquired time-series fluorescence images over the mouse headafter a bolus injection of ICG (Figure 1B). The dynamics in eachpixel typically consisted of the first peak followed by subsequent
peaks formed by systemic recirculation (Figure 1C). The relative
intensities and time points of each peak were distinct in different
tissues. The time point of the first peak over the cerebrum wasearlier than those in the large vein and the extra-cranial skeletal
muscle. The first peak in the skeletal muscle showed lower
intensity than later peaks. We extracted Trising values from the firstpeak as a representative parameter of the status of tissue blood
supply. Trising was calculated by subtracting Tarrival, the time of
appearance of the bolus, from the first peak time (Tpeak)(Figure 1D). The average Trising value over a somatosensory
cortical region of nine mice anesthetized with isoflurane was
2.760.1 s. Trising values in each pixel were reconstructed to createa 2D CBF map (Figure 1E). The CBF map clearly revealed theshape of the mouse brain. This finding indicates that ICG
fluorescence imaging combined with analysis of molecular
dynamics can separate the brain from adjacent tissues using thedifferences in hemodynamic parameters in mice with intact skulls.

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Four Types of Blood Flow Map and Application to
Cerebral Ischemia
We next selected three more parameters in addition to Trisingfrom the ICG time kinetics, with or without intensity information,
to improve the CBF estimates (Table 1). Tarrival was selected as thetime axis. We calculated the mean transit time (MTT) as thecenter of gravity of the dynamic curve and the blood flow index(BFI) as the slope of the first peak [3,15]. Four parameters wereused to generate independent CBF maps. We investigated changes
in CBF maps under cerebral ischemia conditions. We induced
focal cerebral ischemia in mice by middle cerebral artery occlusion(MCAO) surgery. Representative CBF maps for a normal brain
before MCAO and an ischemic brain during MCAO aredisplayed in Figure 3A. All four maps showed altered hemodynamic parameters in the ischemic hemisphere compared to thosein the normal hemisphere. Lateral regions of the cerebral cortex
are supplied primarily by the middle cerebral artery (MCA), butthey also receive a fraction of their blood supply from the anterior
cerebral artery (ACA) on the medial side through anastomoses in
the case of MCAO [34]. In ischemic brains where the MCA is
occluded, flow from the ACA partially recovers the blood supply of2

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Figure 1. Time-series fluorescence imaging of indocyanine green (ICG) on a mouse head and generation of a cerebral blood flow(CBF) map. (A) Schematic of imaging setup. (B) Time-series acquisition of fluorescence images after intravenous bolus injection of ICG. The scalp was
excised and the bregma was positioned at the center of the images (scale bar: 2 mm). (C) ICG dynamics in three pixels after injection of an ICG bolus,
indicated in (B). Different regions of interest are indicated by colored shapes (red circle, cerebrum; green rectangle, large vein; blue triangle, skeletal
muscle). (D) Smoothed and interpolated dynamics were plotted as a solid line, and the arrival time (Tarrival) and the first peak time (Tpeak) for
calculating the rising time (Trising) are indicated on the time axis. (E) A representative CBF map drawn using the Trising parameter. CCD, charge-coupleddevice; LED, light-emitting diode; a.u., arbitrary unit; Tmax, maximum intensity time.
doi:10.1371/journal.pone.0048383.g001

resolution dominantly represent capillary flow (Figure S2), thedelay in the arrival of the bolus through arterial vessels did notmatch the tissue blood flow status estimated by Trising and MTT.
The BFI values, which utilize the rising time as the denominator,
were lower in the lateral regions, indicating a noticeable decreasein blood volume.
Average values of parameters in the regions over left
somatosensory cortices in nine mice were calculated andcompared before and during MCAO (Figure 3B). Because Tarrivalis affected by the time of initiation of the bolus injection, only

lateral regions. Consistent with this notion, the Tarrival map
showed a gradual delay in the arrival of the ICG bolus from themedial to the lateral side. However, in the Trising map, the entire
cortex in the ipsilateral hemisphere showed increased Trisingvalues. An increase in MTT was also observed in the ipsilateralhemisphere. The dynamic analysis clearly indicated that not only
the lateral regions primarily supplied by the MCA but also themedial regions supplied by the ACA received a decreased blood
supply by MCAO, although the ACA flow was not disturbed
directly. Because the dynamics observed in the supra-capillary
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Figure 2. Intracranial blood flow parameters are predominantly measured by dynamic fluorescence imaging analysis. (A) Image of amouse head (in which the extracerebral tissues were totally removed in the left hemisphere) and a Trising map. To remove extracerebral tissues, thescalp was excised, and a craniotomy was applied along with dura mater excision. (B) ICG dynamics of a region from which all extracerebral tissues
were removed (red line) and a contralateral region with intact skull and dura mater (blue line). (C and D) Representative Trising maps of a head with anintact scalp in an ICR mouse (C) and a C57Bl/6 mouse (D). (E and F) Diagrams of the selective block of blood supply to intracerebral (E) andextracerebral (F) tissues. (G and H) Left internal carotid artery (ICA) was ligated, and a nylon suture filament was inserted toward the middle cerebral
artery (MCA) to block the intracerebral blood supply. The external carotid artery (ECA) was ligated to block the extracerebral blood supply. Trisingmaps for the selective block of the intracerebral blood supply in a scalp-removed mouse (G) and an intact-scalp mouse (H). (I and J) Trising maps forthe selective block of the extracerebral blood supply in a scalp-removed mouse (I) and an intact-scalp mouse (J). All mice were anesthetized withketamine and xylazine. CCA, common carotid artery.
doi:10.1371/journal.pone.0048383.g002

indicate that CBF parameters and maps are capable of detecting
cerebral ischemia status.

Trising, MTT, and BFI showed significant differences between
groups (t-test, p,0.001). In contrast, when the parameters in theipsilateral ischemic hemispheres were compared to those in thecontralateral normal hemispheres during MCAO in individual
mice, all four parameters exhibited statistically significant differences (paired t-test, p,0.001) (Figure 3C). These results clearly

Detection of Cerebral Hemodynamic Changes using CBF
Maps
We expanded our application of CBF maps to other experimental conditions accompanying cerebral hemodynamic changes.

Figure 3. Types of CBF maps and a comparison of CBF parameters in normal and ischemic hemispheres. (A) Representative CBF maps
using the four parameters described in Table 1 were generated from a normal condition (left panels) and an ischemic condition in which the left MCA
was occluded (right panels). (B) Average blood flow parameters of regions over left somatosensory cortices of nine mice before and after MCA
occlusion (MCAO) surgery. Trising and blood flow index (BFI) parameters showed significant differences between the normal and ischemic conditions(t-test, ***p,0.001). (C) Average blood flow parameters of regions over ipsilateral ischemic somatosensory cortices were compared to those regions
over contralateral normal cortices in nine mice during MCAO. All four parameters showed significant differences (paired t-test, ***p,0.001). MTT,
mean transit time; Contra, contralateral region; Ipsi, Ipsilateral region.
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Table 1. Definitions and interpretations of cerebral blood flow (CBF) map parameters.

Map parameter

Definition

Interpretation

Reference

Arrival time (Tarrival)

Initiation time of rise of the first passage
after bolus injection.

Tarrival reflects the fastest vascular path from the heart.
Marked occlusion of major arteries will alter Tarrival
maps. Absolute value of Tarrival is affected largely by
the bolus injection method.

[44]

Rising time (Trising)*

Time taken to reachthe first peak after arrival of bolus.
Trising ~Tpeak {Tarrival

Trising may be a primary choice for evaluation of local
tissue perfusion. Volume of blood supply isrepresented in Trising maps with a high contrast.

[26]

Mean transit time (MTT)

Mean detection time of ICG

MTT calculates whole blood supply including delayed
collateral flows. MTT may be a better choice in casesof low signal-to-noise ratios.

[27]

Blood flow index (BFI)*The slope of the first peak in

BFI represents overall blood volume information
with respect to time.

[15,33]P :t I(t)
tparticles in a region of interest. MTT~ PI(t)ta time-intensity curve. BFI~

Ipeak {Iarrival
Tpeak {Tarrival*Other reports may use Tmax and Imax instead of Tpeak and Ipeak, respectively.
t, observation time point; I(t), intensity function of time; Ipeak, intensity of the first peak; Tpeak, time of Ipeak; Iarrival, intensity at Tarrival; Imax, maximum intensity; Tmax, timeof Imax.
doi:10.1371/journal.pone.0048383.t001

in somatosensory regions in the K–X group resulted in significantly increased Tarrival, Trising, and MTT and decreased BFI (ttest, p = 0.001 for Tarrival and p,0.001 for others) (Figure 4B).
Second, we obtained serial CBF maps throughout the timecourse of transient MCAO (Figure 4C). Images were taken before
MCAO surgery, at 15 min after MCAO induction, and at 5 min,and 2, 6, and 24 h after reperfusion following 45 min of occlusion.

First, we anesthetized two groups of mice using different
anesthetics and compared their CBF maps. Inhalation of
isoflurane increases CBF, and injection of a ketamine–xylazine
cocktail (K–X) causes a decrease in CBF through its effects on
systemic hemodynamic functions [35–37]. In Trising maps, theoverall Trising value in mice anesthetized with K–X was larger thanthat in mice anesthetized by isoflurane (Figure 4A). Average values

Figure 4. Detection of cerebral hemodynamic changes using CBF maps. (A) Representative Trising maps for mice anesthetized with either
1.5% isoflurane or 0.1 mg/g ketamine and 0.01 mg/g xylazine. (B) Averaged CBF parameters of regions over left somatosensory cortices of six miceanesthetized with isoflurane and six mice anesthetized with ketamine and xylazine. Tarrival, Trising, and MTT parameters increased significantly, and BFIparameter was decreased significantly, in ketamine and xylazine group (t-test, **p,0.01; ***p,0.001). (C) Timeline of the transient MCAO protocol(upper diagram) and representative Trising maps (lower panels). Reperfusion (RP) was induced at 45 min after MCA occlusion. The six time points forimaging acquisition are indicated under the timeline.
doi:10.1371/journal.pone.0048383.g004

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without eliminating recirculation [39]. MTT is referred to as thetransition time after the bolus arrives at a pixel or a region ofinterest, so it is subtracted by arterial input dynamics suitable forthe pixel or the region. Practically a large artery observed in thesame image is chosen for global use as an arterial input function.
As such large reference arteries are hard to utilize in 2D projected
fluorescence images, we calculated MTT using fixed interval
dynamics after Tarrival without compensating for the arterial input
function. Although this approach may decrease MTT accuracy
compared to that observed in mice using other methods [40], it issufficient to compare region by region in the same hemisphere or
between two hemispheres in an identical mouse. Because a
relatively larger amount of information on the time axis is used to
calculate MTT than other parameters, the MTT is particularly
useful for analyzing low SNR dynamics. BFI is a parameter widelyused to analyze ICG dynamics [15,33]. BFI considers not only
temporal information but also intensity information. The intensity
value is used for blood volume information during derivation of
the BFI [15]. Therefore, the ICG injection volume determines thescale of the BFI, and its control is important for comparisonsamong animals.
Bolus arrival can be greatly affected by injection method, thesize of the animals, and environmental factors such as room
temperature. Automatically injecting the ICG bolus using asyringe pump and triggered initiation of imaging acquisition will
reduces the variability of bolus arrival and enhance the accuracy ofTarrival parameters. Trising, MTT, and BFI are free from theinjection time variation, and they significantly distinguished theischemic hemispheres among individual experiments. However,regardless of variation in injection time, all four parameters aregood candidates for analyzing blood flow in a single mouse tocompare region by region or an ipsilateral hemisphere with acontralateral hemisphere.
We were not only able to detect local remarkable decreases in
CBF such as MCAO using CBF maps, but also global CBFchanges using different anesthetics or subtle changes in local CBF
using serial imaging during the course of transient MCAO
(Figure 4). K–X is expected to decrease CBF compared toisoflurane [37], and all four CBF parameters showed corresponding differences. However, users who compare CBF parameters
among animals anesthetized with different anesthetics must beaware that MTT can be biased by the anesthetic. Decreased
hepatic ICG clearance by isoflurane, in contrast to increased liver
enzyme activity by K–X, may reduce the decrease in late-time
intensity [41,42]. Because MTT takes a larger portion of the
dynamics into account than do the other parameters, the central
gravity of the dynamic might be shifted to the right.
In this study, we used a charge-coupled device (CCD) camera ata low frame rate (580 ms per frame) to enhance the SNR of NIR
fluorescence signals and increased the temporal resolution by postprocessing of dynamics, including smoothing and interpolation.
Signals obtained by high SNR CCD enabled easy and accurate
determinations of the initiation points and the maximum points ofthe first peak rise to calculate Tarrival and Tpeak. Other acquisition
devices with high frame rates could also be used, and they may
provide high temporal resolution. However, fine algorithms todetermine Tarrival and Tpeak might be required in the absence ofproper signal-processing procedures. Imaging with a higher frame
rate system was reported by Hillman et al., and alternative
mathematical approaches to extraction of different dynamic
components from complicated in vivo signals were introduced[21]. The technical aspects of tail-vein injection were also
described in detail in that study. Despite the non-invasivenessand accessibility advantages, CBF optical imaging by analysis of

The CBF status of the two hemispheres could be identified in CBF
maps at each imaging time-point. These results indicate that CBF
maps can be used to monitor CBF status affected by hemodynamic
conditions or CBF changes after cerebral ischemic events.

Discussion
We demonstrated that optical imaging using the NIR fluorescence dye, ICG, combined with a time-series analysis of themolecular dynamics, can be used for noninvasive analysis of CBF.
The patterns of ICG dynamics were distinct in each tissue, and thedifferences could be successfully expressed using four dynamic
parameters including the Tarrival, Trising, MTT, and BFI. Bloodflow maps generated using these parameters clearly identified theshape of the mouse cerebrum and detected changes in CBF during
cerebral ischemia. Hair removal alone was sufficient for imaging of
normal and reduced CBF induced by selective vascular occlusion
surgery. We further confirmed that this technique can be used tocompare CBF status in animals anesthetized with different
anesthetics and to monitor changes in CBF after induction of
cerebral ischemia followed by reperfusion.
Our results clearly demonstrate that the dynamics of theintracerebral CBF are dominantly detected by dynamic fluorescence imaging, even in the presence of extracerebral tissues. Dueto deeper penetration of NIR wavelength light, signals from
sufficiently deep brain tissue are collected, and the portion of
detected signals from the relatively thin extracerebral tissues may
be negligible. We found that the intensity of fluorescence signalsthat originated below the scalp was up to four times higher thanthose from within the scalp. A large portion of extracerebral
signals may be masked by strong intracerebral signals, andqualitative analysis of intracerebral flow may not be practicallyinterrupted, as shown in Figure 2H.
Repetitive ICG injections and CBF measurements were feasible
as shown in Figure 4C. Reinduction of anesthesia after a recovery
period may require repeated catheterization procedures, and thenumber of attempts for venous puncture on tail veins will belimited. This may restrict the practical repeatability of ICG
imaging. During experiments, the injection route was retainedusing catheterization; waiting 10 min was sufficient to carry out
subsequent ICG injections and imaging. However, excessive
repetition of injections should be avoided in a single animal overa short period considering plasma volume overload.
Each of the four blood flow parameters provided uniqueinformation regarding CBF, and the combined analysis enhanced
the interpretation of the CBF condition such as hypoperfusion,anastomoses, and collateral blood supply induced by experimental
intervention of CBF. Tarrival represents the first detectable bolus toarrive through different passages. Under an ischemic condition,
Tarrival could be used to indicate which blood supply feeds theischemic region. Even in the case in which the decrease in tissue
blood flow was not so prominent by a Trising map or a MTT map,
the Tarrival map could be used to determine the success of an
attempt to interrupt arterial sources. The Trising parameter
provides a highest contrast map between ipsilateral and contralateral hemispheres in MCAO and should be the first choice toestimate changes in CBF status. Because the Trising parameter
eliminates the effect of injection time uncertainty by subtracting
Tarrival, a direct comparison of Trising and its derivative, BFI is also
possible for results obtained from independent experiments. MTT
is traditionally introduced in radiological imaging methods [3] andis currently used for analysis of ICG dynamics detected bymethods such as NIRS [27]. MTT is theoretically calculated fromthe first passage of the bolus [38] but can also be calculated
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Noninvasive Optical Imaging of Brain Perfusionarrays emitting light with a peak intensity at 760 nm that passed
through a 760612 nm band-pass filters and a CCD camera with a
25 cm TV lens coupled with an 830612 nm band-pass filter(Figure 1A). Twelve-bit grayscale images were captured every
580 ms for ,3 min, and the initial 40 frames were used togenerate blood flow maps.

the molecular dynamics of ICG has limitations in terms of
dimensionality. The acquired images were projected in two
dimensions, and this method cannot provide 3D blood flow
information, as provided by CT or MRI. According to the central
volume theorem, the regional CBF can be calculated by dividing
the fractional cerebral blood volume by the MTT [3]. However,
the reference arterial voxel required to adjust the MTT and to
calculate the fraction of blood volume cannot be obtained because
the arterial pixel was not identifiable due to poor resolution of thecurrent imaging setting, and the projected pixel contains
summated information from all of the vertically aligned nonarterial voxels. Therefore, several hemodynamic parameters,
which can be calculated in 3D imaging techniques, may not beapplied in this 2D optical imaging method.
Other limitations of the current technique involve quantitative
calculation of CBF. First, Tarrival, Tpeak, Tmax, Trising, and MTT
are not expressed in terms of volume per unit time per unit tissue
weight (e.g., mL/100 g/min), the standard unit of tissue blood
flow. These parameters provide a qualitative rather thanquantitative comparison of CBF status. Especially under ischemic
conditions, a bolus can arrive from more than two arteries withdifferent time kinetics. Thus, the shape and height of the peaks arealtered, making it difficult to estimate the status of CBF using
simple parameters. Therefore, more than one parameter should beconsidered for precise interpretation, and quantitative parametersthat do not depend on the shape of the peaks must eventually bedevised.

Analysis of Fluorescence Dynamics
Time-series fluorescence images were analyzed by custom-built
graphic-user-interface software using Delphi 2009 (EmbarcaderoTechnologies, San Francisco, CA). ICG dynamics in each pixel
were processed to compensate for noise and low temporal
resolution (Figure 1D). They were smoothed using the Savitzky–
Golay algorithm with third order polynomials and interpolated toa resolution of 11.6 ms per frame using the cubic spline method.
The half-intensity time (Thalf) was first identified to find Tarrival and
Tpeak. Thalf was defined as the first time at which the intensity was
equal or greater than the median intensity. Thalf was traced
backward along the downslope until the intensity did not decrease,and the destination time was designated Tarrival. In the samemanner, Thalf was traced forward along the upslope until theintensity did not increase, and the destination time was designatedTpeak. Trising was calculated as the difference between Tarrival and
Tpeak. MTT was calculated as the center of gravity of the dynamic
curve [3]. In this study, signals during the 20 s period after Tarrival
were used in the calculation. After adjusting the baseline intensity
to zero, the following equation was applied;P :t I(t)t
MTT~ PI(t)Materials and Methods
Animal Preparation
Eight-week-old male ICR and C57Bl/6 mice were obtained
from Samtako (Gyeonggi-do, Republic of Korea) and Koatech(Gyeonggi-do, Republic of Korea), respectively. ICR mice wereused for all experiments except imaging with intact scalps, which
was performed using C57Bl/6 mice. Animals were anesthetizedwith either 1.5% isoflurane in 70% N2O and 30% O2 with avaporizer or ketamine (0.1 mg/g wt) and xylazine (0.01 mg/g wt).
Isoflurane was used as a default anesthetic and the use of K-X wasindicated in the figure legends. All efforts were made to minimize
suffering. Body temperatures were monitored by rectal probe andmaintained at 3761uC using a temperature-controlled heatblanket. The scalp was excised for ordinary ICG imaging, andthe scalp hair was shaved when imaging the intact scalp. A 262mm craniotomy was made with a dental drill above the leftparieto-temporal cortex and the dura mater was removed to image
the cerebral cortex without any extracerebral tissue. The head was
fixed in a stereotaxic frame using two ear-bars and a jaw-holder.
All procedures were approved by the Institutional Animal Careand Use Committee of the Korea Advanced Institute of Scienceand Technology (Permit Number: KA2010-39) and performed inaccordance with the ARRIVE guidelines (http://www.nc3rs.org/
ARRIVE).

BFI was defined as the slope of the first peak [15]. The
definitions and calculations of the four parameters are summarizedin Table 1. Blood flow maps were generated by combining eachparameter in each pixel, and the range of the values matching thegrayscale intensity was adjusted and displayed under the map inarbitrary units for BFI or in seconds for other parameters. BFIvalues were normalized for display of each image, but not forcomparative analyses among hemispheres. Regions of 262 mm in
left hemispheres whose centers were positioned 2 mm posteriorand 2 mm lateral from the bregma were used to calculate mean
blood flow parameters. Symmetric regions in the right hemispheres were compared with the ischemic left hemispheres during
MCAO.

Selective Vascular Occlusion Surgery
MCAO surgery was performed to induce focal cerebral
ischemia using a protocol described previously, with somemodifications [43]. The neck was incised along the midline, andthe left common carotid artery (CCA), the external carotid artery(ECA), and the internal carotid artery (ICA) were exposed. The
CCA, ICA, and two positions on the ECA were ligated with 6-0
silk sutures. The ECA was cut partially between two ligatedpositions, and a 5-0 rounded tip monofilament nylon suture wasintroduced through the opening in the CCA. The ECA wascompletely cut, and the suture direction was reversed toward the
ICA. We proceeded with the nylon suture for 9 mm from theinsertion position. Time-series images were obtained before thesurgery and at 15 min after MCAO. The suture was removed at
45 min after its insertion, the ECA was permanently ligated, andthe ligations applied to the ICA and the CCA were released for

ICG Imaging
Tail veins were catheterized using a 30-gauge needle andpolyethylene (PE)-10 tubing. Cardiogreen (I2633; Sigma-Aldrich,
St. Louis, MO) was dissolved in phosphate-buffered saline to aconcentration of 0.5 g/L. A bolus of 1.5 mL/g wt cardiogreen
solution was injected manually through a tail vein catheter after
the start of the time-series imaging experiments. Time-series ICG
fluorescence signals were acquired using a custom-built device
manufactured by Vieworks Corp. (Gyeonggi-do, Republic ofKorea). The device was composed of two light-emitting diode
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ð1Þ

t

8

October 2012 | Volume 7 | Issue 10 | e48383

Noninvasive Optical Imaging of Brain Perfusiontransient MCAO. A series of ICG images was additionally taken at
5 min, and 2, 6, and 24 h after reperfusion. Two positions on the
ICA were ligated to selectively block the intracerebral blood
supply, and a nylon suture, identical to that used for the MCAO,
was inserted between the two positions and proceeded until
bifurcation of the MCA. The ECA was ligated to selectively block
the extracerebral blood supply.

region of an ischemic cortex. (A) Under anesthesia with K-X,
a fluorescence image was taken above an ischemic cortex of amouse that received left MCAO surgery during time-series
imaging. The images were obtained from a thinned skull windowusing intravital fluorescence microscopy after a bolus injection of
2 MDa fluorescein isothiocyanate (FITC)-dextran. The arterial
branching patterns and their direction indicate that the pialarteries shown are branched from the MCA (scale bar: 200 mm).
(B) Fluorescence dynamics of FITC in three arteries (a1, a2, anda3), three regions lacking large vessels (c1, c2, and c3) and threeveins (v1, v2, and v3) are indicated. The averaged fluorescence
dynamics of a region of interest (white circle) and of ICG in thesame region after a bolus injection of ICG were compared.
(TIF)

Statistical Analysis
Data are expressed as means 6 standard errors. Statisticalsignificance was assessed by two-tailed unpaired t-tests to compare
before and after MCAO surgery and between groups anesthetizedwith either isoflurane or K–X. The two-tailed paired t-test wasused to compare the two hemispheres in mice under MCAO.

Methods S1

Supporting Information(DOCX)

Figure S1 Reduction of extracerebral flow by occlusionof the ECA. (A) Under anesthesia with K-X, two round patches
that blocked the ICG fluorescence signal were inserted under thescalp through a midline incision, and the left ECA was ligated. (B)The ECA-ligated (a) and control (b) regions are indicated on the
BFI map. (C) Fluorescence dynamics of the ECA-ligated andcontrol regions. Note that the increase in fluorescence intensity
was attenuated in concordance with the time delay of the
maximum intensity in the ECA-ligated region.
(TIF)

Acknowledgments
We thank Jungsul Lee (Department of Bio and Brain Engineering, KAIST)
for valuable discussion and Min-su Kim and Prof. Yun Seon Song (Collegeof Pharmacy, Sookmyung Women’s University) for setting up the MCAO
surgery.

Author Contributions
Conceived and designed the experiments: TK CC. Performed theexperiments: TK CC. Analyzed the data: TK CC. Contributed
reagents/materials/analysis tools: TK CC. Wrote the paper: TK CC.

Figure S2 Dynamics of the averaged fluorescence
dominantly represents the capillary blood flow in a

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