Lasers in Surgery and Medicine 42:566–576 (2010)

Targeted Increase in Cerebral Blood Flow by Transcranial
Near-Infrared Laser Irradiation
Yoichi Uozumi, MD,1 Hiroshi Nawashiro, MD, DMSc,1* Shunichi Sato, PhD,2 Satoko Kawauchi, PhD,3
Katsuji Shima, MD, DMSc,1 and Makoto Kikuchi, PhD3
1
Department of Neurosurgery, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan
2
Division of Biomedical Information Sciences, National Defense Medical College Research Institute, 3-2 Namiki,
Tokorozawa, Saitama 359-8513, Japan3
Department of Medical Engineering, National Defense Medical College, 3-2 Namiki, Tokorozawa,
Saitama 359-8513, Japan

Background and Objective: Brain function is highly
dependent on cerebral blood flow (CBF). The precise
mechanisms by which blood flow is controlled by NIR laser
irradiation on the central nervous system (CNS) have notbeen elucidated. In this study, we examined the effect of
808 nm laser diode irradiation on CBF in mice.
Study Design/Materials and Methods: We examinedthe effect of NIR irradiation on CBF at three different
power densities (0.8, 1.6 and 3.2 W/cm2) and directly
measured nitric oxide (NO) in brain tissue during NIR
laser irradiation using an amperometric NO-selective
electrode. We also examined the contribution of NO and aneurotransmitter, glutamate, to the regulation of CBF by
using a nitric oxide synthase (NOS) inhibitor, Ng-nitro-Larginine methyl ester hydrochloride (L-NAME), and an
N-methyl-D-aspartate (NMDA) receptor blocker, MK-801,
respectively. We examined the change in brain tissue
temperature during NIR laser irradiation. We also investigated the protection effect of NIR laser irradiation ontransient cerebral ischemia using transient bilateralcommon carotid artery occlusion (BCCAO) in mice.
Results: We showed that NIR laser irradiation (1.6 W/cm2
for 15–45 minutes) increased local CBF by 30% comparedto that in control mice. NIR laser irradiation also induced asignificant increase in cerebral NO concentration. In micethat received L-NAME, NIR laser irradiation did not induce
any increase in CBF. Mice administered MK-801 showed
an immediate increase but did not show a delayed additional increase in local CBF. The increase in brain tissue
temperature induced by laser irradiation was estimated to
be as low as 0.88C at 1.6 W/cm2, indicating that the heating
effect is not a main mechanism of the CBF increase inthis condition. Pretreatment with NIR laser irradiation
improved residual CBF and reduced the numbers of
apoptotic cells in the hippocampus.
Conclusion: Our data suggest that NIR laser irradiation isa promising experimental and therapeutic tool in the fieldof cerebral circulation research. Lasers Surg. Med. 42:566–
576, 2010. ß 2010 Wiley-Liss, Inc.
Key words: cerebral blood flow; near infrared laser;
MK-801; forebrain ischemia; nitric oxide
ß 2010 Wiley-Liss, Inc.

INTRODUCTION
Brain function is highly dependent on cerebral blood flow(CBF). Human brain weight is about 2% of the whole bodyweight. However, CBF reaches about 15% of cardiac
output. Improved outcome results when reduced CBF are
prevented or respond to treatment not only in brainischemia but also in traumatic brain injury, degenerative
disease such as Parkinson’s disease and Alzheimer’s
disease [1–6].
Photobiostimulation effects of near-infrared (NIR) laser
irradiation have been known for almost 40 years [7]. Many
studies have shown increased blood flow in various organs
during and after NIR laser irradiation [7–10]. Recently, it
has been reported that NIR laser irradiation is effective forcontrolling cerebral ischemia in vivo and clinically [11–14].
However, the precise mechanisms by which blood flow iscontrolled by NIR laser irradiation on the central nervous
system (CNS) have not been elucidated. The vasodilatory
action of NIR laser irradiation is most likely mediated bynitric oxide (NO). In this study, we examined the effect of
808 nm laser irradiation on CBF in mice and directly
measured NO in brain tissue during NIR laser irradiation
using an amperometric NO-selective electrode [15]. We also
examined the contribution of NO and a neurotransmitter,glutamate, to the regulation of CBF by using a nitricoxide synthase (NOS) inhibitor, Ng-nitro-L-arginine methyl
ester hydrochloride (L-NAME), respectively. It is currently
accepted that N-methyl-D-aspartate (NMDA) receptor
interacts with NOS in the neurotransmitter pathway
No work resembling the enclosed article has been published oris being submitted for publication elsewhere. We certify that wehave each made a substantial contribution so as to qualify for
authorship. We have disclosed all financial support for our workand other potential conflicts of interest.
All authors have seen and agree with the contents of themanuscript.
*Correspondence to: Hiroshi Nawashiro, MD, DMSc, Department of Neurosurgery, National Defense Medical College, 3-2
Namiki, Tokorozawa, Saitama 359-8513, Japan.
E-mail: nawa1957@ndmc.ac.jp
Accepted 26 April 2010
Published online 15 July 2010 in Wiley InterScience(www.interscience.wiley.com).
DOI 10.1002/lsm.20938

INCREASED CBF BY NEAR-INFRARED LASER IRRADIATION[16]. To gain an insight into potential mechanisms linking
neurotransmission with increased CBF, the effect ofan NMDA receptor blocker, MK-801, was investigated.
The thermal effect has been frequently discussed on themechanisms of the effect of NIR laser irradiation. Therefore, we examined the change in skull surface and brain
tissue temperature during NIR laser irradiation. We also
investigated the protective effect of NIR laser irradiation
on transient cerebral ischemia using transient bilateralcommon carotid artery occlusion (BCCAO) in mice [17,18].
We evaluated the CBF during BCCAO and apoptosis in the
cerebral cortex and dorsal hippocampus 96 hours after thereperfusion by using in situ terminal deoxynucleotidyltransferase (TdT)-mediated dUTP-biotin nick end-labeling(TUNEL) of fragmented DNA.
MATERIALS AND METHODS
Animals
The institutional animal care committees of National
Defense Medical College approved all experimental procedures. Extreme care was taken throughout the study
to minimize pain and discomfort. Male C57BL/6J mice
9–11 weeks in age and weighing 23–27 g (CLEA Japan,
Inc., Tokyo, Japan) were allowed free access to food andwater before experimental use.

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anesthesia was also used for surgery. Eight hundred eightnanometers CW diode laser (B&W Tek, Inc., Newark, DE)
was applied to the left hemisphere transcranially (2 mm
posterior to and 3 mm left of the bregma; Fig. 1a). The
exposure field was set to 3 mm in diameter. The durationof laser irradiation was 45 minutes. To determine theappropriate power density (PD) of NIR laser irradiation, we
irradiated the brain with the laser at three different PDs(0.8 W/cm2, n ¼ 6; 1.6 W/cm2, n ¼ 9; 3.2 W/cm2, n ¼ 6: NIR
laser irradiation group), and laser power was checked usinga photodiode-type laser power meter (PD300; Ophir
Optronics Ltd, Jerusalem, Israel) before and after every
irradiation. CBF in the cortex was measured semiquantitatively for both hemispheres with a non-invasive and noncontact laser Doppler blood perfusion imager (PeriScan
PIM II, PeriMed, Stockholm, Sweden). By scanning thetissue with a low-power laser beam, color images of blood
perfusion in the scanned area were created (Fig. 1b,c). CBF
values were recorded before and at 15, 30, and 45 minutes
after starting NIR laser irradiation. Indirect arterial
blood pressure was monitored by using a non-preheating
blood pressure monitor (MK-2000ST, Muromachi, Tokyo,
Japan). Respiratory rates were also recorded. CBF andphysiological parameters were also measured for shamoperated mice (n ¼ 9: sham group) in the same way as
described above without NIR laser irradiation.

NIR Laser Irradiation and Cerebral Blood
Flow Measurement

Measurement of Brain Tissue Temperature

Mice were administered 20 mg/kg sodium pentobarbital
intraperitoneally. Rectal temperature was maintained at
approximately 37.08C, until consciousness was regained,by using a heating pad (Digital Thermo Control Meter
DT-102; Inter Medical, Nagoya, Japan) connected to arectal thermistor. The head of each mouse was fixed with ahead clamping device (Stereotaxic instrument SR-5M;
Narishige, Tokyo, Japan). The skull was exposed after amidline scalp and periosteum incision with lidocaine local
anesthesia. A mild dose of sodium pentobarbital was used
to minimize its effects on CBF [19,20]. Therefore, local

Information on tissue temperature is essential forunderstanding the mechanisms underlying the effect oflaser irradiation on tissue. The temperature of tissue
subjected to laser irradiation is often measured with athermocouple directly inserted into the tissue exposedto laser light or into tissue in the vicinity of thelaser irradiation area. In these cases, however, scatteredlaser light can directly hit the thermocouple, possibly
causing an error in measurement. Thus, we estimatedthe temperature of brain tissue exposed to laser lightby inserting a thermocouple (diameter, 300 mm; type-T;

Fig. 1. Representative examples of the exposure field and perfusion images. a: The exposure
field was set to 3 mm in diameter (2 mm posterior to and 3 mm left of the bregma).
Scale bar is 5 mm in length. At the time of measurement, perfusion images were obtained(b: pre, c: 15 minutes after NIR laser irradiation). Targeted increase in cerebral blood flow of
the irradiated field was observed (c).

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HYP1-30-1/2-T-G-60-SMP-M Omega, USA) into the brain
tissue at the center of the laser irradiation area immediately (<2 seconds) after turning off the laser at a time ofinterest; the temperature is referred to as Tc. This method
enables accurate temperature estimation for the brain
exposed to laser light, since the response time of thethermocouple (<1 seconds) is much shorter than thethermal relaxation time for the tissue. A cranial window
with a diameter of 5 mm was made in the parietal bone of
the right hemisphere. The exposed cortex was irradiatedwith laser light at a PD of 1.1 or 2.2 W/cm2, corresponding
to 1.6 or 3.2 W/cm2 for transcranial irradiation, respectively, since transmittance of the skull was estimated to
be about 70%. In addition, we continuously monitored
the temperature of tissue in the vicinity of the laser
irradiation area with another thermocouple of the same
type; the temperature is referred to as Tv. As describedlater, we confirmed that Tv was almost the same as Tc under
the present experimental conditions, indicating thatthe effect of scattered laser light on Tv is negligibly
small. We also confirmed that the temperatures obtainedby open-skull measurements described above were almost
equal to those obtained by intact-skull measurements
under corresponding conditions.
Direct Measurement of Nitric Oxide (NO)We directly measured nitric oxide in the brain
tissue during NIR laser irradiation by an amperometricNO-selective electrode (IMN-111, Inter Medical) [15].
All mice were prepared in the same way as that describedabove. A microelectrode sensor of 0.5 mm in diameter
was inserted through the small burr hole of the skull
to the brain 1.5 mm in depth just under the exposure
field of NIR laser irradiation (PD, 1.6 W/cm2, n ¼ 4; PD,
0.8 W/cm2, n ¼ 4). Measurement was started before
irradiating NIR laser. In sham control mice (n ¼ 4), NO
was measured in the same way as that in the NIR laser
irradiation group without laser irradiation. NO was alsomeasured during NIR laser irradiation in Ng-nitro-Larginine methyl ester hydrochloride (L-NAME)-treatedmice (n ¼ 4).
Pharmacological Treatment
L-NAME was obtained from Sigma (St. Louis, MO), andMK-801 was obtained from Banyu (Tokyo, Japan). Micereceived L-NAME (500 mg/L) in their drinking water for
5 weeks (n ¼ 9: NAME group) as described by Deckel et al.
[21]. Next, we investigated the effect of MK-801 on CBF
increase by laser irradiation to examine the relationshipbetween CBF increase and neurotransmission by laser
irradiation. Three different doses of MK-801 (0.1, 1.0, and10 mg/kg; n ¼ 8, n ¼ 9, and n ¼ 9, respectively) were given
intraperitoneally at 1 hour prior to 45-minute NIR laser
irradiation (PD: 1.6 W/cm2). Since MK-801 is known to
induce hypothermia [22], rectal temperature was carefully
monitored before, during and after NIR laser irradiation.
CBF was measured before and at 15, 30, and 45 minutes
after starting NIR laser irradiation.

Surgical Procedures for Transient Bilateral
Common Carotid Artery Occlusion (BCCAO)
Animals were administered 20 mg/kg sodium pentobarbital intraperitoneally. Under local anesthesia, thebilateral common carotid arteries were exposed after aventromedial cervical skin incision. The bilateral common
carotid arteries were encircled loosely with a 4/0 silk thread
to enable later occlusion with a non-traumatic small
aneurysm clip. The distal portions of the arteries were
inspected to confirm the absence of blood flow after applying
the clips. CBF was measured sequentially just before,
during 30 seconds and 1, 2, 5, 10, and 15 minutes, and at
30 seconds and 5 minutes after BCCAO. Rectal temperature, indirect arterial pressure and respiratory rate werealso recorded as described above. To confirm the effectof pretreatment by NIR laser irradiation, we conducted
1.6 W/cm2 NIR laser irradiation to the left hemispheretranscranially for 30 minutes before BCCAO (n ¼ 13, NIR
laser irradiationþBCAAO group). The control mice (n ¼ 13,
BCCAO control group) were also subjected to 15-minute
BCCAO without pretreatment by NIR laser irradiation.
Anatomical Study of Cerebrovasculature
Variation in collateral circulation affects residual CBF
following BCCAO. In all 26 mice that were subjected to
BCCAO, the existence of the posterior communicatingartery (P-com A) which connects the posterior circulation of
the brain from the vertebral arteries with the anterior
circulation from the carotid arteries in the circle of Willis
was examined. The mice with P-com A have a bypass flowfrom posterior circulation through P-com A while conducting BCCAO. Therefore, these animals were excluded fromfurther analysis. Ninety six hours after the reperfusion, themice were given 100 mg/kg sodium pentobarbital used foroverdose, and were perfused through the heart with 30 mlof saline solution (0.9%), followed by 30 ml of paraformaldehyde (4%) in 0.1 mol/L phosphate buffer (pH 7.4), andthen injected with 1.5 ml colored silicon via the left cardiac
ventricle. The brains were removed carefully, and the circleof Willis was examined.
Histological Study
To examine histological change due to NIR
laser irradiation, randomly selected animals that were
irradiated with NIR laser (PD, 1.6 W/cm2, n ¼ 4) were
perfused transcardially in the same way as that describedabove. The brains were removed and embedded in paraffin
after fixation in the same fixative for 24 hours at 48C. Serial
coronal sections (5 mm in thickness) were stained withhematoxylin-eosin.
In Situ Labeling of DNA Fragmentation
To evaluate the effect of NIR laser irradiation against
transient BCCAO in mice, we carried out in situ terminal
deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin
nick end-labeling (TUNEL) of fragmented DNA by using
an apoptosis in situ detection kit (Wako Pure Chemical
Industries, Osaka, Japan). After the anatomical studyof cerebrovasculature, the brains without P-com were

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2

embedded in paraffin after fixation in the same fixative for24 hours at 48C. Serial coronal sections (5 mm in thickness)were made. The procedures were performed as recommended by the manufacturer (http://www.wako-chem.
co.jp/siyaku/info/gene/pdf/apotosisdkit.pdf).
The TUNEL-positive cells in the irradiated cerebral
cortex and CA1 subfield in the dorsal hippocampus werecounted in photomicrographs.

indicated that NIR laser irradiation at 1.6 W/cm could
increase CBF most efficiently. Therefore, we selected the
PD of 1.6 W/cm2 for irradiation in subsequent studies.
Arterial blood pressure and respiratory rates were similarand showed no significant differences among groups.
Histology of the mouse brain with 45-minute NIR laser
irradiation (n ¼ 4) revealed no detectable ablation, coagulation or bleeding change on and around the exposure field.

Statistical Analysis

Brain Tissue Temperature During
Laser Irradiation

All data are expressed as means  SD. All data wereanalyzed by statistical software (Prism4.0; GraphPad
Software, San Diego, CA). The Mann–Whitney U-test
was used to compare CBFs between two groups. Analysis ofvariance followed by Bonferroni correction was employed tocompare the value for physiological parameters. A P-valueof <0.05 was considered statistically significant.

A representative sequence of laser Doppler perfusion
imaging is shown in Figure 1c,d. Targeted increase in CBFof the irradiated field was invariably observed. The effects
of NIR laser irradiation at different PDs on CBF are shownin Figure 2. At PDs of 0.8, 1.6, and 3.2 W/cm2, CBF at
the irradiated hemisphere was increased by 9.1  5.7%,
30.4  3.1%, and 30.5  5.6% respectively. These results

Brain temperature Tv (continuously monitored tissue
temperature in the vicinity of the laser irradiation area)and brain temperature Tc (tissue temperature at the centerof the laser irradiation area immediately after turning off
the laser) were compared at PDs of 1.1 and 2.2 W/cm2,
corresponding to 1.6 and 3.2 W/cm2 for transcranial
irradiation, respectively. Tc and Tv were 33.1 and 33.28C
at 1.1 W/cm2, respectively, and they were 36.7 and 36.28C at
2.2 W/cm2 at the same measurement time point. At bothPDs, the difference between Tv and Tc was within 0.58C,
indicating that the effect of scattered laser light on Tv was
negligibly small and that tissue temperature of the brain
exposed to laser irradiation can be monitored by measuring
Tv. Figure 3 shows a typical time course of the brain
temperature Tv for irradiation at 1.1 W/cm2. Mean Tv forabout 10 minutes before laser irradiation was 32.98C. After
starting irradiation, Tv slightly increased and reached
an equilibrium in about 25 seconds; the rise of mean

Fig. 2. Effect of NIR laser irradiation at three different power
densities (PDs). At PDs of 0.8 (a), 1.6 (b), and 3.2 W/cm2 (c),there were increases in CBF of 9.1  5.7%, 30.4  3.1% and30.5  5.6%, respectively, in the irradiated hemisphere. Eachblack solid line indicates the change in CBF in the irradiatedhemisphere. Each dotted line indicates the change in CBF in
the non-irradiated hemisphere. Each gray solid line indicatesthe changes in CBF in the sham group. There were significant
differences between the BCF of each hemisphere and that of
the sham group (**P<0.01, ***P<0.001).

RESULTS
Effect of NIR Laser Irradiation on CBF,
Physiological Variables, and Histology

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Fig. 3. Change in the brain tissue temperature during NIR laser irradiation. This is atypical time course of the brain temperature Tv for irradiation at 1.1 W/cm2. Mean Tv for about
10 minutes before laser irradiation was 32.98C. After starting irradiation, Tv slightly increased
and reached an equilibrium in about 25 seconds; the rise of mean temperature Tv during laser
irradiation for about 30 minutes was as low as 0.88C.

temperature Tv during laser irradiation for about
30 minutes was as low as 0.88C. At 2.2 W/cm2, on the other
hand, the rise of mean temperature Tv during irradiationfor 45 minutes was 3.88C.

NO Concentration
Changes in NO concentration are shown in Figure 4. In
the sham control mice, NO concentration was unchanged(Fig. 4a). All mice showed an immediate increase in NO

Fig. 4. Representative examples of change in nitric oxide(NO) concentration during NIR laser irradiation. In the shamcontrol mice, NO concentration was unchanged (a). All mice
showed an immediate increase in NO concentration after thestart of NIR laser irradiation at PD of 1.6 W/cm2, and NO

returned to a resting level after the end of NIR laser irradiation(b). NO levels were significantly reduced by L-NAME treatment (d). Mice irradiated with NIR laser at PD of 0.8 W/cm2
showed a small increase in NO concentration, only 15% of thatin mice irradiated with NIR laser at PD of 1.6 W/cm2 (c).

INCREASED CBF BY NEAR-INFRARED LASER IRRADIATION

concentration after the start of NIR laser irradiation at a
PD of 1.6 W/cm2, and NO returned to a resting level after
the end of NIR laser irradiation (Fig. 4b). NO levels weresignificantly reduced by L-NAME treatment (Fig. 4c). Mice
irradiated with NIR laser at a PD of 0.8 W/cm2 showeda small increase in NO concentration, only 15% of thatin mice irradiated with NIR laser at a PD of 1.6 W/cm2(Fig. 4d).
Pharmacological Treatment
The effect of L-NAME on CBF during NIR irradiation. Almost no increase in CBF was observed in the
L-NAME group. The increase in CBF was only 5.0  2.1% at
45 minutes after starting NIR laser irradiation (Fig. 5a).
Deckel et al. [21] investigated NOS activity in mice orally
administered several doses (0, 5, 10, 50, 100, and 500 mg/L)
of L-NAME and they reported that NOS activity was
reduced dose-dependently as L-NAME concentration in
the drinking water was increased. Therefore, we used theconcentration of 500 mg/L in drinking water in this study.
The absence of stimulatory effects of light in L-NAME
treated mice indicates the involvement of NOS in theprocess.
The effect of MK-801 on CBF during NIR laser
irradiation. The CBF was increased by approximately
20% by NIR laser irradiation in the three different MK-801
dose groups dose-independently (Fig. 5b). There was noFig. 5. Effects of L-NAME and MK-801 on CBF during NIR
laser irradiation. Almost no increase in CBF was observed in
the L-NAME group (n ¼ 9). The increase in CBF was only
5.0  2.1% at 45 minutes after NIR laser irradiation (a). CBF
was increased by approximately 20% by NIR laser irradiationin all three MK-801 dose groups (b). Delayed additional
increases in CBF at 30 and 45 minutes after NIR laser571

significant difference among the three groups. Delayed
additional increases in CBF at 30 and 45 minutes after
starting NIR laser irradiation were not observed in thethree groups. There were significant differences in CBF
at 45 minutes after starting NIR laser irradiation in the
MK-801 groups (0.1 and 10 mg/kg) compared with thatin the NIR laser irradiation group. MK-801 is a noncompetitive NMDA receptor blocker, and it suppresses
glutamatergic synaptic transmission. The NMDA receptor
interacts with neuronal NOS in the neurotransmitter
pathway [16]. Suppression of glutamatergic neurotransmission by MK-801 did not influence the early increase but
blunted the delayed additional increase in CBF by NIR
laser irradiation. This result indicates that the delayed
additional increase in CBF by NIR laser irradiation might
be related to neuronal activation.
Arterial blood pressure, rectal temperature and respiratory rate were similar and showed no significant
differences among groups.
Effect of Pretreatment by NIR Laser Irradiationfor BCCAO in Mice
Pretreatment with NIR improves CBF in micesubjected to BCCAO. In the anatomical study of cerebrovasculature, P-com A was detected in 4 mice in the NIR
laser irradiation group and in 5 mice in the control group.
These 9 mice were excluded from the analysis. Mice inirradiation were not observed. CBFs at 45 minutes after
NIR laser irradiation in the MK-801 groups (0.1; n ¼ 8 and
10 mg/kg; n ¼ 9) were significantly different from CBF in the
NIR laser irradiation group (n ¼ 9). (*P<0.05; NIR laser
irradiation group vs. 10 mg/kg MK-801 group, **P<0.01; NIR
laser irradiation group vs. 0.1 mg/kg MK-801 group).

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which P-com A was observed showed high residual CBF
more than 40% of pre-occlusion values in the same
hemisphere. Residual CBF in mice without P-com was lessthan 30% of that in the control group during BCCAO.
Sequential CBF before, during and after BCCAO is shownin Figure 6. Pretreatment by NIR laser irradiation
significantly improved residual CBF during BCCAO in
mice.
Physiological parameters during BCCAO. Physiological parameters are presented in Figure 7. Body temperatures, respiratory rates and heart rates were similarand showed no significant differences among the groups(Fig. 7a–c). However, there was a significant difference inarterial blood pressure (BP) between the laser irradiationgroup and control group (Fig. 7d).
NIR laser irradiation protects against transient
brain ischemia and DNA fragmentation. Neuronaldamage 96 hours after the reperfusion in both groups wasevaluated using DNA fragmentation assay. However thefrequency of damaged cell labeled with TUNEL staining in
the laser irradiation group (n ¼ 9) was lower than that in
the control group (n ¼ 8) (Fig. 8). The percentage of the
TUNEL-positive cells to the total number of cells in thecortex was 9.1  8.3% in the NIR laser irradiation group,and 27.0  20.0% in the control group (P<0.05) (Fig. 8d).
The percentage of the TUNEL-positive cells in the CA1
subfield was 44.8  26.6% in the NIR laser irradiationgroup, and 84.0  19.0% in the control group (P<0.01)(Fig. 8e).
DISCUSSION
Many studies have shown increased blood flow in various
tissues during and after NIR laser irradiation [7]. However,to the best of our knowledge, there has been no study inwhich the relationship between NIR laser irradiation and
CBF was examined. The results of this study indicate that
NIR laser irradiation with the appropriate power density iseffective for increasing CBF and that NO is involved in
the mechanism of CBF increase in response to NIR laser
irradiation, and also suggest a protective effect of NIR laser
irradiation for transient ischemia.
The Effect of NIR on CBF
We examined the effect of NIR laser irradiation on CBF at
three different densities (0.8, 1.6, and 3.2 W/cm2). CBF at
the irradiated hemisphere was increased by 9.1  5.7%,
30.4  3.1%, and 30.5  5.6% respectively. These results
indicated that NIR laser irradiation at 1.6 W/cm2 could
increase CBF most efficiently. The results obtained at
different PDs indicate the possible existence of a thresholdfor a profound induction of NO by NIR irradiation.
Temperature of Irradiated Brain Tissues
The rise in temperature of the brain tissue exposed tolaser light was found to be as low as 18C under irradiation
conditions enabling the most efficient CBF increase(1.6 W/cm2, 30 minutes). Therefore, the CBF increase
would not be due to the heating effect. Many studies
have shown increased blood flow in various organs during
and after NIR laser irradiation [7–10]. The biologicaleffects of NIR laser irradiation are wavelength-specific andthey are not therefore attributable to thermal effects
in many cases. For neuronal tissue, photobiomodulation
effects have been reported under a wide variety oflight irradiation conditions, PD ranging from 7 mW/cm2 to5 W/cm2 and total energy density ranging from 0.9 J/cm2
to 4,300 J/cm2 [11–14,23–25]. For example, NIR laser
irradiation at 808 nm significantly reduced neurologicalFig. 6. Effect of NIR laser irradiation for 15-minute BCCAO. Pretreatment by NIR laser
irradiation increased residual CBF following bilateral carotid occlusion in mice. The reductionin CBF was significantly (*P<0.05, **P<0.01) suppressed compared with that in the control
group during BCCAO. Open triangles indicate the NIR irradiation group. Open squares
indicates the control group. a: Irradiated hemisphere, (b) non-irradiated hemisphere.

INCREASED CBF BY NEAR-INFRARED LASER IRRADIATION

573

Fig. 7. Changes in physiological parameters. Body temperatures (a), respiratory rates (b) and heart rates (c) were similarand showed no significant differences among the groups.
However, there was a significant difference in arterial bloodpressure (d) between the two groups (*P<0.05). In the control
group, the blood pressure increased just after BCCAO andreturned to the baseline value after the reperfusion. On theother hand, in the pretreatment by NIR laser irradiationgroup, the blood pressure increase was mild, and bloodpressure gradually returned to the baseline value before
removal of the clip. Open triangles indicate the NIR irradiationgroup. Open squares indicates the control group.

deficits in animal stroke models and human stroke patientsat 7–25 mW/cm2 and 0.9–15 J/cm2 [11–13,23]. For acuterat spinal cord injury (SCI), NIR laser irradiation at 810 nm
promoted axonal regeneration and functional recovery
at 530 mW/cm2 and 1,600 J/cm2 [24]. For the rat
normal brain, an increase in cerebral ATP content wasobserved with 830-nm laser irradiation at 4.8 W/cm2 and4,300 J/cm2 [25]. In the present study, we observed themost efficient increase in CBF at 1.6 W/cm2 (Fig. 2), at
which the rise in temperature of tissue was as low as 18C.
Thus, heating effect would not be a main mechanism forthe increase in CBF in this condition. At 3.2 W/cm2,
however, the temperature rise reached 48C and heating
effect cannot therefore be excluded in the mechanism of
CBF increase, although thermal damage was not observedin tissue by histological analysis. Further study is needed
to fully understand the mechanism by which CBF is
increased by NIR laser irradiation in the present animalmodels.

Effect of NIR on NO Concentration andPharmacologic Inhibitors
In tissues other than brain, some studies have shown arelationship between the effect of NIR laser irradiation on
blood flow increase and NO [7–10]. In this study, we
examined this relationship for the brain by administering
several inhibitors and directly monitoring NO concentration in the brain tissue.
NO is a free radical gas that is a powerful regulator of
circulation (as an endogenous vasodilator) and a neurotransmitter (helping in the processing of nerve signals as
they cross synapses). Due to its chemical properties, highdiffusibility and short half-life, NO is a unique molecule inthe brain [26]. NO is a gas and thus passes freely throughthe cell membrane. Therefore, neurons without synaptic
connection can be influenced by diffusing NO. However, thehalf-life of NO is very short, that is, a few seconds [27], andthe effect of NO is therefore localized.

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Fig. 8. The effect of NIR laser irradiation against transient ischemia on DNA fragmentation
TUNEL staining in the CA1 subfield of dorsal hippocampus in the control group (a), and in the
NIR irradiation group (b). TUNEL staining in the irradiated cerebral cortex in the NIR
irradiation group indicates no increase in DNA fragmentation (c). A significant reduction in
the percentage of the TUNEL positive cells in the cerebral cortex (d) and in the CA1 pyramidal
cell layers (e) was observed in the NIR irradiation group (n ¼ 9) compared with that in thecontrol group (n ¼ 8).

Mice irradiated with NIR laser at a PD of 1.6 W/cm2
showed a significant increase in CBF (by 30% of control
values). In the L-NAME-treated mice, on the other hand,
the increase in CBF was suppressed to baseline values.
Similarly, NO concentration was increased by NIR laser
irradiation at a PD of 1.6 W/cm2, and this response was also
suppressed by L-NAME. These results indicate that CBF
increase depends on NOS activity and NO concentration.
Photobiomodulation effects on neuronal tissue and cells
often show unique wavelength dependence, and photoactivation of cytochrome c oxidase in mitochondria istherefore considered as the possible mechanism [7,28]. It

is known that NO competes with O2 in the mitochondrial
respiratory chain [29,30], by which respiration is regulated.
Cytochrome c oxidase is a dominant photoreceptor in the
NIR spectral region [31], and NIR laser irradiation canrelease NO from cytochrome c oxidase [7,32,33], resulting
in increased blood flow. This is called the NO hypothesis[32,34] to explain the effect of increased blood flow induced
by NIR laser irradiation; the wavelength-dependent
effect can be interpreted by the absorption spectrum ofcytochrome c oxidase [7,8,28,31]. The present study clearly
showed that NIR laser irradiation was accompanied byincreased NO content in tissue, which supports the NO

INCREASED CBF BY NEAR-INFRARED LASER IRRADIATION

hypothesis for the effect of increasing CBF by NIR laser
irradiation.
There was no significant change of NOS immunoreactivity after NIR (data were not shown). NO may be producedby cells in the absence of NOS (NOS-independent pathway)
as described by Martin et al. [35].
Mice treated with MK-801 at three different doses
showed a significant suppression in delayed additional
increase of CBF compared with that in the control groupdose-independently. There was no significant difference
among these three groups. MK-801 is a non-competitive
NMDA receptor blocker, and it suppresses glutamatergic
synaptic transmission. As Andresen et al. [36] reported intheir article, the mechanism responsible for coupling bloodflow to neural activity is an intense area of investigation,and it is currently accepted that the NMDA receptor
interacts with neuronal NOS in the neurotransmitter
pathway [16]. NIR laser irradiation may influence neurons,
astrocytes, and endothelial cells. Different forms of NOS inthese cells may participate in NO production following
NIR laser irradiation. Suppression of glutamatergic
neurotransmission by MK-801 did not influence the early
increase but blunted the delayed additional increase in
CBF by NIR laser irradiation. This result indicates that thedelayed additional increase in CBF by NIR laser irradiation
might be related to neuronal activation. Our results
suggest that an appropriate duration of NIR laser irradiation activates the CNS.
NIR Pretreatment of BCCAO Treated MiceIn this study, pretreatment by NIR laser irradiation
significantly improved residual CBF during BCCAO andneuronal damage. The increase in CBF induced by NIR
irradiation may be useful for temporal clinical practice(e.g., enhancement of ischemic tolerance before conducting
temporal occlusion of the cerebral artery in microsurgery).
We therefore investigated the effect of pretreatment with
NIR irradiation for BCCAO in mice. For development of ahighly reproducible BCCAO model, it is crucial to confirm
the patency of P-com A, which connects posterior circulation of the brain from the vertebral arteries with anterior
circulation from the carotid arteries in the circle of Willis[17,18,37,38]. In previous studies, P-com A was detected in
20.0–38.9% of C57BL6 mice [17,18,38]. In this study,
we detected P-com A in 9/26 (34.6%) of the mice. Mice with
P-com A showed high residual CBF (more than 40% of
pre-occlusion values) in the same hemisphere, whereas
residual CBF in mice without P-com A was less than 30% in
the control group during BCCAO. These values are higher
than the values measured by a laser Doppler flow meter ina BCCAO model in mice [7,18,37,38]. The blood flow
measurement technique that we used is qualitative andis not good for distinguishing very low blood flows.
However, we confirmed patency of P-com A in all mice byan anatomical study. Therefore, these relative values are
considered acceptable. Moreover, this technique has theadvantage of enabling measurement in a wider area thanthat with a laser Doppler flow meter. This technique thus
enables simultaneous measurements in both hemispheres.

575

Pretreatment by NIR laser irradiation significantly
improved residual CBF during BCCAO in mice not onlyin the irradiated hemisphere but also in the non-irradiatedhemisphere. Thirty-min NIR laser irradiation at a PDof 1.6 W/cm2 increased CBF by 15% in the opposite hemisphere. Although scattered light may possibly influence
the opposite hemisphere, further study is needed to understand the mechanism by which CBF is increased in the
opposite hemisphere. Interestingly, the recovery of CBF
after cessation of BCCAO in the NIR laser-irradiated mice
was more gradual than that in the BCCAO control group.
This result can be partially explained by the fact that NO
surge after the reperfusion was mitigated by pretreatment
with NIR laser irradiation.
Changes in BP were significantly different among thegroups. In the BCCAO control group, BP increased justafter BCCAO and returned to the baseline value after thereperfusion. In the NIR laser irradiationþBCCAO group,on the other hand, the BP increase was mild, and BP
gradually returned to the baseline value before removal of
the clip. The increased residual CBF following NIR laser
irradiation would result in the elimination of systemic
reactive hypertension after BCCAO.
Neuronal damage 96 hours after the reperfusion in bothgroups was evaluated using DNA fragmentation assay.
Excessive NO has negative effects on neurons. However,the number of the TUNEL positive cells in CA1 pyramidal
cell layers and in the irradiated cerebral cortex was
significantly reduced compared with that in the control
group. These results suggest a protective effect of NIR laser
irradiation for transient ischemia. NIR laser irradiation
may have a protective role against ischemic insult.
CONCLUSION
The results of our study suggest that targeted increase of
cerebral blood flow is achieved by NIR laser irradiation andthat it is concerned with NOS activity and NO concentration. NIR laser irradiation under appropriate conditions
would enable activation of the central nervous system. Thiscan hopefully be applied to protect the brain from transientischemia.
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