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Low-Level Laser Therapy Ameliorates Disease Progression
in a Mouse Model of Alzheimer’sDisease
Dorit Farfara &Hana Tuby &Dorit Trudler &
Ella Doron-Mandel &Lidya Maltz &Robert J. Vassar &
Dan Frenkel &Uri Oron
Received: 16 April 2014 /Accepted: 11 June 2014 / Published online: 4 July 2014
#Springer Science+Business Media New York 2014
Abstract Low–level laser therapy (LLLT) has been used to
treat inflammation, tissue healing, and repair processes. We
recently reported that LLLT to the bone marrow (BM) led to
proliferation of mesenchymal stem cells (MSCs) and their
homing in the ischemic heart suggesting its role in regenera-
tive medicine. The aim of the present study was to investigate
the ability of LLLT to stimulate MSCs of autologous BM in
order to affect neurological behavior and β-amyloid burden in
progressive stages of Alzheimer’s disease (AD) mouse model.
MSCs from wild-type mice stimulated with LLLT showed to
increase their ability to maturate towards a monocyte lineage
and to increase phagocytosis activity towards soluble amyloid
beta (Aβ). Furthermore, weekly LLLT to BM of AD mice for
2 months, starting at 4 months of age (progressive stage of
AD), improved cognitive capacity and spatial learning, as
compared to sham-treated AD mice. Histology revealed a
significant reduction in Aβbrain burden. Our results suggest
the use of LLLT as a therapeutic application in progressive
stages of AD and imply its role in mediating MSC therapy in
brain amyloidogenic diseases.
Keywords Amyloid beta (Aβ)bonemarrow(BM) .
Mesenchymal stem cells (MSC) .Alzheimer’s Disease (AD) .
Low-level-laser therapy (LLLT)
Introduction
Alzheimer’s disease (AD) affects more than 18 million people
worldwide and is characterized by progressive memory defi-
cits, cognitive impairment, and personality changes. The main
cause of AD is generally attributed to the increased production
and accumulation of amyloid-beta (Aß), in association with
neurofibrillary tangle (NFT) formation that leads to neuronal
death, which affects the hippocampus which is critical for
learning and memory (Selkoe 2004). The presence of stem
cells in this structure has led to an increased interest in the
phenomenon of adult neurogenesis and its role in hippocam-
pal functioning (Morgan 2007). Many known factors
impacting neurogenesis in the hippocampus are implicated
in the pathogenesis of AD (Rodriguez and Verkhratsky
2011). Since neurogenesis is modifiable, stimulation of this
process, or the potential use of stem cells, recruited endoge-
nously or implanted by transplantation, has been speculated as
a possible treatment for neurodegenerative disorders such as
AD. It was previously demonstrated that the source of a new
brain cells might be either local from the subventricular zone
(SVZ) of the forebrain (Luskin 1993; Alvarez-Buylla et al.
1998) and the subgranular zone (SGZ) of the hippocampus
(Reznikov 1991), or peripheral from the bone marrow (BM)
(Munoz et al. 2005).
The bone marrow (BM) is a complex tissue, featuring
several different types of pluripotent cells: hematopoietic stem
cells, mesenchymal stem cells (MSCs), endothelial progenitor
cells, side population cells, and multipotent adult progenitor
cells. Like other stem cells, MSCs are capable of multilineage
differentiation from a single cell and in vivo functional
D. Farfar a :D. Trudler :E. Doron-Mandel :D. Frenkel
Department of Neurobiology, George S. Wise Faculty of Life
Sciences, Tel Aviv University, Tel Aviv, Israel
H. Tuby :L. Maltz :U. Oron (*)
Department of Zoology, George S. Wise Faculty of Life Sciences,
Tel Aviv University, Tel Aviv 69978, Israel
e-mail: oronu@post.tau.ac.il
D. Trudler :D. Frenkel
Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel
R. J. Vassar
Department of Cell and Molecular Biology, Northwestern University
Feinberg School of Medicine, Chicago, IL 60611, USA
J Mol Neurosci (2015) 55:430–436
DOI 10.1007/s12031-014-0354-z
reconstitution of injured tissues (Uccelli et al. 2008). One of
the properties of stem cells is their capacity to migrate to one
or more appropriate microenvironments (Devine et al. 2001).
Certain stem cells are able to exit their production site and
circulate in the blood before reseeding in their target tissues.
For MSCs, the nature of homing sites and circulation into
peripheral blood is still under debate. However, MSCs have
been found after infusion in multiple tissues, leading to the
hypothesis that they have the ability to home, and that they
adjust their differentiation pathways to diverse tissue micro-
environments (Liechty et al. 2000). It was recently shown that
intracerebral transplantation of bone-marrow-derived MSCs
into the brain of an induced AD model reduced the Aß protein
levels and accelerated the activation of microglia cells when
compared to sham-transplanted animals. Furthermore, it was
suggested that blood-derived microglia-like cells have the
ability to eliminate amyloid deposits by means of a cell-
specific phagocytic mechanism (Simard et al. 2006).
Low-level laser therapy (LLLT) has been found to
photostimulate and modulate various biological processes,
such as increasing mitochondrial respiration and ATP synthe-
sis (Bibikova and Oron 1993), facilitating wound healing, and
promoting the process of skeletal muscle regeneration and
angiogenesis (Bibikova and Oron 1993; Bibikova et al.
1994;Karu2007). It has been previously shown that laser
irradiation induces synthesis of cell-cycle regulatory proteins
in satellite cells from skeletal muscles due to the activation of
early cell-cycle regulatory genes (Shefer et al. 2001,2002). In
an experimental model of the infarcted heart in rats and dogs,
it was demonstrated that LLLT application at optimal power
parameters to the heart significantly reduces infarct size (scar
tissue formation) after myocardial infarction (Oron et al.
2001). The effect of LLLT on the brain has also been exten-
sively investigated. Transcranially applied LLLT has been
shown to have beneficial effects on AD mouse models and
rats and rabbits post stroke (Lapchak et al. 2004;Oronetal.
2006; De Taboada et al. 2011). Furthermore, LLLT was
transcranially applied in a double blinded clinical study in
patients after acute stroke (Lampl et al. 2007). The precise
mechanisms associated with the effect of LLLT on cells and
tissues are not yet clearly understood. There is evidence
suggesting that a primary mitochondrial chromophore for
photobiostimulation is cytochrome c oxidase. In addition to
leading to increased ATP formation, photobiostimulation may
also initiate secondary cell-signaling pathways (Karu 2007).
The effect of photobiostimulation on stem cells or progen-
itor cells has not been extensively studied. A remarkable
increase in stem cells counts was observed on the fourth day
of regeneration, when regenerating Dugesia tigrina (worms)
was stimulated by laser irradiation (de Souza et al. 2005).
Laser application to normal human neural progenitor cells
significantly increases ATP production in these cells (Oron
et al. 2007). LLLT was also found to significantly increase
survival and/or proliferation of MSCs post-implantation into
the ischemic heart, followed by a marked reduction of scarring
and enhanced angiogenesis (Tuby et al. 2009). Furthermore,
LLLT applied to autologous bone marrow (BM) in infarcted
heart of rats caused a 79 % reduction of the extent of scarring
in the heart developed after myocardial infarction and enhance
regeneration (Oron 2011;Tubyetal.2011,2013). This phe-
nomenon could partially be attributed to a higher extent laser
induced MSC from the BM that was mobilized via the circu-
lating blood to the infarcted area in the heart.
The effect of LLLT application to autologous BM in the
progression of AD in AD transgenic mouse model has not
been studied. In this current study, we aimed to determine
whether peripheral LLLT treatment to the BM can activate a
beneficial immune response in progressive disease stages of
AD mouse model.
Materials and Methods
Animals As an AD mouse model, we used 5XFAD transgenic
male mice (Tg6799) that co-overexpress familial AD (FAD)
mutant forms of human APP (the Swedish mutation, K670N/
M671L; the Florida mutation, I716V; and the London muta-
tion, V717I) and PS1 (M146L/L286V) trans-genes, under
transcriptional control of the neuron-specific mouse Thy-1
promoter, obtained from Robert Vassar (Northwestern
University, Chicago) (Oakley et al. 2006). Hemizygous trans-
genic mice were crossed with C57Bl/6 J breeders for at least
seven generations. Genotyping was performed by PCR anal-
ysis of tail DNA as described. The mice were housed in
individual cages in a temperature-controlled facility with a
12 h light/dark cycle. All animal care and experimental use
were in accordance with the Tel Aviv University guidelines
and approved by the University’sanimalcarecommittee.
Object Recognition Test (ORT) At the age of 6 months, male
mice were tested using ORT (Bevins and Besheer 2006).
Object recognition is distinguished by more time spent
interacting with the novel object. Memory was operationally
defined by the discrimination index for the novel object as the
proportion of time the mice spent investigating the novel
object and the familiar one.
Fear-Conditioning Test The contextual fear-conditioning test
(FCT) was conducted as described (Saura et al. 2005). Briefly,
male mice were subjected to an unconditioned electric stimu-
lus (US footshock; 1 mA/1 s) in a pre-session training.
Twenty-four hours later, FCT was measured by scoring freez-
ing behavior (the absence of all but respiratory movement) for
180 s using a FreezeFrame automated scoring system
(Coulbourn Instruments, USA).
J Mol Neurosci (2015) 55:430–436 431
Immunohistology Six-month-old male LLLT- and vehicle-
treated 5XFAD mice were sacrificed (transcardially punctured
and saline-perfused) and their brains rapidly excised and
frozen. The brains (left hemisphere) were cut in 14 μmsagittal
sections using a cryostat at −20 °C and used for histological
examination. The analysis was performed by bordering the
whole hippocampus, including the CA1, CA3, and dentate
gyrus areas. The slices were stained at Bregma −1.58 mm with
Congo-Red staining [Sigma-C6767] and Anti Aβ(6e10)
(SIG-39320500 R&D), and visualized by fluorescence mi-
croscopy for quantification of amyloid depositions.
Quantification of hippocampal Aβburden was performed
blind, using Imaging Research software from the NIH in an
unbiased stereological approach. The results are presented as
the percentage of insoluble total Aβand Congo-red positive
region from the entire hippocampus region.
LLLT Treatment A low-level laser, with a tunable power output
of maximum of 400 mW was used. LLLT to the BM of 5XFAD
and C57/B6 male mice was performed by placing the distal tip of
the fiber optic directly on the middle portion of the medial part of
the tibia after making a small incision in the skin. The beam
diameter of the laser was 0.3 cm on the BM in the tibia. The
power of irradiation of the BM was set to 1 J/cm
2
. Control mice
underwent the same procedure as the laser-irradiated group but
the laser was not turned on. Control- and laser-irradiated mice
were chosen randomly. Mice were treated with LLLT six times
(at 10-day intervals, for 2 months) starting at the age of 4 months
(at this time these mice already have a well-established AD
pathology). Mice were divided into three groups: Group I
(n=8), AD mice treated with LLLT every 10 days, commencing
at 4 month of age (+LLLT); Group II (n=7), sham-operated AD
mice that underwent the same procedure as the laser-irradiated
mice but without the laser irradiation (−LLLT). Control- and
laser-irradiated mice were chosen randomly; and Group III
(n=6), intact WT mice of the same strain as the AD mice.
MSCs Isolation Isolation of MSCs was performed essentially
as described previously (Tuby et al. 2009). In brief, femur and
tibia bones were excised from 10 C57/B6 male mice (8 weeks
old), and the bone marrow was collected using a stainless steel
rod pushed through the marrow cavity. Cells were then seeded
in 24-well culture plates at a concentration of 1.3× 10
6
cell/cm
2
for 1 week (medium was changed 48 h post seeding)
as described previously (Tuby et al. 2009). The Ga-Al-As
laser (Lasotronic Inc. Switzerland) was equipped with fiber
optic was used. The cultured MSCs were exposed to the laser
for 20 s to yield 1.0 J/cm
2
energy density. Another set of 6-
well plates containing MSCs were sham-exposed (control) to
the laser (cells treated as above, but the laser was not turned
on). The laser-treated and the control MSCs were left in the
incubator for 3 days post-laser treatment and then incubated
until 70 % confluence.
Analysis of Aβ(1–42) Phagocytosis MSCs cells were incubat-
ed with 12 μM HilyteFlour TM488-Aβ(1–42) (ANASPEC
#69479) for 2 h. MSCs were labeled with anti-CD11b antibody
(BD Pharmigen 557397) as described (Farfara et al. 2011), and
the percentage of Aβ(1–42) phagocytosis was analyzed by
fluorescence-activated cell sorting (FACS).
Statistical Analysis Data are presented as mean ±Standard
error of the mean (SEM). Student’sTtest was performed
when two groups were compared. One-way analysis of vari-
ance (ANOVA) was followed by Bonferroni’s multiple com-
parisons tests for three samples. Pvalue of <0.05 was consid-
ered significant. *p<0.05, **p<0.01, ***p<0.0001.
Results
Laser-Treated MSCs Increase Phagocytosis of AβProtein
with an Elevation in the Activation State.
We first aimed to evaluate the ability of laser-treated MSCs to
phagocytose Aβproteins. A significant (p=0.041) 35 % in-
crease in phagocytosis of Aß(1–42) was found in the cells that
were laser treated as compared to the non-laser treated
(−LLLT) cells (Fig. 1a). Furthermore, a significant
(p<0.0001) 10 % increase was detected for CD11b activation
marker of monocyte-derived cells (Fig. 1b).
LLLT-Treated Mice Showed an Increased Ability in Cognitive
Tes ts
For AD mouse model, we used the 5XFAD mice that
begin to develop amyloid plaque burdens at 2 months of
age (Oakley et al. 2006). By the age of 4 months, this
mouse model possesses high amyloid load commencing in
the cortex and expanding to the hippocampus. Four-
month-old 5XFAD mice at progressive stage of disease
were treated every 10 days for 2 months with LLLT
(totaling six treatments) to their BM. A non-laser-treated
group (−LLLT) and WT mice served as control. ORT was
performed in all mice at the age of 6 months. The time
spent around a new object is calculated as ratio between
the percentage of total time spent around the new object
and the old object. Application of LLLT to the BM of
5XFAD mice significantly elevated the percentage of time
spent near the new object almost to the level of the WT
mice (Fig. 2). WT 6-month-old mice demonstrated an
average of 73±4.11 % the time spent around a new
object. This value was significantly (p<0.01) reduced to
47.3±5.58 % in the group of the 6-month-old non-laser-
treated 5XFAD mice, (Fig. 2). However, the average
percentage of time spent near a new object in the laser-
432 J Mol Neurosci (2015) 55:430–436
treated mice was 68.7±3 %/s (Fig. 2). There was no
statistical difference in the time spent near a new object
between the WT mice and the 5XFAD mice treated by
LLLT.
At six months of age, mice were subjected to a contextual
fear-conditioning test, a behavioral test widely used to evalu-
ate associative learning and memory. In this test, an automatic
(laser-assisted) system analyzed the length of time that the
mice freeze following an electric shock stimulus (1 mA/s) in a
training pre-session, 1 day earlier. In the first day of training,
mice receive an electric foot stimulus after a 3 min exploration
time. The test is conducted 24 h after the unconditional stim-
ulus, in the same apparatus, in order to evaluate memory
ability of the mice to a prior contextual stimulus. Reduced
freezing indicates memory-loss and cognitive decline.
Figure 3represents the results of the fear test of
LLLT mice compared to non-laser-treated (−LLLT) as
compared to the freezing time of the WT mice.
Untreated 5XFAD mice showed a significant (p<0.001)
reduction in freezing time (11.6±4.6 s) as compared to
WT mice (71.1±4.6 s). Laser-treated 5XFAD mice
showed a significant (p<0.01) increased freezing time
of 40.4±5.28 s, compared to non-laser-treated mice.
These results indicate a significantly enhanced cognitive
ability and memory-gain in the laser-treated BM mice,
compared to non-laser-treated mice.
Reduction of β-Amyloid Burden in 5XFAD Mouse Model
Brain Following LLLT Treatment
5XFAD mice demonstrate amyloid burden starting from
2 months of age, commencing in the cortex. At 6 months of
Fig. 2 Object recognition test (ORT) of BM LLLT-treated 5XFAD mice.
Six-month-old 5XFAD mice were tested for cognition. All tested mice
(non-treated n= 7, treated n=8) were compared to WT mice, which had
shown a high percentage of time spent near the new object (n=6). Results
are expressed as mean± SEM. One-way ANOVA tests (Bonferroni) show
p<0.0011, (**p<0.01)
Fig. 1 Effect of LLLT on the
activation of BM-derived MSCs
to induce phagocytosis of
fluorescent Aß particles evaluated
by FACS. aLabeled Aβ(1–42)
was added for2 h to LLLT-treated
(+LLLT) MSC compared to MSC
from control mice (−LLLT). b
CD11b marker for activated cells
versus MSC control mice. Results
are presented as mean± SEM of
five mice in each group. Ttest
*p<0.05, ***p<0.0001
J Mol Neurosci (2015) 55:430–436 433
age, mice demonstrate a robust amyloid burden both in the
cortex and the hippocampus. To evaluate the amyloid burden
after 2 months treatment of LLLT (+LLLT), mice were
sacrificed at the age of 6 months and their brains were
snapped-frozen and immunostained with anti-Aβ(6E10)
(Fig. 4a). The percentage of Aβburden in the hippocampus
region of the non-laser-treated mice (−LLLT) was 180± 15
(Fig. 4b), while in the laser-treated mice there was a signifi-
cant (p<0.05) reduction of 68 % in Aβburden relative to the
control mice (Fig. 4b).
Discussion
The present study demonstrates that LLLT applied to autolo-
gous BM activate cells towards improving cognitive functions
in progressive stages of AD mouse model most probably by
increasing phagocytosis activity towards toxic Aβin the brain.
It has been suggested that LLLT may affect the immune
system (Novoselova et al. 2006; Assis et al. 2012).
Furthermore, it was shown that LLLT decrease inflammatory
cytokines while up-regulating nitric oxide in
lipopolysacharide (LPS)-treated macrophages (Gavish et al.
2008). Specific wavelengths of light trigger different inflam-
matory pathways of immune cells such as mast cells and
macrophage cells (Dube et al. 2003), which leads to increased
infiltration into the tissues. The ability of macrophages to act
as phagocytes is also modulated under the application of
LLLT (Dube et al. 2003). In addition, LLLT enhances the
proliferation, maturation, and motility of fibroblasts and in-
creases the production of basic fibroblast growth factor
(Hawkins and Abrahamse 2005). Here, we demonstrate a
significant elevation in the activation of immune cells, detect-
ed by CD11b in MSCs of the BM, following LLLT.
Furthermore, we show an increase in MSC reactivity to
phagocytose soluble neurotoxic Aβ, which tends to lead to
toxic oligomers in the brain. Indeed, it was previously sug-
gested that migration of peripherally-derived mononuclear
cells lead to clearance of amyloid load in AD mouse model
and improve cognition (Simard et al. 2006; Butovsky et al.
2007; Frenkel et al. 2008). We have recently shown that LLLT
Fig. 3 Effect of LLLT on 5XFAD mice in a FCT. Cognition in 6-month-
old mice treated with LLLT radiation (+LLLT, n=7) compared to non-
laser-treated (−LLLT, n=11) and WT mice from the same background
(n=9). The results are presented as mean±SEM. One-way ANOVA tests
(Bonferroni) show p<0.0001, (**p<0.01, ***p<0.001)
Fig. 4 Effect of LLLT to BM on
amyloid burden in 5XFAD mice.
aRepresentative staining for total
insoluble Aβwith anti-Aβ
antibody (6E10) in 14 μm sagittal
hippocampal (H) sections,
focusing on the dentate gyrus
(DG) area. Bar= 200 μm. b
Quantitative analysis of Aβ
burden in the sagittal sections of
treated mice (+LLLT) compared
to non-treated mice (−LLLT),
using histomorphological
software (n=6). Results are
expressed as mean± SEM. Ttest
*p<0.05
434 J Mol Neurosci (2015) 55:430–436
applied to autologous BM in infarct heart of rats after MI
caused a 79 % reduction of the extent of scarring in the heart
developed after myocardial infarction (Oron 2011;Tubyetal.
2011). This phenomenon could partially be attributed to laser-
induced MSCs from the BM that migrate to the circulating
blood and home in the infarct area in the heart. In an AD mice
model, it was shown that activation of peripheral monocyte-
derived macrophages can play a role in clearance of brain
amyloids (Miyazawa et al. 1989; Simard and Rivest 2006;
Mildner et al. 2007; Frenkel et al. 2008). Here, we show that
the laser-induced CD11b positive phagocytotic monocyte
cells, which can migrate from the BM to the circulating blood
and finally home to the brain of the 5XFAD mice, leading to a
reduction of brain amyloid load.
It was previously demonstrated that the effect of different
chemical therapeutic compounds reduce amyloid load and
improve cognition in a four months treatment time in the early
stages of the disease in 5XFAD mice (Frydman-Marom et al.
2011; Scherzer-Attali et al. 2012; Avrahami et al. 2013). In
this current study, we found that LLLT treatment leads to
significant reduction in brain amyloid load following short
period of treatment, starting at a late progressive disease stage.
Furthermore, we found that LLLT treatment improves cogni-
tive behavior in the laser-treated 5XFAD mice as compared to
non-treated mice. Our study clearly demonstrates efficacy in
significantly progressive diseased mouse model and in ad-
vanced stage of disease (4 months). These results also corre-
late with the general beneficial effect of applying LLLT
transcranially to AD mice (De Taboada et al. 2011).
However, in this current study, LLLTwas applied for a shorter
period of time and a less frequent application to autologous
BM cells as a target organ. Moreover, in the current study,
LLLT had been applied already in a progressive stage of the
disease (4 month) and not at a significant earlier stage. In
humans, this early stage is not detectable.
The results regarding the enhanced capacity of the laser-
induced CD11b positive cells in the BM to phagocytose
neurotoxic soluble Aβin vitro corroborate the in vivo findings
in this study. The amyloid burden (at 6 months) in the 5XFAD
mouse model was found to be significantly reduced in the
LLLT-treated mice as compared to control mice. The results of
the behavioral tests in this study are in concert with a reduction
of amyloid burden in the brain. They indicate a significantly
improved cognitive ability and memory in the laser-treated
mice over the non-laser-treated ones. It should be noted that
regarding the ORT, the mice that received multiple applica-
tions of LLLT to the BM between 4 and 6 months of age
demonstrated a significant improvement that reached the level
of the cognitive ability of the WT mice.
The present study also has clinical relevance. The safety of
LLLT application (at a similar power density as in the current
study) in experimental animals and in stroked human double-
blind studies has been reported (Ilic et al. 2006; Lampl et al.
2007). Moreover, we recently demonstrated that LLLT appli-
cation even at higher power densities to the BM of mice did
not cause any histological changes in various organs over a
period of almost their entire life-span (Tuby et al. 2013). Thus,
it may be assumed that LLLT to the BM in humans will be
safe. Our ability to show that LLLT application to the BM
improves cognitive brain function and reduces plaque con-
centration in the brain of 5XFAD mice, even when treatment
is commenced at a progressive stage, is of significance. It
suggests that LLLT could be applied to humans with AD,
which is usually diagnosed already in a progressive stage.
In conclusion, our results indicate a novel approach of
applying LLLT to autologous BM of AD mice, which induce
stem cells and immune cells which most probably migrate to
the brain, preventing the progression of the disease in AD
mice. LLLT thus offers a potential therapeutic strategy in
treating symptoms of Alzheimer’s disease and maybe other
neurodegenerative diseases.
These results suggest that laser application to monocytes or
other cell types, among the MSC population in the BM,
demonstrating phagocytotic activity, can cause significant
activation of these cells and, hence, enhance their capacity to
uptake specifically accumulated Aß proteins in the brains of
AD mice.
Acknowledgments This work is supported by grants from the
Alzheimer’s Association NIRG-11-205535 and ISF (to D.F.). There are
no conflicts of interest.
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