brain research ] (]]]]) ]]]–]]]

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Research Report

Therapeutic effects of repetitive transcranial

magnetic stimulation in an animal model of
Parkinson's disease

Ji Yong Leea,b,c,1, Sung Hoon Kimd,1, Ah-Ra Koe, Jin Suk Leeb, Ji Hea Yua,f,
Jung Hwa Seoa,g, Byung Pil Chob,h,n, Sung-Rae Choa,f,g,i,j,nn
a
Department and Research Institute of Rehabilitation Medicine, Yonsei University College of Medicine, Seoul, South Korea
b
Department of Anatomy, Yonsei University Wonju College of Medicine, Wonju, South Korea
c
Graduate School of Medicine, Yonsei University, Seoul, South Korea
d
Department of Rehabilitation Medicine, Yonsei University Wonju College of Medicine, Wonju, South Korea
e
Life and Health Science, UL, South Korea
f
Brain Korea 21 plus Project for Medical Science, Yonsei University, Seoul, South Korea
g
Graduate Program of Nano Science and Technology, Yonsei University, Seoul, South Korea
h
Institute of Lifestyle Medicine, Yonsei University Wonju College of Medicine, Wonju South Korea
i
Yonsei Stem Cell Research Center, Avison Biomedical Research Center, Seoul, South Korea
j
Rehabilitation Institute of Neuromuscular Disease, Yonsei University College of Medicine, Seoul, South Korea

art i cle i nfo ab st rac t

Article history: Repetitive transcranial magnetic stimulation (rTMS) is used to treat neurological diseases
Accepted 26 August 2013 such as stroke and Parkinson's disease (PD). Although rTMS has been used clinically, its
underlying therapeutic mechanism remains unclear. The objective of the present study was

Keywords: to clarify the neuroprotective effect and therapeutic mechanism of rTMS in an animal model

Repetitive transcranial magnetic of PD. Adult Sprague-Dawley rats were unilaterally injected with 6-hydroxydopamine

stimulation (6-OHDA) into the right striatum. Rats with PD were then treated with rTMS (circular coil,

Parkinson's disease 10 Hz, 20 min/day) daily for 4 weeks. Behavioral assessments such as amphetamine-induced

Dopaminergic neuron rotational test and treadmill locomotion test were performed, and the dopaminergic (DA)

Neurotrophic/growth factor
neurons of substantia nigra pas compacta (SNc) and striatum were histologically examined.
Expression of neurotrophic/growth factors was also investigated by multiplex ELISA, western
blotting analysis and immunohistochemistry 4 weeks after rTMS application. Among the
results, the number of amphetamine-induced rotations was significantly lower in the rTMS
group than in the control group at 4 weeks post-treatment. Treadmill locomotion was also

n
Corresponding author at: Institute of Lifestyle Medicine, Department of Anatomy, Yonsei University Wonju College of Medicine, 172,
Ilsan-dong, Wonju 220-702, South Korea.
nn
Corresponding author at: Department and Research Institute of Rehabilitation Medicine, Yonsei University College of Medicine, 134
Shinchon-dong, Seodaemun-gu, Seoul 120-752, South Korea. Fax: þ82 2 363 2795.
E-mail addresses: bpcho@yonsei.ac.kr (B.P. Cho), srcho918@yuhs.ac (S.-R. Cho).
1
Both authors contributed equally to this work.

0006-8993/$ - see front matter & 2013 Published by Elsevier B.V.

http://dx.doi.org/10.1016/j.brainres.2013.08.051

Please cite this article as: Lee, J.Y., et al., Therapeutic effects of repetitive transcranial magnetic stimulation in an animal
model of Parkinson's disease. Brain Research (2013), http://dx.doi.org/10.1016/j.brainres.2013.08.051
2 brain research ] (]]]]) ]]]–]]]

significantly improved in the rTMS-treated rats. Tyrosine hydroxylase-positive DA neurons

and DA fibers in rTMS group rats were greater than those in untreated group in both
ipsilateral SNc and striatum, respectively. The expression levels of brain-derived neuro-
trophic factor, glial cell line-derived neurotrophic factor, platelet-derived growth factor, and
vascular endothelial growth factor were elevated in both the 6-OHDA-injected hemisphere
and the SNc of the rTMS-treated rats. In conclusion, rTMS treatment improved motor
functions and survival of DA neurons, suggesting that the neuroprotective effect of rTMS
treatment might be induced by upregulation of neurotrophic/growth factors in the PD
animal model.
& 2013 Published by Elsevier B.V.

1. Introduction rTMS treatment, the present study investigated motor func-

tions and tyrosine hydroxylase (TH)-immunoreactive DA
Parkinson's disease (PD) occurs as a result of degenerating neurons and DA nerve fibers in the SNc and striatum. In
cell death of dopaminergic (DA) neurons in the substantia addition, the expression of neurotrophic/growth factors was
nigra pars compacta (SNc). PD is characterized by certain examined after rTMS application in an animal model of PD.
motor disturbances such as resting tremor, bradykinesia, and
gait disturbance. Dopamine replacement therapy is an effec-
tive medical treatment for the symptomatic improvement of
PD. In spite of such effects, fluctuations in abnormal invo-
2. Results
luntary movement eventually occur in most patients after
long-term dopaminergic treatment. Consequently, several
2.1. Effects of rTMS on amphetamine-induced rotation test
studies have investigated various methods other than dopa-
minergic drugs for PD treatment (Wu et al., 2008).
Adult Sprague-Dawley rats were unilaterally injected with 6-
Repetitive transcranial magnetic stimulation (rTMS) is a
hydroxydopamine (6-OHDA) into the right striatum. Two
non-invasive therapeutic device that can alter the excitability
weeks after 6-OHDA injection, rats with PD were treated with
of the cerebral cortex or neural network. rTMS has been used
rTMS (circular coil, 10 Hz, 20 min/day) daily for 4 weeks
to treat neuropsychiatric diseases (Zwanzger et al., 2002;
(Fig. 1A–C). Functional benefit in rTMS-treated rats on
Bentwich et al., 2011; Fitzgerald and Daskalakis, 2011) and
amphetamine-induced rotation behavior was evaluated up
stroke (Theilig et al., 2011; Corti et al., 2012). Recently, several
to 4 weeks after treatment (n¼ 12 each). Whereas untreated
clinical trials have revealed that rTMS offers therapeutic
group showed continuous augmentation in rotational beha-
benefit for functional recovery in PD (Siebner et al., 1999;
vior, the increasing rate of amphetamine-induced rotations
Arias-Carrión, 2008; Wu et al., 2008; Yang et al., 2013). rTMS
was significantly lowered in rTMS-treated rats at 4 weeks
treatment has shown to improve motor function and cogni-
after treatment (Fig. 2A). The number of rotations was not
tion in patients with PD (Zamir et al., 2012). Additionally,
significantly different between the rTMS group (387.3743.4)
increased DA levels in serum and subcortical areas have been
and the untreated group (319.2741.3) at 2 weeks post-
observed following rTMS in both PD patients and experimen-
treatment. However, the number of rotations in the rTMS
tal animals (Strafella et al., 2001, 2003; Khedr et al., 2007;
group (386.9743.8) was significantly lower than that in the
Choand Strafella, 2009).
untreated group (549.9733.6) at 4 weeks post-treatment
On the other hand, the underlying mechanism of rTMS (F ¼5.52, p¼ 0.03).
still remains to be elucidated. Recently, several experiments
have shown that rTMS has the ability to mediate neuroplas-
ticity by enhancing the expressions of glutamate neurotrans-
mitters and brain-derived neurotrophic factor (BDNF) in rat 2.2. rTMS improved locomotor function in a PD animal
brains (Müller et al., 2000; Keck et al., 2000; Yue et al., 2009). model
rTMS did not only activate brain regions in terms of immedi-
ate early gene expression, but also increased the expression Treadmill locomotion test was assessed to determine the
of BDNF-TrkB signaling in rats and humans (Ji et al., 1998; effects of the DA lesion on motor functions (n¼ 12 each).
Hausmann et al., 2000; Doi et al., 2001; Wang et al., 2011). The rTMS-treated rats showed improvement in locomotor
Additionally, rTMS modulated neurotrophic factors such as function 2 weeks after treatment (280725.0 mm), compared
BDNF, cholecystokinin and neuropetide tyrosine in healthy with the untreated group (225.1734.1 mm) (Fig. 2B). The
humans and patients with depression and amyotrophic running distance of rTMS-treated rats continuously increased
lateral sclerosis (Angelucci et al., 2004; Yukimasa et al., to a final score of 320.2723.1 mm throughout the treatment
2006; Gedge et al., 2012). However, there are few rTMS studies period, while the distance of untreated groups maintained to
on the effects of various neurotrophic/growth factors in PD. the score of 230735.5 mm at 4 weeks post-treatment (F¼ 3.56,
To clarify the neuroprotective effect of rTMS on SNc DA p ¼0.04), implying that rTMS induced motor improvements in
neurons and the therapeutic mechanism in overall brain after the PD rats.

Please cite this article as: Lee, J.Y., et al., Therapeutic effects of repetitive transcranial magnetic stimulation in an animal
model of Parkinson's disease. Brain Research (2013), http://dx.doi.org/10.1016/j.brainres.2013.08.051
 brain research ] (]]]]) ]]]–]]] 3

6-OHDA
8 weeks 14 weeks
old rTMS old

sacrifice

SNc
ST
MFB
10 days 14 days 2 weeks 4 weeks
Behavioral Behavioral
test test
Amphetamine
6-OHDA -induced
injection rotation test

rTMS treatment

SNc
ST
MFB

Fig. 1 – Experimental design. (A) Schematic diagram of the experimental schedule. PD in rats was confirmed by amphetamine-
induced rotation test 10 days after 6-OHDA injection. In rats with PD, rTMS was treated 2 weeks after 6-OHDA injection, and
behavioral tests were then performed at 2 week intervals. (B) Schematic figure illustrating injection of 6-OHDA into the striatum
using a stereotaxic device. (C) Schematic figure of rTMS treatment on the rat brain. rTMS—repetitive transcranial magnetic
stimulation, 6-OHDA—6-hydroxydopamine, ST—striatum, MFB—medial forebrain bundle and SNc—substantia nigra pars compacta.

Amphetamine-induced Treadmill locomotion

rotation test test
800 400 *
Number of rotations (turns)

Untreated Untreated
Nose tip position (mm)

rTMS * rTMS
600 300

400 200

200 100

0 0
Pretreatment 2 weeks 4 weeks Pretreatment 2 weeks 4 weeks
Post-treatment Post-treatment

Fig. 2 – Behavioral assessment after rTMS treatment in PD rats. (A) Amphetamine-induced rotation test. Rotational behavior in
response to amphetamine (5 mg/kg) was tested before stimulation (pretreatment, 10 days after 6-OHDA injection), 2 weeks
and 4 weeks after rTMS stimulation (n ¼12 each). The increasing rate of amphetamine-induced rotations was significantly
lowered in rTMS-treated rats compared to that in the untreated rats 4 weeks after treatment (*po0.05 compared with
untreated group). (B) Treadmill locomotion test. Data show the distance between the position of rat's nose tip and the rear wall
of treadmill chamber during 20-s treadmill walking periods. The average distance markedly increased after rTMS stimulation,
indicating increased locomotion ability (*po0.05 compared with untreated group). Untreated—PD animals without rTMS
treatments, rTMS—PD animals treated with rTMS.

2.3. Neuroprotective effect of rTMS on DA neurons in the unilateral 6-OHDA injection, the number of THþ neurons
SNc decreased compared to the corresponding contralateral
(non-lesioned) SNc (Table 1). Four weeks after treatment of
The therapeutic effect of rTMS on DA neurons in SNc was rTMS, the number of THþ neurons (210710.0) in the ipsilat-
assessed by immunohistochemistry (n¼ 5 each). Following eral (lesioned) SNc was significantly higher than the number

Please cite this article as: Lee, J.Y., et al., Therapeutic effects of repetitive transcranial magnetic stimulation in an animal
model of Parkinson's disease. Brain Research (2013), http://dx.doi.org/10.1016/j.brainres.2013.08.051
4 brain research ] (]]]]) ]]]–]]]

in untreated group (14477.5) (t¼ 5.34, p ¼0.01) (Fig. 3A, B),
whereas the number of THþ neurons in the contralateral SNc
was not different between the untreated group and rTMS-
treated group (Table 1). In addition, western blotting analysis
demonstrated that the expression of TH increased in the
ipsilateral hemisphere 4 weeks after rTMS treatment (Fig. 3C).
The results suggest that rTMS treatment might preserve or
slow DA neuronal degeneration from 6-OHDA administration.
2.4. Neuroprotective effect of rTMS on DA efferents in the
striatum
The therapeutic effect of rTMS on the striatal DA efferent fibers
was evaluated by striatal TH immunohistochemistry. Repre-
sentative findings of striatal TH staining in the ipsilateral
hemispheres were observed compared with those in the con-
tralateral hemispheres (Fig. 3D). Expressing volume values as

Table 1 – THþ dopaminergic neurons in the SNc at 4 weeks after rTMS treatment.

Contralateral side Ipsilateral side Survival rate (%)

Untreated (n ¼ 5) 1163.4743.5 144.077.5 12.3870.4

rTMS (n ¼ 5) 1205.6741.6 210.879.9* 17.4471.4*

Note: Values are mean7SEM. Survival rate (%) is the percentage of THþ neurons in the ipsilateral SNc relative to those in the contralateral SNc.
TH ¼tyrosine hydroxylase; SNc ¼substantia nigra pars compacta; Untreated ¼ PD animals without rTMS treatments; rTMS ¼ PD animals treated
with rTMS.
n
po0.05 compared with untreated group.

Contralateral Ipsilateral Ipsilateral SNc

250
Untreated rTMS

TH + neurons (number)
200
*
150
Rostral

100

50

0
Untreated rTMS
Caudal

Normal PD
SNc Untreated rTMS
VTA
TH (60 kD)

GAPDH (37 kD)

Contralateral Ipsilateral
Untreated

Ipsilateral Striatum
ST 30
**
TH+ fibers (%)

20

10
rTMS

ST
0
Untreated rTMS

Fig. 3 – THþ neurons after rTMS treatment in PD rats. (A) Representative findings of TH staining in the coronal sections passing
through the rostral and caudal parts of the SNc 4 weeks after rTMS treatment. (B) The number of THþ neurons in the ipsilateral
SNc (n ¼5 each). The total number of THþ neurons of the ipsilateral SNc was significantly greater in the rTMS treatment group
(npo0.05). (C) TH expressions of a normal control, untreated group, and rTMS group in the ipsilateral hemisphere by western
blot analysis. (D) Representative findings of striatal TH staining in the ipsilateral striatum at 4 weeks after rTMS treatment.
(E) The volume of striatal THþ fibers (%) relative to contralateral striatum. Dopaminergic THþ fibers were significantly higher in
the rTMS treatment group than in the untreated group (nnpo0.01). Untreated—PD animals without rTMS treatments, rTMS—
PD animals treated with rTMS, SNc—substantia nigra pars compacta, VTA—ventral tegmental area, TH—tyrosine
hydroxylase, Normal—naïve animal without rTMS treatment, ST—striatum.

Please cite this article as: Lee, J.Y., et al., Therapeutic effects of repetitive transcranial magnetic stimulation in an animal
model of Parkinson's disease. Brain Research (2013), http://dx.doi.org/10.1016/j.brainres.2013.08.051
 brain research ] (]]]]) ]]]–]]] 5

the percentage of THþ fibers in the ipsilateral striatum relative
to those in the contralateral striatum, the volume of dopami-
nergic THþ fibers was significantly higher in the rTMS group
(2277.3%) than in the untreated group (2.070.5%) at 4 weeks
post-treatment (t¼4.73, p¼ 0.003) (Fig. 3E). The results suggest
that more striatal THþ efferent fibers in concurrence with more
THþ neurons in the SNc, relative to the untreated group, could
improve motor performance following rTMS treatment.
2.5. Effects of rTMS on the expression of neurotrophic/
growth factors
neurotrophic factor (GDNF), NGF, PDGF, and VEGF (n¼5 each)
were investigated in both hemispheres by western blotting
analysis. Expressions of neurotrophic/growth factors BDNF
(t¼ 3.14, p¼0.013), GDNF (t¼ 2.28, p¼0.02), NGF (t¼ 3.79,
p¼0.04), PDGF (t¼ 6.15, p¼0.008) and VEGF (t¼3.02, p¼ 0.02)
significantly increased in the rTMS-treated rats compared with
those in untreated group (Fig. 5A–E). The expressions of
neurotrophic/growth factors exhibited a similar pattern to
the multiplex ELISA results in the ipsilateral hemispheres.
On the other hand, the expressions of neurotrophic/growth
factors in the contralateral hemispheres were not significantly
increased at 4 weeks after treatment (Fig. 5A–E).

To identify the growth factors associated with the repair

process of functional recovery and the neuroprotective and/
or neurorestorative effects induced by rTMS, the expressions
of various neurotrophic/growth factors including nerve
growth factor (NGF), ciliary neurotrophic factor (CNTF),
platelet-derived growth factor AA (PDGF-AA), and vascular
endothelial growth factor (VEGF) were investigated in ipsilat-
eral hemispheres by multiplex ELISA assay (n ¼3 each).
Among the factors, NGF (t ¼3.79, p ¼0.03), PDGF (t¼3.12,
p¼ 0.009) and VEGF (t¼ 2.59, p¼ 0.02) levels were significantly
elevated in the 6-OHDA-injected hemispheres of rTMS group
compared with those of the untreated groups at 4 weeks after
treatment, although the change in CNTF expression was not
statistically significant (Fig. 4A–D).
To confirm the expressions of neurotrophic/growth factors,
brain derived neurotrophic factor (BDNF), glial cell line-derived
2.6. Effects of rTMS on the expression of neurotrophic/
growth factors in the SNc
To identify the effect of rTMS on the expression of neuro-
trophic/growth factors in the SNc, the expression levels of
BDNF, GDNF, NGF, PDGF and VEGF were performed using
immunohistochemistry in the ipsilateral SNc at 4 weeks after
treatment (n¼ 4 each). Representative findings of BDNF,
GDNF, PDGF, and VEGF staining in the ipsilateral SNc after
rTMS treatment were shown in comparison with untreated
group (Fig. 6A). According to the density of neurotrophic/
growth factorsþ cells (%), calculated as the immunoreactive
cell area (/mm2) divided by the SNc area (/mm2), the expres-
sions of BDNF (t¼ 4.54, p¼ 0.04), GDNF (t ¼3.03, p¼ 0.03), PDGF
(t¼2.81, p¼ 0.02), and VEGF (t¼2.34, p¼ 0.042, n ¼5) were

80
NGF PDGF
400
* **
PDGF-AA (ρg/ml)
β-NGF (ρg/ml)

60 300

40 200

20 100

0 0
Untreated rTMS Untreated rTMS

VEGF CNTF
4 400

*
VEGF (ρg/ml)

CNTF (ρg/ml)

3 300

2 200

1 100

0 0
Untreated rTMS Untreated rTMS

Fig. 4 – Neurotrophic/growth factors by multiplex ELISA assay. (A–D) The expression levels of neurotrophic/growth factors
were increased in the rTMS treatment group compared with the untreated group (n ¼3 each). The levels of NGF (A), PDGF-AA
(B), and VEGF (C) were significantly higher in the rTMS group than in the untreated group at 4 weeks post-treatment. Change
in CNTF expression (D) was not statistically significant (npo0.05, nnpo0.01). Untreated—PD animals without rTMS treatments,
rTMS—PD animals treated with rTMS, NGF—nerve growth factor, PDGF—platelet-derived growth factor, VEGF—vascular
endothelial growth factor, CNTF—ciliary neurotrophic factor.

Please cite this article as: Lee, J.Y., et al., Therapeutic effects of repetitive transcranial magnetic stimulation in an animal
model of Parkinson's disease. Brain Research (2013), http://dx.doi.org/10.1016/j.brainres.2013.08.051
6 brain research ] (]]]]) ]]]–]]]

BDNF GDNF
3 2.5
Untreated Untreated
rTMS * 2.0 rTMS *

Relative Index
Relative Index
Contralateral Ipsilateral 2
1.5
Untreated rTMS Untreated rTMS
1.0
1
BDNF(28kD) 0.5

0 0.0
Contralateral Ipsilateral Contralateral Ipsilateral

GDNF(21kD)
NGF PDGF
2.0 5
Untreated Untreated **
rTMS rTMS
NGF(27kD) 4

Relative Index

Relative Index
1.5 *
3
1.0
2
PDGF(23kD) 0.5
1

0.0 0
Contralateral Ipsilateral Contralateral Ipsilateral
VEGF(43kD)

VEGF
4
Untreated
GAPDH(37kD) rTMS *
Relative Index

0
Contralateral Ipsilateral

Fig. 5 – Neurotrophic/growth factors by western blotting analysis. (A) Representative expressions of BDNF, GDNF, NGF, PDGF
and VEGF in both contralateral and ipsilateral hemispheres. (B–F) The relative amounts of neurotrophic/growth factors (n ¼5
each). The expression levels of BDNF (B), GDNF (C), NGF (D), PDGF (E) and VEGF (F) were significantly increased in the rTMS
group (npo0.05, nnpo0.01). Untreated—PD animals without rTMS treatments, rTMS—PD animals treated with rTMS, BDNF—
brain-derived neurotrophic factor, GDNF—glial cell-derived neurotrophic factor, NGF—nerve growth factor, PDGF—platelet-
derived growth factor, VEGF—vascular endothelial growth factor.

significantly increased in the rTMS group compared with the
untreated group (Fig. 6B). On the other hand, NGFþ cell
density was not increased after rTMS treatment (data not
shown). Taken together, these results suggest that the upre-
gulation of various neurotrophic/growth factors in rTMS-
treated hemispheres and SNc might preserve DA neuronal
damage, consequently inducing functional recovery in rTMS-
treated PD rats.
3. Discussion
rTMS has been shown to modulate cortical network and
corticomotor excitability, and to improve motor function in
patients with PD (Fisher et al., 2008; Yang et al., 2013).
However, this study focused on the alterations of neuro-
trophic/growth factors after rTMS in a PD animal model
rather than modulation of the cortical network. This study
showed the therapeutic potential of rTMS to improve motor
functions and to preserve DA neuronal survival by upregula-
tion of neurotrophic/growth factors in a 6-OHDA-lesioned PD
condition. This might be the first report to demonstrate
modulation of various trophic factors by rTMS treatment in
PD rats.
We demonstrated that rTMS elicited neuroprotective
effects on functional outcomes in the amphetamine rota-
tional test and treadmill locomotion test. Namely, rTMS
treatment alleviated unilateral 6-OHDA induced rotation 4
weeks after treatment. This result was consistent with a
previous study that demonstrated that the number of rota-
tions was reduced by low-frequency (0.5 Hz) rTMS treatment
(Yang et al., 2010). Treadmill locomotion test also revealed
that rTMS has the capability to improve reciprocal locomo-
tion in unilateral DA-lesioned rats (Pellis et al., 1987; Johnson
et al., 1999; Lee et al., 2012). Treadmill locomotion test
nonspecifically reflects cortical dysfunction as well as dys-
function of basal ganglia loops including striatal dopamine
depletion (Chang et al., 2003), and can immediately control
not only basal ganglia loops but also cortical networks (Arias-
Carrion et al., 2011). On the other hand, amphetamine-
induced rotation results from a persistent and consistent
one side preference, and is thought to be mediated by
lateralized functioning of the dopaminergic nigrostriatal
pathways in brain. Amphetamine rotation preferences of
unilateral PD rats have been shown to be related to not only

Please cite this article as: Lee, J.Y., et al., Therapeutic effects of repetitive transcranial magnetic stimulation in an animal
model of Parkinson's disease. Brain Research (2013), http://dx.doi.org/10.1016/j.brainres.2013.08.051
 brain research ] (]]]]) ]]]–]]] 7

BDNF GDNF PDGF VEGF

Untreated
rTMS

BDNF GDNF PDGF VEGF

40 10
30 80
* *
*

Density in SNc (%)

* 8
Density in SNc (%)

Density in SNc (%)

Density in SNc (%)

30 60
20 6
20 40
4
10
10 20 2

0 0 0 0
Untreated rTMS Untreated rTMS Untreated rTMS Untreated rTMS

Fig. 6 – Neurotrophic/growth factors in the ipsilateral SNc. (A) Representative findings of immunoreactivity with BDNF, GDNF,
PDGF, and VEGF in ipsilateral SNc at 4 weeks after rTMS treatment. (B) The density of neurotrophic/growth factors-
immunoreactive cells (%) (n ¼ 4 each). Expressions of BDNF, GDNF and PDGF were significantly increased in the rTMS
treatment group compared with the untreated group (npo0.05). Untreated—PD animals without rTMS treatments, rTMS—PD
animals treated with rTMS, BDNF—brain-derived neurotrophic factor, GDNF—glial cell-derived neurotrophic factor, NGF—
nerve growth factor, PDGF—platelet-derived growth factor, VEGF—vascular endothelial growth factor.

endogenous asymmetries in striatal dopamine content but also
survived DA neurons on SNc or DA nerve terminals in striatum
in a dopamine-dependent manner (Glick et al., 1986). The PD
animal model of this study, which was made by the 6-OHDA
injected into striatum, was characterized by slowly evolving
and progressive nigrostriatal degeneration until 8 weeks after 6-
OHDA administration and resembles the progressive nature of
the neurodegenerative process of human PD (Sauer and Oertel,
1994; Blandini et al., 2007). We observed that improvement in
treadmill locomotion was observed at 2 weeks after rTMS
treatment (4 weeks after 6-OHDA administration), but amphe-
tamine turning improvements was not shown at the time
point. A possible explanation for the difference between the
amphetamine-induced rotation and treadmill locomotion out-
comes is that the discrepancy might be related to different
mechanism of each test at the time when degeneration of DA
neurons was ongoing in the SNc.
The histological results of this study revealed that rTMS-
treated rats had more striatal THþ efferent fibers as well as
more THþ neurons in the SNc than those of the untreated
group, demonstrating that rTMS has a neuroprotective effects
on survival of DA neurons and DA nerve terminals in the
ipsilateral SNc and striatum of 6-OHDA-induced PD rats.
Our results showed that THþ fibers were primarily increased
in the ventromedial striatum rather than dorsolateral striatum
which plays a key role in motor function. However, previous
studies have shown that ventral striatum was also related with
motor functions such as amphetamine or apomophine rotation
and forepaw motor functions (Grealish et al., 2010; Heuer et al.,
2012; Nozaki et al., 2013). Taken together, our data suggested
that rTMS treatment might preserve or slow neuronal degen-
eration by 6-OHDA administration, and consequently improve
motor performances in an animal model of PD.
Finally, we evaluated endogenous neuroprotective and/or
neurorestorative proteins from the 6-OHDA-lesioned brains
after rTMS treatment. We demonstrated that rTMS promoted
increases in expression levels of neurotrophic/growth factors
such as BDNF, GDNF, NGF, PDGF, and VEGF in ipsilateral
hemispheres and SNc. The present study confirmed that
these factors increased in the rTMS group rather than in
the untreated PD group at 4 weeks after treatment. rTMS can
change signaling pathways and gene transcription, immedi-
ate early gene expression, and initiate the biosynthesis of
new molecules which persist in the tissue beyond the period
of stimulation, consequently increasing the synthesis of
growth factors (Arias-Carrion et al., 2011).

Please cite this article as: Lee, J.Y., et al., Therapeutic effects of repetitive transcranial magnetic stimulation in an animal
model of Parkinson's disease. Brain Research (2013), http://dx.doi.org/10.1016/j.brainres.2013.08.051
8 brain research ] (]]]]) ]]]–]]]
Among these factors, BDNF is expressed by DA neurons in
both the SN and the ventral tegmental area (Baquet et al.,
2005; Peterson and Nutt, 2008). In postmortem samples of PD
patients, BDNF as well as GDNF was lower in the SNc than in
controls (Mogi et al., 1999; Howells et al., 2000; Peterson and
Nutt, 2008). BDNF protected DA neurons in vitro from the
neurotoxic effects of 1-methyl-4-phenylpyridinium ion and
6-OHDA. Intrastriatal injection of BDNF in a rat model prior to
lesioning reduced destruction of DA neurons in the SNc and
decreased the apomorphine-induced rotation (Spina et al.,
1992; Shults et al., 1995; Mogi et al., 1999). rTMS is well known
for changing the expression of BDNF. Previous studies have
reported that high-frequency rTMS increased the level of
BDNF expression in vivo and in patients with neuropsychia-
tric disorders (Kole et al., 1999; Müller et al., 2000; Angelucci
et al., 2004; Yukimasa et al., 2006; Yue et al., 2009). However,
there are few reports that rTMS modulates other neuro-
trophic factors except BDNF.
GDNF has been shown to promote the survival of DA
neurons and has powerful protective effects for neurotoxin-
induced degenerated DA neurons (Lin et al., 1993; Hoffer et al.,
1994; Gill et al., 2003; Slevin et al., 2005; Lang et al., 2006). In the
DA-depleted rat model, GDNF protects 6-OHDA, 1-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine and medial forebrain bundle
axotomy-induced degenerated midbrain DA neurons (Kearns
and Gash, 1995; Tomac et al., 1995; Cohen et al., 2003; Bradley
et al., 2010). In addition, NGF supports sympathetic and
sensory neurons, and functions in the development and
maintenance of cholinergic neurons in the basal forebrain
(Sofroniew et al., 2001). NGF level decreased in experimental
models and patients with PD. DA neurons and NGF are
strongly believed to be functionally related. When NGF was
administrated in a 6-OHDA-lesioned rat model of PD, a
significant increase of THþ neurons was also observed in the
SN, showing potential neuroprotective effects of NGF on
nigrostriatal DA neurons (Lorigados Pedre et al., 2002;
Chaturvedi et al., 2006). PDGF is a major mitogen for fibro-
blasts, smooth muscle cells and other cells (Heldin and
Westermark, 1999). PDGF also has the neuroprotective and
neurotrophic functions such as neuronal survival and neurite
formation not only in cultures of rat DA neurons (Nikkhah
et al., 1993; Othberg et al., 1995; Pietz et al., 1996), but also in
DA neurons in the SN of 6-OHDA-lesioned rats (Funa et al.,
1996). It has been suggested that PDGF agonists may be
potential therapeutic agents for patients with PD (Othberg
et al., 1995). VEGF is an established angiogenic factor with
specificity for endothelial cells (Jin et al., 2000; Rosenstein and
Krum, 2004). Recently, VEGF has been reported to have
neuroprotective effects in cerebral ischemia and ischemic
peripheral neuropathy, and neurogenesis effects in neurologi-
cal disorders (Schratzberger et al., 2000; Yasuhara et al., 2004a).
The increased survival of the DA neurons by VEGF might be
mediated by neuroprotective mechanisms. Appropriate neo-
vascularization could improve microcirculation around DA
fibers in the striatum, and protect DA neurons (Jin et al.,
2000; Yasuhara et al., 2004a, 2004b, 2005). Taken together,
enhanced various neurotrophic/growth factors might protect
neuronal populations against excitotoxic, oxidative, and meta-
bolic insults as described in the previous reports (Mattson
et al., 2002; Mattson and Lindvall, 1997).
Please cite this article as: Lee, J.Y., et al., Therapeutic effects of repetitive transcranial magnetic stimulation in an animal
model of Parkinson's disease. Brain Research (2013), http://dx.doi.org/10.1016/j.brainres.2013.08.051
In addition, there are evidences that a variety of neuro-
chemical factors induced by physical exercise lead to motor
functional improvement as well as neuroprotection in animal
models of PD. (Tuon et al., 2012; Lau et al., 2011). Previous
reports also showed that neurotrophic factors such as BDNF
and NT-3 augment locomotor activity and rotational beha-
vior, and striatal DA turnover, indicating that BDNF enhances
the nigrostriatal DA system function (Shen et al., 1994;
Martin-Iverson et al., 1994; Altar et al., 1994). Therefore, we
considered that neurotrophic factors induced by rTMS might
modulate nigrostriatal DA system, leading to functional
recovery.
As a limitation of this study, we did not elucidate mechan-
isms of rTMS that involve modulation of the cortical network.
Instead of functional alterations of the corticospinal pathway
involving short interval intracortical inhibition and paired-
association stimulation effects (Cantello et al., 2002), this
study focused on the alterations of neurotrophic/growth
factors after rTMS in a PD animal model. Because this study
did not evaluate neurophysiologic function, such as motor
evoked potentials, we could not interpret behavioral out-
comes with respect to modulation of the cortical network or
activation of the nigrostriatal dopaminergic pathway.
The other limitation of this study is that repeated amphe-
tamine administration has been reported to induce sensitiza-
tion and priming effects in hemiparkinson rats (Erhardt et al.,
2004). Therefore, it is possible that rTMS can affect the
priming process instead of behavioral improvement from
preserved DA neuronal damage in the SNc and upregulation
of neurotrophic/growth factors. However, amphetamine-
induced rotation test is a method that has been widely used
to evaluate motor function related to endogenous DA depletion
in hemiparkinson rat models. In this study, amphetamine-
induced rotational test revealed that both groups continuously
increased in the number of rotations until 6 weeks after the 6-
OHDA injection. A possible explanation for the increased
rotations is that this experimental model was characterized
by evolving progressive nigrostriatal degeneration, resembling
the progressive nature of the neurodegenerative process of
human PD (Sauer and Oertel, 1994; Blandini et al., 2007).
Another limitation of this study is that we did not provide
evidence for neurorestorative effects, including the repopula-
tion of neurons, increased endogenous neural stem/progenitor
cells, synaptogenesis and angiogenesis. On the other hand, this
study discerned the upregulation of neurotrophic/growth fac-
tors in rTMS-treated hemispheres and SNc. Recent literature
demonstrated that GDNF involved neurorestorative processes
(Airavaara et al., 2012). VEGF and PDGF were also shown to be
related with neurorestoration by angiogenesis (Zhang and
Chopp, 2009). Therefore, we need to validate the neurorestora-
tive processes described above in future studies.
In conclusion, rTMS treatment improved motor functions
and preserved DA neuronal damage by 6-OHDA administra-
tion in a rat model of PD. Additionally, rTMS promoted
expression of various growth factors such as BDNF, GDNF,
NGF, PDGF-AA, and VEGF. The neuroprotective effects might
be induced by upregulation of neurotrophic/growth factors.
Collectively, our results could support a therapeutic potential
role of rTMS in the treatment of PD, extending its application
to other neurological diseases.
 brain research ] (]]]]) ]]]–]]]
4. Experimental procedure
4.1. Animals
Animal use and care protocols were approved by the Institu-
tional Review Board, and the Institutional Animal Care and
Use Committee. Thirty adult male Sprague-Dawley rats (body
weight, 250–300 g; Charles River Lab., Wilmington, MA, USA)
were used. All efforts were made to minimize the number of
animals required and to ensure minimal suffering. Rats were
housed with free access to food and water in a room
maintained at constant room temperature (20–22 1C) with
alternate 12-h light/dark cycles according to animal protec-
tion regulations.
4.2. 6-hydroxydopamine (6-OHDA) lesion
Animals were pretreated with norepinephrine transporter
blocker desipramine hydrochloride (12.5 mg/kg; Sigma-
Aldrich, St. Louis, MO, USA) 30 min before surgery. For the
surgery, rats were deeply anesthetized with an intraperito-
neal injection of ketamine (70 mg/kg body weight) and xyla-
zine (8 mg/kg body weight). Each animal was positioned
within a stereotaxic apparatus (Stoelting Co., USA). The
20 μg/4 μl/site of 6-OHDA (Sigma-Aldrich, St. Louis, MO, USA)
was injected into two sites (total of 40 μg) in the right striatum
according to the brain atlas (Paxinos and Watson, 2009). The
6-OHDA was injected at the following coordinates relative to
bregma and dura: anterior–posterior (AP) þ0.5 mm, medial–
lateral (ML) 2.5 mm, dorsal–ventral (DV) 5.0 mm and AP
0.5 mm, ML 4.2 mm, and DV 5.0 mm at a rate of 1 μl/min
using a 26 G Hamilton syringe (Fig. 1B). The inserted needle
was withdrawn from each location after 5 min, and the skin
was sutured. Afterward, animals were kept on a heating pad
at 37 1C until they completely recovered. Ten days after the
injections, animals showing more than 100 rotations per
50 min were considered to be successfully altered and were
used for further experiments (n ¼24). Animals were sacrificed
at 6-week time intervals post-lesion. The contralateral side of
the brain was used as the internal control.
4.3. Repetitive transcranial magnetic stimulation
The rats with PD were randomly allocated into the following
two groups: rTMS group and untreated controls (n¼ 12 each).
The rats were immobilized with an acrylic holder, and the
distance between the rat's head and the coil was maintained
at 1 cm. The coil was placed parallel to the skull of the rat.
The rTMS was administrated to awaken animals by using a
round coil (5-cm diameter) and the Bicon-100 (M-cube Tech,
Korea) at a rate of 10 Hz for 1 s with 100% power that
generates a field of approximately 1 T and the duration of
stimulation was 20 min per day. The stimulation intensity
was set to 100% machine output. The treatments were
performed daily for 4 weeks (Fig. 1A).
Please cite this article as: Lee, J.Y., et al., Therapeutic effects of repetitive transcranial magnetic stimulation in an animal
model of Parkinson's disease. Brain Research (2013), http://dx.doi.org/10.1016/j.brainres.2013.08.051
9
4.4. Amphetamine-induced rotation test
Rotational behaviors were evaluated in response to intraper-
itoneal injection of D-amphetamine (5 mg/kg; Sigma-Aldrich,
St. Louis, MO, USA) on day 10 after the 6-OHDA injection.
Animals were injected with the amphetamine and placed into
an automated multichannel rotometer system (ROTORAT,
MED Associates, Inc., St Albans, USA). Each animal was placed
into a cylindrical test chamber for 50 min. Animals with more
than 100 asymmetric turns per 50 min were used for experi-
ments. Amphetamine-induced rotation test was performed at
2 and 4 weeks after treatment.
4.5. Treadmill locomotion test
The rats were placed on a treadmill which was covered with a
see-through box with lines drawn from 0 to 40 cm. The
treadmill belt moved at 72 rpm during the on cycles. After
the rats adapted to running for 1 min, the lengths from the
beginning of the box to the end of the rats' noses were
measured. One cycle was defined as 20 s of running and 20 s
of rest. The treadmill locomotion test measurement was
repeated five times, and the average result was used.
4.6. Immunohistochemistry
Six rats of each group were deeply anesthetized as described
above, and the thorax was opened. The rats were perfused
transcardially with 100 ml of 0.9% sodium chloride, then with
200 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB,
pH 7.4). The brain was carefully removed from the skull, fixed
for 12 h in the same fixative, and infiltrated with 30% sucrose
solution at 4 1C until they sank. The brains were then placed
on a brain blocker (David Kopf Instruments, St. Tujunga, USA)
and sliced into a block containing the striatum or SNc, based
on the atlas of Paxinos and Watson (2009). The block was
then rapidly frozen in 2-methylbutane, chilled on dry ice, and
mounted in Tissue-Tek OCT compound (Sakura Finetechnical
Co., Tokyo, Japan). Serial coronal sections 40 μm in thickness
were obtained using a cryostat microtome (Leica Microsys-
tems Inc., Wetzlar, Germany) and were distributed sequen-
tially into a set of 12 tissue wells containing 0.1 M PB. After a
brief wash with 0.1 M PB, the brain sections were transferred
to another set of 12 tissue wells containing cryoprotection
solution and then stored in a freezer at a temperature below
20 1C until use. The sections were washed three times with
0.1 M PBS and treated with 3% hydrogen peroxide for 15 min
to suppress endogenous peroxidase activity. After washing
with 0.1 M PBS, the sections were treated for 1 h with 5%
normal serum obtained from the same host species to reduce
non-specific binding and were then incubated with rabbit
anti-TH monoclonal antibody (1:1000; Chemicon, Temecula,
CA, USA), rabbit anti-BDNF (1:200; Abcam, Cambridge, UK),
rabbit anti-GDNF (1:200; Abcam, Cambridge, UK), rabbit anti-
NGF (1:200; Abcam, Cambridge, UK), rabbit anti-PDGF (1:200;
Abcam, Cambridge, UK) and rabbit anti-VEGF(1:200; Abcam,
Cambridge, UK) diluted in 0.1 M PBST for 12 h. Sections were
washed in PBS and incubated for 1 h with biotinylated goat
anti-rabbit IgG (1:200; Vector, Burlingame, CA, USA) for TH
and goat-anti rabbit Alexa-488 for TH and growth factors.
10 brain research ] (]]]]) ]]]–]]]
After rinsing, sections were exposed to avidin–biotin perox-
idase complex (Vector) for 1 h prior to incubation. For perox-
idase detection, chromogens including 3,3′-diaminobenzidine
tetrahydrochloride (DAB; Sigma-Aldrich, St. Louis, MO, USA)
and DAB-nickel (Vector) chromogen were used for 3–5 min.
4.7. Image analysis
TH positive neurons on both sides of the SNc were counted
with the analySIS image analyzer system (Olympus Soft
Imaging System, Münster, Germany), and the percentage of
THþ neurons was calculated by dividing the average number
of THþ neurons in the ipsilateral SNc by the number of THþ
neurons in the contralateral SNc. Light photomicrographic
images were acquired on a Nikon Optiphot microscope
(Nikon Inc., Tokyo, Japan) fitted with a Nikon digital camera
(DXM1200), using Nikon ACT-1 image capture software
(ver. 2.2). TH positive neurons on both sides of the striatum
were measured using the MetaMorph Imaging System (Mole-
cular Device, Sunnyvale, CA), and the percentage of THþ fibers
was calculated by dividing the average volume of THþ fibers in
the ipsilateral striatum by the volume of fibers in the contral-
ateral striatum. The area of the SNc was obtained using the
MetaMorph Imaging System (Molecular Device, Sunnyvale, CA)
and converted to volume by multiplying the area by the section
thickness (40 μm). BDNFþ, GDNFþ, NGFþ, PDGFþ and VEGFþ
cells (%) in the SNc (/mm2) were quantified using the Meta-
Morph Imaging System (Molecular Device, Sunnyvale, CA). The
images were imported into Adobe Photoshop (ver. 7.0, Adobe
Systems Inc., San Jose, CA, USA) and were adjusted for bright-
ness and contrast to optimize photographic representation of
the images obtained by the microscope.
4.8. Western blotting
To compare the expression levels of neurotrophic factors,
ipsilateral hemispheres (n¼ 5 per group) were lysed in 500 μl
of cold RIPA buffer (50 mM Tris–HCl, pH 7.5, 1% Triton X-100,
150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), and 1%
sodium deoxycholate) with a protease inhibitor cocktail
(Sigma-Aldrich, St. Louis, MO, USA). Tissue lysate was centri-
fuged at 13,000  g for 15 min at 4 1C. The supernatant was
harvested, and protein concentration was analyzed using a
Qunt-iT protein assay kit (Molecular Probes, Eugene, Oregon,
USA). For electrophoresis, 50 μg of protein was dissolved in
sample buffer (60 mM Tris–HCl, pH 6.8, 14.4 mM β-mercap-
toethanol, 25% glycerol, 2% SDS, and 0.1% bromophenol blue),
boiled for 10 min and separated on a 10% SDS reducing gel.
Separated proteins were transferred onto polyvinylidene
difluoride (PVDF) membranes (Invitrogen, Carlsbad, CA) using
a trans-blot system. Blots were blocked for 1 h in Tris-buffered
saline (TBS) (10 mM Tris–HCl, pH 7.5, 150 mM NaCl) containing
5% nonfat dry milk at room temperature, washed three times
with TBS and incubated at 4 1C overnight with an anti-rabbit
polyclonal BDNF (1:1000, Abcam, Cambridge, MA, USA), anti-
rabbit glial cell-derived neurotrophic factor (GDNF, 1:1000,
Abcam), anti-rabbit NGF (1:1000; Abcam, Cambridge, UK), anti-
rabbit PDGF (1:1000; Abcam, Cambridge, UK), anti-rabbit VEGF
(1:1000; Abcam, Cambridge, UK) and anti-rabbit TH (1:1000;
Abcam, Cambridge, UK) and anti-GAPDH (1:3000, Cell signaling,
Please cite this article as: Lee, J.Y., et al., Therapeutic effects of repetitive transcranial magnetic stimulation in an animal
model of Parkinson's disease. Brain Research (2013), http://dx.doi.org/10.1016/j.brainres.2013.08.051
Boston, MA, USA) antibody in TBST (10 mM Tris, pH 7.5, 150 mM
NaCl, and 0.02% Tween 20) containing 5% nonfat dry milk. On
the next day, blots were washed three times with TBST and
incubated for 1 h with horseradish peroxidase-conjugated sec-
ondary antibodies (1:3000, Santa Cruz Biotech, Santa Cruz, CA,
USA) in TBST containing 3% nonfat dry milk at room tempera-
ture. After washing three times with TBST, proteins were
visualized with an ECL detection system (Amersham Pharmacia
Biotech, Piscataway, NJ, USA).
4.9. Multiplex ELISA
To identify growth factors regulated by rTMS, a multiplex
ELISA assay (Quantibodys array, RayBio-tech, Norcross, GA)
was used to determine which of the following growth factors
were detectable in the ipsilateral hemispheres (n¼ 3 per
group): NGF, CNTF, PDGF-AA and VEGF. Expression of growth
factors was detected using an array scanner (Gene PIX™
4000B, Axon instruments, USA).
4.10. Data analysis
Statistical analysis was performed using the t-test (Prism
Graph Pad Software, San Diego, CA, USA) and two-way
repeated ANOVA, followed by a Bonferroni post-hoc compar-
ison (SAS version 9.2, SAS Institute Inc., Cary, NC, USA). The
data were expressed as a mean7standard error of the mean
(SEM). The significance level was assumed at po0.05, unless
otherwise indicated.
Author contributions
Lee JY: Conception and design, collection and/or assembly of
data, manuscript writing; Kim SH: Conception and design,
data analysis and interpretation, manuscript writing; Ko AR,
Lee JS,Yu JH, Seo JH: Collection and/or assembly of data; Cho
BP: Administrative support, conception and design, data
analysis and interpretation; Cho SR: Data analysis and inter-
pretation, manuscript writing, final approval of manuscript.
Acknowledgments
This study was supported by grants from the National
Research Foundation Research 2009-0077194; 2010-0020408;
2010-0024334) funded by the Ministry of Education, Science
and Technology, Republic of Korea, and the Korea Health
Technology R&D project, Ministry of Health & Welfare
(A100054).
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Please cite this article as: Lee, J.Y., et al., Therapeutic effects of repetitive transcranial magnetic stimulation in an animal
model of Parkinson's disease. Brain Research (2013), http://dx.doi.org/10.1016/j.brainres.2013.08.051