翻页电子书制作,电子书制作,电子杂志制作

欢迎来到云展网,国内唯一的3D翻页电子书免费发布、阅读平台。上传PDF即可转换为翻页电子书!

15. Brain lipid changes after repetitive transcranial magnetic stimulation potential links to therapeutic effects—翻页版预览

阅读,搜索本杂志文字内容 点击阅读
阅读云展网其他3D杂志 点击阅读
8787 上传于 2018-09-07 12:43:24

15. Brain lipid changes after repetitive transcranial magnetic stimulation potential links to therapeutic effects

Metabolomics (2012) 8:19–33
DOI 10.1007/s11306-011-0285-4

ORIGINAL ARTICLE

Brain lipid changes after repetitive transcranial magnetic
stimulation: potential links to therapeutic effects?

Lynette Hui-Wen Lee • Chay-Hoon Tan •
Yew-Long Lo • Akhlaq A. Farooqui • Guanghou Shui •
Markus R. Wenk • Wei-Yi Ong

Received: 29 October 2010 / Accepted: 28 January 2011 / Published online: 12 February 2011
Ó Springer Science+Business Media, LLC 2011

Abstract Repetitive transcranial magnetic stimulation were harvested 1 week after rTMS and lipid profiles ana-
(rTMS) is increasingly used in the management of neuro- lyzed by tandem mass spectrometry. rTMS resulted in
logic disorders such as depression and chronic pain, but little changes mainly in the prefrontal cortex. There were signif-
is known about how it could affect brain lipids, which play icant alterations in plasmalogen phosphatidylethanolamines,
important roles in membrane structure and cellular func- phosphatidylcholines, and increases in sulfated galactosyl-
tions. The present study was carried out to examine the ceramides or sulfatides. Plasmalogen species with long chain
effects of rTMS on brain lipids at the individual molecular polyunsaturated fatty acids (PUFAs) showed decrease in
species level using the novel technique of lipidomics. Rats abundance together with corresponding increase in lyso-
were subjected to high frequency (15 Hz) stimulation of the phospholipid species suggesting endogenous release of long
left hemisphere with different intensities and pulses of chain fatty acids such as docosahexaenoic acid (DHA) in
rTMS. The prefrontal cortex, hippocampus and striatum brain tissue. The hippocampus showed no significant chan-
ges, whilst changes in the striatum were often opposite to that
L. H.-W. Lee Á C.-H. Tan of the prefrontal cortex. It is postulated that changes in brain
Department of Pharmacology, National University of Singapore, lipids may underlie some of the clinical effects of rTMS.
Singapore 119260, Singapore
Keywords Transcranial magnetic stimulation Á Sulfatide Á
Y.-L. Lo Plasmalogens Á Lipids Á Polyunsaturated fatty acids Á
Department of Neurology, National Neuroscience Institute, Depression Á Pain Á Alzheimer’s disease Á Frontal cortex
Singapore 169608, Singapore
1 Introduction
A. A. Farooqui
Department of Molecular and Cellular Biochemistry, The Ohio Repetitive transcranial magnetic stimulation (rTMS) is
State University, Columbus, OH 43210, USA increasingly used in the management of neurologic disor-
ders such as depression and chronic pain. It involves the
G. Shui Á M. R. Wenk application of a magnetic flux to induce secondary currents
Department of Biochemistry, National University of Singapore, in the brain and action potentials in neurons to induce
Singapore 119260, Singapore changes in cortical function and plasticity (Bestmann
2008). Depending on stimulation parameters, induced
M. R. Wenk electrical currents depolarize neurons resulting in changes
Department of Biological Sciences, National University of in cortical function and behavior (Ziemann 2004) and
Singapore, Singapore 119260, Singapore muscle activation (Kapogiannis and Wassermann 2008).
High frequency rTMS stimulation ([5 Hz) enhances motor
W.-Y. Ong (&) excitability, whereas low frequency stimulation (1 Hz)
Department of Anatomy, National University of Singapore, depresses cortical excitability (Rossi et al. 2000). The
Singapore 119260, Singapore
e-mail: wei_yi_ong@nuhs.edu.sg

M. R. Wenk Á W.-Y. Ong
Ageing/Neurobiology Research Programme, National University
of Singapore, Singapore 119260, Singapore

123

20 L. H.-W. Lee et al.

effects of rTMS are not confined to the stimulated cortex, groups consisting of 4 rats per group. Rats were anesthetized
but also involve other connected areas within a functional by intraperitoneal injection of ketamine (75 mg/kg) ?
network (Koch and Rothwell 2009; Lefaucheur 2009). xylazine (10 mg/kg) cocktail and rTMS was carried out using
Small-scale studies suggest that rTMS could be useful in a MagstimÒ QuadroPulseTM Model 500 stimulator (Magstim,
the management of depression and chronic pain (Lefauc- Whitland, United Kingdom) capable of delivering a maxi-
heur et al. 2006; O’Reardon et al. 2007; Avery et al. 2008; mum output of 2.5 Tesla via pulses of 100 ls duration. A 7 cm
Bloch et al. 2008; Fitzgerald 2008; George et al. 2010). A diameter figure of eight coil was used. The center of the coil
recent meta-analysis concluded that patients with depres- was positioned over the left cerebral hemisphere (Sachdev
sion treated with high-frequency rTMS over the left dor- et al. 2007) with reference to an atlas (Paxinos and Watson
solateral prefrontal cortex (DLPFC) show improved 1986). The handle of the coil was parallel to the spine of the
outcomes compared to control subjects (Schutter 2009). In rat, and the surface of the coil was placed just touching the fur
addition, rTMS rescued defects in long term potentiation in and tangential to the scalp, but not pressing on the skull. Rats
rats subjected to the forced swim test, suggesting that were administered with either 50 pulses of TMS at 30%
rTMS treatment rescued impaired synaptic efficiency intensity (30% of the maximum output of the stimulator) or
caused by depression (Kim et al. 2006). Increased levels of 200 pulses of TMS at 100% intensity per day for five con-
brain-derived neurotrophic factor (BDNF) were detected in secutive days. Each pulse consisted of four ‘high frequency’
the rat brain after long-term rTMS, similar to the effect of (15 Hz) trains i.e., rat received a total number of 200 or 800
antidepressant drug treatment (Muller et al. 2000). stimuli per day. The stimuli were delivered over 30–45 min.
The treatment doses used may be relevant to humans, since
Lipidomics is a systems level analysis and involves higher rTMS intensities have been shown to have stronger
characterization of lipids and their interacting moieties antidepressive effects in patients (Padberg et al. 2002). Con-
(Wenk 2005; Adibhatla et al. 2006). Tandem mass spec- trol rats were anesthetized the same way as treated ones, but
trometry analysis (MS/MS) together with multiple reaction pulses were delivered at a distance of more than 10 cm from
monitoring (MRM) is used for quantitative analyses of the head (Ji et al. 1998).
lipids of known fragmentation profiles with up-front liquid
chromatography (Watson 2006). Brain lipids play impor- Rats were sacrificed 1 week after the last day of rTMS or
tant roles in membrane structure and cell signaling (Adi- sham treatment, a time when therapeutic effects are expected
bhatla and Hatcher 2007), but thus far little is known about in human patients receiving rTMS for depression (Pascual-
possible changes in individual lipid species after rTMS. In Leone et al. 1996; Eschweiler et al. 2000). They were deeply
this study, we carried out rTMS on rats by simulating a anaesthetized, decapitated, and the left and right prefrontal
protocol that is effective for treatment of depression in cortex, hippocampus and striatum were dissected out, snap
human patients (Pascual-Leone et al. 1996; Eschweiler frozen in liquid nitrogen, and kept in a -80°C freezer until
et al. 2000), followed by lipidomic analyses to elucidate analyses. All procedures involving animals were approved
changes in lipids at the individual molecular species level, by the Institutional Animal Care and Use Committee of the
in regions of the brain thought to be important in depres- National University of Singapore, and in accordance with the
sion, i.e., the prefrontal cortex (Drevets 2000), hippocam- guidelines of the National Advisory Committee for Labo-
pus (Sapolsky 2001) and striatum (Aizenstein et al. 2005). ratory Animal Research (NACLAR).
We show changes in relative abundance of lipids, in par-
ticular, sulfated galactosylceramides or sulfatides (SLs) 2.2 Lipidomic analyses
which are a class of lipids found in myelin and neurons
(Farooqui 1981; Eckhardt 2008) and plasmalogens after 2.2.1 Lipid extraction
rTMS, which might be related to the endogenous release of
long chain fatty acids, and beneficial effects of rTMS. A widely used modified protocol of Bligh and Dyer was used
(Bligh and Dyer 1959). Tissues were homogenized in 750 ll
2 Materials and methods of chloroform–methanol, 1:2 (v/v) using Tissue TearorTM
(Biospec. Inc, Bartlesville, USA). Samples were vortexed and
2.1 Rats and rTMS incubated on ice for 15 min with vortexing done at every
5 min interval. Subsequently, 250 ll of chloroform and
Male Wistar rats weighing around 200–250 g each were used 450 ll of 0.88% potassium chloride (KCl) were added.
in this study. They were housed under defined conditions Samples were vortexed and incubated on ice for 1 min. Lipids
(room temperature 22°C, relative humidity 65%, lighting were isolated from the organic phase after centrifugation
12 h/day) with free access to food and water. Rats were (7000 g, 4°C, 2 min). Samples were then vacuum dried
randomly divided into rTMS and sham control treatment (Thermo Savant SpeedVac, Waltham, MA), resuspended in
chloroform–methanol (1:1 v/v) and used for analyses.

123

Lipidomics after rTMS 21

2.2.2 Internal standards 2.3 Real-time RT-PCR

Levels of individual lipid levels were quantified using A further four rats which received 800 rTMS stimuli at 100%
spiked internal standards including C14-phosphatidylcho- intensity and four sham controls were used for this portion of
line dimyristoyl phosphatidylcholine (28:0-PC), dimyri- the study. The rTMS treated rats were sacrificed 1 week from
stoyl phosphatidylethanolamine (28:0-PE), dimyristoyl the last day of rTMS treatment. Total RNA was extracted
C14-phosphatidylserine (28:0-PS), dimyristoyl phosphati- from the prefrontal cortex using TRIzol reagent (Invitrogen,
dylglycerol (28:0-PG), dimyristoyl phosphatidic acid CA, USA) according to the manufacturer’s protocol and
(28:0-PA) and C19-ceramide (C19-CER), which were RNeasyÒ MiniKit (Qiagen, Inc., CA, USA) was used to
obtained from Avanti Polar Lipids (Alabaster, AL, USA). purify the RNA. The samples were then reverse transcribed
Dioctanoyl phosphatidylinositol (PI, 16:0-PI) was used for using the High-Capacity cDNA Reverse Transcription Kits
phosphatidylinositol quantitation and obtained from Eche- (Applied Biosystems, CA, USA). Reaction conditions were
lon Biosciences, Inc. (Salt Lake City, UT, USA). Sulfatides as follows: 25°C for 10 min, 37°C for 120 min and 85°C for
were analyzed using normalized intensity as well as C14- 5 s. Subsequently, real-time PCR amplification was carried
phosphatidylserine (28:0-PS) as an internal standard. out via the 7500 Realtime PCR system (Applied Biosystems,
CA, USA) using TaqManÒ Universal PCR MasterMix
2.2.3 Analysis of lipids using high performance liquid (Applied Biosystems, CA, USA) and probes for the mRNA
chromatography/mass spectrometry of enzymes along the SL biosynthetic or breakdown path-
way, serine C-palmitoyltransferase (SPT), 3-dehydrosp-
An Agilent high performance liquid chromatography hinganine reductase (3KDAR), UDP-glycosyltransferase 8
(HPLC) system coupled with an Applied Biosystem Triple (UDP-8), galactosylceramide (GAL-3) and arylsulfatase-A
Quadrupole/Ion Trap mass spectrometer (4000Qtrap, Fos- (ARS) were utilized according to the manufacturers’
ter City, California, USA) was used for quantification of instructions. b-actin was used as an internal control and all
individual polar lipids. Samples were introduced into the primers and probes were synthesized by Applied Biosystems.
mass spectrometer by loop injections with chloro- The PCR conditions were: an initial incubation of 50°C for
form:methanol (1:1) as a mobile phase for positive ESI 2 min and 95°C for 10 min followed by 40 cycles of 95°C for
mode and chloroform:methanol:200 mM piperidine 15 s and 60°C for 1 min. All reactions were carried out in
(1:1:0.1) as a mobile phase for negative ESI mode, triplicates. The threshold cycle, CT, which correlates inver-
respectively, both at a flow of 250 ll min-1 (Shui et al. sely with the levels of target mRNA, was measured as the
2007). Based on product ion and precursor ion analyses of number of cycles at which the reporter fluorescence emission
head groups, two comprehensive sets of multiple reaction exceeds the preset threshold level. The amplified transcripts
monitoring (MRM) transitions were set up for quantitative were quantified using the comparative CT method as
analysis of various lipids including phosphatidylcholines described previously (Livak and Schmittgen 2001) with the
(PCs), phosphatidylethanolamines (PEs), phosphatidylse- formula for relative fold change = 2-DDCT. The mean was
rines (PSs), phosphatidylinositols (PIs), phosphatidylgly- calculated and possible significant differences between the
cerols (PGs), phosphatidic acids (PAs), sphingomyelins prefrontal cortex of rTMS-treated and control specimens
(SMs), ceramides (Cers) and sulfatides (SLs). The signal were analyzed using the Student’s t-test. P \ 0.05 was con-
intensity of each MRM value was normalized using Eq. 1. sidered significant. Outliers identified by the Real-time PCR
system were also removed prior to statistical calculations.
Relative abundance of lipid 1
3 Results and discussion
¼P Signal intensity of lipid 1
3.1 Results
Signal intensity of all MRM transition measured
3.1.1 Effects of rTMS on lipids in the ipsilateral (left)
ð1Þ hemisphere

2.2.4 Statistical analysis Left prefrontal cortex (Tables 1, 2; Fig. 1, 2, 3) All sig-
nificant changes in lipids were only observed at the highest
The data were tested for normality using SPSS statistical dose of rTMS, namely 800 rTMS stimuli at 100% intensity.
software version 16.0 and based on the Shapiro–Wilk PEs showed significant changes in relative abundance in
significance value were shown to be normally distributed. the prefrontal cortex. Increase in relative abundance was
Comparisons of means were carried out using one-way
analysis of variance (ANOVA) followed by False Dis-
covery Rate (FDR) analysis. FDR \ 0.05 was considered
statistically significant.

123

22 L. H.-W. Lee et al.

observed with PE34p:1, PE36p:2, PE36p:1, PE36:1 and PIs (PI34:1, PI36:4, PI36:2, PI36:1, PI38:5, PI38:4,
PE38p:1, whilst decrease in relative abundance was PI38:3, PI40:5, PI40:4) showed decreases in relative
observed with majority of the PEs, especially those with abundance after rTMS treatment whilst PSs (PS36:2,
longer chains (PE34:1, PE36p:4, PE36:4, PE36:2, PE38p:6, PS36:1, PS38:5, PS40:7, PS40:6, PS40:5,and PS40:4)
PE38:6, PE38:4, PE40p:6, PE40:6, PE40:5 and PE40:4) showed a trend to an increase after rTMS treatment
(Table 1; Fig. 1). There was increased abundance in most (Table 2). This was opposite to that observed in the pre-
lysoPEs as compared to controls after treatment with frontal cortex, where most of the PSs showed a trend to a
rTMS, namely lysoPE16:0p, lysoPE16:0, lysoPE18:1p, decrease.
lysoPE18:0p and lysoPE22:6 (Table 1; Fig. 2).
The changes in relative abundance of SLs in the striatum
Increases in PCs were generally detected after the were opposite to that in the prefrontal cortex. SLs showed
highest dose of rTMS. PC34:2p, PC34:1p, PC34:0p, large decreases in relative abundance (more than 50% as
PC36:4p, PC36:3p, PC36:2p, PC36:1p, PC36:0p, PC36:2, compared to controls). All species were involved, i.e., SL
PC36:1, PC38:4p, PC38:3p, PC38:2p, PC38:3, PC40:4p, d18:1/18:1 h, SL d18:1/20:0, SL d18:1/22:1, SL d18:1/
PC40:3p, PC40:2p and PC40:1p showed significant 22:0, SL d18:1/24:1, SL d18:1/24:0, SL d18:1/24:1 h, and
increases in relative abundance (Table 1). Some other PCs SL d18:1/24:0 h (Table 2).
such as PC32:2, PC32:1, PC32:0, PC34:1, PC36:4 and
PC38:6 were significantly decreased (Table 1). On the 3.2 Effects of rTMS on lipids in the contralateral
other hand, lysoPCs mainly increased in relative abundance (right) hemisphere
after rTMS, namely lysoPC18:1, lysoPC18:0, lysoPC20:0
and lysoPC20:4 (Table 1). Right prefrontal cortex (Tables 3, 4) Similar changes in
relative abundance of lipid were observed in the right
A general trend to decrease in relative abundance was prefrontal cortex as compared to the left prefrontal cortex,
exhibited in PIs and PSs, with some of the species being except that there were fewer significant results. Shorter
significant (PI36:4, PI36:3, PI38:5, PI38:4, PI38:3, PI40:6, chains PEs (PE34p:1, PE36p:2, PE36p:1, PE36:1 and
lysoPS16:1, PS34:1, PS36:2, PS38:5, PS40:7, PS40:6, PE38p:1) showed significant increases in relative abun-
PS40:5) (Table 2). dance, whilst decreases in relative abundance were
observed in the longer chains PEs (PE38p:6, PE38:6,
SLs showed large increases in relative abundance after PE40p:6, PE40:6, PE40:5 and PE40:4) (Table 3). There
the highest dose of rTMS treatment, with all species of SLs was also an increase in abundance in lysoPEs as compared
showing significant increases (SL d18:1/18:1 h, SL d18:1/ to controls after treatment with rTMS, namely lys-
20:0, SL d18:1/22:1, SL d18:1/22:0, SL d18:1/24:1, SL oPE18:1p, lysoPE18:0p and lysoPE22:6 (Table 3).
d18:1/24:0, SL d18:1/24:1 h and SL d18:1/24:0 h)
(Table 2, Fig. 3). The increase in relative abundance was Increases in PCs were generally detected after rTMS.
more than 100% compared to controls. PC34:1p, PC34:0p, PC36:3p, PC36:2p, PC36:1p, PC36:0p,
PC36:2, PC36:1, PC38:4p, PC38:3p, PC38:2p, PC38:3,
Left hippocampus No significant changes in lipids were PC40:4p, PC40:3p, PC40:2p and PC40:1p showed signifi-
found in the left hippocampus. cant increase after rTMS (Table 3). Other PCs such as
PC32:2, PC32:1, PC32:0, PC34:1 and PC36:4 were
Left striatum (Tables 1, 2) The changes in lipid species decreased significantly. No significant changes were seen
in the striatum were, in general, opposite to that detected in in lysoPCs (Table 3).
the prefrontal cortex. Similarly, significant lipid changes
were only observed at the highest dose of rTMS. Increases PSs exhibited a general trend to decrease in relative
in relative abundance of PE38p:6, PE40:6 and PE40:4 were abundance, with PS34:1, PS38:5, PS40:6 and PS40:5
observed in the striatum after treatment with rTMS, showing significant decreases after rTMS (Table 4). SLs
although these lipids were decreased in the prefrontal showed large increases in relative abundance after rTMS
cortex after the highest dose of rTMS (Table 1). Lys- treatment, with SL d18:1/22:1, SL d18:1/22:0, SL d18:1/
oPE16:0 and lysoPE20:4 showed increases in relative 24:1, SL d18:1/24:0, SL d18:1/24:1 h and SL d18:1/24:0 h
abundance after rTMS (Table 1). showing significant increase (Table 4). Similar to the left
prefrontal cortex, the increase in relative abundance was
PC34:2p, PC38:3p, PC38:2p, PC40:4p, PC40:2p and more than 100% when compared to controls.
PC40:1p showed decreases in relative abundance after
rTMS (Table 1). This was in contrast to the increases in Right hippocampus No significant changes in lipids
relative abundance seen in the prefrontal cortex. PC36:4, were found in the right hippocampus.
PC38:6, PC40:7 and PC40:6 showed significant increases
after rTMS, opposite to that in the prefrontal cortex Right striatum (Tables 3, 4) The right striatum showed a
(Table 1). LysoPCs were decreased after rTMS, with ly- similar trend to the left striatum and opposite to that
soPC16:0, lysoPC18:2 and lysoPC18:0 showing significant
differences (Table 1).

123

Lipidomics after rTMS 23

Table 1 Changes in relative abundance of PEs, lysoPEs, PCs and lysoPCs in the left prefrontal cortex/left striatum after high dose rTMS

Left brain regions

Lipid species Treatment
Relative abundance (910-3)

Prefrontal cortex Striatum

Control 800 stimuli (100% intensity) Control 800 stimuli (100% intensity)

Phosphatidylethanolamines

PE34p:1 18.94 ± 2.06 35.66 ± 2.65* 57.05 ± 4.30 43.66 ± 0.80*
11.15 ± 0.57* 10.50 ± 1.02 9.71 ± 0.62
PE34:1 13.48 ± 0.77 29.89 ± 1.62* 38.35 ± 3.27
53.34 ± 6.42* 73.91 ± 8.86 45.10 ± 4.08
PE36p:4 36.12 ± 1.07 45.40 ± 3.83* 63.88 ± 2.80 67.44 ± 5.14
10.34 ± 0.54* 54.31 ± 2.28*
PE36p:2 27.08 ± 1.24 2.12 ± 0.57 7.39 ± 0.10
21.54 ± 1.38* 1.82 ± 0.07 8.24 ± 1.04
PE36p:1 23.55 ± 1.14 45.69 ± 2.75* 22.03 ± 0.87 2.25 ± 0.12*
7.07 ± 1.42* 53.02 ± 3.36 22.26 ± 0.61
PE36:4 12.17 ± 0.31 32.12 ± 2.42* 9.42 ± 0.57 61.03 ± 3.32*
90.44 ± 3.79* 20.93 ± 1.96 9.25 ± 1.57
PE36:3 1.99 ± 0.21 129.97 ± 8.37* 68.75 ± 4.98 21.53 ± 2.57
12.68 ± 0.69* 87.61 ± 6.35 75.18 ± 5.74
PE36:1 18.02 ± 1.09 14.81 ± 1.18* 8.73 ± 0.64 107.47 ± 6.42*
12.85 ± 0.99 10.64 ± 1.42
PE38p:6 55.93 ± 2.08 4.50 ± 0.59* 16.17 ± 0.53*
1.44 ± 0.10* 6.84 ± 0.65
PE38p:1 2.81 ± 0.41 11.60 ± 2.34* 0.92 ± 0.16 6.98 ± 0.61
11.01 ± 0.91* 20.90 ± 3.94 1.61 ± 0.32*
PE38:6 41.75 ± 0.97 0.86 ± 0.16 15.77 ± 1.73 18.49 ± 3.05
0.60 ± 0.20* 0.41 ± 0.17 14.48 ± 1.87
PE40p:6 111.49 ± 6.13 0.32 ± 0.06 1.35 ± 0.20*
3.65 ± 0.17* 0.87 ± 0.22
PE40:6 158.34 ± 6.59 31.05 ± 0.86* 3.82 ± 0.10
212.87 ± 4.53* 29.96 ± 0.29 3.66 ± 0.10
PE40:5 15.08 ± 0.39 0.23 ± 0.01* 185.80 ± 10.71 28.53 ± 1.05
3.67 ± 0.20* 203.39 ± 6.39
PE40:4 17.08 ± 1.14 27.04 ± 3.49* 0.38 ± 0.05
343.03 ± 8.89* 6.18 ± 0.78 0.27 ± 0.01*
Lysophosphatidylethanolamines 0.92 ± 0.03* 33.67 ± 3.76 5.13 ± 0.36
2.23 ± 0.16* 324.45 ± 1.75 31.44 ± 2.87
LysoPE16:0p 2.63 ± 0.25 2.45 ± 0.19* 1.91 ± 0.22 330.08 ± 7.83
6.80 ± 0.85* 2.70 ± 0.43 2.09 ± 0.18
LysoPE16:0 1.07 ± 0.15 9.25 ± 0.95* 3.52 ± 0.54 2.42 ± 0.24
32.02 ± 1.10* 9.86 ± 1.11 2.92 ± 0.34
LysoPE18:1p 4.33 ± 0.33 20.31 ± 0.96* 11.19 ± 1.26 7.75 ± 0.99
93.49 ± 6.05* 27.57 ± 1.83 9.87 ± 0.74
LysoPE18:0p 6.92 ± 0.46 1.14 ± 0.08* 21.32 ± 0.36 31.95 ± 1.37*
1.34 ± 0.04* 106.37 ± 7.53 22.11 ± 1.08
LysoPE20:4 1.49 ± 0.05 0.67 ± 0.09* 1.43 ± 0.19 99.08 ± 4.47
23.91 ± 1.13* 1.79 ± 0.12 1.41 ± 0.05
LysoPE22:6 0.22 ± 0.02 1.54 ± 0.20 1.46 ± 0.13*
21.44 ± 1.01 0.96 ± 0.13*
Phosphatidylcholines 24.03 ± 1.03*

PC32:2 6.11 ± 0.17

PC32:1 39.39 ± 1.23

PC32:0 242.91 ± 6.36

PC34:2p 0.14 ± 0.02

PC34:1p 1.68 ± 0.10

PC34:0p 10.93 ± 0.87

PC34:1 361.74 ± 2.50

PC36:4p 0.85 ± 0.02

PC36:3p 1.36 ± 0.13

PC36:2p 1.08 ± 0.08

PC36:1p 2.54 ± 0.12

PC36:0p 4.94 ± 0.12

PC36:4 36.22 ± 1.36

PC36:2 17.11 ± 0.21

PC36:1 62.97 ± 2.34

PC38:4p 0.72 ± 0.06

PC38:3p 0.83 ± 0.06

PC38:2p 0.29 ± 0.03

PC38:6 26.41 ± 0.63

123

24 L. H.-W. Lee et al.

Table 1 continued Treatment
Left brain regions Relative abundance (910-3)
Lipid species

Prefrontal cortex Striatum
Control
Control 800 stimuli (100% intensity) 800 stimuli (100% intensity)

PC38:3 4.78 ± 0.15 5.50 ± 0.39* 6.73 ± 0.46 6.58 ± 2.97
0.59 ± 0.03* 0.94 ± 0.14 0.64 ± 0.06*
PC40:4p 0.32 ± 0.04 0.45 ± 0.03* 0.65 ± 0.17 0.50 ± 0.06
0.29 ± 0.01* 0.78 ± 0.06 0.44 ± 0.10*
PC40:3p 0.30 ± 0.02 0.51 ± 0.05* 1.04 ± 0.07 0.75 ± 0.13*
4.47 ± 0.30 5.23 ± 0.28 6.03 ± 0.15*
PC40:2p 0.11 ± 0.01 16.39 ± 0.99 14.86 ± 1.41 18.71 ± 0.30*

PC40:1p 0.21 ± 0.06 0.91 ± 0.06 1.03 ± 0.03 0.84 ± 0.05*
0.27 ± 0.10 0.84 ± 0.16 0.30 ± 0.08*
PC40:7 4.78 ± 0.14 0.71 ± 0.02* 0.92 ± 0.06 0.73 ± 0.12
0.87 ± 0.11* 1.10 ± 0.05 0.81 ± 0.12*
PC40:6 17.33 ± 0.45 0.21 ± 0.03* 0.53 ± 0.20 0.23 ± 0.07
0.15 ± 0.00* 0.19 ± 0.04 0.15 ± 0.02
Lysophosphatidylcholines

LysoPC16:0 0.98 ± 0.08

LysoPC18:2 0.39 ± 0.12

LysoPC18:1 0.59 ± 0.04

LysoPC18:0 0.74 ± 0.05

LysoPC20:0 0.14 ± 0.03

LysoPC20:4 0.12 ± 0.02

All values are expressed as mean relative abundance ± SD. Results for low dose rTMS are not shown. Data were analyzed by one-way ANOVA
followed by FDR analysis. * FDR \ 0.05 as compared with sham controls

detected in the right prefrontal cortex. Again, increase in d18:1/22:1, SL d18:1/22:0, SL d18:1/24:1, SL d18:1/24:0,
relative abundance in PE38p:6 and PE40:6 were observed SL d18:1/24:1 h, and SL d18:1/24:0 h (Table 4).
in the right striatum after treatment with rTMS although
these lipids showed a trend to a decrease in the right pre- 3.2.1 Real time RT-PCR analysis of enzymes involved
frontal cortex (Table 3). LysoPE20:4 showed increase in in the SL biosynthetic pathway
relative abundance after rTMS (Table 3).
No significant differences in mRNA expression of enzymes
Few changes were seen in PCs and lysoPCs. PC34:2p, involved in SL biosynthetic or catabolic pathways were
PC38:3p, PC38:2p, PC40:4p, PC40:1p and lysoPC20:0 detected after rTMS as analyzed by Student’s t test (data
showed significant decreases in relative abundance after not shown).
rTMS (Table 3). This was in contrast to the increase in
relative abundance in the right prefrontal cortex. PC36:4, 3.3 Discussion
PC38:6, PC40:7 and PC40:6 showed significant increases
after rTMS (Table 3). The present study aimed to elucidate the effect of rTMS on
global lipid profiles in different parts of the brain important
PIs (PI34:1, PI36:4, PI36:3, PI36:2, PI36:1, PI38:5, in depression. Lipidomic analyses of different parts of the
PI38:4, PI38:3, PI40:6) showed significant decreases in rat brain was carried out after 5 days of rTMS treatment
relative abundance, whilst PSs (PS38:5, PS38:4 PS40:7, followed by an interval of 7 days, to mimic a treatment
PS40:6, PS40:5,and PS40:4) showed a trend to an increase protocol that has been shown to be effective in the man-
after rTMS treatment (Table 4). LysoPSs on the other agement of major depression in human patients, where a
hand, exhibited a reduction in relative abundance with minimum course of 5 days rTMS was used and effects
lysoPS16:0, lysoPS18:1 and lysoPS18:0 being significant expected after a week (Pascual-Leone et al. 1996; Esc-
(Table 4). hweiler et al. 2000). SLs showed large and significant
increases in relative abundance after 800 stimuli of rTMS
The changes in SLs in the right striatum were opposite at 100% intensity. SLs are important constituents of myelin
to that in the right prefrontal cortex. SLs showed a large
decrease in relative abundance with all species being
involved, i.e., SL d18:1/18:1 h, SL d18:1/20:0, SL

123

Lipidomics after rTMS 25

Table 2 Changes in relative abundance of PIs, PS, lysoPSs, SLs, SMs and Cers in the left prefrontal cortex/left striatum after high dose rTMS

Left brain regions

Lipid species Treatment
Relative abundance (910-3)

Prefrontal cortex Striatum

Control 800 stimuli (100% intensity) Control 800 stimuli (100% intensity)

Phosphatidylinositols 0.33 ± 0.05 0.33 ± 0.03 0.34 ± 0.03 0.22 ± 0.02*
PI34:1 3.07 ± 0.19 2.60 ± 0.17* 2.21 ± 0.28 1.51 ± 0.11*
PI36:4 0.37 ± 0.04 0.28 ± 0.03* 0.27 ± 0.06 0.18 ± 0.02
PI36:3 0.09 ± 0.01 0.09 ± 0.02 0.10 ± 0.01 0.07 ± 0.01*
PI36:2 0.14 ± 0.01 0.17 ± 0.03 0.22 ± 0.02 0.13 ± 0.02*
PI36:1 2.44 ± 0.14 1.95 ± 0.12* 1.68 ± 0.12 1.15 ± 0.10*
PI38:5 18.94 ± 1.29 16.22 ± 0.74* 17.44 ± 1.63 11.77 ± 0.69*
PI38:4 2.15 ± 0.10 1.86 ± 0.15* 2.18 ± 0.20 1.42 ± 0.09*
PI38:3 0.42 ± 0.05 0.31 ± 0.04* 0.21 ± 0.05 0.14 ± 0.02
PI40:6 0.14 ± 0.02 0.12 ± 0.02 0.11 ± 0.03 0.06 ± 0.01*
PI40:5 0.10 ± 0.01 0.13 ± 0.02 0.10 ± 0.02 0.06 ± 0.00*
PI40:4 0.03 ± 0.00 0.04 ± 0.01 0.04 ± 0.00 0.03 ± 0.01
PI40:3
0.80 ± 0.04 0.50 ± 0.02* 0.37 ± 0.01 0.47 ± 0.06
Phosphatidylserines 1.94 ± 0.09 1.54 ± 0.04* 1.23 ± 0.07 1.74 ± 0.17*
PS34:1 5.68 ± 0.21 7.83 ± 0.69* 7.87 ± 0.26 11.25 ± 1.66*
PS36:2 0.33 ± 0.03 0.27 ± 0.02* 0.18 ± 0.01 0.32 ± 0.05*
PS36:1 2.20 ± 0.17 2.54 ± 0.11* 2.28 ± 0.11 2.93 ± 0.29
PS38:5 0.53 ± 0.04 0.69 ± 0.05* 0.61 ± 0.03 0.90 ± 0.20
PS38:4 0.64 ± 0.06 0.39 ± 0.05* 0.28 ± 0.05 0.57 ± 0.05*
PS38:3 31.37 ± 1.81 21.90 ± 1.32* 16.13 ± 0.62 26.92 ± 1.56*
PS40:7 6.04 ± 0.29 4.54 ± 0.31* 3.27 ± 0.21 5.04 ± 0.35*
PS40:6 2.80 ± 0.19 2.61 ± 0.15 1.94 ± 0.06 3.08 ± 0.31*
PS40:5
PS40:4 0.04 ± 0.01 0.01 ± 0.01* 0.00 ± 0.00 0.01 ± 0.01
0.08 ± 0.02 0.10 ± 0.01 1.18 ± 0.36 0.05 ± 0.01*
Lysophosphatidylserines 0.20 ± 0.03 0.16 ± 0.03 0.25 ± 0.06 0.13 ± 0.03*
LysoPS16:1 0.45 ± 0.03 0.62 ± 0.07 0.38 ± 0.14 0.30 ± 0.04
LysoPS16:0
LysoPS18:1 0.24 ± 0.06 0.36 ± 0.06* 0.63 ± 0.11 0.25 ± 0.05*
LysoPS18:0 2.26 ± 0.12 2.46 ± 0.08* 3.00 ± 0.23 1.31 ± 0.15*
0.28 ± 0.01 0.58 ± 0.10* 1.04 ± 0.10 0.43 ± 0.04*
Sulfatides 0.93 ± 0.12 2.45 ± 0.39* 4.34 ± 0.79 1.46 ± 0.22*
SL d18:1/18:1 h 5.12 ± 0.40 12.55 ± 2.05* 20.77 ± 2.81 8.17 ± 1.68*
SL d18:1/20:0 4.29 ± 0.54 10.14 ± 1.54* 15.93 ± 3.03 6.28 ± 0.94*
SL d18:1/22:1 1.38 ± 0.15 3.22 ± 0.51* 4.54 ± 0.74 1.85 ± 0.34*
SL d18:1/22:0 2.76 ± 0.24 6.16 ± 0.66* 8.37 ± 1.48 3.18 ± 0.63*
SL d18:1/24:1
SL d18:1/24:0 2.20 ± 0.15 1.66 ± 0.09* 1.83 ± 0.16 1.66 ± 0.23
SL d18:1/24:1 h 3.77 ± 0.14 2.47 ± 0.14* 2.86 ± 0.26 2.50 ± 0.07
SL d18:1/24:0 h 48.62 ± 1.33 36.87 ± 2.10* 40.28 ± 0.95 36.77 ± 1.09*
2.47 ± 0.23 4.14 ± 0.46* 7.72 ± 0.99 5.90 ± 0.85
Sphingomyelins
SM18/16:0
SM18/18:1
SM18/18:0
SM18/24:1

123

26 L. H.-W. Lee et al.

Table 2 continued Treatment
Left brain regions Relative abundance (910-3)
Lipid species

Prefrontal cortex Striatum
Control
Control 800 stimuli (100% intensity) 800 stimuli (100% intensity)

SM18/24:0 1.68 ± 0.11 2.80 ± 0.32* 4.56 ± 0.39 3.29 ± 0.55*
Ceramides
0.41 ± 0.07 0.69 ± 0.15* 1.12 ± 0.52 0.72 ± 0.30
Cer d18:1/22:0 9.05 ± 1.56 15.80 ± 1.67* 22.80 ± 3.32 12.82 ± 1.59*
Cer d18:1/24:1 2.02 ± 0.50 5.13 ± 0.86* 3.16 ± 0.31*
Cer d18:1/24:0 5.24 ± 0.78

All values are expressed as mean relative abundance ± SD. Results for low dose rTMS are not shown. Data were analyzed by one-way ANOVA
followed by FDR analysis. * FDR \ 0.05 as compared with sham controls

Fig. 1 Graph showing changes A Relative abundance of PEs in the left prefrontal cortex with varying
in relative abundance of PEs in doses of rTMS
the left prefrontal cortex after 0.07
varying doses of rTMS. Data Relative abundance 0.06 * Control
were analyzed by one-way 0.05
ANOVA followed by FDR 0.04 * 200TMS (30%
analysis. * P \ 0.05 as 0.03 intensity)
compared with sham controls 0.02
0.01
* 800TMS (100%
0 * intensity)

*

* **

PE34p:1
PE34:2
PE34:1
PE34:0
PE36p:4
PE36p:3
PE36p:2
PE36p:1
PE36:4
PE36:3
PE36:2
PE36:1

B Relative abundance * Lipid species *
**
0.18 **
0.16 *
0.14 *
0.12

0.1
0.08
0.06
0.04
0.02

0

PE38p: 6
PE38p:5
PE38p: 4
PE38p:1
PE3 8:7
PE38: 6
PE3 8:5
PE38: 4
PE40p:6
PE4 0p:5
PE40p:4
PE4 0:6
PE40: 5
PE4 0:4

Lipid species

for structural stabilization, and mediate diverse biological plasticity (Ishizuka 1997; Honke et al. 2004). Reduction in
processes including the regulation of cell growth, protein SLs is one of the earliest detectable lipid changes in the
trafficking, signal transduction, cell adhesion and neuronal brain and is thought to contribute to AD progression (Han

123

Lipidomics after rTMS 27

Fig. 2 Graph showing changes A Relative abundance of lysoPEs in the left prefrontal cortex with varying
in relative abundance of doses of rTMS
lysoPEs in the left prefrontal 0.016
cortex after varying doses of 0.014 * Control
rTMS. Data were analyzed by 0.012 * 200TMS (30%
one-way ANOVA followed by intensity)
FDR analysis. * P \ 0.05 as 0.01 Relative abundance
compared with sham controls 0.008 LysoPE16:0p 800TMS (100%
0.006 LysoPE16:0
0.004 LysoPE18:1pintensity)
0.002 LysoPE18:0p
LysoPE18:0*
0 LysoPE20:4
*

Lipid species

B Relative abundance *
LysoPE16:1
0.0012 LysoPE18:2
0.001 LysoPE18:1
LysoPE20:0p
0.0008 LysoPE22:6

0.0006
0.0004
0.0002

0

Lipid species

Fig. 3 Graph showing changes Relative abundance of SLs in the left prefrontal cortex with varying doses
in relative abundance of SLs in
the left prefrontal cortex after 0.016 of rTMS Control
varying doses of rTMS. Data 0.014
were analyzed by one-way *
ANOVA followed by FDR
analysis. * P \ 0.05 as Relative abundance 0.012 * 200TMS(30%
compared with sham controls intensity)

0.01 800TMS (100%
0.008 intensity)
0.006
*

0.004 * *

*

0.002 *

0 *

SL d18:1/18:1h
SLd18:1/20:0
SLd18:1/22:1
SLd18:1/22:0
SLd18:1/24:1
SLd18:1/24:0
SL d18:1/24:1h
SL d18:1/24:0h

Lipid species

et al. 2002). SLs aid in the clearance of amyloid-beta (Ab) mice (Han et al. 2003). We postulate that increased SLs
and substantially lower amounts of SLs were found in could underlie the improvement in cognitive performance
human apoE4 transgenic mice as compared to wild type after rTMS in patients with AD (Cotelli et al. 2006). On the

123

28 L. H.-W. Lee et al.

Table 3 Changes in relative abundance of PEs, lysoPEs, PCs and lysoPCs in the right prefrontal cortex/right striatum after high dose rTMS

Right brain regions

Lipid species Treatment
Relative abundance (910-3)

Prefrontal cortex Striatum

Control 800 stimuli (100% intensity) Control 800 stimuli (100% intensity)

Phosphatidylethanolamines

PE34p:1 18.19 ± 2.52 35.77 ± 1.01* 56.00 ± 2.83 46.47 ± 4.06*
11.66 ± 0.50 10.79 ± 1.00 10.47 ± 0.96
PE34:1 12.81 ± 0.47 31.45 ± 3.37 35.97 ± 6.27 44.20 ± 3.17
55.53 ± 1.90* 81.15 ± 8.85 67.40 ± 7.75
PE36p:4 36.72 ± 3.68 45.20 ± 2.98* 67.23 ± 8.99 60.09 ± 1.11
10.41 ± 0.43
PE36p:2 22.89 ± 3.92 2.10 ± 0.11 7.44 ± 1.24 7.84 ± 1.02
21.35 ± 1.42* 2.48 ± 0.32 1.63 ± 0.25*
PE36p:1 20.07 ± 1.88 48.19 ± 3.04* 23.16 ± 1.24 20.50 ± 2.00
6.74 ± 0.61* 47.92 ± 2.99 65.66 ± 1.13*
PE36:4 12.10 ± 0.80 30.80 ± 2.74* 10.62 ± 1.38 11.07 ± 0.90
89.00 ± 2.10* 19.74 ± 1.92 19.61 ± 1.20
PE36:3 2.66 ± 0.35 120.25 ± 2.16* 65.00 ± 3.98 70.78 ± 3.37
11.73 ± 0.46* 80.18 ± 7.65 105.58 ± 7.78*
PE36:1 16.54 ± 0.71 15.49 ± 1.21* 8.32 ± 1.24 9.67 ± 1.82
10.73 ± 1.53 13.88 ± 2.08
PE38p:6 55.98 ± 1.74 4.12 ± 0.46
1.28 ± 0.20 7.16 ± 0.62 8.19 ± 1.10
PE38p:1 2.56 ± 0.60 10.74 ± 0.63* 1.24 ± 0.43 1.32 ± 0.26
11.03 ± 0.82* 20.31 ± 1.84 19.89 ± 0.53
PE38:6 39.98 ± 1.66 1.39 ± 0.26 16.34 ± 1.34 15.34 ± 1.04
0.86 ± 0.15* 0.60 ± 0.12 1.83 ± 0.20*
PE40p:6 106.68 ± 6.13 0.52 ± 0.18 0.63 ± 0.06
3.75 ± 0.39*
PE40:6 154.35 ± 3.07 31.92 ± 2.02* 3.92 ± 0.47 3.22 ± 0.28
222.62 ± 2.69* 29.53 ± 1.67 27.57 ± 1.17
PE40:5 16.26 ± 0.71 0.21 ± 0.02 177.59 ± 13.67 183.69 ± 11.29
3.80 ± 0.21*
PE40:4 18.58 ± 1.04 26.90 ± 1.16* 0.36 ± 0.04 0.28 ± 0.02*
335.05 ± 6.89* 6.82 ± 1.57 5.17 ± 0.21
Lysophosphatidylethanolamines 0.82 ± 0.06 41.00 ± 7.98 34.19 ± 1.57
2.33 ± 0.13* 324.09 ± 15.16 336.25 ± 8.88
LysoPE16:0p 3.16 ± 0.47 2.63 ± 0.09* 1.91 ± 0.27 2.26 ± 0.14
7.05 ± 0.22* 2.92 ± 0.48 2.37 ± 0.21
LysoPE16:0 1.16 ± 0.14 8.95 ± 0.43* 3.79 ± 0.69 2.95 ± 0.29
32.58 ± 0.99* 10.55 ± 1.47 8.93 ± 0.21
LysoPE18:1p 4.21 ± 0.53 20.29 ± 0.99* 12.14 ± 1.84 10.58 ± 0.52
89.91 ± 5.95* 28.09 ± 1.44 32.23 ± 0.50*
LysoPE18:0p 7.36 ± 0.53 1.20 ± 0.10* 22.62 ± 1.31 22.90 ± 0.96
1.32 ± 0.18* 112.88 ± 11.06 103.34 ± 3.51
LysoPE20:4 0.82 ± 0.22 0.67 ± 0.06* 1.41 ± 0.19 1.47 ± 0.04
24.25 ± 0.97 1.80 ± 0.12 1.50 ± 0.14*
LysoPE22:6 0.44 ± 0.12 1.70 ± 0.20 1.02 ± 0.06*
20.63 ± 0.76 23.09 ± 0.71*
Phosphatidylcholines

PC32:2 5.69 ± 0.33

PC32:1 38.21 ± 1.69

PC32:0 238.54 ± 8.47

PC34:2p 0.17 ± 0.03

PC34:1p 1.64 ± 0.22

PC34:0p 11.34 ± 1.28

PC34:1 372.50 ± 4.40

PC36:4p 0.85 ± 0.05

PC36:3p 1.31 ± 0.14

PC36:2p 0.92 ± 0.14

PC36:1p 2.35 ± 0.37

PC36:0p 4.80 ± 0.61

PC36:4 35.57 ± 1.26

PC36:2 16.13 ± 0.98

PC36:1 61.60 ± 2.90

PC38:4p 0.78 ± 0.06

PC38:3p 0.97 ± 0.07

PC38:2p 0.29 ± 0.03

PC38:6 24.77 ± 2.00

123

Lipidomics after rTMS 29

Table 3 continued Treatment
Right brain regions Relative abundance (910-3)
Lipid species

Prefrontal cortex Striatum
Control
Control 800 stimuli (100% intensity) 800 stimuli (100% intensity)

PC38:3 4.88 ± 0.26 5.74 ± 0.10* 6.62 ± 0.38 6.50 ± 0.34
0.64 ± 0.04* 0.86 ± 0.07 0.67 ± 0.05*
PC40:4p 0.39 ± 0.07 0.48 ± 0.01* 0.65 ± 0.12 0.54 ± 0.02
0.29 ± 0.02* 0.74 ± 0.17 0.41 ± 0.04
PC40:3p 0.33 ± 0.03 0.52 ± 0.07* 1.13 ± 0.13 0.72 ± 0.08*
4.52 ± 0.14 4.89 ± 0.45 6.13 ± 0.54*
PC40:2p 0.13 ± 0.02 15.91 ± 0.22 15.51 ± 1.34 18.80 ± 0.49*

PC40:1p 0.20 ± 0.04 0.99 ± 0.06 0.99 ± 0.10 0.86 ± 0.02
0.22 ± 0.01 0.45 ± 0.17 0.30 ± 0.06
PC40:7 4.59 ± 0.31 0.67 ± 0.03 0.96 ± 0.14 0.78 ± 0.02
0.79 ± 0.05 1.24 ± 0.23 0.88 ± 0.02
PC40:6 15.94 ± 0.83 0.19 ± 0.05 0.36 ± 0.02 0.16 ± 0.05*
0.13 ± 0.01 0.15 ± 0.06 0.16 ± 0.03
Lysophosphatidylcholines

LysoPC16:0 0.89 ± 0.08

LysoPC18:2 0.44 ± 0.13

LysoPC18:1 0.58 ± 0.08

LysoPC18:0 0.67 ± 0.07

LysoPC20:0 0.18 ± 0.06

LysoPC20:4 0.14 ± 0.03

All values are expressed as mean relative abundance ± SD. Results for low dose rTMS are not shown. Data were analyzed by one-way ANOVA
followed by FDR analysis. * FDR \ 0.05 as compared with sham controls

other hand, excessive levels of SL may be harmful to the docosahexaenoic acid (DHA) at the sn-2 position. PS34:1,
brain. Accumulation of SL due to a deficiency in arylsul- PS38:5, PS40:6 and PS40:5 were also significantly
fatase A causes the severe lysosomal storage disease decreased in both left and right sides of the prefrontal cortex
metachromatic leukodystrophy (Ramakrishnan et al. 2007; indicating the stimulation of enzymes associated with
Eckhardt 2008). Elevated levels of SLs in neurons in degradation of PS. It is possible that the decrease in PSs is
transgenic animals have also been shown to be associated due to decarboxylation to form PEs (Vance 2008; Mozzi
with lethal audiogenic seizures (van Zyl et al. 2010). We and Buratta 2010).
surveyed the mRNA expression of enzymes involved in
synthesis or breakdown of SLs after rTMS using real-time In contrast to the increase in the PEs and PCs mentioned
RT-PCR, but did not detect significant changes. Increase in above, ethanolamine plasmalogens with long chain poly-
SL levels may be due to rTMS-mediated increase in ATP unsaturated fatty acid chains (PE38p:6 and PE40p:6)
(Feng et al. 2008), which is necessary for synthesis of 30- showed significant decrease in relative abundance in the
phosphoadenosine 50-phosphosulfate (PAPS), a reactant prefrontal cortex after high dose rTMS. Based on the
required for the synthesis of SLs in brain tissues (Farooqui corresponding increase in lysophospholipid species (lys-
1981). oPE16:0p, lysoPE16:0, lysoPE18:1p, lysoPE18:0p,
lysoPE22:6), we can deduce the identity of the fatty acid
rTMS treatment also resulted in significant increase in side chain being released. For instance, PE38p:6 -
many PEs and PCs in the left and right prefrontal cortex. lysoPE16:0p = 22:6 (docosahexaenoic acid, or DHA),
The species involved included PE34p:1, PE36p:2, PE36p:1, PE38p:6 - lysoPE18:1p = 20:5 (eicosapentaenoic acid,
PE36:1, PE38p:1, lysoPE16:0p, lysoPE16:0, lysoPE18:1p, or EPA) (Table 5). The results are consistent with
lysoPE18:0p, lysoPE22:6, PC34:2p, PC34:1p, PC34:0p, increased activity of plasmalogen selective phospholipase
PC36:4p, PC36:3p, PC36:2p, PC36:1p, PC36:0p, PC36:2, A2 (PlsEtn-PLA2), which acts on the sn-2 position of eth-
PC36:1, PC38:4p, PC38:3p, PC38:2p, PC38:3, PC40:4p, anolamine plasmalogens to produce DHA (Farooqui and
PC40:3p, PC40:2p and PC40:1p. The majority of the PCs Horrocks 2001). Long chain fatty acids such as DHA could
and PEs exhibiting changes were of the plasmalogen class be metabolized to resolvins and neuroprotectins that have
(e.g., 34p:1, the symbol ‘‘p’’ representing a vinyl ether neuroprotective effects (Bazan 2009; Farooqui 2009). Our
linkage to the glycerol chain at the sn-1 position). Plas- previous study has indicated possible involvement of
malogens usually contain arachidonic acid (AA) or intracellular PLA2s (probably iPLA2) and release of long

123

30 L. H.-W. Lee et al.

Table 4 Changes in relative abundance of PIs, PS, lysoPSs, SLs, SMs and Cers in the right prefrontal cortex/right striatum after high dose rTMS

Right brain regions

Lipid species Treatment
Relative abundance (910-3)

Prefrontal cortex Striatum

Control 800 stimuli (100% intensity) Control 800 stimuli (100% intensity)

Phosphatidylinositols 0.33 ± 0.04 0.33 ± 0.02 0.36 ± 0.04 0.20 ± 0.03*
PI34:1 3.12 ± 0.06 2.76 ± 0.33 2.50 ± 0.31 1.44 ± 0.14*
PI36:4 0.35 ± 0.02 0.32 ± 0.04 0.33 ± 0.08 0.17 ± 0.02*
PI36:3 0.09 ± 0.02 0.09 ± 0.01 0.13 ± 0.02 0.07 ± 0.02*
PI36:2 0.15 ± 0.01 0.17 ± 0.03 0.25 ± 0.04 0.13 ± 0.03*
PI36:1 2.49 ± 0.23 2.19 ± 0.25 1.82 ± 0.28 1.11 ± 0.12*
PI38:5 19.05 ± 0.90 17.30 ± 2.27 19.24 ± 2.31 11.54 ± 0.51*
PI38:4 2.20 ± 0.18 2.05 ± 0.28 2.39 ± 0.26 1.34 ± 0.11*
PI38:3 0.31 ± 0.02 0.35 ± 0.08 0.25 ± 0.05 0.13 ± 0.01*
PI40:6 0.11 ± 0.02 0.12 ± 0.03 0.09 ± 0.01 0.06 ± 0.01
PI40:5 0.10 ± 0.01 0.12 ± 0.03 0.11 ± 0.02 0.08 ± 0.01
PI40:4 0.04 ± 0.01 0.03 ± 0.01 0.04 ± 0.01 0.02 ± 0.00
PI40:3
0.70 ± 0.05 0.51 ± 0.04* 0.42 ± 0.07 0.50 ± 0.04
Phosphatidylserines 1.64 ± 0.05 1.53 ± 0.18 1.30 ± 0.23 1.56 ± 0.05
PS34:1 5.05 ± 0.85 7.83 ± 0.69* 9.25 ± 1.52 11.02 ± 0.37
PS36:2 0.34 ± 0.02 0.26 ± 0.02* 0.20 ± 0.01 0.26 ± 0.02*
PS36:1 1.98 ± 0.20 2.41 ± 0.21 2.25 ± 0.16 2.78 ± 0.21*
PS38:5 0.50 ± 0.07 0.70 ± 0.07* 0.66 ± 0.08 0.74 ± 0.06
PS38:4 0.58 ± 0.06 0.43 ± 0.07 0.37 ± 0.06 0.50 ± 0.02*
PS38:3 29.54 ± 1.04 22.61 ± 1.70* 17.73 ± 2.05 24.79 ± 0.53*
PS40:7 5.84 ± 0.33 4.65 ± 0.47* 3.37 ± 0.39 4.46 ± 0.31*
PS40:6 2.83 ± 0.22 2.75 ± 0.26 2.09 ± 0.15 2.82 ± 0.17*
PS40:5
PS40:4 0.04 ± 0.02 0.01 ± 0.00* 0.00 ± 0.00 0.00 ± 0.00
0.20 ± 0.09 0.16 ± 0.13 1.02 ± 0.12 0.04 ± 0.01*
Lysophosphatidylserines 0.25 ± 0.10 0.19 ± 0.04 0.22 ± 0.02 0.11 ± 0.02*
LysoPS16:1 0.60 ± 0.15 0.59 ± 0.13 0.38 ± 0.04 0.22 ± 0.03*
LysoPS16:0
LysoPS18:1 0.26 ± 0.08 0.40 ± 0.02 0.85 ± 0.10 0.29 ± 0.01*
LysoPS18:0 2.28 ± 0.31 2.38 ± 0.22 3.69 ± 0.44 1.31 ± 0.18*
0.30 ± 0.10 0.61 ± 0.08* 1.43 ± 0.33 0.49 ± 0.07*
Sulfatides 1.01 ± 0.29 2.41 ± 0.41* 5.34 ± 1.47 1.68 ± 0.16*
SL d18:1/18:1 h 5.50 ± 1.41 12.46 ± 1.66* 28.63 ± 6.81 9.02 ± 0.66*
SL d18:1/20:0 4.71 ± 1.13 10.06 ± 1.96* 20.74 ± 5.03 6.83 ± 0.63*
SL d18:1/22:1 1.66 ± 0.42 3.13 ± 0.48* 5.82 ± 1.08 1.97 ± 0.15*
SL d18:1/22:0 3.21 ± 0.77 6.46 ± 0.96* 10.53 ± 2.55 3.53 ± 0.43*
SL d18:1/24:1
SL d18:1/24:0 2.00 ± 0.11 1.57 ± 0.13* 1.90 ± 0.13 1.63 ± 0.06
SL d18:1/24:1 h 3.83 ± 0.22 2.47 ± 0.27* 2.63 ± 0.22 2.43 ± 0.09
SL d18:1/24:0 h 49.67 ± 1.99 35.10 ± 0.86* 37.03 ± 2.51 35.32 ± 2.04
2.30 ± 0.35 3.92 ± 0.06* 8.61 ± 1.92 5.89 ± 0.19
Sphingomyelins
SM18/16:0
SM18/18:1
SM18/18:0
SM18/24:1

123

Lipidomics after rTMS 31

Table 4 continued Treatment
Right brain regions Relative abundance (910-3)
Lipid species

Prefrontal cortex Striatum
Control
Control 800 stimuli (100% intensity) 800 stimuli (100% intensity)

SM18/24:0 1.55 ± 0.26 2.75 ± 0.11* 5.02 ± 0.51 3.72 ± 0.10*
Ceramides
0.31 ± 0.06 0.82 ± 0.22 0.86 ± 0.56 0.81 ± 0.20
Cer d18:1/22:0 7.43 ± 1.34 16.42 ± 2.71* 20.14 ± 6.30 17.37 ± 0.72
Cer d18:1/24:1 2.97 ± 0.19 4.65 ± 0.75* 4.71 ± 0.69
Cer d18:1/24:0 3.99 ± 0.90

All values are expressed as mean relative abundance ± SD. Results for low dose rTMS are not shown. Data were analyzed by one-way ANOVA
followed by FDR analysis. * FDR \ 0.05 as compared with sham controls

Table 5 Examples of possible phospholipid hydrolysis by PLA2 in glial cells (Fujiki and Steward 1997) and this may
enzymes to release long chain fatty acids and lysophospholipids contribute to changes in lipids.

Phospholipid Lysophospholipid Fatty acid side chain being Rats were administered rTMS while anaesthetized to
minimize restraint stress. Since there is a possibility that
released cleaved off via PLA2 activity anaesthetics might affect brain lipid composition, we have
similarly anaesthetized sham treated controls, to ensure that
PE38p:6 16:0p 38p:6 - 16:0p = 22.6 (DHA) any changes observed were due to rTMS treatment. It is
PE38:6 16:0 38:6 - 16:0 = 22:6 (DHA) noted, however, that the results may not be exactly compa-
PE40p:6 18:0p 40p:6 - 18:0p = 22:6 (DHA) rable to humans who are not anaesthetized during rTMS.
PE38p:6 18:1p 38p:6 - 18:1p = 20:5 (EPA) The results of rTMS treatment were different from that
observed after excitotoxicity induced by kainate injections
chain fatty acids in the mouse prefrontal cortex after (Guan et al. 2006). Kainate injections resulted in seizures
chronic antidepressant treatment (Lee et al. 2009). It is and decrease in phospholipids species with polyunsaturated
possible that treatment modalities of depression may fatty acyl chains but increases in ceramide and lysophos-
involve the induction of PLA2 isoforms to endogenously pholipid species, indicating increase in cytosolic phospho-
release long chain fatty acids such as DHA, which have lipase A2 (cPLA2) activity (Guan et al. 2006). In comparison
antidepressant effects (DeMar et al. 2006; Rao et al. 2007). seizures were not observed and general increase rather than
decrease in phospholipids was detected after rTMS.
Interestingly, many of the lipid changes in the striatum
were opposite to that of the prefrontal cortex. PEs and PCs 4 Conclusions
species that were increased in the prefrontal cortex, were
decreased in the striatum after rTMS. In particular, the The present study showed significant changes in rat brain
level of SLs decreased in the left and right striatum after lipids after rTMS. Appropriate control of the proportion of
rTMS, as opposed to its large increases in the left and right false positive results was carried out using false discovery
prefrontal cortices. The reason for the opposing changes in rate to ensure that the multiple comparisons were valid.
the striatum compared to the prefrontal cortex is unknown, While recognizing that rat frontal cortex structure and
but is postulated to be due to the large numbers of medium connectivity to other brain areas differs from the human
spiny GABAergic neurons in these nuclei. These neurons frontal cortex and the results may not be completely
not only send projections to the globus pallidus externa or extrapolated to humans, it is hoped that these findings
the globus pallidus interna/substantia nigra pars reticulata, could provide some insights into possible changes in brain
but also recurrent projections to neighboring medium spiny lipids that contribute to the effects of rTMS in the man-
neurons (Koos and Tepper 1999). It is postulated that agement of neurological disorders. Further studies are
rTMS could result in net inhibition of the striatum due to necessary to elucidate possible changes in signaling path-
stimulation of recurrent GABAergic collaterals of medium ways of SL, plasmalogens, and DHA metabolites after
spiny neurons. In contrast, the treatment is expected to rTMS.
result in excitation of the prefrontal cortex, in view of its
predominant glutamatergic cell population (Celada et al.
2001). Besides neurons, rTMS could affect gene expression

123

32 L. H.-W. Lee et al.

Acknowledgments This work was supported by the National Feng, H. L., Yan, L., & Cui, L. Y. (2008). Effects of repetitive
Medical Research Council (R-181-000-125-275 and R-183-000-224- transcranial magnetic stimulation on adenosine triphosphate
213), National Research Foundation (CRP Award No. 2007-04), content and microtubule associated protein-2 expression after
Biomedical Research Council (R-183-000-211-305) and the Aca- cerebral ischemia-reperfusion injury in rat brain. Chinese
demic Research Fund (R-183-000-160-112). There are no conflicts of Medical Journal (English), 121, 1307–1312.
interest.
Fitzgerald, P. (2008). Brain stimulation techniques for the treatment
References of depression and other psychiatric disorders. Australas Psychi-
atry, 16, 183–190.
Adibhatla, R. M., & Hatcher, J. F. (2007). Role of lipids in brain
injury and diseases. Future Lipidology, 2, 403–422. Fujiki, M., & Steward, O. (1997). High frequency transcranial
magnetic stimulation mimics the effects of ECS in upregulating
Adibhatla, R. M., Hatcher, J. F., & Dempsey, R. J. (2006). Lipids and astroglial gene expression in the murine CNS. Brain Research
lipidomics in brain injury and diseases. AAPS Journal, 8, E314– Molecular Brain Research, 44, 301–308.
E321.
George, M. S., Lisanby, S. H., Avery, D., et al. (2010). Daily left
Aizenstein, H. J., Butters, M. A., Figurski, J. L., Stenger, V. A., prefrontal transcranial magnetic stimulation therapy for major
Reynolds, C. F., I. I. I., & Carter, C. S. (2005). Prefrontal and depressive disorder: A sham-controlled randomized trial.
striatal activation during sequence learning in geriatric depres- Archives of General Psychiatry, 67, 507–516.
sion. Biological Psychiatry, 58, 290–296.
Guan, X. L., He, X., Ong, W. Y., Yeo, W. K., Shui, G., & Wenk, M.
Avery, D. H., Isenberg, K. E., Sampson, S. M., et al. (2008). R. (2006). Non-targeted profiling of lipids during kainate-
Transcranial magnetic stimulation in the acute treatment of induced neuronal injury. FASEB Journal, 20, 1152–1161.
major depressive disorder: Clinical response in an open-label
extension trial. Journal of Clinical Psychiatry, 69, 441–451. Han, X., Cheng, H., Fryer, J. D., Fagan, A. M., & Holtzman, D. M.
(2003). Novel role for apolipoprotein E in the central nervous
Bazan, N. G. (2009). Cellular and molecular events mediated by system. Modulation of sulfatide content. The Journal of Biolog-
docosahexaenoic acid-derived neuroprotectin D1 signaling in ical Chemistry, 278, 8043–8051.
photoreceptor cell survival and brain protection. Prostaglandins
Leukotrienes and Essential Fatty Acids, 81, 205–211. Han, X., Holtzman, D. M., McKeel, D. W., Jr., Kelley, J., & Morris, J.
C. (2002). Substantial sulfatide deficiency and ceramide eleva-
Bestmann, S. (2008). The physiological basis of transcranial magnetic tion in very early Alzheimer’s disease: Potential role in disease
stimulation. Trends in Cognitive Sciences, 12, 81–83. pathogenesis. Journal of Neurochemistry, 82, 809–818.

Bligh, E. G., & Dyer, W. J. (1959). A rapid method of total lipid Honke, K., Zhang, Y., Cheng, X., Kotani, N., & Taniguchi, N. (2004).
extraction and purification. Canadian Journal of Biochemistry Biological roles of sulfoglycolipids and pathophysiology of their
and Physiology, 37, 911–917. deficiency. Glycoconjugate Journal, 21, 59–62.

Bloch, Y., Grisaru, N., Harel, E. V., et al. (2008). Repetitive transcranial Ishizuka, I. (1997). Chemistry and functional distribution of sulfo-
magnetic stimulation in the treatment of depression in adolescents: glycolipids. Progress in Lipid Research, 36, 245–319.
An open-label study. Journal of ECT, 24, 156–159.
Ji, R. R., Schlaepfer, T. E., Aizenman, C. D., et al. (1998). Repetitive
Celada, P., Puig, M. V., Casanovas, J. M., Guillazo, G., & Artigas, F. transcranial magnetic stimulation activates specific regions in rat
(2001). Control of dorsal raphe serotonergic neurons by the brain. Proceedings of the National Academy of Sciences of the
medial prefrontal cortex: Involvement of serotonin-1A, United States of America, 95, 15635–15640.
GABA(A), and glutamate receptors. Journal of Neuroscience,
21, 9917–9929. Kapogiannis, D., & Wassermann, E. M. (2008). Transcranial
magnetic stimulation in clinical pharmacology. Central Nervous
Cotelli, M., Manenti, R., Cappa, S. F., et al. (2006). Effect of System Agents in Medicinal Chemistry, 8, 234–240.
transcranial magnetic stimulation on action naming in patients
with Alzheimer disease. Archives of Neurology, 63, 1602–1604. Kim, E. J., Kim, W. R., Chi, S. E., et al. (2006). Repetitive
transcranial magnetic stimulation protects hippocampal plastic-
DeMar, J. C., Jr., Ma, K., Bell, J. M., Igarashi, M., Greenstein, D., & ity in an animal model of depression. Neuroscience Letters, 405,
Rapoport, S. I. (2006). One generation of n-3 polyunsaturated 79–83.
fatty acid deprivation increases depression and aggression test
scores in rats. Journal of Lipid Research, 47, 172–180. Koch, G., & Rothwell, J. C. (2009). TMS investigations into the task-
dependent functional interplay between human posterior parietal
Drevets, W. C. (2000). Functional anatomical abnormalities in limbic and motor cortex. Behavioural Brain Research, 202, 147–152.
and prefrontal cortical structures in major depression. Progress
in Brain Research, 126, 413–431. Koos, T., & Tepper, J. M. (1999). Inhibitory control of neostriatal
projection neurons by GABAergic interneurons. Nature Neuro-
Eckhardt, M. (2008). The role and metabolism of sulfatide in the science, 2, 467–472.
nervous system. Molecular Neurobiology, 37, 93–103.
Lee, L. H., Shui, G., Farooqui, A. A., Wenk, M. R., Tan, C. H., &
Eschweiler, G. W., Wegerer, C., Schlotter, W., et al. (2000). Left Ong, W. Y. (2009). Lipidomic analyses of the mouse brain after
prefrontal activation predicts therapeutic effects of repetitive antidepressant treatment: Evidence for endogenous release of
transcranial magnetic stimulation (rTMS) in major depression. long-chain fatty acids? The International Journal of Neuropsy-
Psychiatry Research, 99, 161–172. chopharmacology, 10, 1–12.

Farooqui, A. A. (1981). Metabolism of sulfolipids in mammalian Lefaucheur, J. P. (2009). Methods of therapeutic cortical stimulation.
tissues. Advances in Lipid Research, 18, 159–202. Neurophysiologie Clinique, 39, 1–14.

Farooqui, A. A. (2009). Lipid mediators in the neural cell nucleus: Lefaucheur, J. P., Hatem, S., Nineb, A., et al. (2006). Somatotopic
Their metabolism, signaling, and association with neurological organization of the analgesic effects of motor cortex rTMS in
disorders. Neuroscientist, 15, 392–407. neuropathic pain. Neurology, 67, 1998–2004.

Farooqui, A. A., & Horrocks, L. A. (2001). Plasmalogens, phospho- Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene
lipase A2, and docosahexaenoic acid turnover in brain tissue. expression data using real-time quantitative PCR and the 2(-
Journal of Molecular Neuroscience, 16, 263–272. (discussion Delta Delta C(T)) method. Methods, 25, 402–408.
279–284).
Mozzi, R., & Buratta, S. (2010). Brain phosphatidylserine: Metab-
olism and functions. In A. Lajtha, G. Tettamanti, & G. Goracci
(Eds.), Handbook of neurochemistry and molecular neurobiol-
ogy: Neural lipids (pp. 39–59). New York: Springer.

123

Lipidomics after rTMS 33

Muller, M. B., Toschi, N., Kresse, A. E., Post, A., & Keck, M. E. related cortical activity in humans. Cerebral Cortex, 10,
(2000). Long-term repetitive transcranial magnetic stimulation 802–808.
increases the expression of brain-derived neurotrophic factor and Sachdev, P. S., Loo, C. K., Mitchell, P. B., McFarquhar, T. F., &
cholecystokinin mRNA, but not neuropeptide tyrosine mRNA in Malhi, G. S. (2007). Repetitive transcranial magnetic stimulation
specific areas of rat brain. Neuropsychopharmacology, 23, for the treatment of obsessive compulsive disorder: A double-
205–215. blind controlled investigation. Psychological Medicine, 37,
1645–1649.
O’Reardon, J. P., Solvason, H. B., Janicak, P. G., et al. (2007). Sapolsky, R. M. (2001). Depression, antidepressants, and the
Efficacy and safety of transcranial magnetic stimulation in the shrinking hippocampus. Proceedings of the National Academy
acute treatment of major depression: A multisite randomized of Sciences of the United States of America, 98, 12320–12322.
controlled trial. Biological Psychiatry, 62, 1208–1216. Schutter, D. J. (2009). Antidepressant efficacy of high-frequency
transcranial magnetic stimulation over the left dorsolateral
Padberg, F., Zwanzger, P., Keck, M. E., et al. (2002). Repetitive prefrontal cortex in double-blind sham-controlled designs: A
transcranial magnetic stimulation (rTMS) in major depression: meta-analysis. Psychological Medicine, 39, 65–75.
Relation between efficacy and stimulation intensity. Neuropsy- Shui, G., Bendt, A. K., Pethe, K., Dick, T., & Wenk, M. R. (2007).
chopharmacology, 27, 638–645. Sensitive profiling of chemically diverse bioactive lipids.
Journal of Lipid Research, 48, 1976–1984.
Pascual-Leone, A., Rubio, B., Pallardo, F., & Catala, M. D. (1996). van Zyl, R., Gieselmann, V., & Eckhardt, M. (2010). Elevated
Rapid-rate transcranial magnetic stimulation of left dorsolateral sulfatide levels in neurons cause lethal audiogenic seizures in
prefrontal cortex in drug-resistant depression. Lancet, 348, mice. Journal of Neurochemistry, 112, 282–295.
233–237. Vance, J. E. (2008). Phosphatidylserine and phosphatidylethanol-
amine in mammalian cells: Two metabolically related amino-
Paxinos, G., & Watson, C. (1986). The rat brain in stereotaxic phospholipids. Journal of Lipid Research, 49, 1377–1387.
coodinates. Sydney: Academic Press. Watson, A. D. (2006). Thematic review series: Systems biology
approaches to metabolic and cardiovascular disorders. Lipido-
Ramakrishnan, H., Hedayati, K. K., Lullmann-Rauch, R., et al. mics: A global approach to lipid analysis in biological systems.
(2007). Increasing sulfatide synthesis in myelin-forming cells of Journal of Lipid Research, 47, 2101–2111.
arylsulfatase A-deficient mice causes demyelination and neuro- Wenk, M. R. (2005). The emerging field of lipidomics. Nature
logical symptoms reminiscent of human metachromatic leuko- Reviews Drug Discovery, 4, 594–610.
dystrophy. Journal of Neuroscience, 27, 9482–9490. Ziemann, U. (2004). TMS induced plasticity in human cortex.
Reviews in the Neurosciences, 15, 253–266.
Rao, J. S., Ertley, R. N., Lee, H. J., et al. (2007). n-3 polyunsaturated
fatty acid deprivation in rats decreases frontal cortex BDNF via a
p38 MAPK-dependent mechanism. Molecular Psychiatry, 12,
36–46.

Rossi, S., Pasqualetti, P., Rossini, P. M., et al. (2000). Effects of
repetitive transcranial magnetic stimulation on movement-

123

点击阅读翻页书版本