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!

14. Systems wide analyses of lipids in the brainstem during inflammatory orofacial pain - evidence of increased phospholipase A(2) activity.—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
点击进入免费阅读翻页书版本
喜欢这样的3D电子杂志?您也可以在几分钟内把文档免费上传到云展网变成翻页书![点击上传我的文档]
8787 上传于 2018-09-07 12:42:30

14. Systems wide analyses of lipids in the brainstem during inflammatory orofacial pain - evidence of increased phospholipase A(2) activity.

ORIGINAL ARTICLE

Systems wide analyses of lipids in the brainstem during
inflammatory orofacial pain – Evidence of increased
phospholipase A2 activity

M.-T. Ma1, J.-F. Yeo1, G. Shui2,5, M.R. Wenk2,3,5, W.-Y. Ong4,5

1 Department of Oral and Maxillofacial Surgery, National University of Singapore, Singapore
2 Department of Biochemistry, National University of Singapore, Singapore
3 Department of Biological Sciences, National University of Singapore, Singapore
4 Department of Anatomy, National University of Singapore, Singapore 119260, Singapore
5 Neurobiology and Ageing Research Programme, National University of Singapore, Singapore

Correspondence Abstract
Wei-Yi Ong
Department of Anatomy, National University Recent studies suggest that CNS phospholipase A2 (PLA2) isoforms play a
of Singapore, Singapore 119260, Singapore. role in nociception, but until now, direct evidence of increased brain PLA2
Tel.: +65 65163662; fax: +65 67787643. activity during allodynia or hyperalgesia is lacking. The present study was
E-mail: wei_yi_ong@nuhs.edu.sg carried out, using lipidomics or systems wide analyses of lipids using
tandem mass spectrometry, to elucidate possible changes in rat brain lipids
Funding sources after inflammatory pain induced by facial carrageenan injection. The
None caudal medulla oblongata showed decreases in phospholipids including
phosphatidylethanolamine and phosphatidylinositol species, but increases
Conflicts of interest in lysophospholipids, including lysophosphatidylethanolamine, lysophos-
None declared phatidylinositol and lysophosphatidylserine, indicating increased PLA2
activity and release of arachidonic acid after facial carrageenan injection.
Accepted for publication These changes likely occur in the spinal trigeminal nucleus which relays
23 June 2011 nociceptive input from the orofacial region. High levels of sPLA2-III, sPLA2-
XIIA, cPLA2 and iPLA2 mRNA expression were detected in the medulla
doi:10.1016/j.ejpain.2011.06.013 oblongata. Increase in sPLA2-III mRNA expression was found in the caudal
medulla of carrageenan-injected rats, although no difference in sPLA2-III
protein expression was detected. The changes in lipids as determined by
lipidomics were therefore consistent with an increase in PLA2 enzyme
activity, but no change in enzyme protein expression. Together, these
findings indicate enhanced PLA2 activity in the caudal medulla oblongata
after inflammatory orofacial pain.

1. Introduction COX-2 to prostaglandins which regulate inflammation
and signal transduction.
Recent studies have emphasized the importance of
specific phospholipase A2 (PLA2) isoforms in neurode- cPLA2 is expressed at low levels in the forebrain, but
generative and neuropsychiatric conditions (Ong at higher levels in the brainstem and spinal cord (Kim
et al., 2010; Sun et al., 2010). PLA2 are esterases that et al., 2008; Ong et al., 1999, 2010). This isoform is
specifically cleave the acyl ester bond at the sn-2 posi- involved in acute and chronic neurodegenerative
tion of glycerophospholipids to produce a free fatty diseases and spinal nociceptive processing. Enzyme
acid e.g. arachidonic acid (AA) and lysophospholipid. activity in the spinal cord and pain level after carragee-
The isoforms include cytosolic PLA2 (cPLA2), calcium- nan and formalin injections to the hind paw
independent PLA2 (iPLA2) and secretory PLA2 (sPLA2). are significantly reduced after intrathecal injection
AA is metabolized by cyclooxygenase-1 (COX-1) or of cPLA2 inhibitors (Lucas et al., 2005). Moreover,
knockdown of cPLA2 in the rat spinal cord with

38 Eur J Pain 16 (2012) 38–48 © 2011 European Federation of International Association for the Study of Pain Chapters

M.-T. Ma et al. Systems wide analyses of lipids in the brainstem during inflammatory orofacial pain

antisense oligonucletotide significantly attenuates tails, to allow the behavioral responses of an indi-
formalin-induced hyperalgesia (Kim et al., 2008). vidual rat to be followed at different time intervals.
Another isoform, iPLA2 is highly expressed in the CNS
(Ong et al., 2005, 2010). This enzyme is required for Treated rats received facial injections of lambda car-
repair of basal lipid peroxidation and maintenance of rageenan, Sigma-Aldrich, St. Louis, MI, USA (0.6 ml
mitochondrial function after oxidative stress (Kinsey of 80 mg/ml in total, injected over the cutaneous dis-
et al., 2008). Treatment of PC12 cells with the iPLA2 tribution of the trigeminal nerve on the right side).
inhibitor bromoenol lactone results in loss of mito- The rats were anesthetized with an intraperitoneal
chondrial membrane potential and excessive exocyto- injection of ketamine and xylazine cocktail and carra-
sis (Ma et al., 2010b). sPLA2 consists of a family of low geenan was injected in the subcutaneous tissue on the
molecular weight (14–19 kDa) enzymes, sPLA2-IB, right side of the face above the eye, on the upper jaw
-IIA, -IIC, -IID, - IIE, -IIF, -III, -V, -X and -XIIA (Gelb below the eye, and on the lower jaw. The carrageenan
et al., 2000; Ho et al., 2001; Six and Dennis, 2000). injection produced local inflammation and swelling of
sPLA2-IIA is found in snake venom whilst sPLA2-III is approximately 4 mm in diameter at each injected site,
present in bee venom (Telleria-Diaz et al., 2010). and allodynia in the days following the injection
sPLA2 is associated with physiological, pathological, (Ng and Ong, 2001). The rats were assessed for
and toxic processes. For example, sPLA2-IIA from responses to von Frey hair stimulation of the face
human synovial fluid plays a role in inflammation, in before injections, and from 1 to 8 days after injections
rheumatoid arthritis (Jacques and Berenbaum, 1996). (Poh et al., 2009). All procedures involving rats were
in accordance with guidelines of the Committee for
Mice that were intracerebroventricularly injected Research and Ethical Issues of IASP and approved by
with cPLA2 or sPLA2 inhibitors showed decreased the Institutional Animal Care and Use Committee of
responses to von Frey hair stimulation after facial NUS.
carrageenan injection (Yeo et al., 2004). Similarly,
intrathecal injection of the sPLA2 inhibitor LY311727 2.2 Assessment of responses to
reduced nociception after inflammation-induced mechanical stimulations
hyperalgesia of the rat hind paw (Svensson et al.,
2005). The above studies point to an important role of All assessment procedures were carried out in a blinded
CNS PLA2 in nociception. This implies changes in brain manner as previously described (Vos et al., 1994). Rats
lipids, in particular, phospholipids and lysophospho- were tested individually in a deep rectangular stainless
lipids, but until now, direct evidence of increased brain steel tank (60 ¥ 40 ¥ 25 cm). The experimenter
PLA2 activity during hyperalgesia is lacking. reached into the tank with a von Frey hair to habituate
the rats to the reaching movements for 5–10 min
The present study was carried out using lipidomics, before testing. The rats were observed during this time,
or systems wide analysis and characterization of lipids to ensure that they were able to move freely, and had
by tandem mass spectrometry analyses with multiple no obvious motor deficits. The test stimulations were
reaction monitoring, to elucidate possible changes in administered when the rats were in a no-locomotion
brain lipids at the individual molecular species level state, with four paws placed on the ground, neither
(Adibhatla et al., 2006; Watson, 2006; Wenk, 2005), moving nor freezing, but exhibiting sniffing behavior. A
after inflammatory pain induced by facial carrageenan new stimulus was applied only when the rats resumed
injection. Possible changes in gene expression of PLA2 this position after the preceding stimulation. The
isoforms in medulla oblongata were also elucidated. carrageenan-injected area of the face was probed 20
times with a von Frey hair (Touch-Test Sensory Evalu-
2. Materials and methods ator, North Coast Medical, Morgan Hill, USA). A fila-
ment delivering 60 g force (5.88 log units) was used.
2.1 Time course study of pain responses after The responses after each stimulus were scored based on
facial carrageenan injection the criteria for “increasing” nociceptive responses (Vos
et al., 1994). These were, “no response” (score of 0),
Sixteen adult male Wistar rats weighing 200 g each “non-aversive response” or detection (score of 1), “mild
were used for this portion of the study. The rats were aversive response” or withdrawal (score of 2), “strong
purchased from the Laboratory Animal Centre, Sin- aversive response” or attack/escape (score of 3) and
gapore and divided into two groups of eight right facial “prolonged aversive response” or facial scratching/
carrageenan-injected and eight uninjected controls. grooming (score of 4). Since the number of face
All rats were labeled with a coded number tag on their scratches after each stimulus was also indicative of the

Eur J Pain 16 (2012) 38–48 © 2011 European Federation of International Association for the Study of Pain Chapters 39

Systems wide analyses of lipids in the brainstem during inflammatory orofacial pain M.-T. Ma et al.

degree of nociception these were counted, and multi- USA). Samples were vortexed and incubated on ice for
plied by the score of four (Vos et al., 1994). The scores 15 min with vortexing done at every 5 min interval.
after each probe were added, and the total score after 20 250 ml of chloroform and 450 ml of 0.88% KCl were
probes calculated to obtain the response of each rat. The then added, and samples vortexed and incubated on ice
mean and standard deviation of the total number of for 1 min. Lipids were isolated from the organic phase
responses of eight rats in each treatment group was after centrifugation (9000 rpm, 4 °C, 2 min). Samples
then calculated for each post-injection time interval, were then vacuum dried (Thermo Savant SpeedVac,
and possible significant differences between the Waltham, MA, USA), resuspended in chloroform-
carrageenan- injected and untreated groups elucidated methanol (1 : 1 v/v), and used for analysis.
using two-tailed unpaired Student’s t-test. P < 0.05 was
considered significant. 2.3.3 Analysis of lipids using liquid
chromatography/mass spectrometry
2.3 Lipidomic analyses
An Agilent high performance liquid chromatography
Fourteen rats consisting of seven right facial (HPLC) system coupled with an Applied Biosystem
carrageenan-injected and seven uninjected controls Triple Quadrupole/Ion Trap mass spectrometer
were used for this part of the study. The former were (4000Qtrap, Foster City, CA, USA) was used for quan-
sacrificed at 3 days post-carrageenan injection i.e. the tification of individual polar lipids. Samples were
estimated time of peak responses after facial carragee- introduced into the mass spectrometer by loop injec-
nan injection. The rats were anesthetized with an tions with chloroform : methanol (1 : 1) as a mobile
intraperitoneal injection of ketamine and xylazine phase for positive ESI mode and chloroform : metha-
cocktail and killed by decapitation. The medulla nol: 200 mM piperidine (1 : 1 : 0.1) as a mobile phase
oblongata was divided into a rostral portion (RM) and for negative ESI mode, respectively, both at a flow of
caudal portion (CM) which included the spinal 250 ml min-1 (Shui et al., 2007). Based on product ion
trigeminal nucleus for relay of nociceptive inputs from and precursor ion analyses of head groups, two com-
the orofacial region, by the caudal limit of the fourth prehensive sets of multiple reaction monitoring tran-
ventricle. The right half of the RM or CM were dis- sitions were set up for quantitative analysis of various
sected out, snap frozen in liquid nitrogen and kept at lipids including phosphatidylcholines (PCs), phos-
-80 °C till analyses. phatidylethanolamines (PEs), phosphatidylserines
(PSs), phosphatidylinositols (PIs), phosphatidylgly-
2.3.1 Internal standards cerols (PGs), phosphatidic acids (PAs), sphingomyelins
(SMs), ceramides (Cers) and sulfatides (SLs) (Fei et al.,
Levels of individual lipids were quantified using spiked 2008). The signal intensity of each MRM value was
internal standards including C14-phosphatidylcholine normalized using the following equation:
dimyristoyl phosphatidylcholine (28:0-PC), dimyris-
toyl phosphatidylethanolamine (28:0-PE), dimyristoyl Relative abundance of lipid 1
C14-phosphatidylserine (28:0-PS), dimyristoyl phos-
phatidylglycerol (28:0-PG), dimyristoyl phosphatidic ∑= Signal intensity of lipid 1
acid (28:0-PA) and C19-ceramide (C19-CER), which Signal intensity of all MRM transitions measured
were obtained from Avanti Polar Lipids (Alabaster, AL,
USA). Dioctanoyl phosphatidylinositol (PI, 16:0-PI) The mean and standard deviation of the relative
was used for phosphatidylinositol quantitation and abundance of lipid of seven rats from each treatment
obtained from Echelon Biosciences, Inc. (Salt Lake group was calculated, and possible significant differ-
City, UT, USA). Sulfatides were analyzed using nor- ences between carrageenan-injected and untreated
malized intensity as well as C14-phosphatidylserine rats analyzed using two-tailed unpaired Student’s
(28:0-PS) as an internal standard. t-test. P < 0.05 was considered significant.

2.3.2 Lipid extraction 2.4 Real-time RT-PCR

A widely-used protocol for lipid extraction was used Six facial carrageenan-injected and six uninjected con-
(Bligh and Dyer, 1959). The tissue samples were trols were used for this part of the study. The former
homogenized in 750 ml of chloroform–methanol, 1 : 2 were sacrificed at 3 days post-carrageenan injection.
(v/v) using Tissue Tearor™ (Biospec. Inc., Bartlesville, The rats were anesthetized and killed by decapitation.
The right half of the RM or CM were quickly removed

40 Eur J Pain 16 (2012) 38–48 © 2011 European Federation of International Association for the Study of Pain Chapters

M.-T. Ma et al. Systems wide analyses of lipids in the brainstem during inflammatory orofacial pain

and immersed in RNAlater (Ambion, TX, USA), snap polyvinylidene difluoride (PVDF) membrane (Amer-
sham Pharmacia Biotech, Little Chalfont, UK). Non-
frozen in liquid nitrogen and kept at -80 °C till analy- specific binding sites on the PVDF membrane were
blocked by incubation with 5% non-fat milk in 0.1%
ses. Total RNA was extracted and isolated using Tween-20 TBS (TTBS) for 1 h. The PVDF membrane
was incubated overnight at 4 °C with goat polyclonal
TRizol reagent (Invitrogen, CA, USA) according to antibody to sPLA2-III (Santa Cruz, Delaware, CA,
USA) diluted to 1 : 500 in 5% non-fat milk in TTBS.
the manufacturer’s protocol. RNeasy1 Mini Kit Negative controls were carried out by incubation with
sPLA2-III antigen-absorbed antibody, prepared by
(Qiagen, Inc., CA, USA) was used to purify the preincubating sPLA2-III antibody with 20 times con-
centration of immunizing peptide (Santa Cruz), over-
RNA. The samples were then reverse transcribed using night. After washing with TTBS, the membrane
was incubated with horseradish peroxidase conju-
High-Capacity cDNA Reverse Transcription Kits gated anti-goat immunoglobulin IgG (Amersham,
Pharmacia Biotech) for 1 h at room temperature. The
(Applied Biosystems, CA, USA). Reaction conditions protein was visualized with an enhanced chemilumi-
nescence kit (Pierce, Rockford, IL, USA) according to
were 25 °C for 10 min, 37 °C for 120 min and 85 °C the manufacturer’s instructions. The densities of the
sPLA2-III bands were normalized against those of
for 5 s. Real-time PCR amplification was then b-actin, and the mean ratios calculated. Possible sig-
nificant differences between carrageenan-injected
carried out in the 7500 Real time PCR system using and untreated rats were analyzed using two-tailed
unpaired Student’s t-test. P < 0.05 was considered
TaqMan1 Universal PCR Master Mix (Applied Biosy- significant.

stems), and sPLA2-IB (Rn00580896_m1), sPLA2-IIA 2.6 Immunohistochemistry of sPLA2-III

(Rn00580999_m1), sPLA2-IIC (Rn01520676_m1), Four facial carrageenan-injected and four uninjected
controls were used for this part of the study. The
sPLA2-IID (Rn01520520_m1), sPLA2-IIE(Rn01516481 former were sacrificed at 3 days post-carrageenan
injection. The rats were deeply anesthetized and per-
_m1), sPLA2-IIF (Rn01516171_m1), sPLA2-III fused through the left ventricle with a solution of 4%
paraformaldehyde in 0.1 M phosphate buffer (pH 7.4).
(Rn01442985_m1), sPLA2-V (Rn00567782_m1), The CM was removed and sectioned coronally at
100 mm using a vibrating microtome. The sections
sPLA2-X (Rn00691350_m1), sPLA2-XIIA were washed for 3 h in phosphate-buffered saline con-
taining. They were then incubated overnight with
(Rn01518649_m1), cPLA2 (Rn00591916_m1), iPLA2 goat anti-sPLA2-III antibody (Santa Cruz) diluted to
(Rn00588064_m1) or rat b-actin probes, according to 1 : 50 in 0.15 M NaCl in 0.05 M Tris buffer, pH 8.6,
containing 1% bovine serum albumin, followed by
the manufacturer’s instructions. The PCR conditions three washes in PBS and incubation for 1 h at room
temperature in a 1 : 200 dilution of biotinylated horse
were: an initial incubation of 50 °C for 2 min and anti-goat IgG (Vector, Burlingame, CA, USA). The
sections were reacted for 1 h at room temperature
95 °C for 10 min followed by 40 cycles of 95 °C for with an avidin-biotinylated horseradish peroxidase
complex, and visualized by treatment for 5 min in
15 s and 60 °C for 1 min. All reactions were carried 0.05% 3,3-diaminobenzidine tetrahydrochloride solu-
tion in Tris buffer containing 0.05% hydrogen pero-
out in triplicate. The threshold cycle, CT, which corre- xide. The color reaction was stopped with several
washes of Tris buffer. Some sections were mounted on
lates inversely with the levels of target mRNA, was glass slides and lightly counterstained with methyl
green before coverslipping. Control sections were
measured as the number of cycles at which the

reporter fluorescence emission exceeds the preset

threshold level. Amplified transcripts were quantified

using the comparative CT method (Livak and Schmit-

tgen, 2001), with relative fold change = 2-DDCT. The

mean and standard deviation of the relative fold

were calculated, and possible significant differences

between carrageenan-injected and untreated rats

analyzed using two-tailed unpaired Student’s t-test.

P < 0.05 was considered significant.

2.5 Western blot analysis

Four facial carrageenan-injected and four uninjected
controls were used for this part of the study. The
former were sacrificed at 3 days post-carrageenan
injection. The right half of the CM was dissected out
and homogenized in 10 volumes of ice-cold buffer
containing 0.32 M sucrose, 4 mM Tris–Cl, pH 7.4,
1 mM EDTA, and 0.25 mM dithiothreitol. After cen-
trifugation at 12,000 g for 30 min, the supernatant
was collected and protein concentrations in the prepa-
ration were measured using the BioRad protein assay
kit (Bio-Rad Laboratories, CA, USA). Total proteins
(40 mg) were resolved in 10% SDS polyacrylamide gels
under reducing conditions and electrotransferred to a

Eur J Pain 16 (2012) 38–48 © 2011 European Federation of International Association for the Study of Pain Chapters 41

Systems wide analyses of lipids in the brainstem during inflammatory orofacial pain M.-T. Ma et al.

Figure 1 Responses to von Frey hair stimula-
tion of the face after tissue inflammation
induced by carrageenan injection. The Y-axis
represents the total number of responses to
von Frey hair stimulation of the carrageenan-
injected areas of the face. BI: before injection,
1D–8D refers to 1 day to 8 days after injec-
tion. U: untreated control. C: after facial car-
rageenan injection. Analyzed by two-tailed
unpaired Student’s t-test. Asterisks indicate
significant difference (P < 0.05) between
untreated and carrageenan-injected rats.

incubated with antigen-absorbed antibody instead of injected rats compared to controls. This was
primary antibody. accompanied by significant increase in relative abun-
dance of lysoPE18:0p, lysoPE18:1 and lysoPE18:0,
3. Results lysoPI18:0 and lysoPI20:4 indicating increased PLA2
activity (Fig. 2; Table 1).
3.1 Time course study of pain responses after
facial carrageenan injection (Fig. 1) Carrageenan-injected rats showed decrease in PC
species (PC36:4p), but this was not accompanied by
Increase in the total number of responses to mechani- increase in lysoPC species observed in the CM.
cal stimulation was detected after facial carrageenan LysoPS18:0 showed increase in relative abundance
injection (n = 8) with peak responses at three post- after treatment (Fig. 3; Table 1).
injection days, as compared to uninjected controls
(n = 8). The number of responses declined to baseline 3.3 mRNA expression of PLA2 isoforms in the
by the 8th post-injection day (Fig. 1). medulla oblongata (Fig. 4)

3.2 Lipidomic analyses (Figs. 2 and 3 and The mRNA expression of various PLA2 isoforms
Table 1) were normalized to the lowest level of message
among the isoforms (apart from sPLA2-X, which
Significantly increased response to mechanical stimu- was nearly undetectable) i.e. the value for sPLA2-IIF
lation was observed in rats which were sacrificed at 3 in untreated rats, to indicate relative expression
days post-carrageenan injection (data not shown). of the isoforms, as well as between untreated and
Reduction in the relative abundance of PE (PE38p:6, carrageenan-injected rats (Fig. 4A and B). sPLA2-III
PE38p:4, PE38:7) (p indicating plasmalogen species) showed the highest mRNA expression among the
PS (PS38:6, PS40:7) and PC species (PC36:3p and PLA2 isoforms in the CM of control rats (n = 6).
PC36:4p), and increase in abundance of PE44:9 and Its expression was increased by 1.3 folds in rats
PE44:8 were detected in the RM of carrageenan- that received facial carrageenan injection (n = 6)
treated rats (n = 7) compared to controls (n = 7). (Fig. 4B).
Decrease in relative abundance of lysoPI20:0,
lysoPC20:4 were observed after carrageenan treat- 3.4 sPLA2-III protein expression and
ment. Cer species, Cer d18:1/24:1 showed increase localization in the CM (Fig. 5)
after treatment.
The antibody to sPLA2-III detected a band at 19 kDa in
Reduction in the relative abundance of the PE homogenates from the CM, consistent with the
species (PE34p:1, PE34:1, PE36p:4, PE38p:6, expected molecular weight of the active sPLA2 domain
PE38p:5, PE38p:4, PE38:7, PE38:6, PE42p:6, PE42:4, of sPLA2-III. High level of expression was detected in
PE44:10) and increase in abundance of PI species both untreated (n = 4) and carrageenan-injected rats
PI36:1 were detected in the CM of carrageenan- (n = 4). No significant difference in protein expression

42 Eur J Pain 16 (2012) 38–48 © 2011 European Federation of International Association for the Study of Pain Chapters

M.-T. Ma et al. Systems wide analyses of lipids in the brainstem during inflammatory orofacial pain

Figure 2 Lipidomic analysis of changes in selected lipids in right half of caudal medulla oblongata (CM) sacrificed at 3 days post-carrageenan injection.
Increased PLA2 activity was observed in the CM of carrageenan-injected rats which showed significant decrease in phospholipids including
PE and PI species, but increase in lysoPE and lysoPI species compared to the untreated rats. Analyzed by two-tailed unpaired Student’s t-test.
Asterisks indicate significant difference (P < 0.05) between untreated and carrageenan-injected rats. LysoPE, lysophosphatidylethanolamine;
LysoPI, Lysophosphatidylinositol.

Eur J Pain 16 (2012) 38–48 © 2011 European Federation of International Association for the Study of Pain Chapters 43

Systems wide analyses of lipids in the brainstem during inflammatory orofacial pain M.-T. Ma et al.

Figure 3 Lipidomic analysis of changes in selected lipids in the caudal medulla oblongata (CM) sacrificed at 3 days post-carrageenan injection. The
carrageenan-injected rats showed significant decrease in phospholipids PC species and significant increase in lysoPS species were observed. Analyzed by
two-tailed unpaired Student’s t-test. Asterisks indicate significant difference (P < 0.05) between untreated and carrageenan-injected rats.
LysoPC, lysophosphatidylcholine; LysoPS, lysophosphatidylserine.

44 Eur J Pain 16 (2012) 38–48 © 2011 European Federation of International Association for the Study of Pain Chapters

M.-T. Ma et al. Systems wide analyses of lipids in the brainstem during inflammatory orofacial pain

Table 1 Changes in selected lipids in the rostral medulla oblongata and was found between the two groups (Fig. 5A). No
caudal medulla oblongata after facial carrageenan-injection. Analyzed by bands were detected in blots incubated with antigen-
two-tailed unpaired Student’s t-test. Asterisks indicate significant differ- absorbed antibody, indicating specificity of the anti-
ence (P < 0.05) between untreated and carrageenan-injected rats. body (Fig. 5A).

Relative abundance (mean Ϯ standard deviation ¥ 10-3) Dense immunostaining to sPLA2-III was observed in
the spinal trigeminal nucleus of sections from
Lipid species Untreated Carrageenan untreated (n = 4) (Fig. 5B) or facial carrageenan
injected injected rats (n = 4) (Fig. 5C). No difference in staining
was detected between the two groups. Sections incu-
Rostral medulla oblongata 7.70 Ϯ 0.24 7.20 Ϯ 0.25* bated with antigen-absorbed antibody showed
Phosphatidylethanolamines 20.48 Ϯ 0.50 19.04 Ϯ 0.77* absence of labeling (Fig. 5D).

PE38p:6 0.56 Ϯ 0.03 0.48 Ϯ 0.02* 4. Discussion
PE38p:4 1.24 Ϯ 0.17 1.45 Ϯ 0.07*
PE38:7 1.56 Ϯ 0.27 1.95 Ϯ 0.15* The present study was carried out to elucidate possible
PE44:9 changes in brain lipids and changes in expression of
PE44:8 0.47 Ϯ 0.02 0.42 Ϯ 0.03* PLA2 isoforms in the medulla oblongata after inflam-
Phosphatidylserines 0.36 Ϯ 0.02 0.32 Ϯ 0.01* matory pain induced by facial carrageenan injection.
PS38:6 Facial injection of carrageenan resulted in increased
PS40:7 0.50 Ϯ 0.17 0.15 Ϯ 0.13* nociception, as demonstrated by increased responses
Lysophosphatidylinositols to von Frey hair stimulation. The peak responses
LysoPI20:0 2.24 Ϯ 0.25 1.90 Ϯ 0.13* occurred at 3 days post-injection and declined to base-
Phosphatidylcholines 1.22 Ϯ 0.07 1.0 Ϯ 0.15* line values by the 8th post-injection day. The remain-
PC36:3p ing carrageenan-injected rats were sacrificed and brain
PC38:4p 0.14 Ϯ 0.03 0.09 Ϯ 0.01* samples obtained at three post-injection days.
Lysophosphatidylcholines
LysoPC20:4 1.86 Ϯ 0.33 2.86 Ϯ 0.32* Lipidomic analysis showed significant decrease in
Ceramides phospholipids including PE and PI species, but
Cer d18:1/24:1 32.3 Ϯ 0.54 30.5 Ϯ 1.21* increase in lysoPE and lysoPI species in the CM, com-
Caudal medulla oblongata 2.31 Ϯ 0.13 2.12 Ϯ 0.10* pared to control rats. The decrease in phospholipids but
Phosphatidylethanolamines 6.77 Ϯ 0.36 5.70 Ϯ 0.26* increase in the corresponding lysophospholipids pro-
PE34p:1 7.26 Ϯ 0.27 6.21 Ϯ 0.28* vides evidence of increased PLA2 activity after carragee-
PE34:1 12.34 Ϯ 1.02 10.97 Ϯ 0.37* nan treatment. The changes likely occur in the spinal
PE36p:4 20.71 Ϯ 1.31 18.18 Ϯ 0.73* trigeminal nucleus which relays nociceptive inputs
PE38p:6 0.51 Ϯ 0.04 0.44 Ϯ 0.04* from the orofacial region, and occupies a large propor-
PE38p:5 3.65 Ϯ 0.20 3.26 Ϯ 0.17* tion of the CM in rats. Based on the decrease in PE
PE38p:4 0.67 Ϯ 0.04 0.61 Ϯ 0.02* species (PE38p:6, PE38p:5, PE38p:4, PE38:7, PE38:6,
PE38:7 0.32 Ϯ 0.03 0.27 Ϯ 0.03* PE42p:6, PE42:4, PE44:10) but increases in lysophos-
PE38:6 0.71 Ϯ 0.06 0.61 Ϯ 0.04* pholipid species, i.e. lysoPE18:0p, lysoPE18:1 and
PE42p:6 lysoPE18:0 in the CM of the carrageenan treated rats,
PE42:4 12.47 Ϯ 0.60 13.73 Ϯ 1.04* the identity of the fatty-acid side-chain being released
PE44:10 0.43 Ϯ 0.03 0.50 Ϯ 0.04* can be deduced. A major fatty acid released is:
Lysphosphatidylethanolamines 0.85 Ϯ 0.08 1.04 Ϯ 0.08* PE38p:4–lysoPE18:0p = 20:4 or arachidonic acid (AA).
LysoPE18:0p The latter is processed by cyclooxygenase-1 (COX-1)
LysoPE18:1 0.09 Ϯ 0.00 0.11 Ϯ 0.01* or COX-2 to generate prostaglandins (Capper and
LysoPE18:0 Marshall, 2001; Kujubu et al., 1991).
Lysophosphatidylserines 0.45 Ϯ 0.03 0.50 Ϯ 0.02*
LysoPS18:0 COX-1 mRNA expression was significantly upregu-
Phosphatidylinositol 0.22 Ϯ 0.03 0.29 Ϯ 0.04* lated in the spinal cord after formalin injection into
PI36:1 0.04 Ϯ 0.00 0.05 Ϯ 0.00* the hind paw (Zhang et al., 2007). Likewise, COX-2
Lysophosphatidylinositols was upregulated in the spinal cord of rats after periph-
LysoPI18:0 0.37 Ϯ 0.05 0.29 Ϯ 0.02* eral inflammation induced by complete Freund’s adju-
LysoPI20:4 vant injection into the hind paw (Seybold et al.,
Phosphatidylcholines 2003). Expression was induced by the p38 mitogen-
PC36:4p activated kinase pathway (Fitzsimmons et al., 2010).

*Significant difference compared to untreated rats (P < 0.05 by two-tailed
unpaired Student’s t-test).

Eur J Pain 16 (2012) 38–48 © 2011 European Federation of International Association for the Study of Pain Chapters 45

Systems wide analyses of lipids in the brainstem during inflammatory orofacial pain M.-T. Ma et al.

Figure 4 Real-time RT PCR analysis of differ-
entially expressed PLA2 subgroups in the
rostral medulla oblongata (RM) (A) and caudal
medulla oblongata (CM) (B). The values were
normalized to the lowest level of message
among the isoforms in the normal medulla
oblongata. Data represents the mean and
standard deviation of six rats. However, due
to small standard deviation, they are not
visible. Analyzed by two-tailed unpaired
Student’s t-test. Asterisks indicate significant
difference (P < 0.05) between untreated and
carrageenan-injected rats.

Intrathecal injection of PLA2 and COX-2 inhibitors able to cause inflammation at nanomolar and micro-
attenuated hyperalgesia produced by peripheral molar concentrations (Saxena, 2000; Wise, 2006;
inflammation (Samad et al., 2001; Svensson and Mechiche et al., 2003). Together with the present
Yaksh, 2002; Telleria-Diaz et al., 2010). Carrageenan results, these findings indicate increased CNS PLA2
injection to the hind paw resulted in increased lipid activity, resulting in generation of metabolites that
metabolites of the COX and 12-lipoxygenase pathways contribute to allodynia after peripheral inflammation.
(Buczynski et al., 2010). AA and its metabolites such
as PEG2 and leukotrienes are biologically active and Studies using inhibitors to PLA2 isoforms, in
particular cPLA2 and sPLA2 in pain models have

Figure 5 A: Western blot analyses of sPLA2-
III protein expression in different parts of the
rat caudal medulla oblongata (CM). The anti-
body to sPLA2-III detects a band at 19 kDa in
homogenates from CM (A) consistent with
the expected molecular weight of the active
form of sPLA2-III. Right panel indicates the
negative control incubated with sPLA2-III
antigen-absorbed antibody. (B and C) Light
micrographs of sPLA2-III immunolabeled sec-
tions from untreated and carrageenan-
injected rat CM. Dense staining is observed in
the spinal trigeminal nucleus in both normal
(B, asterisk) and treated (C, asterisk) sections
while section incubated with blocking peptide
of sPLA2-III, showing absence of labeling (D,
asterisk). V, spinal trigeminal nucleus. Scale:
200 mm.

46 Eur J Pain 16 (2012) 38–48 © 2011 European Federation of International Association for the Study of Pain Chapters

M.-T. Ma et al. Systems wide analyses of lipids in the brainstem during inflammatory orofacial pain

generally implicated these isoforms in nociception References
(Lucas et al., 2005; Svensson et al., 2005; Yeo et al.,
2004). cPLA2 activity in the spinal cord was Adibhatla RM, Hatcher JF, Dempsey RJ. Lipids and lipido-
enhanced after nerve injury-induced neuropathic mics in brain injury and diseases. AAPS J 2006;8:E314–21.
pain, and neuropathic pain-like behaviors were
attenuated by intrathecal injection of, an inhibitor of Bligh EG, Dyer WJ. A rapid method of total lipid extraction
cPLA2 arachidonyl trifluoromethylketone (AACOCF3) and purification. Can J Biochem Physiol 1959;37:911–7.
(Ma et al., 2010a). In addition, mice that were
intracerebroventricularly injected with cPLA2 or Buczynski MW, Svensson CI, Dumlao DS, Fitzsimmons BL,
sPLA2 inhibitors showed significantly decreased Shim JH, Scherbart TJ, et al. Inflammatory hyperalgesia
responses to von Frey hair stimulation after facial induces essential bioactive lipid production in the spinal
carrageenan injection, compared to controls without cord. J Neurochem 2010;114:981–93.
inhibitors (Yeo et al., 2004). In view of this, we
carried out analyses of a complete set of PLA2 iso- Capper EA, Marshall LA. Mammalian phospholipases A(2):
forms including sPLA2-IB, sPLA2-IIA, sPLA2-IIC, mediators of inflammation, proliferation and apoptosis.
sPLA2-IID, sPLA2-IIE, sPLA2-IIF, sPLA2-III, sPLA2-V, Prog Lipid Res 2001;40:167–97.
sPLA2-XIIA, cPLA2 and iPLA2 in the medulla oblon-
gata to determine possible changes in expression Fei W, Shui G, Gaeta B, Du X, Kuerschner L, Li P, et al.
after allodynia. mRNA expression of all PLA2 iso- Fld1p, a functional homologue of human seipin, regulates
forms except sPLA2-X was detected in rat medulla the size of lipid droplets in yeast. J Cell Biol 2008;180:473–
oblongata. Of these, sPLA2-III mRNA showed the 82.
greatest expression in the medulla oblongata, and its
expression was increased in the CM of facial Fitzsimmons BL, Zattoni M, Svensson CI, Steinauer J, Hua
carrageenan-injected rats compared to untreated XY, Yaksh TL. Role of spinal p38alpha and beta MAPK in
controls. inflammatory hyperalgesia and spinal COX-2 expression.
Neuroreport 2010;21:313–7.
Previous results demonstrated increased activity of
cPLA2 but no change in its mRNA or protein expres- Gelb MH, Valentin E, Ghomashchi F, Lazdunski M, Lambeau
sion in the spinal cord after hyperalgesia induced by G. Cloning and recombinant expression of a structurally
carrageenan injection to the hind paw (Lucas et al., novel human secreted phospholipase A2. J Biol Chem
2005). We verified that sPLA2-III protein was highly 2000;275:39823–6.
expressed in the CM, with the detected band at 19 kDa
being consistent with the active form of the enzyme Ho IC, Arm JP, Bingham 3rd CO, Choi A, Austen KF,
(its sPLA2 domain) (Masuda et al., 2008). However, in Glimcher LH. A novel group of phospholipase A2s prefer-
contrast to mRNA expression, no increase in protein entially expressed in type 2 helper T cells. J Biol Chem
level was detected by Western blots or immunohis- 2001;276:18321–6.
tochemistry, compared to controls. These results indi-
cate that sPLA2-III mRNA expression is highest among Kim DH, Fitzsimmons B, Hefferan MP, Svensson CI,
PLA2 isoforms in the untreated CM, and the small Wancewicz E, Monia BP, et al. Inhibition of spinal cytoso-
increase in sPLA2-III mRNA level after facial carrag- lic phospholipase A(2) expression by an antisense oligo-
eenan injection is not translated to a detectable nucleotide attenuates tissue injury-induced hyperalgesia.
increase in enzyme protein expression in Western Neuroscience 2008;154:1077–87.
blots. The changes in lipids as detected by lipidomics
are therefore consistent with an increase in enzyme Kinsey GR, Blum JL, Covington MD, Cummings BS,
activity, but no change in enzyme protein expression. McHowat J, Schnellmann RG. Decreased iPLA2gamma
Together, these findings indicate enhanced PLA2 acti- expression induces lipid peroxidation and cell death and
vity in the caudal medulla oblongata after inflamma- sensitizes cells to oxidant-induced apoptosis. J Lipid Res
tory orofacial pain. 2008;49:1477–87.

Acknowledgments Kujubu DA, Fletcher BS, Varnum BC, Lim RW, Herschman
HR. TIS10, a phorbol ester tumor promoter-inducible
This work was supported by grants from the National mRNA from Swiss 3T3 cells, encodes a novel prostaglandin
University of Singapore (JFY) and the National synthase/cyclooxygenase homologue. J Biol Chem 1991;
Medical Research Council of Singapore (WYO). 266:12866–72.

Jacques C, Berenbaum F. Role of type II secreted phospho-
lipase A2 in inflammatory processes. C R Seances Soc Biol
Fil 1996;190:437–43.

Livak KJ, Schmittgen TD. Analysis of relative gene expres-
sion data using real-time quantitative PCR and the
2(-Delta Delta C(T)) method. Methods 2001;25:402–8.

Lucas KK, Svensson CI, Hua XY, Yaksh TL, Dennis EA. Spinal
phospholipase A2 in inflammatory hyperalgesia: role of
group IVA cPLA2. Br J Pharmacol 2005;144:940–52.

Ma L, Uchida H, Nagai J, Inoue M, Aoki J, Ueda H. Evidence
for de novo synthesis of lysophosphatidic acid in the spinal
cord through phospholipase A2 and autotaxin in nerve
injury-induced neuropathic pain. J Pharmacol Exp Ther
2010a;333:540–6.

Eur J Pain 16 (2012) 38–48 © 2011 European Federation of International Association for the Study of Pain Chapters 47

Systems wide analyses of lipids in the brainstem during inflammatory orofacial pain M.-T. Ma et al.

Ma MT, Yeo JF, Farooqui AA, Ong WY. Role of calcium Shui G, Bendt AK, Pethe K, Dick T, Wenk MR. Sensitive
independent phospholipase A(2) in maintaining mito- profiling of chemically diverse bioactive lipids. J Lipid Res
chondrial membrane potential and preventing excessive 2007;48:1976–84.
exocytosis in PC12 cells. Neurochem Res 2010b;36:347–
54. Six DA, Dennis EA. The expanding superfamily of phospho-
lipase A(2) enzymes: classification and characterization.
Masuda S, Yamamoto K, Hirabayashi T, Ishikawa Y, Ishii T, Biochim Biophys Acta 2000;1488:1–19.
Kudo I, et al. Human group III secreted phospholipase A2
promotes neuronal outgrowth and survival. Biochem J Sun GY, Shelat PB, Jensen MB, He Y, Sun AY, Simonyi A.
2008;409:429–38. Phospholipases A2 and inflammatory responses in the
central nervous system. Neuromolecular Med 2010;12:
Mechiche H, Naline E, Candenas L, Pinto FM, Birembault P, 133–48.
Advenier C, et al. Effects of cysteinyl leukotrienes in small
human bronchus and antagonist activity of montelukast Svensson CI, Lucas KK, Hua XY, Powell HC, Dennis EA,
and its metabolites. Clin Exp Allergy 2003;33:887–94. Yaksh TL. Spinal phospholipase A2 in inflammatory hype-
ralgesia: role of the small, secretory phospholipase A2.
Ng CH, Ong WY. Increased expression of gamma- Neuroscience 2005;133:543–53.
aminobutyric acid transporters GAT-1 and GAT-3 in the
spinal trigeminal nucleus after facial carrageenan injec- Svensson CI, Yaksh TL. The spinal phospholipase-
tions. Pain 2001;92:29–40. cyclooxygenase-prostanoid cascade in nociceptive process-
ing. Annu Rev Pharmacol Toxicol 2002;42:553–83.
Ong WY, Farooqui T, Farooqui AA. Involvement of cytosolic
phospholipase A(2), calcium independent phospholipase Telleria-Diaz A, Schmidt M, Kreusch S, Neubert AK, Schache
A(2) and plasmalogen selective phospholipase A(2) in F, Vazquez E, et al. Spinal antinociceptive effects of
neurodegenerative and neuropsychiatric conditions. Curr cyclooxygenase inhibition during inflammation: involve-
Med Chem 2010;17:2746–63. ment of prostaglandins and endocannabinoids. Pain
2010;148:26–35.
Ong WY, Sandhya TL, Horrocks LA, Farooqui AA. Distribu-
tion of cytoplasmic phospholipase A2 in the normal rat Vos BP, Strassman AM, Maciewicz RJ. Behavioral evidence
brain. J Hirnforsch 1999;39:391–400. of trigeminal neuropathic pain following chronic constric-
tion injury to the rat’s infraorbital nerve. J Neurosci
Ong WY, Yeo JF, Ling SF. Farooqui AA: distribution of 1994;14:2708–23.
calcium-independent phospholipase A2 (iPLA 2) in
monkey brain. J Neurocytol 2005;34:447–58. Watson AD. Thematic review series: systems biology
approaches to metabolic and cardiovascular disorders.
Poh KW, Lutfun N, Manikandan J, Ong WY, Yeo JF. Global Lipidomics: a global approach to lipid analysis in biological
gene expression analysis in the mouse brainstem after systems. J Lipid Res 2006;47:2101–11.
hyperalgesia induced by facial carrageenan injection –
evidence for a form of neurovascular coupling? Pain Wenk MR. The emerging field of lipidomics. Nat Rev Drug
2009;142:133–41. Discov 2005;4:594–610.

Samad TA, Moore KA, Sapirstein A, Billet S, Allchrone A, Wise H. Lack of interaction between prostaglandin E2 recep-
Poole S, et al. Interleukin-1beta-mediated induction of tor subtypes in regulating adenylyl cyclase activity in cul-
Cox-2 in the CNS contributes to inflammatory pain hyper- tured rat dorsal root ganglion cells. Eur J Pharmacol
sensitivity. Nature 2001;410:471–5. 2006;535:69–77.

Saxena NC. Inhibition of GABA(A) receptor (GABAR) cur- Yeo JF, Ong WY, Ling SF, Farooqui AA. Intracerebroven-
rents by arachidonic acid in HEK 293 cells stably trans- tricular injection of phospholipases A2 inhibitors modu-
fected with alpha1beta2gamma2 GABAR subunits. lates allodynia after facial carrageenan injection in mice.
Pflugers Arch 2000;440:380–92. Pain 2004;112:148–55.

Seybold VS, Jia YP, Abrahams LG. Cyclo-oxygenase-2 con- Zhang FY, Wan Y, Zhang ZK, Light AR, Fu KY. Peripheral
tributes to central sensitization in rats with peripheral formalin injection induces long-lasting increases in
inflammation. Pain 2003;105:47–55. cyclooxygenase 1 expression by microglia in the spinal
cord. J Pain 2007;8:110–7.

48 Eur J Pain 16 (2012) 38–48 © 2011 European Federation of International Association for the Study of Pain Chapters

点击阅读翻页书版本