Role of phospholipase a2 in orofacial pain and synaptic transmission 2

Chapter 3
Role of Group III sPLA
2
in nociception and synaptic transmission in the CNS


101

3.4. Discussion
This part of the study aimed to examine sPLA
2
-III expression profile in the
rat CNS and its effects on exocytosis to elucidate its role in nociceptive
transmission. Expression of sPLA
2
-III was analyzed in entire regions of the rat
brain including olfactory bulb, striatum, cortex, hippocampus,
thalamus/hypothalamus, cerebellum, brainstem and cervical, thoracic and lumbar
spinal segments using real-time RT-PCR. Among these regions, sPLA
2
-III
showed the highest level of mRNA expression in the brainstem and cervical,
thoracic and lumbar spinal segments suggesting that this sPLA
2
-III plays a role in
the ascending pain pathway. Similarly, using Western blot analysis, protein
expression sPLA
2
-III was observed to be at the highest level in homogenates of
the spinal segments, consistent with the findings in the Chapter 2. The
immunoreactivity of sPLA

2
-III was also observed to be high in the spinal
trigeminal nucleus and dorsal horn of the spinal segments. This protein was
localized to dendrites, the postsynaptic terminal in neurons, reflecting the
possible role of sPLA
2
-III in nociception. These findings are supported by
previous studies which showed that sPLA
2
-III was expressed in the neuronal
cells, such as peripheral neuronal fibres, spinal dorsal root ganglia neurons and
cerebellar Purkinje cells (Masuda et al. 2008).
The findings above supported the results in the Chapter 2 which showed
an increased sPLA
2
-III mRNA expression in the CM region after peripheral
inflammation induced by facial CA injection, indicating the role of this isozyme in
nociception. sPLA
2
-III has also been associated to various diseases such as
Chapter 3
Role of Group III sPLA
2
in nociception and synaptic transmission in the CNS


102

atherosclerosis and cancer (Murakami et al. 2005; Sato et al. 2008) thus leading
it to be a likely target for drugs. sPLA

2
-III could regulate nociception in mammals
as subcutaneous injection of PLA
2
-related peptide isolated from the bee venom
led to nociceptive paw flinches (Chen et al. 2006; Chen and Lariviere 2010). Both
sPLA
2
-III that was expressed in mouse skin and Tg mice overexpressing human
sPLA
2
-III could lead to spontaneous development of inflammation in the skin.
This was accompanied by entry of neutrophils and macrophages and augmented
levels of pro-inflammatory cytokines, chemokines and PGE
2
were observed (Sato
et al. 2009). Studies have also shown the association of sPLA
2
-III with
microvascular endothelium in human tissues after inflammation and ischemic
injury as sPLA
2
-III expression is induced by pro-inflammatory cytokines
(Murakami et al. 2005). Moreover, the enzyme transcription was upregulated by
the production of cytokines such as IL-1 α, TNF- α and interferon-γ by immune
cells at the site of inflammation (Farroqui et al. 2002). sPLA
2
-III when injected
into cervical dorsolateral funiculus, it showed dose-dependent demyelination, and
axonopathy, thus indicating its role in pain transmission (Titsworth et al. 2007).

After the N- and C-terminal of sPLA
2
-III are proteolytically cleaved, it
allows for the production of sPLA
2
only domain in almost all cell types (Murakami
et al. 2005). sPLA
2
domain alone is adequate to conduct catalytic activity and
generate PGE
2
in various cell types (Murakami et al. 2005), contributing to
inflammatory pathway. However, the mechanism by which this enzyme is
proteolytically processed in cells is still not known (Murakami and Kudo 2004).
Even so, it has been suggested that the proteolytic cleavage of the enzyme is
Chapter 3
Role of Group III sPLA
2
in nociception and synaptic transmission in the CNS


103

likely to occur prior to the secretion of the sPLA
2
domain of the sPLA
2
-III into the
extracellular space (Murakami et al. 2005). The presence of either C or N or both
terminals of the sPLA

2
-III results in the cytoplasmic localization of the enzyme,
whereas in the absence of both, the enzyme was found to be distributed mainly
on the plasma membrane of the cell (Murakami et al. 2003).
Upon the release of the enzyme into the extracellular space, the sPLA
2
-III
will act upon plasma membrane of neighboring cells, giving rise to various
catalytic products of membrane phospholipids, possibly suggesting its function in
nociceptive transmission. In this study, it was observed in PC-12 cells that there
was an increase in capacitance measurement indicating exocytosis, under
voltage clamp conditions after addition of sPLA
2
-III in this study. Exocytosis
induced by sPLA
2
-III was attenuated by pretreatment with MBCD suggesting
dependence on integrity of lipid rafts on the cell membrane which are membrane
domains in which neurotransmitter signaling could take place via clustering of
receptors and components of receptor-activated signaling cascade (Allen et al.
2007). Moreover, treatment of cells with thapsigargin and recording in zero Ca
2+

conditions, or treatment of cells with lanthanum chloride and recording in external
solution containing Ca
2+
, resulted in attenuation of sPLA
2
-III induced
exocytosis.sPLA

2
-III also induced rise in [Ca
2+
]i, and this effect was abolished in
cells which had been pre-incubated with MBCD, or cells that were pre-treated
with lanthanum chloride and recorded in external solution containing Ca
2+
.
Together, the results indicated that neurotransmitter release triggered by sPLA
2
-
III could possibly be dependent on the integrity of cholesterol rich lipid domains
Chapter 3
Role of Group III sPLA
2
in nociception and synaptic transmission in the CNS


104

on cellular membranes and a rise in [Ca
2+
]i concentration, through influx via Ca
2+

channels.
Various studies have also shown the involvement of bee venom in
exocytosis in tissues and PC-12 cells (Kurihara et al. 1986; Ray et al. 1997; You
et al. 2008). Evidence from previous study showed that application of PLA
2

activators, melittin and mastoparan on rat anterior pituitary gland cells induced
hormonal release (Kurihara et al. 1986). The bee venom test, using bee venom
or components of bee venom is also a well established experimental animal
model for pain to show its role in nociceptive transmission (Mogil et al. 1999;
Lariviere and Melzack 2000; Lariviere et al. 2002). Peripheral bee venom
injection induced changes in synaptic transmission of the anterior cingulate
cortex which plays an important role in the affective dimension of pain (Gong et
al. 2010), indicating the important role of bee venom in nociceptive transmission.
The findings in this section of the study, together with the results from the
Chapter 2, it suggests the role of sPLA
2
-III in nociceptive transmission. Besides
sPLA
2
-III, sPLA
2
-IIA also participated in neurotransmission in the hippocampal
cultured neurons and PC-12 cells (Wei et al. 2003). Therefore in the next chapter,
the role of sPLA
2
-IIA in nociception by elucidating its expression and localization
in the CNS will be further explored.

Chapter 4
Role of Group IIA sPLA
2
in nociception

105









CHAPTER 4
ROLE OF GROUP IIA sPLA
2
IN NOCICEPTION


Chapter 4
Role of Group IIA sPLA
2
in nociception


106

4.1. Introduction
Mammalian sPLA
2
consists of enzymes which have low molecular masses
(13–19 kDa) (Yang et al. 2009) and their require mM range of Ca
2+

for their
activities (Six and Dennis 2000; Valentin and Lambeau 2000; Farooqui and
Horrocks 2004). These isozymes include sPLA
2
-IB, IIA, IIC, V and X and are
involved in multiple physiological and pathological processes via release of AA
from membrane phospholipids or specific binding to membrane receptors (Suzuki
et al. 2000). Total sPLA
2
activity is highest in the medulla oblongata, pons, and
hippocampus, moderate in the hypothalamus, thalamus, and cerebral cortex, and
low in the cerebellum and olfactory bulb (Thwin et al. 2003).
sPLA
2
has a well-established role in inflammation and inflammatory
diseases (Nevalainen et al. 2000) and thus, inhibition of sPLA
2
would prevent the
formation of inflammatory eicosanoids prior to the COX reaction in which PLA
2
is
the rate limiting precursor in AA production (Schaefers et al. 1996). Therefore its
blockade should eliminate the need for COX-1 versus COX-2 specificity in anti-
inflammatory therapeutics. sPLA
2
activity is elevated in several body fluids of
patients with acute pancreatitis (Makela et al. 1990). Synovial fluid from arthritic
joints of rheumatic patients contains sPLA
2
-IIA (Kramer et al. 1989; Seilhamer et

al. 1989) while the total PLA
2
activity and sPLA
2
-IIA is also enhanced in
bronchoalveolar lavage fluids from patients with adult respiratory distress
syndrome (Kim et al. 1995a). TNFα, IL-1, and LPS were shown to induce sPLA
2
-
IIA production in cultured astrocytes and direct injection of LPS into brain
increased sPLA
2
-IIA mRNA (Oka and Arita 1991).
Chapter 4
Role of Group IIA sPLA
2
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107

sPLA
2
-IB mRNA is present in the human brain and its distribution is mainly
neuronal. It is highly expressed in the cerebral cortex and hippocampus. sPLA
2
-
IB mRNA has been identified in rat cerebral neurons, cells of neurodermal origin
(Kolko et al. 2005; Kolko et al. 2007) and the normal spinal cord by quantitative
PCR (Lucas et al. 2005). sPLA

2
-IIA is ubiquitously expressed in the rat brain
(Molloy et al. 1998) and spinal cord (Lucas et al. 2005). sPLA
2
-IIA is associated
with the endoplasmic reticulum in perinuclear regions of Purkinje cell somata
(Shirai and Ito 2004). Increased sPLA
2
-IIA mRNA and immunoreactivity is
present in the rat brain after cerebral ischemia and in Alzheimer’s disease
(Lauritzen et al. 1994; Lin et al. 2004; Moses et al. 2006; Adibhatla and Hatcher
2007). Moreover, sPLA
2
-IIA is upregulated by cytokines including TNF-α and IL-
1α/β (Adibhatla and Hatcher 2007). sPLA
2
-IIC mRNA expression is low in
peripheral tissues but is found in all parts of the brain (Molloy et al. 1998) and
spinal cord (Lucas et al. 2005). sPLA
2
-V mRNA is expressed in the cerebral
cortex and hippocampus while present at low levels in most areas of the brain
(Molloy et al. 1998; Kolko et al. 2006) and is also identified in the rat cerebellum
by immunostaining and in situ hybridization histochemistry and localized in
Bergmann glia cells (Shirai and Ito 2004). sPLA
2
-V is also present in the rat
spinal cord as shown by quantitative PCR and Western blot analysis (Lucas et al.
2005; Svensson et al. 2005). sPLA
2

-X is expressed in the rat brain and in primary
neuronal cell cultures, and is expressed at low levels in the cerebral cortex
(Kolko et al. 2006). Having shown in the previous chapters that sPLA
2
-III was the
particular isozyme which was significantly increased after orofacial pain induced
Chapter 4
Role of Group IIA sPLA
2
in nociception


108

by facial CA, the important role of sPLA
2
isoforms with strong secretory signals in
nociceptive transmission could not be neglected especially when it has been
shown that addition of sPLA
2
-IIA which has strong secretory signal was able to
induce exocytosis in hippocampal neurons (Wei et al. 2003). In this part of the
study the expression profile of multiple sPLA
2
isoforms in the rat CNS was
elucidated with focus on sPLA
2
-IIA in the brainstem and spinal cord.

Chapter 4
Role of Group IIA sPLA
2
in nociception


109

4.2. Materials and methods
4.2.1. Real-time RT-PCR
sPLA
2
isoforms with strong secretory signal (P > 0.6), and which could be
released as a neuromodulator were selected for analyses using SignalP 3.0
Server, which predicts the presence and location of signal peptide cleavage sites
in amino acid sequences from different organisms. These included sPLA
2
-IB (P =
0.82), -IIA (P = 0.67), -IIC (P = 0.7), and -X (P = 0.63). sPLA
2
-V (P = 0.4) was
also analysed for comparison. Four uninjected adult male Wistar rats weighing
approximately 200 g each were used for this portion of the study. The rats were
anesthetized with an intraperitoneal injection of ketamine and xylazine cocktail
and killed by decapitation. Adequate measures were taken to minimize pain and
discomfort, and procedures involving rats were approved by the Institutional
Animal Care and Use Committee.
Various regions of the brain including olfactory bulb, cerebral neocortex,
hippocampus, striatum, thalamus/hypothalamus, cerebellum, brainstem and
cervical, thoracic and lumbar spinal segments were quickly removed and

immersed in RNAlater (Ambion, TX,USA), snap frozen in liquid nitrogen and kept
at -80
o
C till analyses. Total RNA was extracted and isolated using TRizol
reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol.
RNeasy1 Mini Kit (Qiagen, Inc., CA, USA) was used to purify the RNA. The
samples were then reverse transcribed using High-Capacity cDNA Reverse
Transcription Kits (Applied Biosystems, CA, USA). Real-time PCR amplification
was then carried out in the 7500 Real time PCR system using TaqMan1
Chapter 4
Role of Group IIA sPLA
2
in nociception


110

Universal PCR Master Mix, and sPLA
2
-IB (Applied Biosystems ID
Rn00580896_m1), sPLA
2
-IIA (Rn00580999_m1), sPLA
2
-IIC (Rn01520676_m1),
sPLA
2
-V (Rn00567782_m1), sPLA
2
-X (Rn00691350_m1) or rat β-actin probes,

according to the manufacturer’s instructions. All reactions were carried out in
triplicate. The amplified transcripts were quantified using the comparative CT
method (Livak and Schmittgen 2001), with the formula for relative fold change =
2
ΔΔCT
. The fold change for each sPLA
2
subgroup expression in different parts of
the brain was observed.

4.2.2. Western blot analysis
Four uninjected Wistar rats of 200 g each were used for this portion of the
study. The animals were anesthetized and killed as described above. The
olfactory bulb, cerebral neocortex, hippocampus, striatum,
thalamus/hypothalamus, cerebellum, brainstem and cervical, thoracic, and
lumbar spinal segments were dissected out and homogenized in 10 volumes of
ice-cold buffer containing 0.32 M sucrose, 4 mM Tris–HCl, pH 7.4, 1 mM EDTA,
and 0.25 mM dithiothreitol. After centrifugation at 1000 g for 30 min, the
supernatant was collected and protein concentrations in the preparation
measured using the BioRadprotein assay kit (Bio-Rad Laboratories). Total
proteins were resolved in 10% SDS polyacrylamide gels under reducing
conditions and electrotransferred to a PVDF membrane (Amersham Pharmacia
Biotech). Nonspecific binding sites on the PVDF membrane were blocked by
incubation with 5% non-fat milk for 1 h. The PVDF membrane was then
Chapter 4
Role of Group IIA sPLA
2
in nociception

111

incubated overnight with sPLA
2
-IIA antibody diluted 1:5000 in TTBS at 4
o
C. The
antibody was prepared by immunizing a rabbit with purified recombinant rat
sPLA
2
-IIA as described earlier (Nyman et al. 2000). After washing with TTBS, the
membrane was incubated with horseradish peroxidase conjugated anti-rabbit
immunoglobulin IgG (Amersham) for 1 h at room temperature. The protein was
visualized with an enhanced chemiluminescence kit (Pierce, Rockford, IL)
according to the manufacturer’s instructions.

4.2.3. Immunohistochemistry
A further three uninjected male Wistar rats were used for this portion of
the study. The animals were anesthetized as described above and perfused
through the left ventricle with a solution of 4% paraformaldehyde in 0.1 M
phosphate buffer (pH 7.4). Brains were removed and sectioned coronally at 100
mm using a Vibratome. The sections were washed for 3 h in PBS. They were
then incubated overnight with sPLA
2
-IIA antibody (diluted to 1:500 in 0.15 M
NaCl in 0.05 M Tris buffer, pH 8.6, and containing 1% bovine serum albumin).
This antibody has been characterized previously, and used for
immunohistochemical labeling of sPLA
2
-IIA in the rat ileum (Nyman et al. 2000).

The sections were then washed in PBS and incubated for 1 h at room
temperature in a 1:200 dilution of biotinylated horse anti-rabbit IgG (Vector). The
sections were reacted for 1 h at room temperature with an avidin-biotinylated
horseradish peroxidase complex, and visualized by treatment for 5 min in 0.05%
DAB solution in TBS 0.05% hydrogen peroxide. The color reaction was stopped
Chapter 4
Role of Group IIA sPLA
2
in nociception


112

with several washes of TBS. Some sections were mounted on glass slides and
lightly counterstained with methyl green before coverslipping. The remaining
sections were processed for electron microscopy. Control sections were
incubated with preimmune rabbit serum (1:500 dilution) instead of primary
antibody.

4.2.4. Electron microscopy
Electron microscopy was carried out by subdissecting the immunolabeled
spinal cord sections into smaller portions that included the dorsal or ventral horn.
The sections were soaked in PBS overnight at 4
o
C before processing. Following
which, the sections were post fixed in 1% osmium tetroxide, pH 7.4 for 1 h at
room temperature. This was followed by washing the sections with PBS two
times for 5-10 min at room temperature. The sections were then dehydrated
through a series of ascending ethanol series at room temperature. Finally the
sections were embedded in araldite and left to polymerise at 60

o
C for 24 h.
Semi-thin sections were obtained from the first 5 µm of the sections, mounted on
Formvar-coated copper grids, and stained with lead citrate. They were viewed
using a Jeol 1010EX electron microscope.






Chapter 4
Role of Group IIA sPLA
2
in nociception


113

4.3. Results

4.3.1. Real-time RT-PCR (Fig. 2.4.1., Table 2.4.1.)

The expression of various sPLA
2
isoforms were normalized to the lowest
level of message among the isoforms in the different brain regions (apart from
sPLA
2
-X, which was nearly undetectable), i.e., the value for sPLA

2
-IB in the
cerebral neocortex, to give an indication of relative expression in different parts of
the brain and spinal cord. sPLA
2
-IB expression was low throughout the CNS,
sPLA
2
-IIC expression was relatively high in the cerebral neocortex, hippocampus
and thalamus/hypothalamus; sPLA
2
-V showed greatest expression in the
olfactory bulb and cerebellum, and moderate expression in the spinal cord, and
sPLA
2
-X was expressed at very low levels in the CNS. sPLA
2
-IIA showed
greatest expression in the brainstem and spinal cord; the brainstem and spinal
cord also contained highest levels of this isoform (Fig. 2.4.1., Table 2.4.1.).

Chapter 4
Role of Group IIA sPLA
2
in nociception


114

Fig.2.4.1. A: Real-time RT-PCR analysis of differentially expressed sPLA
2
subgroups in the CNS.
The values were normalized to the lowest level of message among the isoforms in the different
brain regions, i.e., the value for sPLA
2
-IB in the cerebral neocortex, to give an indication of
relative expression in different parts of the brain and spinal cord. sPLA
2
-IB expression was low
throughout the CNS, sPLA
2
-IIC expression was relatively high in the cerebral neocortex,
hippocampus and thalamus/hypothalamus; sPLA
2
-V showed greatest expression in the olfactory
bulb and cerebellum, and moderate expression in the spinal cord, and sPLA
2
-X was expressed at
very low levels in the CNS (not visible in this scale). sPLA
2
-IIA showed greatest expression in the
brainstem and spinal cord; conversely, the brainstem and spinal cord contained highest levels of
this isoform. Data represent the mean and standard deviation of four rats.

0
10
20
30
40
50
sPLA2-IB sPLA2-IIA sPLA2-IIC sPLA2-V sPLA2-X
Relative Expression
Olfactory Bulb Cortex
Hippocampus Striatum
Thalamus/hypothalamus Cerebellum
Brainstem Cervical
Thoracic Lumbar
A

Chapter 4
Role of Group IIA sPLA
2
in nociception


115

0
2
4
6
8

10
Olfactory bulb
Cortex
Hippocampus
Thalamus
Striatum
Cerebellum
Brainstem
Cervical
Thoracic
Lumbar
Relative Expression

Fig.3.4.1. B: Real-time RT-PCR analysis of sPLA
2
-IIA in the various regions of CNS. The values
were normalized to the lowest level of message in the different brain regions, i.e., the value for
sPLA
2
-IIA in the striatum, to give an indication of relative expression in different parts of the brain
and spinal segments. sPLA
2
-IIA showed highest level of expression in the hindbrain. Data
represent the mean and standard deviation of four rats.

B
Chapter 4
Role of Group IIA sPLA
2
in nociception

116

Table 2.4.1. Comparison of sPLA
2
isoforms mRNA and protein expression in rat CNS between previous reports and current findings.


+++, high expression; ++, moderate expression; +, low expression; N.S. not studied.

sPLA
2

isoforms
Olfactory
Bulb
Cortex Hippocampus Striatum
Thalamus/
hypothalamus
Cerebellum Brainstem
Spinal
cord
Detected
Expression
(mRNA/protein)
References


N.S. +++ +++ N.S. N.S. +++ N.S. N.S. mRNA, protein

Kolko et al., 2007
+ + + + + + + N.S. mRNA
Molloy et al., 1998
N.S. N.S. N.S. N.S. N.S. N.S. N.S. +++ mRNA
Lucas et al., 2005
+ + + + + + + + mRNA Current data
sPLA
2
-IB

N.S. + + + + + +++ N.S. mRNA
Molloy et al., 1998
N.S. N.S. N.S. N.S. N.S. N.S. N.S. +++ mRNA
Lucas et al., 2005
N.S. N.S. N.S. N.S. N.S. N.S. N.S. +++ protein
Svensson et al., 2005
+ + + + + + ++ +++ mRNA, protein Current data
sPLA
2
-IIA

N.S. +++ + +++ ++ + + N.S. mRNA
Molloy et al., 1998
N.S. N.S. N.S. N.S. N.S. N.S. N.S. +++ mRNA
Lucas et al., 2005
+ +++ +++ + +++ + + + mRNA Current data
sPLA
2
-IIC

+ + +++ + + + + N.S. mRNA
Molloy et al., 1998
N.S. N.S. N.S. N.S. N.S. N.S. N.S. +++ mRNA
Lucas et al., 2005
N.S. N.S. N.S. N.S. N.S. N.S. N.S. +++ protein
Svensson et al., 2005
N.S. +++ +++ N.S. N.S. N.S. N.S. N.S. mRNA, protein
Kolko et al., 2006
+++ + + + + +++ + ++ mRNA Current data
sPLA
2
-V

N.S. + ++ N.S. N.S. N.S. N.S. N.S. mRNA, protein
Kolko et al., 2006
+ + + + + + + + mRNA Current data
sPLA
2
-X

Chapter 4
Role of Group IIA sPLA
2
in nociception


117

4.3.2. Western blot analysis (Fig. 2.4.2.)

The antibody to sPLA
2
-IIA detected a band at 14 kDa in homogenates
from brainstem and various parts of spinal cord consistent with the expected
molecular weight of sPLA
2
-IIA. A dimerised band of sPLA
2
-IIA with molecular
weight of approximately 30 kDa was also detected in the cervical, thoracic and
lumbar spinal segments. High level of expression was detected in the brainstem,
cervical, thoracic and lumbar spinal segments whilst low level of expression was
found in the olfactory bulb, cerebral neocortex, hippocampus, striatum, thalamus
and hypothalamus and cerebellum (Fig. 2.4.2.).

Fig.2.4.2. Western blot analyses of sPLA
2
-IIA protein expression in different parts of the rat CNS.
The antibody to sPLA
2
-IIA detects a band at 14 kDa in homogenates from brainstem and various
parts of spinal cord consistent with the expected molecular weight of the active form of sPLA
2
-IIA.
A dimerised band of sPLA
2
-IIA with molecular weight of approximately 30 kDa is also detected in
the cervical, thoracic and lumbar spinal segments. High level of expression was detected in the
brainstem (lane 7), cervical (lane 8), thoracic (lane 9) and lumbar (lane 10) spinal segments. In
contrast, low level of expression was found in the olfactory bulb (lane 1), cerebral neocortex (lane

2), hippocampus (lane 3), striatum (lane 4), thalamus and hypothalamus (lane 5) and cerebellum
(lane 6).





1

2

3

4

6

5

14kD
a

~30kDa

8

7

10

9

β-actin

Chapter 4
Role of Group IIA sPLA
2
in nociception


118

4.3.3. Immunohistochemistry (Figs. 2.4.3., 2.4.4. and 2.4.5.)

Sections incubated with sPLA
2
-IIA antibody showed very little labeling in
the forebrain including the cerebral neocortex (Fig. 2.4.3A) and hippocampus
(Fig. 2.4.3B). Little labeling was observed in thalamus (Fig. 2.4.3C) and light
labeling was observed in Purkinje cells in the cerebellar cortex (Fig. 2.4.3D).




Fig.2.4.3. Light micrographs of sPLA
2
-IIA immunolabeled sections from a normal rat CNS. Very
little labeling is observed in the cerebral neocortex (A) and hippocampus (B). Little labeling was
observed in thalamus (C) and light labeling was observed in Purkinje cells in the cerebellar cortex
(D). Abbreviations: CX, cerebral neocortex; HC, hippocampus; TH, thalamus; CCX, cerebellar

cortex. Scale: 200 μm.

CX HC
TH
CCX
B
A
C
D
Chapter 4
Role of Group IIA sPLA
2
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119

In contrast to the forebrain, dense labeling for sPLA
2
-IIA was detected in
the brainstem and spinal cord. Dense labeling was observed in neurons of the
central grey matter in the brainstem (Fig. 2.4.4A), spinal trigeminal nucleus (Fig.
2.4.4B) and facial motor nucleus (Fig. 2.4.4C). Dense sPLA
2
-IIA labeling was
also observed in the neurons in the dorsal- (Fig. 2.4.4D) and ventral horns of the
spinal cord (Fig. 2.4.4E). Control sections incubated with preimmune rabbit
serum showed only background labeling (Fig. 2.4.4F).

Chapter 4
Role of Group IIA sPLA
2
in nociception


120


Fig.2.4.4. Light micrographs of sPLA
2
-IIA immunolabeled sections from a normal rat CNS. Dense
staining is observed in the central gray (A, asterisk), spinal trigeminal nucleus (B, asterisk), facial
motor nucleus (C, asterisk), dorsal horn of the cervical spinal cord (D, asterisk), ventral horn of
the lumbar spinal cord (E, asterisk). (F) Section through the ventral horn of lumbar spinal
segment, incubated with preimmune rabbit serum, showing background labeling (asterisk).
Abbreviations: CG, central gray matter; V, spinal trigeminal nucleus; VII, facial motor nucleus; DH,
dorsal horn of spinal cord; VH, ventral horn of spinal cord; VH (C), ventral horn of spinal cord
(control). Scale: 200 μm.




*

*

*

*

*

D
*

CG
V
VII
DH
VH
VH (C)
B
A
F
E

C