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Anovel role of phospholipase A2 in mediating spinal cord secondary injury.

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ORIGINAL ARTICLES
A Novel Role of Phospholipase A2 in
Mediating Spinal Cord Secondary Injury
Nai-Kui Liu, MD, PhD,1 Yi Ping Zhang, MD,1 William Lee Titsworth, BS,1 Xiaoyan Jiang, MD,2
Shu Han, PhD,2 Pei-Hua Lu, MD,2 Christopher B. Shields, MD,1 and Xiao-Ming Xu, PhD1,2
Objective: To investigate whether phospholipase A2 (PLA2) plays a role in the pathogenesis of spinal cord injury (SCI).
Methods: Biochemical, Western blot, histological, immunohistochemical, electron microscopic, electrophysiological, and
behavior assessments were performed to investigate (1) SCI-induced PLA2 activity, expression, and cellular localization
after a contusive SCI; and (2) the effects of exogenous PLA2 on spinal cord neuronal death in vitro and tissue damage,
inflammation, and function in vivo. Results: After SCI, both PLA2 activity and cytosolic PLA2 expression increased
significantly, with cytosolic PLA2 expression being localized mainly in neurons and oligodendrocytes. Both PLA2 and
melittin, an activator of endogenous PLA2, induced spinal neuronal death in vitro, which was substantially reversed by
mepacrine, a PLA2 inhibitor. When PLA2 or melittin was microinjected into the normal spinal cord, the former induced
confined demyelination and latter diffuse tissue necrosis. Both injections induced inflammation, oxidation, and tissue
damage, resulting in corresponding electrophysiological and behavioral impairments. Importantly, the PLA2-induced
demyelination was significantly reversed by mepacrine. Interpretation: PLA2, increased significantly after SCI, may play
a key role in mediating neuronal death and oligodendrocyte demyelination following SCI. Blocking PLA2 action may
represent a novel repair strategy to reduce tissue damage and increase function after SCI.
Ann Neurol 2006;59:606 – 619
There are two mechanisms of damage to the spinal
cord after injury: a primary mechanical injury and a
secondary injury mediated by multiple injury processes
including inflammation, free radical–induced cell
death, and glutamate excitotoxicity.1– 4 Because multiple harmful substances are involved in the secondary
spinal cord injury (SCI), it is unlikely that blocking
one substance or mechanism would significantly prevent the course of secondary injury. However, it is possible that these different substances and mechanisms
may share a common pathway to exert their detrimental effects. If so, blocking such a common pathway
should be more effective than blocking a single pathway. A candidate molecule that could serve as a common or converging mediator for the secondary SCI is
phospholipase A2 (PLA2). PLA2 is a diverse family of
enzymes that hydrolyze the acyl bond at the sn-2 position of glycerophospholipids to produce free fatty acids and lysophospholipids.5,6 These products are precursors of bioactive eicosanoids and platelet-activating
factor, which are well-known mediators of inflammation and tissue damage implicated in pathological states
of several acute and chronic neurological disorders.6 – 8
PLA2 contributes to these disorders by attacking cellular membranes, releasing proinflammatory lipid mediators, and generating free radicals.6,9
PLA2 could serve as a common mediator in the
pathogenesis of SCI because it can be induced by multiple harmful substances including inflammatory cytokines such as tumor necrosis factor-␣ (TNF-␣) and
interleukin-1␤ (IL-1␤),5,10 free radicals such as those
induced by H2O2,11 and excitatory amino acids such
as glutamate,9,12,13 and because its metabolic products
are involved in multiple injury processes such as inflammation,5,7 oxidation,5,7 and neurotoxicity.7,14 The
PLA2 family of enzymes is normally present in the
mammalian brain15,16 and spinal cord.17 In pathological conditions such as ischemia,18 –20 closed head injury,21 kainate-induced brain lesion,13 and multiple
sclerosis–like disease,22 increased PLA2 activity or expression, or both, was found.
Although PLA2 may play an important role after
SCI, no information is available concerning its action
and underlying mechanisms after SCI. Here, we provide evidence that PLA2 is rapidly activated and is expressed for a prolonged period after SCI. The findings
From the 1Departments of Neurological Surgery and Anatomical
Sciences and Neurobiology, Kentucky Spinal Cord Injury Research
Center, University of Louisville School of Medicine, Louisville, KY;
and 2Department of Neurobiology, Shanghai Second Medical University, Shanghai, China.
Published online Feb 23, 2006 in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.20798
Received Jul 19, 2005, and in revised form Dec 12. Accepted for
publication Dec 13, 2005.
606
Address correspondence to Dr Xu, Department of Neurological Surgery, University of Louisville School of Medicine, 511 S. Floyd
Street, MDR 616, Louisville, KY 40292.
E-mail: xmxu0001@louisville.edu
© 2006 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
that PLA2 was expressed mainly in neurons and oligodendrocytes and that it induced neuronal death and
oligodendrocyte demyelination collectively indicate
that these two cell types are particularly vulnerable to
PLA2-medated cytotoxicity. Thus, blocking PLA2 expression and activity may represent a novel strategy to
reduce multiple damaging processes in the course of
secondary SCI.
Materials and Methods
Animals
A total of 174 adult female Sprague–Dawley rats (Harlan,
Indianapolis, IN), weighing 210 to 230gm, were used in this
study. These included 108 rats that received contusive SCI
and 66 rats that received injections of PLA2 or melittin. All
surgical interventions and postoperative animal care were
performed in accordance with the Guide for the Care and
Use of Laboratory Animals (National Research Council, National Academy Press, Washington, D.C., 1996) and the
Guidelines of the University of Louisville Institutional Animal Care and Use Committee.
Contusive Spinal Cord Injury
Contusive SCI was performed using a New York University
Impactor, as described previously.23 In brief, rats were anesthetized with pentobarbital (50mg/kg intraperitoneally) and
received a laminectomy at the T9-10 level. The SCI was performed at T10 by dropping a 10gm rod (2.5mm in diameter) onto the dorsal surface of the cord from a height of
12.5mm. For the sham-operated control rats, the animals
underwent a T9-10 laminectomy without the weight-drop
injury.
Phospholipase A2 Activity Assay
A 10mm spinal cord segment containing the injury epicenter
was homogenized in 50mM Hepes, pH 7.4, containing
1mM EDTA and centrifuged at 10,000 g for 15 minutes at
4°C. Supernatant was removed for PLA2 activity analysis according to the protocol in the PLA2 Assay Kits (Cayman
Chemical Company, Ann Arbor, MI).
Western Blotting
Western blotting followed a previously described procedure.23 In brief, proteins were extracted from a 10mm spinal
cord segment containing the injury epicenter. Forty microgram proteins were electrophoresed on a 7.5% sodium dodecyl sulfate-polyacrylamide gel, transferred onto a polyvinylidene diflouride membrane, and immunoblotted with the
primary mouse monoclonal anti–cytosolic PLA2 (cPLA2) antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA)
and a secondary horseradish peroxidase–conjugated donkey
anti–mouse IgG antibody (1:5,000; Amersham Pharmacia
Biotech, Piscataway, NJ). The blot was visualized using the
ECL Plus Detection Kit (Amersham Pharmacia Biotech). For
the negative control, the primary antibody was omitted.
NIH/3T3 whole-cell lysates (Santa Cruz Biotechnology)
were used as a positive control for the expression of cPLA2.
Immunohistochemistry
After perfusion, a 15mm-long spinal cord segment containing the injury epicenter of each rat was removed, cryoprotected in 30% sucrose buffer, cut transversely and serially at
20␮m on a cryostat, and mounted on Superfrost/Plus slides
(Fisher Scientific, Pittsburgh, PA) in five identical sets. One
set of the sections was incubated with primary polyclonal
rabbit anti-cPLA2 (1:100) antibody (Santa Cruz Biotechnology) overnight at 4°C. On the second day, the sections were
incubated with secondary biotinylated goat anti–rabbit IgG
antibody (1:400; Vector Laboratories, Burlingame, CA) for 1
hour at room temperature. The reaction product was shown
by incubation for 5 minutes with 0.02% diaminobenzidine
tetrahydrochloride and 0.003% H2O2 in 0.05M Tris-HCl
(pH 7.6). Slides were examined using an Olympus BX60
light microscope (Olympus America, Inc., Mellville, NY).
Primary antibody omission controls were used to further
confirm the specificity of the immunohistochemical labeling.
Immunofluorescence Double Labeling
Immunofluorescence double labeling has been described in a
previous publication.23 In brief, a mixture of rabbit polyclonal anti-cPLA2 (1:100; Santa Cruz Biotechnology) and
mouse anti-NeuN (1:100; Chemicon, Temecula, CA), antiSMI-31 (1:2,000; Sigma, St. Louis, MO), anti-CC1 (APC-7,
1;100; Calbiochem, San Diego, CA ), anti-OX-42 (1:100;
Harlan Sera-lab, Sussex, United Kingdom), or anti–glial
fibrillary acidic protein (1:100; Sigma) antibodies were used
to examine colocalization of cPLA2 in neurons, axons, oligodendrocytes, microglia, or astrocytes, respectively. The paired
antibody solutions were applied to the sections overnight at
4°C. On the following day, the sections were incubated with
fluorescein-conjugated goat anti–rabbit (1:100; ICN Biochemicals, Aurora, OH) and rhodamine-conjugated rabbit
anti–mouse (1:100; ICN Biochemicals) antibodies. Primary
antibody omission controls and normal mouse, rabbit, and
goat serum controls were used to further confirm the specificity of the immunofluorescence double labeling. Slides were
examined with an Olympus Optical Fluoview confocal microscope (Olympus America, Inc., Melville, NY).
Spinal Cord Neuronal Culture, Cell Treatment, and
Viability Assessment
Cells obtained from embryonic (E) day 14 rat spinal cords
were dissociated by incubation in 0.05% trypsin/EDTA followed by gentle trituration. The cells were grown in serumfree neurobasal medium supplemented with 2% B27 and
2mM glutamine. Three days later, 5␮M cytosine-␤-Darabinofuranoside (Sigma) was added to the medium for 24
hours to inhibit nonneuronal cell division. Under this culture condition, a purity of greater than 85% spinal cord neuronal population was obtained at the seventh day in vitro.
The cultures were then treated with PLA2 (0, 1, 2, 4, 6nM),
melittin (0, 100, 500, 1,000, 2,000nM), PLA2 (1 nM) ⫹
mepacrine (50 ␮M), or melittin (500 nM) ⫹ mepacrine (50
␮M), respectively. The cultures were maintained for an additional 24 hours, and the culture medium of each well was
removed for lactate dehydrogenase release assay using a lactate dehydrogenase cytotoxicity assay kit (Biovision Inc.,
Mountain, CA). In a subset of cultures, spinal cord neurons
Liu et al: PLA2 in Spinal Cord Injury
607
treated with PLA2 (6nM) or vehicle were fixed and prepared
for immunofluorescent labeling of ␤-tubulin III (1:800;
Sigma) counterstained with Hoechst 33342 (Molecular
Probes, Eugene, OR), a fluorescent nuclear dye. This was
used to assess nuclear fragmentation or condensation, a phenomenon of apoptosis, between the two experimental
groups.
Microinjections of Phospholipase A2 or Melittin into
the Normal Adult Rat Spinal Cord
A total of 66 rats were used in this study. Among them, 42
received phosphate-buffered saline, PLA2 (at three doses:
0.05, 0.1, or 0.5␮g; type III; Cayman Chemical Company,
Ann Arbor, MI), or melittin (at three doses: 1, 2.5, or 5␮g;
Sigma) and were kept for 4 weeks after the injection. The
amount of PLA2 injection was determined by measuring the
peak of PLA2 activity at 4 hours after SCI. Specifically, a
dose of 0.05 or 0.1␮g PLA2 injected into the spinal cord was
equivalent to the PLA2 activity of 2.09 or 4.00 (2.84 ⫾
0.85) ␮mol/min/gm protein, respectively, within a 10 mmlong cord segment containing the injury epicenter. In brief, a
beveled glass micropipette (external diameter, 10 –20␮m),
loaded with 4␮l phosphate-buffered saline, PLA2, or melittin
was injected bilaterally into the spinal cord (2␮l/side) at
0.6mm lateral to the midline and 1.5mm ventral to the dorsal surface of the cord for 10 minutes. The remaining 24 rats
received phosphate-buffered saline, PLA2 (0.05␮g), PLA2
(0.05␮g) ⫹ mepacrine (5mg/kg), or melittin (5␮g) (n ⫽ 6
per group). Mepacrine was injected intraperitoneally at 24
hours and 30 minutes before the injection of PLA2. A previous study suggests that this dose of mepacrine is metabolically stable and can cross the blood–brain barrier.24
Measurements of Axonal Conduction
Transcranial magnetic motor-evoked potentials (tcMMEPs)
were performed at 2, 5, 7, 14, 21, and 28 days after injections, using a method described previously.25 The tcMMEP
responses were elicited by the activation of subcortical structures with an electromagnetic coil placed over the cranium.
Action potentials descend in the ventral spinal cord and synapse onto motoneuron pools in which output signals can be
recorded from both of the gastrocnemius muscles.
Behavioral Assessments
Behavioral assessments were performed using the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale, a 21point scale (0 –21) based on the observation of hind-limb
movements of a rat freely moving in an open field.26,27 The
BBB was evaluated at 2 days, 1, 2, 3, and 4 weeks after
injections. During the evaluation, animals were allowed to
walk freely on the open-field surface for 4 minutes while being observed by two scorers lacking knowledge of the experimental groups.
Histological Assessments
At 24 hours or 4 weeks after injections, the cord segments
were isolated, embedded, and sectioned at 20␮m in 5 identical sets. Two sets of the sections were stained for myelin
with Luxol fast blue and counterstained with cresyl violeteosin. Area of lesion was outlined and quantified using an
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Olympus BX60 microscope equipped with a Neurolucida
system (MicroBrightField, Colchester, VT). Area of demyelination was quantified using National Institutes of Health
image software. The other three sets of sections from rats
perfused at 24 hours after injection were immunostained
with 4-hydroxy-2(E)-nonenal (4-HNE), TNF-␣, and IL-1␤.
The primary antibodies used included the polyclonal rabbit
anti– 4-HNE (1:500; Alpha Diagnostic, San Antonio, TX),
goat anti–TNF-␣ (1:50; Santa Cruz Biotechnology), or rabbit anti–IL-1␤ (1:100; Santa Cruz Biotechnology). The secondary antibodies used included the biotinylated goat anti–
rabbit IgG (1:400) or biotinylated rabbit anti–goat IgG
antibody (1:4,000; Vector Laboratories). The immunoreactive signals were quantified using Image-Pro Plus software
(Meida Cybernetics, Silver Spring, MD).
Toluidine Blue Staining and Electron Microscopy
The toluidine blue staining and electron microscopy were
described previously.28 In brief, spinal cord segments were
fixed overnight in the solution containing 2% glutaraldehyde
and 5% sucrose in 0.1M sodium cacodylate buffer, pH 7.4,
followed by 1% osmium tetroxide in the same buffer for 1
hour. The tissue was embedded in Spurr’s epoxy resin and
cured at 70°C. Transverse semithin sections (1␮m) were
stained with a mixture of 1% toluidine blue and 1% sodium
borate. Ultrathin sections (70 –90nm) were collected on copper mesh grids with 600 bars per inch, subsequently counterstained with 4% uranyl acetate in 50% ethanol and Reynolds’ lead citrate, and examined using a Philip CM10
electron microscope (Philips, Einhoven, The Netherlands).
Statistical Analysis
Data are presented as mean ⫾ standard error of the mean
values. One-way analysis of variance with post hoc Tukey t
test was used to determine statistical significance. A p value
less than 0.05 was considered statistically significant.
Results
Phospholipase A2 Activity and Expression Were
Increased in the Spinal Cord after Injury
We first investigated the change of total PLA2 activity
after a moderate contusive SCI. PLA2 activity was
markedly increased at 2 hours after injury (1.99-fold;
p ⬍ 0.01), reached its peak (2.6-fold; p ⬍ 0.001) at 4
hours, and started to decrease at 8 hours. At 7 days
after injury, the PLA2 activity markedly declined from
its peak, although the difference remained statistically
significant compared with the control (1.29-fold; p ⬍
0.05; Fig 1A). We next studied the expression of cytosolic PLA2 (cPLA2) after the same injury. The PLA2
enzymes can be broadly classified into two main
groups: secreted PLA2 and cPLA2. The secreted form is
a low molecular weight form (14kDa) that has no preference for the type of fatty acid at the sn-2 position of
phospholipids.5,7 Members of the cytosolic form have a
higher molecular mass (85–110kDa) and selectively hydrolyze phospholipids containing arachidonic acid
(AA).5,7 We chose to study cPLA2 expression because
immunoreactivity (IR) was detectable at a low level in
both the gray and white matter (Fig 2A). In contrast,
cPLA2 IR was markedly increased in both the injured
gray and white matter (see Figs 2B, C) at 7 days after
injury. Although the labeling was the strongest at the
lesion epicenter, increased cPLA2 IR was found
throughout the entire length (15mm in length, centered at the injury epicenter) of the specimen examined
(see Fig 2C). At high magnifications, increased cPLA2
IR was localized in neurons in the gray matter (see Fig
2D) and glial cells (see Fig 2E) in the white matter. In
sections where the primary antibody was omitted, no
labeling was found, further confirming the specificity
of the antibody (data not shown).
Fig 1. Temporal changes of phospholipase A2 (PLA2) activity
and cytosolic (cPLA2) expression in the spinal cord after a
moderate contusive spinal cord injury (SCI). (A) SCI induced
a rapid and significant increase of total PLA2 activity that
peaked at 4 hours after injury. (B) SCI induced a prolonged
and significant increase in cPLA2 expression that peaked at 3
and 7 days after injury. The top panel in B shows a representative time course of cPLA2 expression. The bottom panel in B
shows compiled results in a bar graph for each time point.
n ⫽ 6 rats per group. *p ⬍ 0.05; **p ⬍ 0.01 versus sham.
Cont ⫽ control.
it is one of the most important PLA2 isozymes involved in regulating lipid mediator generation29 –31 and
it selectively hydrolyzes phospholipids containing arachidonic acid.5,29 Western blot analysis showed that
cPLA2 expression markedly increased and peaked at 3
(6.2-fold; p ⬍ 0.01) and 7 (6.85-fold; p ⬍ 0.01) days
after SCI compared with the control (see Fig 1B). Although cPLA2 expression declined 7 days after SCI, it
remained at significantly high levels for up to 28 days
( p ⬍ 0.01) (see Fig 1B).
To confirm the Western blot results and determine
the spatial distribution of cPLA2 after SCI, we performed immunohistochemistry for the cPLA2 at 7 days
after injury, a time point when cPLA2 was maximally
expressed. In a sham-operated control, cPLA2-
Cytosolic Phospholipase A2 Immunoreactivity Was
Localized Mainly in Neurons and Oligodendrocytes
after Spinal Cord Injury
Although immunohistochemistry confirmed increased
expression of cPLA2 in the injured cord, it did not allow the identification of specific cell types that express
cPLA2. We thus performed immunofluorescence double labeling to localize specific cell types that express
cPLA2 at 7 days after injury. Coexistence of cPLA2 and
NeuN, a neuronal marker, was observed in neurons in
the spinal gray matter after SCI (Figs 3A–C). cPLA2
IR was also detected in some axons, particularly those
showing axonal swelling (SMI-31 IR; see Figs 3D–F).
Significantly, oligodendrocytes (CC1 IR; see Figs
3G–I) were cPLA2-positive. In contrast, only a subpopulation of microglia (OX-42 IR; see Figs 3J–L) expressed cPLA2, and none of the astrocytes (GFAP IR;
see Figs 3M–O) was positive for cPLA2. As expected,
in sham-operated control rats, a basal level of cPLA2 IR
was observed that was localized mainly in neurons and
oligodendrocytes (data not shown). These results, together with the immunohistochemical results described
earlier, showed that cPLA2 is increasingly expressed after SCI and is mainly expressed in neurons and oligodendrocytes in areas close to a contusive SCI.
Phospholipase A2–Induced Spinal Cord Neuronal
Death in a Dose-Dependent Manner In Vitro
Since we observed that both PLA2 activity and expression increased after a contusive SCI, the next question
would be: Could an elevated level of PLA2 induce spinal cord neuronal death? To address this issue, we first
established a spinal cord neuron-enriched cell culture
where greater than 85% spinal neurons in culture was
achieved (Fig 4A). Treatments of PLA2 or melittin, a
potent activator of endogenous PLA2, resulted in massive neuronal death (see Fig 4B), and many dying neurons showed nuclear fragmentation, an indicator of apoptosis (see insert in Fig 4B). We then determined
neuronal death of these cultures by measuring the release of lactate dehydrogenase, which is a stable enzy-
Liu et al: PLA2 in Spinal Cord Injury
609
Fig 2. The spatial distribution of cytosolic phospholipase A2 (cPLA2) immunoreactivity (IR) is shown in the spinal cord at 7 days
after a moderate contusive spinal cord injury (SCI). (A) A baseline amount of cPLA2 IR was detected in a T10 spinal cord cross
section of a sham-operated rat. (B) SCI induced a marked increase in cPLA2 IR at the injury epicenter (asterisk) particularly in
the spared white matter (arrows). (C) In a longitudinal montage photomicrograph, increased cPLA2 IR was found to extend from
the injury epicenter (asterisk) to the distal spinal cord for a considerable distance. (D, E) High magnification of demarcated areas
in c shows that cPLA2 IR was localized in neurons (D, white arrows) and glial cells (E, arrowheads), respectively. Bars ⫽ 2 mm
(A); 1mm (C); 100␮m (D, E).
matic marker that correlates linearly with cell death.32
The results showed that both PLA2 and melittin induced cultured spinal neuronal death in a dosedependent manner (see Fig 4C). Importantly, such
PLA2- or melittin-mediated neuronal death could be
significantly reversed by mepacrine, a PLA2 inhibitor
(see Fig 4D).
Phospholipase A2–Induced Spinal Cord Tissue
Damage in a Dose-Dependent Manner
To further assess whether an increased level of PLA2
could cause tissue damage in vivo, we microinjected
PLA2 (three doses at 0.05, 0.1, or 0.5␮g) or melittin
(three doses at 1, 2.5, or 5␮g) into the normal spinal
cord of adult rats. Although both PLA2 and melittin
induced spinal cord tissue damage, the histopathological characteristics induced by the two molecules were
somewhat different at 24 hours after injection. Although PLA2 injection induced a confined hemorrhage
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Fig 3. Cellular localization of cytosolic phospholipase A2
(cPLA2) expression is shown in the spinal cord at 7 days after
a moderate spinal cord injury (SCI). (A–C) cPLA2 immunoreactivity (IR) (A, arrows) was localized in neurons with
NeuN IR (B, arrows), which can be appreciated in the
merged image (C, arrows). (D–F) cPLA2 IR (D, arrows) was
localized in a subpopulation of axons with SMI-31 IR (E,
arrows), as seen in the merged image (F, arrows). Note that
glial cells that are positive for cPLA2 (D, F, arrowheads) were
negative for SMI-31 (E). (G–I) cPLA2 IR (G, arrows) was
localized in all oligodendrocytes with CC1 IR (H, arrows), as
seen in the merged image (I, arrows). (J–L) cPLA2 IR (J,
arrows) was colocalized only in a subpopulation of microglial
cells with OX-42 IR (K, arrows), and some of these cells (K,
L, arrowheads) were negative for cPLA2 (J–L). (M–O) Interestingly, astrocytes, identified with glial fibrillary acidic protein
(GFAP) IR (N, O, arrowheads), were cPLA2-negative. The
presence of cPLA2-positive but GFAP-negative cells in these
panels further confirms the specificity of these antibodies (M–
O). Bars ⫽ 20␮m.
‹
Figure 3
surrounding the site of injection (Figs 5F, J), melittin
injection induced a massive infiltration of inflammatory cells that spread far beyond the original injection
site (see Figs 5H, K). In both injections, infiltration of
inflammatory cells was found, and most of them were
polymorphonuclear neutrophils (see insets in Figs 5J,
Liu et al: PLA2 in Spinal Cord Injury
611
Fig 4. Effects of phospholipase A2 (PLA2) on spinal cord neuronal death in vitro. (A) A representative photomicrograph shows a
typical spinal cord neuron-enriched culture with the majority of cells being neurons positive for ␤-tubulin, a neuronal marker. The
cells in culture were counterstained with Hoechst 33342, a fluorescent nuclear dye. (B) Administration of PLA2 (6nM for 24
hours) induced massive neuronal death resulting in reduced neuronal density in the culture. In addition, many surviving neurons
showed nuclear fragmentation (inset), indicating they were dying in the form of apoptosis. (C) Lactate dehydrogenase (LDH) release
assay showed that PLA2 and melittin induced cultured spinal cord neuronal death in a dose-dependent manner (n ⫽ 8 per group;
**p ⬍ 0.01, ***p ⬍ 0.001, #p ⬍ 0.05, ##p ⬍ 0.01, ###p ⬍ 0.001, vs the vehicle control). (D) Importantly, PLA2- (1nM) or
melittin-induced (0.5␮M) neuronal death could be significantly reversed by mepacrine (50␮M), a PLA2 inhibitor (n ⫽ 8 per
group; ***p ⬍ 0.001 vs the vehicle control; ###p ⬍ 0.001 vs the PLA2 or melittin group). Mel ⫽ melittin; Mep ⫽ mepacrine.
K). As expected, no apparent tissue damage was found
in rats that received vehicle injection (see Fig 5E). Significantly, PLA2 injection induced a massive demyelination in the ventrolateral white matter radiating from
the site of injection, evidenced by the Luxol fast blue
staining (see Fig 5B). Except for the demyelination, tissue in this area appeared to be well intact, as was
shown in the neighboring cresyl violet-eosin–stained
section (see Fig 5F). Importantly, such PLA2-induced
ventrolateral white matter demyelination could be significantly and almost completely reversed by mepacrine, the PLA2 inhibitor (see Figs 5C, I). A toluidine
blue–stained section further confirmed a significant
loss of myelinated axons in the ventrolateral white mat-
Fig 5. Phospholipase A2 (PLA2) or melittin induced tissue damage at 24 hours after injection in vivo. (A, E) Luxol fast blue (A) and
cresyl violet-eosin (E) stainings showed no tissue damage or demyelination in a vehicle-injected spinal cord. (B, F) Injections of PLA2
(0.05␮g) into the normal spinal cord induced a confined demyelination in the ventrolateral white matter (B) that otherwise appeared
rather intact except for the presence of localized hemorrhagic changes at the site of injection (F). (C, G) Significantly, such focal demyelination and hemorrhagic changes, induced by PLA2, could be substantially reversed by mepacrine (5mg/kg), a PLA2 inhibitor. (D, H) In contrast with the PLA2 injection, injections of melittin (5␮g) induced a broader myelin breakdown (D) and tissue necrotic changes (H). (I)
Bar graph shows the percentage area of myelination per total spinal cord cross-sectional area after treatments with vehicle, PLA2, or
PLA2 ⫹ mepacrine (n ⫽ 6; **p ⬍ 0.01 vs vehicle; #p ⬍ 0.05 vs PLA2). Mepacrine markedly and significantly reduced PLA2-induced
demyelination at 24 hours after PLA2 injection. (J, K) High magnification of boxed areas in F and H showed that in both PLA2- and
melittin-injected groups, infiltration of inflammatory cells (arrows) at or close to the injection site was observed. Many of these were polymorphonuclear leucocytes (insets). (L–N) Toluidine blue–stained semithin cross sections showed intact (L), demyelinated (M), and degenerated (N) axons in the ventral white matter in animals that received injections of vehicle, PLA2, and melittin, respectively. (P–Q) Electron
micrographs further confirmed the massive demyelination (P) and axonal degeneration (Q) in animals that received PLA2 and melittin
injections, respectively. Note that in animals treated with PLA2 (P), axons remained intact despite massive myelin breakdown induced by
PLA2. Bars ⫽ 500␮m (A–H); 100␮m (J, K); 10␮m (insets in J, K); 100␮m (L–N); 5␮m (O–Q).
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‹
Figure 5
ter at 24 hours after the PLA2 injection (see Fig 5M)
compared with the vehicle-injected control rats (see Fig
5L). At the ultrastructural level, the widespread demyelination, induced by PLA2, was clearly seen (see Fig
5P). The myelin sheaths were dismantled, and lamellae
appeared loosely arranged, separated to form large,
clear spaces and degenerated into small clusters of ves-
icles (see Fig 5P). Clearly, most of the axons surrounded by these degenerating myelin sheaths appeared
to be morphologically normal (see Fig 5P). In contrast
with PLA2, melittin-induced changes were more necrotic in nature (see Figs 5N, Q). These changes included axonal swelling, fragmentation, breakdown, and
increased space between axons. The swelling and thin-
Liu et al: PLA2 in Spinal Cord Injury
613
ning of myelin sheaths were prominent, and in many
cases, they contained no axons (see Fig 5Q).
At 4 weeks after the PLA2 injection, a spread of tissue damage from the injury epicenter to the periphery
and to the rostral and caudal segments of the spinal
cord was found (Fig 6A, rows 2– 4). The area of tissue
damage could be well demarcated by the presence of
numerous dark-stained inflammatory cells. The degree
and extent of tissue damage in response to increased
PLA2 were dose dependent (see Fig 6A). Luxol fast
blue staining further showed that the PLA2 injection
resulted in a white matter demyelination in a dosedependent manner (see Fig 6B). A similar pattern of
tissue damage was observed in the spinal cord after the
melittin injection, as is summarized in the crosssectional measure of tissue damage at the injury epicenter (see Fig 6C). As anticipated, in the vehicleinjected control, only slight damage along the needle
track of the injection was observed at 4 weeks after
injection (see Fig 6A, first row).
Phospholipase A2–Induced Spinal Cord Functional
Impairment in a Dose-Dependent Manner
To determine whether injections of PLA2 and melittin
also induce functional impairments, we performed
both BBB locomotor rating scale and tcMMEP analyses. The BBB scale is a sensitive and reliable method
for detecting differences in locomotion across multiple
injury severities after SCI.26,33 In accordance with the
histological data, PLA2 or melittin injection induced a
corresponding locomotor impairment (Fig 7). BBB
scores were reduced after the injection of PLA2 (see Fig
7A) or melittin (see Fig 7B). Clearly, the higher the
doses of PLA2 or melittin used, the lower the BBB
scores obtained. The differences in BBB scores between
the vehicle-treated group and those groups treated with
high doses of PLA2 or melittin were statistically significant across all the time points studied. Similarly, tcMMEP was performed as an in vivo electrophysiological
measure of the integrity of descending motor pathways
in the ventrolateral funiculus. After injections of PLA2
or melittin into the spinal cord, peak-to-trough amplitudes of tcMMEP recordings were substantially decreased (see Figs 7C, D). Figure 7E shows a progressive
decrease in tcMMEP amplitudes in response to increases of administered PLA2.
Phospholipase A2–Induced Inflammatory Cytokine
Expression and Lipid Peroxidation
In an initial attempt to explore mechanisms by which
PLA2 or melittin mediates tissue damage, we investigated whether PLA2 or melittin could induce the activation of inflammatory and oxidative responses. We
found that injections of PLA2 or melittin resulted in a
marked increase in both TNF-␣ (Figs 8B, C, M) and
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IL-1␤ (see Figs 8E, F, N) expression, respectively, at
and near the site of the injections at 24 hours after
SCI, compared with the vehicle-injected group (see
Figs 8A, D). Injections of PLA2 (see Fig 8H) or melittin (see Fig 8I) also resulted in a marked increase in the
expression of 4-HNE (see Figs 8H, L, O), an aldehydic
product of lipid peroxidation and a marker for oxygen
free radical–mediated membrane injury.34,35 The highest 4-HNE IR was found in axons of the ventral white
matter after both injections (see Figs 8K, L). As anticipated, no 4-HNE IR was detected in the vehicleinjected control rats.
Discussion
To our knowledge, this is the first study demonstrating
an important role of PLA2 in the pathogenesis of spinal
cord secondary injury. We demonstrated that both
PLA2 activity and expression increased significantly in
the spinal cord after injury, and that neurons and oligodendrocytes are the two major sources of increased
levels of cPLA2. We also demonstrated that administration of exogenous PLA2 or melittin induced cultured spinal cord neuronal death in a dose-dependent
manner, an effect that could be substantially reversed
by mepacrine, a PLA2 inhibitor. We further demonstrated that microinjections of a single source of PLA2
or melittin into the spinal cord induced tissue damage
and functional impairment. Remarkably, the PLA2induced central demyelination could be significantly
reversed by the mepacrine. Lastly, we demonstrated
that PLA2 or melittin induced tissue inflammation and
oxidation, the two well-characterized mechanisms that
lead to spinal cord secondary injuries.
Although both PLA2 activity and expression increased after SCI, they peaked at different time points.
Whereas PLA2 activity peaked at 4 hours after SCI, its
expression reached the maximum at 3 days. One possible explanation for the difference between the two
peaks is that the PLA2 activity that we measured was
the total activity of PLA2 (including both cPLA2 and
secreted PLA2), whereas the cPLA2 expression that we
measured was only a subclass of the total PLA2. Alternatively, the activity of the PLA2 enzyme responded
quicker than the expression of PLA2 because the accumulation of the latter may require more time in response to SCI.
A significant finding in this study is that PLA2 induced spinal cord neuronal death in both the in vitro
and in vivo experimental paradigms in which PLA2 was
used as the only source of a damaging factor. Given
that SCI induces a rapid increase and prolonged expression of PLA2 and that neurons are among the two
major cell types that express high levels of PLA2 after
SCI, it is conceivable that increased levels of PLA2 may
directly induce neuronal death. This is supported by
the observation that the PLA2-induced neuronal death
Fig 6. Injections of phospholipase A2 (PLA2) or melittin into the normal spinal cord resulted in tissue damage in a dose-dependent
manner. (A) Representative cresyl violet-eosin stainings showed tissue damage increased transversely and longitudinally with increases
in doses of PLA2. (B) Representative photographs of Luxol fast blue–stained sections showed that PLA2 induced demyelination in a
dose-dependent manner. (C) A bar graph is used to show PLA2 or melittin injections induced increases in lesion areas in a dosedependent manner (n ⫽ 6; *p ⬍ 0.05, **p ⬍ 0.01, vs vehicle). Bars ⫽ 1mm.
could be substantially reversed by mepacrine. In addition to its direct effect on neuronal death, PLA2 may
trigger a cascade of downstream events that lead to indirect neuronal death. Indeed, both previous and current studies demonstrated that PLA2 could induce inflammation and oxidation,6,7,36 which, in turn, induce
neuronal death indirectly.6,7 Thus, PLA2 likely serves
as a key molecule that mediates neuronal death in both
direct and indirect manners.
A striking finding of this study is that PLA2 injection induced a rapid and confined demyelination. This
is evidenced by the lack of myelin staining in the ventrolateral white matter surrounding the injection site,
the reduction of myelinated axons in the same region
after toluidine blue staining, and the presence of a large
quantity of myelin breakdown at the electron microscopic level. Remarkably, such PLA2-induced demyelination could be significantly reversed by mepacrine, in-
Liu et al: PLA2 in Spinal Cord Injury
615
Fig 7. Behavioral and electrophysiological changes are displayed after injections of phospholipase A2 (PLA2) or melittin into the normal
spinal cord of adult rats. (A, B) The Basso, Beattie, and Bresnahan (BBB) locomotion rating scale showed that BBB scores decreased in
response to increased doses of PLA2 or melittin (n ⫽ 6; *p ⬍ 0.05, **p ⬍ 0.01, ***p ⬍ 0.001, vs vehicle). (C, D) Transcranial
magnetic motor-evoked potential (tcMMEP) recordings showed that injections of PLA2 or melittin resulted in decreases in peak-totrough (P-T) amplitude at various time points after injection, indicating that PLA2 and melittin could directly cause functional impairments of the injected rats (n ⫽ 6; *p ⬍ 0.05, **p ⬍ 0.01, ***p ⬍ 0.001, vs vehicle). (E) Representative tcMMEP recordings
showed that P-T amplitude decreased in response to increased doses of PLA2 in a dose-dependent manner at 28 days after injection.
dicating that PLA2 could be a key molecule that causes
myelin breakdown. Except for the phenomenon of demyelination, tissues in this area appeared to be well
intact and axons morphologically normal. These results, together with the observation that the oligodendrocyte is another cell type that expressed cPLA2 after
SCI, collectively imply that PLA2 is a molecule that
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could induce central nervous system demyelination. It
remains unclear, however, whether this demyelination
results from a direct attack of PLA2 on the central nervous system myelin or an indirect effect caused by
PLA2-mediated oligodendrocyte cell death.
The reason PLA2 induces myelin breakdown may be
explained by its enzymatic activity.37 This enzyme hy-
Fig 8. Expression of cytokines and 4-hydroxy-2(E)-nonenal (4-HNE), a marker of lipid peroxidation, at 24 hours after phospholipase A2 (PLA2) or melittin injections is shown. (A–F) Increased expression of tumor necrosis factor-␣ (TNF-␣) (B, C) or
interleukin-1␤ (IL-1␤) (E, F) after injections of PLA2 (0.05␮g; B, E) or melittin (5␮g; C, F) compared with vehicle-injected control animals (A, D), respectively. (G–L) Increased expression of 4-HNE was localized mainly in axons after the PLA2 (H) or melittin (I) injection, which could be clearly seen at higher magnifications (K, L) compared with a vehicle-injected control animals (G,
J). (M–O) PLA2 and melittin injections induced increases in the expression of TNF-␣ (M), IL-1␤ (N), or 4-HNE (O) (n ⫽ 6;
***p ⬍ 0.001 vs vehicle). Bars ⫽ 100␮m. VWM ⫽ vanishing white matter.
drolyzes phospholipids to produce lysophospholipids
and a free fatty acid.38 If the phospholipid is phosphatidylcholine, PLA2 can induce lysophosphatidylcholine and arachidonic acid. Lysophosphatidylcholine acts
as a detergent to cause myelin breakdown.39 – 41 Indeed, expression of PLA2 was found to correlate well
with the rate of myelin breakdown associated with
Wallerian degeneration in both the central and peripheral nervous systems, although the rates between the
two were somewhat different.37
Our data confirmed the presence of cPLA2 in neurons and glial cells in the normal rat spinal cord, as
reported previously.17,42 We further demonstrated that
neurons and oligodendrocytes are the two major cell
types that expressed cPLA2 after SCI. Interestingly,
only a subset of microglia and none of the astrocytes
expressed cPLA2 at and near the site of SCI. These
data, together with the observation that PLA2 induced
spinal cord neuronal death and oligodendrocyte demyelination, collectively indicate that the expression of
Liu et al: PLA2 in Spinal Cord Injury
617
cPLA2 in neurons and oligodendrocytes is highly specific, and that the two cell types may be more vulnerable to PLA2-mediated cell death than other cell types.
Because both neurons and oligodendrocytes play critical roles in normal spinal cord circuits and function,
repair strategies targeting against PLA2-mediated neuronal death and axonal demyelination may be effective
approaches to restore anatomical and functional connections after SCI.
Although both PLA2 and melittin induced neuronal
death and tissue damage, differences between the two
were noted. For example, whereas PLA2 microinjection
induced a defined local demyelination at 24 hours after
injection, melittin-induced changes were more necrotic
in nature. One possible explanation for the difference
in histopathological changes after the two types of injections is that the changes induced by PLA2 represent
damage induced by a subclass of PLA2, namely, secreted PLA2, whereas those induced by melittin represent damage induced by all PLA2s, activated by melittin. An alternative explanation is that the
histopathological differences seen after the two molecule injections may simply reflect the difference in
doses used between the two types of injections. Although the similarities and differences between the two
types of injections remain to be investigated further,
the different histopathological outcomes induced by
them may provide two interesting injury models for
the studies of PLA2-mediated spinal cord secondary injury because the sources of damage are relatively clear.
Although a significant increase in PLA2 activity and
expression after SCI was observed in this study, the
mechanism(s) by which they increase remains unclear.
Previous studies using other models, however, showed
that PLA2 activity or expression, or both, was induced
by harmful substances such as inflammatory cytokines,5,10 free radicals,11 and excitatory amino acids.9,12,13 Given that these substances all have been
demonstrated to increase in the injured spinal cord, it
is conceivable that PLA2 may serve as a converging
molecule that mediates the pathogenesis of multiple injury mechanisms associated with spinal cord secondary
injury.1–3 Furthermore, increased levels of PLA2 and
their metabolites may also induce inflammation, oxidation, and neurotoxicity, as demonstrated by previous6 – 8 and current studies, which could further exacerbate the injury. Thus, PLA2, a molecule that could
be induced by multiple injury pathways and, in turn,
enhance these pathways, may serve as a central or convergence molecule that mediates multiple injury mechanisms during the progression of spinal cord secondary
injury. Blocking the activity and expression of PLA2
may represent a novel and more efficient strategy to
block multiple damaging pathways, and therefore
achieve better tissue protection and functional recovery.
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April 2006
This work was supported by the NIH (National Institute of Neurological Disorders and Stroke, NS36350, NS52290), the Kentucky
Spinal Cord and Head Injury Research Trust, (416) the Daniel
Heumann Fund for Spinal Cord Research, (X.M.X.) and the Paralysis Project of America (GRNT050293, N.K.L.).
We thank A. Puckett for care of animals, D. Burke and K. Fentress
for behavioral and electrophysiological assessments, G. Harding for
confocal imaging, and C. Caple for electron microscopy. We also
thank the Norton Healthcare, Kentucky Spinal Cord and Head Injury Research Trust Board, State of Kentucky Bucks for Brains Program, and University of Louisville through the James R. Petersdorf
Endowment. We also appreciate the use of the core facility of the
Kentucky Spinal Cord Injury Research Center, which is supported
by the NIH (COBRE RR15576).
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