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(Stroke. 1996;27:527-535.)
© 1996 American Heart Association, Inc.

Articles

Reactive Glia Express Cytosolic Phospholipase A2 After Transient Global Forebrain Ischemia in the Rat

James A. Clemens, PhD; Diane T. Stephenson, BS; E. Barry Smalstig, BS; Edda F. Roberts, MS; Edward M. Johnstone, MS; John D. Sharp, PhD; Sheila P. Little, PhD Ruth M. Kramer, PhD

From Eli Lilly and Company, CNS and Cardiovascular Divisions, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Ind.

Correspondence to Dr James A. Clemens, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Background and Purpose Phospholipid breakdown has been reported to be an early event in the brain after global cerebral ischemia. Our earlier observations showing the localization of cytosolic phospholipase A₂ (cPLA₂) to astrocytes in aged human brains and the intense glial activation observed after global forebrain ischemia prompted us to investigate the cellular localization of cPLA₂ in the rat brain subjected to global ischemia.

Methods Immunohistochemistry was performed in sections through the dorsal hippocampus in rats subjected to 30 minutes of four-vessel occlusion. PLA₂ was localized with the use of a highly selective antiserum. Double immunofluorescent localization was performed to colocalize cPLA₂ with various glial cell types. cPLA₂ levels were also measured by enzymatic assay and Western blot analysis.

Results A marked induction of cPLA₂ was observed in activated microglia and astrocytes in the CA1 hippocampal region at 72 hours after ischemia. Only a subset of astrocytes and microglia were immunoreactive for cPLA₂. Twenty-four hours after ischemia, numerous cPLA₂ immunoreactive astrocytes were observed. Western blot analysis of hippocampal homogenates at 72 hours after ischemia showed induction of a 100-kD band that comigrated with purified human cPLA₂, and a threefold induction in cPLA₂ activity was demonstrated by enzymatic assay.

Conclusions These results indicate that both reactive astrocytes and microglia contain elevated levels of cPLA₂. Induction of cPLA₂ was confined to areas of neurodegeneration and likely precedes its onset. The results suggest that reactive glia may play a role in the pathophysiology of delayed neuronal death after transient global forebrain ischemia.

Key Words: astrocytes • cerebral ischemia, global • occlusion • phospholipids • rats

	Introduction

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After short periods of global forebrain ischemia, the hippocampal CA1 layer undergoes a process of delayed cell death^{1 2} in all mammalian species thus far examined. Despite years of research into the precise mechanisms that are responsible for delayed cell death, which occurs over a period of days, the causes of this phenomenon are still unknown. Studies with pharmacological agents have shown either exacerbation or amelioration of hippocampal CA1 neuronal cell death after global ischemia and as a result have led to proposals of several different mechanisms that could be responsible for the delayed neuronal cell death. Close examination of the neuropathology of global ischemia revealed that intense glial cell reactivity occurred in and around the hippocampal CA1 layer.^{3 4 5} Much of the glial cell activation occurred before histological evidence of neuronal cell death.^{4 5} Other well-documented observations after transient global forebrain ischemia are elevations of free fatty acids,^{6 7 8} products of the arachidonic acid cascade,^{9 10 11 12} and PAF.¹³ Products of lipid peroxidation have also been reported, which are likely to have resulted from oxygen radical attack on free fatty acids.¹⁴ Because of increases in free fatty acids, eicosanoids, and products of lipid peroxidation, PLA₂ has been proposed to be one of the factors that is at least in part responsible for the events leading up to neuronal cell death after cerebral ischemia.¹⁵ Although it is not known whether the Ca²⁺-sensitive cPLA₂ is directly involved in causing damage to neuronal membranes, it is involved in the release of arachidonic acid from membrane phospholipids and the initiation of the arachidonic acid cascade.^{16 17}

Several different forms of PLA₂ exist. Two types of mammalian PLA₂ have recently been purified, cloned, and sequenced, namely the 14-kD sPLA₂^{18 19} and the 85-kD cPLA₂.^{20 21 22 23} Although sPLA₂ and cPLA₂ perform the same catalytic function, their structural and biochemical properties differ greatly. Thus, cPLA₂ has a marked preference for arachidonic acid esterified at the sn-2 position of phospholipids and is active at submicromolar concentrations of Ca²⁺. In contrast, the sPLA₂ does not exhibit a fatty acid preference and requires millimolar concentrations of Ca²⁺ for catalytic activity. Ca²⁺-independent PLA₂ activities have also been purified and characterized, but their primary structures have not been delineated.^{24 25} We recently reported the localization of cPLA₂ in a subset of human brain astrocytes.²⁶ Because of the intense astrocytic activation in the hippocampus of animals subjected to global ischemia, we decided to investigate the localization of cPLA₂ in the rat brain subjected to global ischemia with an antiserum recently developed that cross-reacts with rodent cPLA₂.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Transient forebrain ischemia was induced by 4-VO according to the method of Pulsinelli and Brierley.²⁷ Briefly, male Wistar rats (weight, 250 to 270 g) (Hilltop Laboratories, Scottsdale, Pa) were prepared for forebrain ischemia under 2% halothane inhalation anesthesia by electrocauterizing the bilateral vertebral arteries and placing atraumatic clasps around the common carotid arteries without interrupting the arterial blood flow. On the following day, forebrain ischemia was induced by tightening the clasps for 30 minutes. The animals were unanesthetized during the ischemia. Body temperature was maintained at 37°C throughout the duration and for 30 minutes after the ischemia. Twenty-four 4-VO animals were evaluated in this study. Seven sham-operated animals were evaluated as controls. The animal procedures were performed in accordance with institutional guidelines. At 24 (n=6) and 72 hours (n=14) after ischemia, animals were perfused with phosphate-buffered saline followed by ice-cold 4% buffered paraformaldehyde. Brains were excised and processed as described below.

Forebrains were postfixed by immersion for 24 hours in fixative and cryoprotected in 30% sucrose. Tissues were rapidly frozen in isopentane chilled with dry ice and serially sectioned in the coronal plane throughout the rostrocaudal extent of the hippocampus. Twenty-micron-thick sections were thaw-mounted onto gelatin-coated slides and stored at -70°C until evaluation. For anatomic localization of the lesioned areas, tissue sections were stained with cresyl violet for Nissl substance. Regions evaluated included coronal sections of the dorsal hippocampus. Immunocytochemistry of cPLA₂ was achieved with the use of a polyclonal antiserum raised against human cPLA₂ purified from a baculovirus insect cell expression system.¹⁶

Immunocytochemistry was performed with the use of the avidin-biotin peroxidase system (ABC kit, Vector Labs), and peroxidase enzymatic activity was revealed with 0.5 mg/mL 3,3'-diaminobenzidene in the presence of 0.003% H₂O₂ (DAB substrate kit, Vector Labs). Immunoperoxidase-stained sections were lightly counterstained with hematoxylin to delineate cytoarchitecture. To ensure accuracy when results from immunostained slides were compared, multiple sections from both 4-VO and control brains were stained in parallel on the same day. Development times in 3,3'-diaminobenzidene were held constant for all sections. These experimental conditions reduced the possibility that differences in immunoreactivity profiles might be accounted for on the basis of experimental variables (eg, antibody titer/affinity, tissue postfixation, 3,3'-diaminobenzidene development times). Quantitative immunocytochemistry with the use of image analysis was not performed on these slides because of the robust differences in specific staining between ischemic versus control animals.

The distribution of cPLA₂ was compared with that of glial cells by performing immunoperoxidase staining on adjacent sections. To identify astrocytes, antisera directed to GFAP (Biogenex Labs), an intermediate filament protein unique to astrocytes,²⁸ and a monoclonal antibody directed against the cytosolic protein S-100 (Biogenex Labs) were used. For localization of microglia, monoclonal antibodies OX42 (Bioproducts for Science) and ED1 were used. OX42 identifies the CR3 complement receptor that is expressed on activated microglia,^{5 29} and the antibody ED1 defines a cytosolic protein that is present within all cells of the rat monocyte/macrophage lineage.³⁰ ED1 recognizes end-stage amoeboid microglia in the brain³¹ and does not stain resting microglia.³² For colocalization experiments on the same tissue section, double immunofluorescence techniques were used. Double staining was performed sequentially as follows: sections were stained first with cPLA₂ antiserum, which was localized with biotin anti-rabbit followed by avidin-Texas red (Vector Labs), then with one of the above monoclonal antibodies for astrocytes or microglia. The monoclonal antibody was localized with goat anti-mouse immunoglobulins (high fluorescent conjugate, Antibodies Inc). Sections were examined with a Nikon Microphot microscope equipped with epifluorescence and Nomarski optics.

Positive and negative controls were conducted in parallel with cPLA₂-stained sections or cells in each experiment. Staining of sections with commercially available antibodies served as the positive control. Negative controls included staining tissue sections with omission of the primary antibody. In addition, sections were stained with anti-cPLA₂ antiserum that had been preadsorbed with an excess of cPLA₂ purified from a baculovirus cell expression system.¹⁶

PLA₂ activity was assayed in microdissected dorsal hippocampal tissue excised from four sham-operated controls and from four rats at 72 hours after 4-VO. Sonicated liposomes containing 1-palmitoyl-2-[¹⁴C]arachidonoyl-sn-glycero-3-phosphocholine (50 mCi/mmol; DuPont-NEN) and sn-1,2-dioleoyl glycerol (Avanti Polar Lipids) at a molar ratio of 2:1 were prepared as previously described.²² These liposomes are an extremely poor substrate for the 14-kD sPLA₂.³³ The assay buffer consisted of 1 mmol/L dithiothreitol, 150 mol/L NaCl, and 50 mmol/L HEPES, pH 7.5, containing 1 mg/mL of bovine serum albumin, 1 mmol/L CaCl₂, 2 µmol/L [¹⁴C]arachidonoyl-phosphatidylcholine (50 000 dpm)/1 µmol/L dioleoyl glycerol, and incubations were performed at 37°C for 15 minutes. To evaluate the contribution of Ca²⁺-independent PLA₂ activities, we included 10 mmol/L EDTA in the assay buffer. This assay system is specific for cPLA₂, as previously shown with platelets (R.M.K. et al, unpublished data, 1993).

All procedures were performed at 4°C. Microdissected dorsal hippocampus (40 to 70 mg) was homogenized in 500 µL lysis buffer (5 mmol/L EDTA, 150 mmol/L NaCl, 25 mmol/L Tris-HCl, pH 7.5, containing 0.2 mmol/L Na₃VO₄, 1 mmol/L Pefabloc [Bachem], 0.06 mg/mL aprotinin, 0.05 mg/mL leupeptin, 100 nmol/L microcystin, 10.2 µmol/L pepstatin A) with the use of a Polytron homogenizer. Homogenates were briefly sonicated (ten 2-second bursts) with a Heat Systems Microson cell disrupter. The sonicates were then centrifuged for 30 minutes at 100 000g with a Sorvall RC-M120EX centrifuge. Supernatants were recovered and concentrated approximately 10-fold with Centricon 10 microconcentrators (Amicon). After volumes were adjusted to 100 µL with lysis buffer, cytosolic extracts were assayed for PLA₂ activity (10 µL) and protein content (1 µL) and subjected to SDS-PAGE/immunoblotting analysis (4 µL) as detailed below.

After addition of Tricine SDS sample buffer (Novex) (twice) and dithiothreitol to a final concentration of 100 mmol/L, brain cytosolic concentrates were boiled for 5 minutes. Samples were electrophoresed in 7.5% polyacrylamide Tris-HCl gels (0.75 mm thick, Daiichi) at 50 mA for 1.3 hours at room temperature with the use of an Integrated Separation Systems apparatus and Integrated Separation Systems premixed anode buffer (200 mmol/L Tris-HCl, pH 8.9) and cathode buffer (100 mmol/L Tris-base, pH 8.25, 100 mmol/L Tricine, and 0.1% SDS). Standard proteins were Rainbow Markers (range, 14.3 to 200 kD; Amersham), human cPLA₂ purified from a baculovirus/insect cell expression system, or rat platelet lysate containing cPLA₂. After electrophoresis, the gel was equilibrated in electroblotting buffer (10 mmol/L 3-(cyclohexylamino)-1-propanesulfonic acid, pH 11.0, 0.01% SDS, and 1% methanol) for 5 minutes, followed by two more washes. Proteins were electroblotted onto polyvinylidene difluoride (Biorad, 0.2 µm) at 500 mA constant current and nonlimiting voltage for 1 hour at 20°C with the use of the Hoefer tank system. After the transfer, the PVDF membranes were dried, wetted in methanol and water, and incubated for 1 hour at room temperature in blocking buffer TBST (25 mmol/L Tris-HCl, pH 7.5, 137 mmol/L NaCl, 2.6 mmol/L KCl, 0.1% Tween-20, and 0.2% I-Block [Torpix]). The blots were incubated for 1 hour with rabbit anti-cPLA₂ antiserum diluted 1:5000 in TBST and were washed three times quickly, followed by three more 5-minute washes in TBST. Membranes were then incubated for 30 minutes with goat anti-rabbit IgG(H+L)/horseradish peroxidase conjugate (Jackson ImmunoResearch) diluted 1:5000 into TBST and then were washed three times quickly, followed by three 5-minute washes with TBST. The blots were washed three times quickly, followed by one 5-minute wash with TBST omitting the I-Block and were then developed with the enhanced chemiluminescence detection system (Amersham). Quantitation was performed by phosphoimager analysis.

Protein measurements were made in the presence of 0.05% SDS with the use of Coomassie Plus protein reagent (Pierce), with bovine serum albumin as a reference standard.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Sections throughout the level of the dorsal hippocampus from twenty 4-VO rats were evaluated and compared with sections from sham-operated controls. Thirty minutes of transient global forebrain ischemia resulted in selective necrosis of neurons in the hippocampal CA1 region at 72 hours after ischemia (Fig 1), consistent with previous reports. Immunocytochemistry was performed with the use of a highly specific antisera made to Ca²⁺-sensitive cPLA₂. cPLA₂ immunostaining revealed a striking pattern in brains from animals subjected to 4-VO. Intense labeling was observed throughout the CA1 region at 72 hours after ischemia (Fig 1D). This staining was not observed in brains from sham-operated controls (Fig 1B). Immunoreactivity was abolished by incubating and labeling sections with antiserum preadsorbed with excess purified cPLA₂.

View larger version (125K): [in this window] [in a new window]   Figure 1. cPLA2 immunoreactivity is expressed in the CA1 region of the hippocampus after transient global forebrain ischemia. Adjacent sections through the dorsal hippocampus are from a control rat (A, B) and from a rat subjected to 30 minutes of 4-VO (C, D, E, F). A, C, and E, Nissl staining; B, D, and F, cPLA2 immunoperoxidase staining. A through D, CA1 region; E and F, CA1-CA2 boundary (arrow). cPLA2 staining is expressed in the area of neurodegeneration. Bar=50 µm.

cPLA₂ was selectively expressed in areas of neurodegeneration. In the hippocampus of animals subjected to 4-VO, cPLA₂ immunoreactivity was observed in multiple layers of the CA1 region including stratum pyramidale, stratum radiatum, stratum oriens, and lacunosum moleculare. Other hippocampal regions displayed relatively little staining. cPLA₂ immunostaining came to an abrupt halt at the boundary between CA1 and CA2 (Fig 1F). This boundary precisely defines the region of selective neuronal degeneration. At this border zone, intact-appearing Nissl-stained neurons of CA2 were observed immediately adjacent to necrotic CA1 neurons that lacked Nissl substance (Fig 1E).

High magnification of cPLA₂ immunoperoxidase-stained sections from 4-VO brains revealed distinctive labeling of cells. The distribution pattern appeared granular in many immunoreactive profiles (Fig 2C, arrows). Many stained structures resembled morphologically identifiable astrocytes. Indeed, some immunopositive processes were found to terminate on blood vessels, a feature unique to these cells. Other immunopositive structures assumed a rounded amoeboid morphology characteristic of brain macrophages. Immunoreactivity was not detected within vascular endothelial cells. In our experience, endothelial cells are not a particularly prominent source of cPLA₂ and are unlikely to significantly contribute to the observed PLA₂ activity and protein (R.M.K. et al, unpublished data, 1993). Control brains exhibited rare cPLA₂ immunoreactive cells scattered throughout the hippocampus that were presumptive astrocytes (Fig 2A). These cells were not observed at low magnification (Fig 1B). When compared with presumptive astrocytes detected in control brains, cPLA₂ immunopositive astrocytes in 4-VO brains exhibited morphological features consistent with an activated state, including swollen cell bodies and enlarged processes.

View larger version (70K): [in this window] [in a new window]   Figure 2. cPLA2 immunoperoxidase staining in the CA1 region from a nonischemic control (A) and from rats at 24 hours (B) and 72 hours (C) after 4-VO. High magnification illustrates rare immunoreactivity present in morphologically identifiable astrocytes (A and B, arrow) and astrocyte processes (A and B, arrowhead) in sham controls and at 24 hours after ischemia. At 72 hours after ischemia, some immunoreactive profiles resemble astrocytes (C, arrow) while others exhibit a rounded or amoeboid morphology (C, curved arrows). Immunoreactivity is concentrated within stratum pyramidale but is also present in stratum oriens and stratum radiatum. At 72 hours after ischemia, cPLA2 staining often displays a punctate or granular distribution (C). Bar=10 µm.

Six animals were evaluated at 24 hours after ischemia. At this time point, there is no histological evidence of neuronal damage in the CA1 region.^{34 35 36} cPLA₂ staining revealed immunoreactive astrocytes scattered throughout the CA1 region (Fig 2B). The intensity and number of immunopositive cells were significantly less than those observed at 72 hours yet more than those observed in sham controls.

To determine the identity of the cells that contain cPLA₂ immunoreactivity, double immunofluorescent staining was performed in the hippocampus from animals subjected to 4-VO. Several markers of both astrocytes and microglia were evaluated. In the CA1 region, cPLA₂ immunopositive cells were found to colocalize with GFAP (Fig 3A and 3B) and with S-100 immunoreactive astrocytes (Fig 3C and 3D). Colocalization of cPLA₂ immunoreactivity with these astrocyte markers was most often observed to be coincident in cells that could be identified as astrocytes by their morphology alone. Not all astrocytes identified by these markers were double labeled with cPLA₂ antiserum. A distinct population of stained cells in this region that did not exhibit astrocyte-like morphology were immunoreactive for the microglial cell marker OX42 (Fig 3E and 3F). The rounded amoeboid-appearing cells were most often colocalized with the macrophage marker ED1 (Fig 3G and 3H). Punctate cPLA2 staining was most often associated with cells that also expressed the microglial markers. Only a subset of microglia, defined by either OX42 or ED1, was positive for cPLA₂ immunoreactivity. Thus, both astrocytes and microglia expressed cPLA₂ immunoreactivity at 72 hours after 4-VO.

View larger version (0K): [in this window] [in a new window]   Figure 3. Double immunofluorescence staining of cPLA2 and various glial cell markers in the CA1 region of rat hippocampus at 72 hours after ischemia. cPLA2 immunoreactivity (A, C, E, G) is colocalized with GFAP (B), S-100 (D), OX42 (F), and ED1 (H) immunopositive glial cells. Arrows indicate double-labeled cells. cPLA2 immunoreactivity was localized with the use of a Texas red label, while astrocytes and microglia were identified with the use of fluorescein isothiocyanate as the fluorophore. In the photomicrographs counterstained with the microglial cell markers OX42 and ED1, cPLA2 immunoreactive structures can be identified in the fields that are not double labeled. Morphologically, these profiles correspond to presumptive astrocytes (arrowheads). Note the cell surface staining of the CR3 receptor protein defined by OX42 (F). Bar=10 µm.

To further examine the presence of cPLA₂ in rat dorsal hippocampus, we performed assays for PLA₂ activity and subjected hippocampal extracts to immunoblotting analysis. We found that the enzymatic activity of cPLA₂ was increased threefold in cytosolic extracts of dorsal hippocampus from ischemic rats compared with sham-operated control rats (Fig 4A). Consistent with the Ca²⁺ requirement of cPLA₂, this enzymatic activity was inhibited in the presence of the chelating agent EDTA. The activity observed is due to cPLA₂ for the following reasons: the assay is performed in the presence of dithiothreitol (known to inactivate sPLA₂) and uses sonicated liposomes consisting of phosphatidylcholine and dioleoyl glycerol previously shown to be an extremely poor substrate for sPLA₂ (see Reference 33). Since the activity is greatly reduced in the presence of EGTA, there clearly is no involvement of a Ca²⁺-independent PLA₂.

View larger version (25K): [in this window] [in a new window]   Figure 4. Induction of cPLA2 activity and protein after global forebrain ischemia. Brain dorsal hippocampal cytosolic extracts were prepared from sham-treated control and ischemic rats as detailed in "Materials and Methods." A, Determination of PLA2 activity in extracts (10 µL) in the presence of 1 mmol/L Ca2+ (+Ca2+) or 10 mmol/L EDTA (+EDTA). The PLA2 activity is shown as mean±SEM (of four different animals) performing duplicate determinations. B, SDS-PAGE/immunoblot probing with rabbit anti-cPLA2 antiserum. Extracts (4 µL) were electrophoresed on 7.5% gels as described in "Materials and Methods." Lane 1, Rat platelets (rPlat); lanes 2 through 5, sham-operated control animals; lanes 6 through 9, ischemic animals; and lane 10, purified human cPLA2 (hcPLA2). Extracts of four different animals (for each control and ischemic rats) were subjected to the immunoblotting analysis.

We detected an approximately 100-kD major immunoreactive band indicative of cPLA₂ in cytosolic extracts subjected to immunoblotting and probed with anti-cPLA₂ antiserum (Fig 4B). This band comigrated with purified human cPLA₂ (purified from a baculovirus expression system) and rat platelet cPLA₂ and could not be detected when the antiserum was preadsorbed with purified cPLA₂. It is well known that cPLA₂ migrates abnormally due to proline-rich residues within the enzyme. Depending on the electrophoresis and gel system used, values for its apparent molecular mass may vary between 85 and 110 kD. On a 7.5% minigel, as used in the present study, cPLA₂ typically runs with an apparent mass of 100 kD. Lane 1 contains activated rat platelet cPLA₂ that shows slightly decreased electrophoretic mobility due to phosphorylation; lane 9 contains cPLA₂ from an ischemic animal; and lane 10 contains purified human cPLA₂ (expressed in a baculovirus/insect cell expressed system) that typically runs slower than rat cPLA₂.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Global cerebral ischemia results in the death of selected neuronal subpopulations in the brain, with the hippocampal CA1 neurons being most susceptible. Marked changes in CA1 hippocampal neurons are not typically observed until 72 hours after reperfusion, whereas ischemic cell changes are seen within 24 hours in striatal and cortical neurons.^{2 36} The temporal profile of glial cell response to ischemia has been fully characterized.^{3 4 5 37 38} The mechanism of delayed neuronal death in the hippocampal region of rats subjected to 4-VO has been a topic of intense investigation.

The results of this study demonstrate that after 30 minutes of global forebrain ischemia induced by 4-VO, cPLA₂ immunoreactive glia appear throughout the CA1 region of the hippocampus.

Moreover, induction of PLA₂ enzymatic activity due to cPLA₂ occurred in microdissected dorsal hippocampus from ischemic animals. Immunoreactivity as well as enzymatic activity was strongly induced at 72 hours after ischemia, the time point of neurodegeneration in this region. A slight increase in cPLA₂ immunoreactivity was already observed at 24 hours after 4-VO, before the onset of histologically evident neuronal cell death. cPLA₂ immunoreactivity in the CA1 region colocalized with both astrocyte and microglial markers; however, only a subset of microglia and astrocytes was found to express cPLA₂ immunoreactivity. cPLA₂ expression in astrocytes occurred as early as 24 hours after 4-VO; few sparsely distributed resting astrocytes were found to express cPLA₂ immunoreactivity in control tissue. Expression of cPLA₂ in microglial cells was not observed until 72 hours after 4-VO, a time that was coincident with neuronal death. Notably, microglia are known to change their morphology from ramified to rounded or amoeboid at this time point.^{4 5} It has been recently proposed that microglia and macrophages may contribute to delayed neuronal cell death after ischemia.^{39 40 41} The demonstration that cPLA₂ is expressed in reactive microglia in this model supports this hypothesis.

The observation that cPLA₂ immunoreactivity is specifically localized to glia is supported by the following observations: (1) cPLA₂ immunoreactivity is colocalized with GFAP, S-100, OX42, and ED1 immunoreactivities in the CA1 region after ischemia; (2) immunoadsorption with purified cPLA₂ enzyme abolishes staining; and (3) cPLA₂ immunoreactivity is present in human brain astrocytes.²⁶ This latter study used a monoclonal antibody and demonstrated cPLA₂-specific immunostaining in human cortical astrocytes in situ and in human astrocytomas in vitro. The fact that both the same cell type as well as the same distribution of stain was observed with two different antibodies in two different species is strong evidence that this enzyme is expressed in glia. Furthermore, the increased cPLA₂ levels in dorsal hippocampal extracts, as demonstrated by Western blot analysis and cPLA₂ enzyme assay, provide strong evidence that the increased cPLA₂ immunoreactivity observed in glia by histochemistry is authentic cPLA₂. Stimulation of endothelial cells in vitro has been shown to induce cPLA₂ enzyme activity; basic fibroblast growth factor⁴² and the state of proliferation⁴³ both affect activation of cPLA₂ in cultured endothelial cells. In our in vivo study, there was no evidence that endothelial cell cPLA₂ contributed to the enzyme reactivity by immunocytochemistry, although we cannot exclude the possibility that cPLA₂ in brain capillary endothelial cells contributed to the increased enzyme activity (by cPLA2 activity measurements) and enzyme levels (observed by Western blots).

Rat cPLA₂ mRNA has been recently reported to be induced in the hippocampus after 4-VO.⁴⁴ With the use of in situ hybridization, a strong induction of cPLA₂ mRNA was observed from 6 to 24 hours in the dentate granule cell layer after 10 minutes of ischemia, while the CA1 layer showed mRNA at moderate levels at 6 hours, modest levels at 12 hours, and virtually none at 24 hours. Although these data support our observations of an increase in cPLA₂ in this model, the cell type expressing the mRNA and that expressing the protein are not identical nor in the same anatomic location. The apparent discrepancy may be due to the different durations of ischemia in the two studies (10 minutes versus 30 minutes) or the different time points evaluated after ischemia. Alternatively, this may be another example illustrating a mismatch between mRNA expression and protein localization.

Although it was reported previously that Ca²⁺-dependent PLA₂ activities are increased in the ischemic/reperfused brain, the PLA₂ activity in these studies was measured after short periods (minutes) of ischemia and reperfusion. Thus, Edgar et al⁴⁵ reported that in ischemic gerbil brain the rise in PLA₂ activity is transient, peaking at 1 minute, and reversible, returning to normal control levels after 5 minutes. Rordorf et al,⁴⁶ on the other hand, found that in gerbil brain PLA₂ activity was increased twofold after 10 minutes of common carotid occlusion followed by 10 minutes of reperfusion. Our study demonstrates that the levels of cPLA₂ are increased in dorsal hippocampus from ischemic compared with control rats after 72 hours. The immunolocalization data suggest that there is increased expression of cPLA₂ in selected cell types in the CA1 hippocampal region of ischemic brain. Our enzymatic and immunochemical analyses confirm that this is accompanied by an increase in the enzymatic activity of cPLA₂ and the protein level of cPLA₂ in cytosolic extracts of dorsal hippocampus from ischemic brain.

It is well documented that brain free fatty acids increase rapidly during ischemia, with arachidonic acid showing the most prominent relative increase.⁶ It was proposed that this early increase in free fatty acids may be due to the action of PLA₂ and/or the combined action of phospholipase C, diacylglycerol lipase, and monoacylglycerol lipase.^{7 8} Notably, cPLA₂ is the only PLA₂ known to date that has a high preference for arachidonic acid esterified at the 2-position of phospholipids. Lipid mediators derived from products of PLA₂ activity, including prostaglandins, leukotrienes, thromboxanes, and PAF,^{11 13 47} are also transiently increased in the brain within an hour after ischemia and reperfusion. This prominent early rise in fatty acids, eicosanoids, and PAF may be the result of the marked increase in cytosolic free [Ca²⁺] known to trigger the activity of phospholipases.

It is tempting to try to relate glial cPLA₂ expression to neuronal degeneration. There remains the possibility that the increase in cPLA₂ is a result of neuronal death. However, we do not believe this is the case because we observed an increase in immunoreactivity in astrocytes at 24 hours after ischemia, before the onset of neuronal necrosis, albeit not as intense as the response observed at 72 hours. Furthermore, we found a profound induction of cPLA₂ immunoreactivity in astrocytes surrounding axotomized motor neurons after facial nerve axotomy, in which neurons never undergo necrosis (D.T.S. and J.A.C., unpublished data, 1995). If the reactive astrocytes containing cPLA₂ are not induced by neuronal cell death, then perhaps they might play a role in some aspect of neuroprotection or synaptic modification. If we consider that astrocytes have profuse connections with neurons and the possibility that astrocytic neuronal signaling is mediated through intercellular connections rather than synaptically,⁴⁸ it then follows that astrocytic products could have a crucial role in neuronal survival. On the other hand, several studies have implicated microglia as a class of cells that can mediate neurodegeneration by producing neurotoxins such as superoxide, nitric oxide, and other unidentified agents.

Several lines of evidence suggest that oxidative damage may underlie the mechanism triggering increased cPLA₂ activity after ischemia. Toxic reactive oxygen species have long been implicated as important mediators of ischemic brain injury.⁴⁹ Lipid peroxidation was reported to be increased in vulnerable brain regions in rats after transient global ischemia between 8 and 72 hours after reperfusion.¹⁴ Hyslop et al⁵⁰ recently measured elevated H₂O₂ levels by microdialysis in vulnerable brain regions from animals subjected to global ischemia and reperfusion. H₂O₂ has been shown to activate the cytosolic form of PLA₂ in vascular smooth muscle cells by promoting its phosphorylation.⁵¹ Under pathological conditions, oxygen radicals can be produced from arachidonate metabolism.⁵² Oxygen radical generation produces lipid peroxides and stimulates PLA₂ activity.^{53 54 55} These data fit well with the hypothesis that reactive oxygen species may induce cPLA₂ activity. However, at the present time it is difficult to assign a specific role for glial cPLA₂ in the neurodegenerative process. It could have a neuroprotective role by producing mediators that cause the production of protective factors; more likely it could potentiate neurodegeneration by local release of agents such as lysophospholipids, eicosanoids, or PAF. Experiments evaluating the efficacy of selective cPLA₂ inhibitors in this model will help address this issue.

	Selected Abbreviations and Acronyms

 

cPLA2 = cytosolic phospholipase A2 GFAP = glial fibrillary acidic protein PAF = platelet-activating factor PAGE = polyacrylamide gel electrophoresis PLA2 = phospholipase A2 SDS = sodium dodecyl sulfate sPLA2 = secretory phospholipase A2 4-VO = four-vessel occlusion

Received June 27, 1995; revision received October 18, 1995; accepted November 8, 1995.

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