(The FASEB Journal. 2006;20:1162-1175.)
© 2006 FASEB
Activation of cerebral peroxisome proliferator-activated receptors gamma promotes neuroprotection by attenuation of neuronal cyclooxygenase-2 overexpression after focal cerebral ischemia in rats
Yi Zhao,
Andreas Patzer,
Thomas Herdegen,
Peter Gohlke and
Juraj Culman1
Institute of Pharmacology, University Hospital of Schleswig-Holstein, Campus Kiel, Kiel, Germany
1Correspondence: Institute of Pharmacology, University Hospital of Schleswig-Holstein, Campus Kiel Hospitalstrasse 4 24105 Kiel, Germany. E-mail: juraj.culman{at}pharmakologie.uni-kiel.de
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ABSTRACT
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Up-regulation of cyclooxygenase (COX)-2 exacerbates neuronal injury after cerebral ischemia and contributes to neuronal cell death. The present study clarifies the function of cerebral peroxisome-proliferator-activated receptor(s) gamma (PPAR
) in the expression of COX-2 in neurons of the rat brain after middle cerebral artery occlusion (MCAO) with reperfusion by immunohistochemistry, Western blot, and immunofluorescence staining. In peri-infarct cortical areas the PPAR was located in both microglia and neurons, whereas COX-2 was almost exclusively expressed in neurons. PPAR immunolabeling reached the peak 12 h after MCAO, whereas the number of COX-2 immunostained cells gradually rose and reached its peak at 48 h. Intracerebroventricular infusion of pioglitazone, an agonist of the PPAR, over a 5-day period before and 2 days after MCAO, reduced the infarct size, the expression of tumor necrosis factor 
(TNF-), COX-2, and the number of cells positively stained for COX-1 and COX-2 in the peri-infarct cortical regions. COX-2 induction was also attenuated in the ipsilateral but not in the contralateral hippocampus. In primary cortical neurons expressing the PPAR, pioglitazone suppressed COX-2 expression in response to oxidative stress. This protective effect was reversed after cotreatment with GW 9662, a selective antagonist of the PPAR, clearly demonstrating a PPAR-dependent mechanism. Our data provide evidence that activation of neuronal PPAR considerably contributes to neuroprotection by prevention of COX-2 up-regulation in vitro and in peri-infarct brain areas.—Zhao, Y., Patzer, A., Herdegen, T., Gohlke, P., Culma, J. Activation of cerebral peroxisome proliferator-activated receptors gamma (PPAR) promotes neuroprotection by attenuation of neuronal cyclooxygenase-2 overexpression after focal cerebral ischemia in rats.
Key Words: cerebral ischemia • PPAR • pioglitazone • cyclooxygenase-2 • cyclooxygenase-1 • rat
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INTRODUCTION
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ISCHEMIC BRAIN INJURY caused by a transient or permanent reduction of cerebral blood flow results from a complex of pathological events. Neurons localized in the ischemic core, where the blood flow is severely reduced, die within minutes after the ischemic episode (1
, 2
). A number of studies have demonstrated the crucial role of inflammation in the progression of neuronal loss and brain injury. Postischemic inflammation is triggered by activation of proinflammatory genes and expression of adhesion molecules and cytokines, such as tumor necrosis factor (TNF-) and interleukin-1ß (IL-1ß), promoting accumulation of neutrophils, macrophages and activated microglia (1)
. Inflammatory reactions result in an expansion of the tissue injury, which gradually develops at the periphery of the ischemic core over hours and days after ischemic event. Ischemic neurons increasingly express cyclooxygenase (COX)-2, an enzyme that worsens the ischemic injury by producing superoxide and prostaglandins (1)
. Such reactions are deleterious to the damaged brain tissue as anti-inflammatory interventions result in alleviation of neurological deficits and reduction of the infarct volume (3)
.
All isoforms of COX, COX-1, COX-2, and COX-3, the latter being encoded by the COX-1 gene, are expressed in the brain (4
, 5
). Although the chemical properties of COX-1 and COX-2 are similar, these two enzymes may subserve different functions after ischemic injury. COX-2 has attracted more attention as it can directly damage neurons (5
, 6
). COX-2, which is up-regulated in neurons after ischemic injury, has been associated with excitotoxicity mediated by N-methyl-D-aspartate (NMDA) receptors as well as with neuronal cell death (7)
. Prostaglandin synthesis and free radical-mediated lipid peroxidation induced by in vivo activation of NMDA receptors in the hippocampus are also dependent on COX-2 activity (8)
. Selective inhibition of COX-2 attenuated the postischemic prostaglandin accumulation and reduced ischemic damage (9
, 10
). Similarly, COX-2-deficient mice showed reduced susceptibility to ischemic brain injury and the NMDA-mediated neurotoxicity (11
, 12
). While COX-2 directly contributes to the delayed neuronal cell death after ischemic injury, the function of COX-1 appears to be restricted to inflammatory processes mediated by infiltrated monocytes and glial cells (6
, 13
).
The peroxisome proliferator-activated receptor(s) gamma (PPAR) has been primarily implicated in anti-inflammatory processes. The PPAR acts as a negative regulator of macrophage activation and PPAR agonists inhibit the production of inflammatory cytokines in monocytes (14
, 15
). In cortical neuron-glia cocultures, PPAR agonists abolished both LPS-stimulated expression of the inducible NOS (iNOS) in microglia and the NO release and COX-2 expression in neurons (16)
. Similarly, PPAR ligands reduced the iNOS expression and attenuated cell death in cerebellar granule cells (17
, 18
).
Systemic treatment with PPAR agonists, such as pioglitazone or troglitazone, improves the recovery from cerebral ischemia (19
, 20
). We recently demonstrated that it is the intracerebral action of pioglitazone that provides neuroprotection (21)
. The PPAR in microglia/macrophages has been suggested to mediate these beneficial effects, as treatment with PPAR agonists attenuated microglia/macrophage accumulation and reduced the expression of proinflammatory mediators (20
, 21
). However, the role of neuronal PPAR in neuroprotection after ischemic injury and their effects on COX-2 expression in neurons have not yet been addressed. Therefore, we investigated the role of the PPAR in modulation of COX-2 expression in ischemic neurons and the impact on the progression of cerebral damage after ischemic insult. In the present study, we assessed the localization of the PPAR and COX-2 in the peri-infarct cortical area after transient unilateral occlusion of the middle cerebral artery (MCA). We demonstrate that intracerebroventricular (ICV) treatment with pioglitazone, an agonist of the PPAR, confers neuroprotection by suppression of COX-2 induction in neurons after cerebral ischemia. Experiments carried out in cortical cell culture provide evidence that the neuroprotective effects of pioglitazone are directly mediated by the neuronal PPAR.
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MATERIALS AND METHODS
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Induction of cerebral ischemia and implantation of osmotic minipumps
Focal cerebral ischemia was induced in male, normotensive Wistar rats (body wt 200–220 g; Charles River, Sulzfeld, Germany) by occlusion of the MCA for 90 min with subsequent reperfusion as described previously (22
, 54
). Regional cerebral blood flow (rCBF) was continuously monitored at one point (1 mm posterior to the bregma, 6 mm from the midline) on the surface of each hemisphere by laser-Doppler-flowmetry (Periflux system 5000, PERIMED) (23)
. Abrupt reduction in rCBF by 
75% to 90% indicated a successful occlusion of the MCA. Body temperature was maintained at 37°C with a heating pad. For ICV infusions of pioglitazone or vehicle, osmotic minipumps (ALZET Model No.2002, Charles River, Sulzfeld, Germany), delivering solutions at a rate of 0.5 µl/h, were implanted subcutaneously (s.c.) as described in detail elsewhere (22)
.
Measurement of infarct volume
Rats were deeply anesthetized and transcardially perfused with ice-cold PBS, pH 7.4, followed by 4% paraformaldehyde. The whole brains were removed, postfixed in 4% paraformaldehyde overnight and cryoprotected in 30% sucrose at 4°C for 72 h. Serial coronal sections (40 µm) were cut in a cryostat from the level bregma +3.7 mm to the concentration bregma –6.7 mm. The sections were used to determine the infarct volume and immunohistochemical evaluation of PPAR, COX-1, and COX-2. To measure the infarct size, 15 sections from different brain levels were stained with cresyl violet. Slice images were digitalized, and the area of the infarct was determined in each slice (Leica QWin image analysis system) (24)
.
Immunohistochemical detection and morphometric evaluation of PPAR, COX-1, and COX-2
Coronal free-floating brain sections (40 µm) were incubated in PBS containing 0.1% Triton X-100 (PBST) for 5 min, followed by incubation in 0.03% H2O2 in methanol for 10 min at room temperature to quench the endogenous peroxidase and in PBST with 5% normal goat serum (NGS) for 1 h to block unspecific binding. After additional washing, the sections were incubated with primary antibodies specific for PPAR (rabbit anti-rat PPAR polyclonal antibody (pAb), 1: 100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), COX-1 (rabbit anti-rat COX-1 pAb, 1:1000, Cayman Chemical, Ann Arbor, MI, USA) or COX-2 (rabbit anti-rat COX-2 pAb, 1:600, Cayman Chemical) overnight at 4°C. The slices were then washed and incubated with a biotinylated secondary antibody (Ab) (goat anti-rabbit Ab, 1:200, Vector Laboratories, Burlingame, CA, USA) at 37°C for 1 h, followed by incubation with avidin-conjugated peroxidase at 37°C for 45 min (Vectastain avidin-biotin complex peroxidase kit). Immunolabeling was visualized using 3, 3'-diaminobenzidine as chromogen. For the morphometric evaluations, three random and nonoverlapping areas (0.125 mm2 per area) were chosen in the boundary zone of the ischemic core in the frontoparietal cortex as described previously (25)
. The quantification of positively stained cells was carried out using Leica image analyzing software (Leica Qwin).
Immunoflorescence staining
Serial 8 µm coronal sections (corresponding to coronal coordinates bregma ±0 to –2 mm) were fixed with cold acetone and incubated in PBST for 5 min. Afterward, the sections were incubated in 5% NGS in PBST at room temperature for 30 min. Cultured primary neuronal cells, grown on glass cover slips, were fixed with 4% paraformaldehyde in PBS for 1 h at 4°C, washed, and permeabilized with 0.3% Triton X-100 in PBS for 5 min. Subsequently brain sections or primary neuronal cells were incubated with the first primary Ab (rabbit anti-rat COX-2, 1:600 or rabbit anti-rat PPAR, 1:50 at 4°C) overnight, followed by incubation with the second primary Ab at 4°C for 4 h [mouse monoclonal antibody (mAb) against neuronal nuclei (NeuN), 1:100 (Chemicon, Temecula, USA), mouse mAb against rat CD 68, 1:100 (Serotec Ltd, Serotec, Oxford, UK), or goat pAb against glial fibrillary acidic protein (GFAP), 1:800, Santa Cruz Biotechnology]. After washing with PBST, slices were incubated with the corresponding secondary Ab in 1% BSA at 37°C for 1 h (Alexa Fluor®546-conjugated goat anti-rabbit Ab, Alexa Fluor®488-conjugated goat antimouse Ab or Alexa Fluor®546-conjugated donkey anti-goat Ab, Molecular Probes, Leiden, The Netherlands). After washing in PBS, the sections were mounted in Slow Fade® Light antifade reagent (Molecular Probes, Leiden, The Netherlands). For qualitative analysis, stained sections or primary neuronal cells were analyzed with a Leica DMR fluorescence microscope.
Western blot analysis
The dissected brain areas (cortical and hippocampal tissues; see the protocols) were homogenized in liquid nitrogen with a denaturing lysis buffer containing 20 mM Tris (pH 7.4), 2% SDS supplemented with 1% phosphatase inhibitor Cocktail II (Sigma-Aldrich, St. Louis, MO, USA) and protease inhibitor mixture (Roche, Indianapolis, IN, USA). Cultured primary neuronal cells were directly lysed in the lysis buffer. After a short incubation (5 min at 95°C), the lysates were briefly sonicated and centrifuged (15,000 g at 4°C for 15 min) to remove insoluble materials. The protein concentration in the supernatant was measured using the BCA protein assay Kit (Pierce, Rockford, IL, USA). Equivalent amounts of protein (15 µg per sample for COX-2 and 40 µg per sample for TNF-) were separated on 12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride transfer membranes (Millipore Corporation, Bedford, MA, USA). The membranes were blocked with 4% nonfatty dry milk in Tris-buffered saline containing Tween-20 (TTBS) and incubated with the primary Ab (rabbit anti-COX-2 pAb, 1:10 000 or goat anti-rat TNF- pAb, 1:1 000) at 4°C overnight. After three washing steps with TTBS, the membranes were incubated with horseradish peroxidase-conjugated donkey anti-rabbit secondary Ab (1:3000, Amersham, Piscataway, NJ, USA) for 1 h at room temperature. The signal was visualized using the enhanced chemiluminescence (ECL) detection system and ECL hyperfilm (Amersham). All membranes were finally stained with Ponceau S (Sigma-Aldrich) to verify equal protein loading. The TNF- and COX-2 bands were scanned and analyzed using the quantification software (Quantity one, Bio-Rad laboratories, Hercules, CA, USA). Protein levels were expressed as arbitrary units. ß-Actin was used as a loading control and the bands were scanned. Since the obtained values were very consistent, we did not normalize the TNF- and COX-2 signals to the ß-actin.
Primary neuronal cell culture
Mixed cortical cultures containing both neurons and astrocytes were prepared as described recently (26)
. Briefly, the cerebral cortices from 0 to 1 day neonatal Wistar rats were dissected and dispersed using 0.25% trypsin, followed by gentle trituration to release cells. After washing with neurobasal medium (Invitrogen, Carlsbad, CA, USA), cells were counted and plated onto poly-L-lysine- (0.1 mg/ml) precoated cover slips in 4-well plates or Petri culture dishes (35 mm) at a density of 1.5 x 105 cells for immunofluorescence staining, or 2 x 106 cells for Western immunoblotting. Cells were maintained in the neurobasal medium containing 2% B27 supplement, 0.5 mm L-glutamine, 100 U/ ml penicillin, and 100 µg/ml streptomycin (Invitrogen) in a humidified atmosphere of 5% CO2-95% air, at 37°C. Every 3 days, half of the culture medium was changed. Seven days after plating, the cultures consisted of 70–80% neurons and 20–30% astrocytes as evaluated using a mAb against NeuN (Chemicon, Temecula, CA, USA) and a goat polyclonal antiserum against GFAP (Santa Cruz Biotechnology), respectively.
Chemicals
Pioglitazone (Calbiochem, Merck Biosciences GmbH, Schwalbach an Taunus, Germany) (6 mm) for ICV infusion was prepared as described (21)
. For in vitro experiments (primary cell cultures), pioglitazone was dissolved in DMSO (1/4 of the final volume) and an equal volume of PBS was added to obtain the final concentration. The selective PPAR antagonist GW 9662 (Cayman Chemical) was dissolved in DMSO (10% of the final vol) and PBS was added to obtain the final concentration.
Experimental protocols
PPAR- and COX-2-positive cells in the peri-infarct frontoparietal cortex after cerebral ischemia
Rats subjected to MCAO for 90 min were deeply anesthetized and transcardially perfused (see above) at the following times after the onset of reperfusion: 12 h, 1, 2, 3, 5, and 7 days (n=4–5 rats/group). Sham-operated rats (n=4) were treated ICV with vehicle and underwent the same surgical procedures except for the occlusion of the MCA. Coronal brain sections (40 µm) were used for immunohistochemical detection of the PPAR and COX-2 and serial 8 µm coronal sections (corresponding to coronal coordinates bregma 0 to –2 mm) to detect the PPAR in neurons, microglia, and astrocytes by double-immunoflorescence staining.
Effect of pioglitazone on TNF-, COX-1, and COX-2 expression in response to cerebral ischemia
Vehicle (control group, n=15) or pioglitazone (n=14) were delivered into the lateral brain ventricle by means of osmotic minipumps, which were implanted s.c. 5 days before MCAO. On day 6, the MCA was occluded for 90 min in all rats. Without interruption, ICV infusions of vehicle and pioglitazone were continued for 2 consecutive days. Sham-operated rats were treated ICV with vehicle and underwent the same surgical procedures except for the occlusion of the MCA. Forty-eight hours after MCAO, the brains were removed, postfixed, and cryoprotected as described above. Coronal brain sections (40 µm) were used to measure infarct volume (all brains) and to detect COX-1 and COX-2 immunostained cells. Their quantification (sham: n=4; vehicle: n=6, pioglitazone: n=6) was carried out on three consecutive slices obtained at the following brain levels: 1) bregma ±0 mm, 2) bregma –1.3 mm, and 3) bregma –2.3 mm. Serial 8 µm coronal sections were used for immunofluorescence staining for COX-2 in cells localized at the border of the infarct region.
For Western blot analysis of TNF- and COX-2, additional groups of rats were used. Rats were pretreated ICV with either vehicle or pioglitazone and exposed to MCAO (see above). TNF- was analyzed in brains isolated 24 h after MCAO (vehicle: n=6; pioglitazone: n=5). Brain samples for COX-2 determination were obtained 48 h after MCAO (vehicle: n=5; pioglitazone: n=5). The brains were placed on their dorsal surface in a plastic rat brain matrix with a coronal slice orientation (World Precision Instruments, Inc., Sarasota, FL, USA) and cut into 7 serial 2 mm thick coronal sections between + 4 and –8 mm relative to the bregma. Western blot analysis of TNF- was carried out in the frontoparietal cortex adjacent to the ischemic core. The following tissue samples were isolated and used for Western blot analysis of COX-2: 1) frontoparietal cortical tissue adjacent to the area of damage, 2) the corresponding cortical area in the contralateral hemisphere, and 3) the hippocampus from both sides.
Effect of pioglitazone on COX-2 in primary neurons exposed to oxidative damage
All experiments on mixed cortical cultures were carried out on day 7 after plating. The PPAR in primary neuronal cells and astrocytes was detected by double-immunofluorescence staining carried out as described above. The optimal concentration and the duration of the exposure of cells to H2O2 to induce oxidative neuronal damage were tested in preliminary experiments. Cellular toxicity was assessed by measuring the lactate dehydrogenase (LDH) release into the culture medium after exposure of cells to 3 different concentrations of H2O2 (100, 200, and 400 µM) for 2, 4, 8, or 24 h. Exposure to 100 µM H2O2 for 24 h produced a 4-fold increase in LDH (data not shown). To test the effects of PPAR ligands on COX-2 expression, primary neurons were exposed to 100 µM H2O2 for 24 h. Cells were treated with vehicle or pioglitazone (1 µM) in the presence or absence of the PPAR antagonist, GW 9662 (1 µM), 30 min prior to H2O2 exposure (100 µM). Twenty-four hours after the treatment, cells were either fixed with 4% paraformaldehyde in PBS and used for immunofluorescence staining or lysed to obtain protein extracts for Western blot. A 24 h exposure of cells to pioglitazone or GW 9662 alone did not induce any toxic effects as revealed by measurement of LDH release into the culture medium.
The animal protocols were approved by the Governmental Committee for the Ethical Use of Animals in the German Federal State Schleswig-Holstein.
Statistical analyses
All values are expressed as means ± SE. Comparisons of infarct areas between the vehicle-treated and the pioglitazone-treated groups of rats were analyzed by repeated measures of ANOVA with two independent groups of subjects, followed by a post hoc Bonferroni test for pairwise comparisons. The effects of ischemia on the time courses of PPAR and COX-2 immunostained cells in the frontoparietal cortex were analyzed by 1-way ANOVA, followed by a post hoc Dunnett test (comparisons vs. sham, 0 days). Comparisons of the numbers of cells positively stained for COX-1 and COX-2 in the vehicle- and pioglitazone-treated groups of rats were carried out by 1-way ANOVA, followed by a post hoc Bonferroni test. Student’s t test for unpaired samples was used to compare the data on TNF- and COX-2 expression in the cortex and hippocampus between the vehicle- and pioglitazone-treated groups of rats. Statistical analysis of COX-2 expression in primary neuronal cell culture (Western blot analysis) was carried out by 1-way ANOVA, followed by a post hoc Bonferroni test. Statistical significance was accepted at P < 0.05.
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RESULTS
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Regional cerebral blood flow
A reduction of rCBF by >75% of the baseline indicates a successful occlusion of the middle cerebral artery. rCBF reductions during MCAO and rCBF values during reperfusion were identical in both groups of rats (vehicle-treated: n=15; pioglitazone-treated: n=14) at any time during MCAO and reperfusion (10 min MCAO: vehicle-treated: –83%, pioglitazone-treated: –83%; 30 min MCAO: vehicle-treated: – 80%; pioglitazone-treated: –81%; 60 min MCAO: vehicle-treated: –79%, pioglitazone-treated: –80%; 30 min reperfusion: vehicle-treated: –34%, pioglitazone-treated: –31%).
PPAR- and COX-2-positive cells in the frontoparietal cortex after focal cerebral ischemia
We first examined the time course of PPAR- and COX-2 expression in cells after cerebral ischemia. The highest accumulation of cells positively stained for the PPAR was observed in the frontoparietal cortex of the ischemic side. MCAO with reperfusion significantly changed the density of these cells (F6,18=6.735; P < 0.001). The number of PPAR immunoreactive cells dramatically increased 12 h after MCAO (P<0.01), then returned to basal values and remained unchanged until the end of the observation period of 7 days (Fig. 1
). Double immunofluorescence staining showed that almost all microglial cells displayed an intense PPAR immunoreactivity (Fig. 2
). However, the PPAR was also localized in neurons as identified by the neuron-specific marker NeuN (Fig. 3
), whereas only few astrocytes (GFAP-positive cells) were immunopositive for PPAR (data not shown).
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Figure 1. Time-dependent expression of the PPAR (upper panel) and cyclooxygenase-2 (COX-2, lower panel) in cells localized in the peri-infarct cortical area. Rats (n=4–5) were killed at the indicated times after middle cerebral artery occlusion with reperfusion. Data are expressed as the means ± SE. Statistical comparison with sham-operated animals (0 days): *P < 0.05; **P < 0.01, calculated by ANOVA, followed by a post hoc Dunnett test.
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Basal COX-2 immunostaining was present in the frontoparietal cortex of sham-operated animals (Fig. 1)
. In rats exposed to cerebral ischemia, the highest accumulation of COX-2-positive cells was detected at very close vicinity to the ischemic core. The density of these cells significantly changed after MCAO with reperfusion (F6,37=28.843; P<0.001). The number of COX-2 immunostained cells had already increased at 12 h, reached a peak at 48 h after MCAO, and remained elevated until the end of the observation period of 7 days (see Figs. 1
, 5
). Immunofluorescence staining of brain sections revealed that COX-2 was localized mainly in neurons (see
Fig. 7
, below).
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Figure 6. Cyclooxygenase-2 (COX-2) protein expression is decreased in ischemic brains of rats treated with pioglitazone. Western blot analysis of COX-2 in the frontoparietal cortex (upper panel) and in the hippocampus (lower panel) ipsilateral (ips) and contralateral (ctr) to the ischemic injury. Representative blots are shown on the right upper side; histograms are on the lower left side of each panel. The density analysis was performed on the COX-2 bands. ß-Actin was used as a loading control. Pioglitazone (hatched columns) attenuated the COX-2 expression in the ipsilateral frontoparietal cortex and in the hippocampus when compared to the vehicle-treated group (solid columns). There was no significant difference in COX-2 induction in the contralateral frontoparietal cortex and hippocampus. Results are expressed as the means ± SE. Statistical comparison with the vehicle-treated group: ***P < 0.001 calculated by Student’s t test for unpaired samples.
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Figure 7. Expression of cyclooxygenase-2 (COX-2) in neurons localized in the peri-infarct frontoparietal cortex in vehicle-treated and pioglitazone-treated rats. Double immunofluorescence staining on the same sections against A) NeuN as a marker for neurons (green), and B) COX-2 (red). C) Overlapping of COX-2 immunoreactivity in neurons (yellow) in rats treated intracerebroventricularly with vehicle shows that almost all neurons at the edge of the infarct core were positively stained for COX-2. D) Double immunofluorescence staining for NeuN/COX-2 (yellow) in rats treated ICV with pioglitazone demonstrates that less than half of the neurons in the peri-infarct frontoparietal cortex express COX-2.
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Activation of the PPAR reduces the expression of TNF-, COX-1, and COX-2 and protects brain tissue after focal cerebral ischemia
Since pioglitazone reaches very low concentrations in the brain soon after its systemic application, a long-term ICV infusion of pioglitazone was chosen to ensure an effective activation of brain PPAR and to exclude any contribution of peripheral effects of the PPAR ligand.
Injured brain cells increasingly produce IL-1ß and TNF- early after ischemic insult. Both cytokines contribute to the ischemic injury (1)
. Twenty-four hours after MCAO, the expression of IL-1ß was inhibited in ischemic brains of rats treated systemically with troglitazone (20)
. In the present study, pioglitazone down-regulated TNF- in the frontoparietal cortex adjacent to the ischemic core 24 h after MCAO when compared with vehicle-treated rats. (Fig. 4
).
Transient occlusion of the MCA induced COX-2 as evidenced by accumulation of COX-2-positive cells in the peri-infarct cortical areas 2 days after MCAO (bregma ±0.0 mm: F2,12=27.16; P<0.001; bregma –1.3 mm: F2,12=41.08, P<0.001, and bregma –2.3 mm: F2,13=29.45, P<0.001). Pioglitazone significantly reduced the number of COX-2 immunostained cells at all three levels of the brain (Fig. 5
). Western blot analysis of COX-2 expression in the frontoparietal cortex confirmed the immunohistochemical data. Pioglitazone suppressed the COX-2 induction in the peri-infarct cortical area and in the ipsilateral hippocampus, which was not directly damaged by a reduction in blood flow during ischemia (Fig. 6
). In contrast, pioglitazone did not affect COX-2 levels in the contralateral cortex or hippocampus (Fig. 6)
.
The majority of cells in the boundary zone to the ischemic core expressed COX-2. Double immunofluorescence staining revealed that COX-2 was localized mainly in neurons (Fig. 7
) and only sparsely in microglial cells (Fig. 8
), whereas astrocytes were not stained (data not shown). In vehicle-treated rats exposed to MCAO, COX-2 was induced in the majority of neurons localized in the peri-infarct cortex. In contrast, activation of cerebral PPAR considerably reduced the number of COX-2 immunoreactive cortical neurons, indicating that pioglitazone prevented neuronal damage (Fig. 7)
.
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Figure 8. Expression of cyclooxygenase-2 (COX-2) in microglia in the peri-infarct frontoparietal cortex in vehicle-treated rats. Double immunofluorescence staining on the same sections against A) CD 68 as a marker for microglial cells (green), and B) COX-2 (red). C) Overlapping of COX-2 immunoreactivity in microglial cells (yellow) shows that only few of these cells are positively stained for COX-2.
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Compared to COX-2, the density of cells expressing COX-1 in the boundary zone to the infarct core was much lower (Fig. 5)
. Transient cerebral ischemia did not significantly change the number of COX-1-positive cells at the level of the bregma (P=0.21), whereas an increase was observed at two brain levels caudally to the bregma (bregma –1.3: F2,13=9.14, P<0.01; bregma –2.3: F2,13=3.84, P<0.05). As with COX-2, pioglitazone reduced the density of COX-1-positive cells in these areas (Fig. 5)
.
Two days after focal cerebral ischemia, severe unilateral injury was clearly detected as area of pallor that was sharply demarcated from the adjacent tissue. The total infarct volume in pioglitazone-treated rats was 154 ± 19 mm3, which was significantly lower than that in vehicle-treated rats (244±17 mm3, P<0.05). The decrease in infarct size was significant in 7 of 15 brain sections examined (F=9.36; P<0.01) (Fig. 9
).
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Figure 9. Activation of cerebral PPAR reduces the infarct size after focal cerebral ischemia. Rats were treated intracerebroventricularly with vehicle (n=15, solid circles) or with pioglitazone (n=14, empty circles) over a 5-day period before and 2 days after middle cerebral artery occlusion (MCAO) for 90 min. Total (cortical and subcortical) areas of injury are shown. Pioglitazone significantly reduced the infarct size. The x axis shows the anterior (A) -posterior (P) distance from the bregma, Results are expressed as the means ± SE. Statistical comparison with the vehicle-treated group: *P < 0.05 and **P < 0.01, calculated by 2-way ANOVA, followed by a post hoc Bonferroni test.
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The PPAR reduces COX-2 of primary neurons and supports their survival after oxidative damage
Experimental data indicate that PPAR ligands protect neurons rather indirectly, through effects on monocytes and microglia (16)
. In the last set of experiments, we investigated whether a direct activation of the PPAR in neurons can modify the expression of COX-2 triggered by oxidative damage. The culture conditions resulted in a mixed cell population with <30% of astroglia and >70% neurons (data not shown). Double immunofluorescence staining showed that the PPAR was localized in almost all neurons (Fig. 10
), but only sparsely in astrocytes (<25%) (Fig. 11
). A 24 h exposure of neurons to H2O2 induced a robust expression of COX-2. Double immunofluorescence staining revealed that the majority of neurons were positively stained for COX-2 (Fig. 12
and Fig. 13
). Pioglitazone added to primary neuronal cells prior to H2O2 significantly reduced the number of COX-2-positive neurons. Blockade of PPAR by cotreatment with the selective PPAR antagonist, GW 9662, reversed the effect of pioglitazone (Fig. 13)
. Western blot analysis confirmed the results obtained in immunohistochemical experiments. The changes in COX-2 expression in primary neuronal cell culture exposed to H2O2 with or without pioglitazone/GW 9662 were highly significant (F3,32=7.165; P<0.001). H2O2-induced COX-2 expression was suppressed in the presence of pioglitazone, and GW 9662 reversed the pioglitazone-induced reduction in COX-2 expression indicating that the effects of pioglitazone were indeed mediated by PPAR (Fig. 13)
.
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Figure 12. Cyclooxygenase-2 (COX-2) is induced in primary neuronal cells exposed to oxidative damage. Seven days after plating, primary cortical cells were exposed to hydrogen peroxide (H2O2) (100 µM) to induce oxidative damage. Representative immunofluorescence staining for A) neurons (green), B) COX-2 (red), and C) overlapping of COX-2 immunoreactivity in primary neuronal cells (yellow) shows a robust induction of COX-2 in all neurons.
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Figure 13. Activation of PPAR in primary neuronal cells reduced cyclooxygenase-2 (COX-2) induction in response to oxidative damage. Seven days after plating, primary cortical cells were exposed to A) vehicle or B) incubated with hydrogen peroxide (H2O2) (100 µM) alone, or C) in the presence of pioglitazone (1 µM), or (D) pioglitazone + GW 9662 (1 µM). Pioglitazone and the PPAR antagonist, GW 9662, were added 30 min prior to H2O2. Upper panel: A robust induction of COX-2 (fluorescent red) was observed in neurons exposed to H2O2. Pioglitazone added to primary neuronal cells prior to H2O2 reduced the number of COX-2-positive neurons and this effect was reversed by cotreatment with GW 9662. Lower panel: Western blot analysis of COX-2 in primary neuronal cells. Pioglitazone suppressed the H2O2- induced COX-2 expression and GW 9662 reversed the pioglitazone-induced reduction in COX-2 expression. The density analysis was performed on the COX-2 bands. ß-Actin was used as a loading control. A representative blot is shown on the right lower side of the figure. Results are expressed as the means ± SE. *P < 0.05, **P < 0.01, statistical comparisons to vehicle-treated cells, #P < 0.05 and  P < 0.05, statistical comparisons to H2O2-treated and H2O2 + pioglitazone + GW 9662-treated cells, respectively, calculated by ANOVA, followed by a post hoc Bonferroni test.
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DISCUSSION
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The results of the present study provide novel insight into the mechanisms of the PPAR-mediated neuroprotection. The PPAR has been detected in several areas of the adult brain and spinal cord and its presence was observed in both neuronal and glial cells (27
28
29
30
31)
. The expression of PPAR in microglia is tightly regulated and dependent on microglial functional state (28)
. Consistent with the up-regulation of PPAR immunoreactivity and mRNA in the ischemic brain (32
and references therein), few hours after MCAO, PPAR immunolabeling increased in the cortical peri-infarct areas mainly in microglial cells and, to a lesser extent, in neurons. The number of PPAR-positive cells then dramatically declined and the lowest values were reached 2 days after ischemic injury, when the postischemic inflammation had already developed and the peri-infarct region is strongly infiltrated with invading macrophages and activated microglia (24)
. This observation is consistent with the finding of a down-regulation of the PPAR expression during microglia activation (28)
. TNF- is a potent suppressor of the PPAR expression in adipocytes, and such antagonism also appears to occur in inflammatory cells (33)
. Consequently, the dramatic drop in the initially increased number of PPAR-positive cells was most likely induced by a rapid down-regulation of PPAR during microglia activation initiated by TNF- or by other mediators of inflammation.
We demonstrate that activation of the PPAR suppresses the COX-2 induction. COX-2, which is constitutively expressed throughout the brain in discrete populations of neurons (34)
, plays a crucial role in the progression of brain injury after ischemia (6)
. COX-2 mRNA levels in the cortical peri-infarct area reach their highest levels 12 h after ischemic injury, and the extent of COX-2 mRNA induction correlates with the severity of tissue damage (9
, 35
, 36
). Here we report that the number of COX-2-positive cells in the peri-infarct cortex gradually increased, reaching the maximum 48 h after the ischemic insult. In line with previous findings, COX- 2 expression occurred mainly in neurons (9)
, although a few positively stained microglial cells were also detected. A robust induction of COX-2 in peri-infarct neurons and infiltrating neutrophils was reported in humans who died 1–2 days after ischemic insult in the territory supplied by the MCA (37
, 38
).
Beneficial effects of systemically administered PPAR agonists in ischemic stroke have been recently demonstrated (19
, 20
). Pioglitazone may improve the recovery from ischemic stroke by reduction of oxidative stress and increased production of NO in endothelial cells, which in turn can modulate cerebral blood flow during ischemia and reperfusion period (39)
. Activation of PPAR in the brain may improve the recovery from stroke by blocking of pathophysiological processes and events, such as inflammatory reactions and apoptosis. Thus, the beneficial effects of systemically administered PPAR agonists in stroke result from activation of both peripheral and central PPAR. In the present study, we sought to investigate the effect of an activation of brain PPAR on postischemic expression of COX-1 and COX-2 in neuronal and glial cells. To achieve an exclusive and selective activation of brain PPAR, pioglitazone was continuously infused ICV over a 5-day period before and during 1 or 2 consecutive days after MCAO with reperfusion. We have reported that pioglitazone infused ICV at the given dose attenuated accumulation of inflammatory cells in the peri-infarct area and improved the recovery from ischemic stroke. A similar dose of another PPAR agonist, troglitazone, reduced the neuronal death in response to bacterial LPS when microinjected into the cerebellum (21
and references therein).
Pioglitazone substantially reduced the number of COX-2-positive cells and the COX-2 expression in peri-infarct cortical tissue. In vitro studies and ex vivo experiments in animals have conclusively demonstrated that augmented COX-2 activity within ischemic neurons exacerbates ischemic injury and promotes neuronal cells death (6)
. In primary neuronal cell cultures, inhibition of COX-2 ameliorated the NMDA- induced cell injury and protected neurons from the LPS-induced neuronal death (40
, 41
). Selective COX-2 inhibitors reduced the infarct volume after focal cerebral ischemia and the COX-2-deficient mice showed reduced susceptibility to ischemic brain injury (9
, 11
, 12
). In view of these findings, the observed reduction in the COX-2 expression in pioglitazone-treated rats strongly indicates that activation of cerebral PPAR protects neurons against ischemic injury induced by excitotoxicity and anoxia, and prevents neuronal loss in the periphery of the infarct, which is the potential target for therapeutic intervention.
The decrease in COX-2 expression in peri-infarct cortical tissue in pioglitazone-treated rats may result from the inhibition of macrophage accumulation and microglia activation. There is now compelling evidence that proinflammatory cytokines IL-1ß and TNF- play a central role in the progression of postischemic brain injury. Increased production of IL-1ß and TNF- by injured brain cells stimulates the expression of adhesion molecules including intercellular adhesion molecule 1 (ICAM 1) and vascular adhesion molecule (VCAM) on the endothelial cell surface, which mediates the adhesion and migration of neutrophils and macrophages into the ischemic brain parenchyma. Infiltrating cells and activated microglia increasingly produce inflammatory cytokines that further exacerbate postischemic inflammation and contribute to neuronal damage (1
, 2
). PPAR agonists can regulate the expression of adhesion molecules, cytokines, and chemokines and other inflammatory mediators through multiple mechanisms. For instance, PPAR ligands inhibit the increase in ICAM 1 and VCAM and suppress the production of inflammatory cytokines, such as IL-1ß and TNF- (see ref 32
for a review). Indeed, systemically applied troglitazone decreased IL-1ß levels in the ischemic brains (20)
. Pioglitazone in the present study was infused ICV, therefore, we do not assume that the PPAR ligand could interfere with the production of adhesion molecules in endothelial cells. However, pioglitazone reduced the expression of TNF- in peri-infarct cortical tissue that, together with the decreased levels of IL-1ß (20)
, may account for the lower densities of reactive microglial cells and macrophages in this area observed in pioglitazone-treated rats after MCAO (21)
. The down-regulation of the neutrophilic and reactive microglia infiltration and production of toxic mediators may in turn reduce the expression of COX-2 in injured neurons and prevent neuronal cell death.
Pioglitazone also attenuated the induction of COX-2 in the hippocampus. The blood flow in the hippocampus is usually not reduced during MCAO, and neuronal COX-2 is induced by activation of NMDA receptors (7)
. In rodents, COX-2 was particularly expressed in the vulnerable hippocampal CA1 neurons and contributed to the delayed neuronal death after cerebral ischemia (12
, 42
, 43
). Our data indicate that activation of brain PPAR reduces COX-2 expression and ameliorates neuronal injury and cell death in both peri-focal areas, in which neurons are at a high risk to die, and extrafocal sites, where neurons are not directly damaged by a local reduction or an arrest of blood flow. Profound beneficial effects of pioglitazone on the neuronal survival most likely contribute to the improved outcome of the ischemic damage, such as the reduction of the infarct size. Systemic treatment with PPAR ligands confers neuroprotection after ischemic stroke (19
, 20
). Intracerebral application of pioglitazone exerts beneficial effects similar to those observed after systemic treatment (21)
. Taken together, our findings provide evidence that the neuroprotective actions of PPAR agonists are mediated by intracerebral PPAR and are independent of peripheral mechanisms.
Some of the anti-inflammatory and neuroprotective effects of PPAR ligands do not require activation of the PPAR (32
, 44
45
46
). To define more precisely the role of PPAR in the reduction of COX-2 expression in ischemic tissue, we examined the effects of pioglitazone and the selective PPAR antagonist, GW 9662, on the expression of COX-2 in neonatal rat primary neurons in response to reactive oxygen species (ROS). Oxidative damage mediated by ROS during the reperfusion further exacerbates ischemic injury and results in a series of molecular events leading to neuronal cell death (1
, 2
). H2O2 is a major ROS generated as a by-product of cellular metabolic events. The beneficial effects of pioglitazone in primary cortical neurons exposed to H2O2 namely, the attenuation of COX-2 expression and the prevention of neuronal cell death, were completely reversed by GW 9662, clearly indicating a PPAR-dependent mechanism.
Data from experimental studies on neuroinflammation suggest that PPAR ligands protect neurons indirectly, through effects on monocytes/microglia (46
, 47
). In neuron-microglia cocultures, PPAR ligands inhibited LPS-induced neuronal death by blocking the production of neurotoxic molecules in microglia (16)
. We hypothesize that in addition to the anti-inflammatory actions, activation of PPAR in neurons is decisive for the neuroprotective actions of PPAR ligands, as 1) primary neuronal cells express PPAR and 2) pioglitazone substantially prevents neuronal damage and cell death in response to oxidative injury in the absence of microglia as demonstrated in the present experiments. Similarly, other PPAR agonists, such as thiadiazolidinones, protected primary cortical neurons from apoptosis induced by cell-free medium from LPS-activated microglial cultures (48)
. These findings convincingly demonstrate that, apart from the PPAR-mediated inhibition of microglia activation, it is the activation of the PPAR in neurons that inhibits COX-2 expression and confers neuroprotection against cerebral ischemia and noxious stimuli. Recent findings have demonstrated that activation of the PPAR promotes neuroregeneration and neurite outgrowth in cultured cells of neuronal origin (49)
. These neurotrophic effects may also contribute to neuroprotective actions of thiazolidinediones in ischemic brain.
Pioglitazone significantly reduced the number of cells in the peri-infarct cortical areas that stained positively for COX-1. In contrast to COX-2, the role of COX-1 in ischemic stroke is controversially discussed (6
, 13
). COX-1 plays a critical role in the maintenance of resting CBF and in the vasodilation induced by endothelium-dependent vasodilators (50)
. The COX-1-deficient mice were more susceptible to brain damage after cerebral ischemia due to a more pronounced CBF reduction during ischemia (51)
. Moreover, COX-1 gene transfer augments prostacyclin PGI2 and reduces leukotriene productions (52)
. In apparent contrast to these studies, COX-1 has been demonstrated to exacerbate neuronal damage and to promote neuronal death following global ischemia (53)
. Thus, the observed inhibition of COX-1 induction by pioglitazone may confer additional protection against ischemic neuronal damage.
The present data favor the view that besides the PPAR-mediated suppression of inflammation and invasion of microglia/macrophages (20
, 21
), activation of the PPAR in neurons promotes neuroprotection. Activation of PPAR attenuated TNF- production, which may contribute considerably to the observed inhibition of COX-2 expression and reduction of ischemic injury. Hosomi et al. (55)
recently demonstrated that blockade of TNF- by neutralizing antibodies reduces the infarct size and edema after transient focal cerebral ischemia. On the other hand, activation of PPAR in neurons can directly decrease COX-2 production, as PPAR ligands were shown to suppress transcriptional activation of COX-2 in various cell types (56
57
58)
.
Our findings also address an important issue about the potential therapeutic use of PPAR agonists in the prevention and treatment of ischemic stroke. Thiazolidinediones lower glucose (Glc) concentrations by ameliorating insulin resistance, improve endothelial dysfunction, reduce blood pressure, and ameliorate dyslipidemia (59)
. A recent clinical study has demonstrated that pioglitazone reduces the composite of all-cause mortality, nonfatal myocardial infarction, and stroke by 16% in patients with type 2 diabetes who had extensive evidence of macrovascular disease (60)
. Therefore, patients with type 2 diabetes may benefit from treatment with thiazolidinedione, since these drugs lower Glc concentrations and prevent serious cardiovascular events, including stroke. On the other side, nondiabetic patients who have a high risk of stroke should not necessarily be treated with pioglitazone or similarly acting drugs, because such treatment has been associated with serious side effects, including higher incidence of heart failure, edema not attributable to heart failure and increases in body weight (60)
. Results of the present and other studies (19
, 20
) lead one to assume that PPAR agonists might be beneficial when given after ischemic injury. The most serious damaging events in ischemic brain tissue develop within few hours or days after ischemic insult (1)
. Treatment of patients with PPAR agonists during this short period may improve the outcome of ischemic stroke without serious side effects, such as heart failure, which are generally observed after a lengthy treatment. In a recent experimental study, troglitazone proved beneficial effects in rats when injected 1 h after the onset of MCAO (20)
. Unfortunately, the rats were examined only once, 24 h after MCAO, so the effects of a long-lasting activation of PPAR after stroke could not be ascertained. New synthetic PPAR agonists with higher availability in the brain and follow-up studies in experimental animals treated after ischemic insult may provide the rationale for the treatment of patients suffering from stroke with PPAR agonists.
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ACKNOWLEDGMENTS
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The authors thank Jan Brdon for his excellent technical assistance. Supported by the Deutsche Forschungsgemeinschaft, SFB 415 (project A12).
Received for publication October 6, 2005.
Accepted for publication February 10, 2006.
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TOP
ABSTRACT
INTRODUCTION
