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Protective role of hematopoietic prostaglandin D synthase in transient focal cerebral ischemia in mice
Neuroscience 163 (2009) 296 –307
PROTECTIVE ROLE OF HEMATOPOIETIC PROSTAGLANDIN D SYNTHASE IN TRANSIENT FOCAL CEREBRAL ISCHEMIA IN MICE M. LIU,a N. EGUCHI,b Y. YAMASAKI,c Y. URADE,b N. HATTORId AND T. URABEd*
rophages in ischemic peri-area and penumbra (P<0.0001) at 72 h and 7 days after reperfusion, suggesting involvement of monocytes/macrophages in HQL-79-induced expansion of ischemic injury. Our results demonstrated that the neuroprotective effects of HPGDS in our model are mediated by suppression of activation and infiltration of inflammatory cells. © 2009 IBRO. Published by Elsevier Ltd. All rights reserved.
a Research Institute for Disease of Old Age, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan b Department of Molecular Behavioral Biology, Osaka Bioscience Institute, Osaka 565-0874, Japan c Hanno Research Center, Taiho Pharmaceutical Co. Ltd., Saitama 357-8257, Japan
Key words: PGD2, PGDS, HQL-79, microglia/macrophage, inflammation, ischemic stroke.
d
Department of Neurology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
There is increasing evidence that inflammation plays an important role in CNS ischemia. Experimentally and clinically, stroke is followed by acute and prolonged inflammatory response characterized by an extremely rapid (within hours) activation of microglia/macrophages (Kato et al., 1996; Lehrmann et al., 1997). Resident microglial cells and infiltrating hematogenous macrophages play an important role during the pathogenetic cascade following cerebral ischemia since they express a plethora of growth factors, chemokines and regulatory cytokines as well as free radicals and other toxic mediators (Raivich et al., 1999) that are involved in secondary infarct expansion (Del Zoppo et al., 2000; Hallenbeck, 2002). Hematopoietic prostaglandin D synthase (HPGDS) is a key enzyme in the production of prostaglandin D and its J metabolites. Previous studies showed that PGD2 is spontaneously converted to 15-deoxy-⌬12,14-PGJ2 (15d-PGJ2) by non-enzymatic dehydration (Shibata et al., 2002; Gilroy et al., 1999). Recent results indicate that prostaglandin D2 protects neonatal mouse brain from hypoxic ischemic injury (Taniguchi et al., 2007) and inhibits the expression of inducible nitric oxide synthase (iNOS) in rat vascular smooth muscle cells (Nagoshi et al., 1998). Moreover, Trivedi et al. (2006) demonstrated that HPGDS⫺/⫺ mice bearing a delayed type hypersensitivity reaction display an exaggerated inflammatory response that fails to resolve. Although, 15d-PGJ2 inhibits the production of pro-inflammatory cytokines and iNOS in activated monocytes (Rossi et al., 2000; Castrillo et al., 2000), to our knowledge no report has described the role of HPGDS in cerebral ischemia/reperfusion injury. We reported previously marked induction of HPGDS after ischemia/reperfusion injury, which was mainly produced by endogenous microglia (Liu et al., 2007). In the present study, we tested the hypothesis that HPGDS is neuroprotective in cerebral ischemia/reperfusion injury using middle cerebral artery (MCA) occlusion (MCAO)/reperfusion model of C57BL/6 mice and bone-marrow chimera mice (Komine-Kobayashi et al., 2006). These mice are known to express enhanced green fluorescent protein
Abstract—Cerebral ischemia/reperfusion injury is characterized by the development of inflammatory response, in which vascular macrophages and endogenous microglia are involved. Recent studies showed marked induction of hematopoietic prostaglandin D synthase (HPGDS) after ischemic/reperfusion injury and its localization in microglia, but the molecular mechanism(s) of HPGDS actions in cerebral ischemia is not clear. To clarify the role of HPGDS in cerebral ischemia, C57BL/6 mice and bone marrow chimera mice with cerebral ischemia/reperfusion injury were treated with (4-benzhydryloxy-(1) {3-(1H-tetrazol-5-yl)-propyl}piperidine (HQL-79), a specific inhibitor of HPGDS. The bone marrow chimera mice exhibit expression of enhanced green fluorescent protein (EGFP) in bone marrow/blood-derived monocytes/macrophages. Mice were subjected to ischemia/reperfusion and either treated with HQL-79 (nⴝ44) or vehicle (nⴝ44). Brain sections prepared at 72 h and 7 days after reperfusion were analyzed for neuronal nuclei (NeuN), HPGDS, ionized calcium-binding adapter molecule 1 (Iba1), inducible NO synthase (iNOS), nitrotyrosine, nuclear factor kappa B (NF-kB) and cyclooxygenase-2 (COX-2). The mortality rate (80%) and infarct size were larger in HQL-79- than vehicle-treated mice (58.7ⴞ8.5 versus 45.2ⴞ4.9 mm3; meanⴞSEM, P<0.0001) at 7 days after reperfusion. HQL-79 reduced NeuN expression in the transition area and Iba1 expression (P<0.0001) in the ischemic peri- and penumbra area, but increased COX-2 (P<0.05) and NF-kB expression (P<0.05) in ischemic penumbra and increased formation of nitrotyrosine (P<0.0001) and iNOS (P<0.0001) in the ischemic core area at 72 h and 7 days after reperfusion. In EGFP chimera mice, HQL-79 increased the migration of Iba1/EGFPpositive bone marrow-derived monocytes/macrophages, and simultaneously upregulated iNOS expression in the ischemic core area (P<0.0001), but increased intrinsic microglia/mac*Corresponding author. Tel: ⫹81-3-3813-3111; fax: ⫹81-3-5684-0476. E-mail address: [email protected] (T. Urabe). Abbreviations: Co, ischemic core; COX-2, cyclooxygenase-2; EGFP, enhanced green fluorescent protein; GFAP, glial fibrillary acidic protein; HPGDS, hematopoietic prostaglandin D synthase; HQL-79, 4-benzhydryloxy-(1) {3-(1H-tetrazol-5-yl)-propyl}piperidine; Iba1, ionized calcium-binding adapter molecule 1; IKK, IkB kinase; iNOS, inducible nitric oxide synthase; MCA, middle cerebral artery; MCAO, middle cerebral artery occlusion; NF-kB, nuclear factor kappa B; PBS, phosphate-buffered saline; Pe, ischemic peri-infarct area; rCBF, regional cerebral blood flow; Tr, ischemic transition area; 15d-PGJ2, 15-deoxy-⌬12,14-PGJ2.
0306-4522/09 $ - see front matter © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2009.06.027
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(EGFP) in bone marrow/blood-derived monocytes/macrophages.
EXPERIMENTAL PROCEDURES Experimental protocol The study protocol was approved by the Committee on Animal Experimental Guidelines of Juntendo University School of Medicine. Adult 8-week old male C57BL/6 mice (Charles River Japan Inc., Kanagawa, Japan), were divided at random into two groups. Mice of the 4-benzhydryloxy-(1) {3-(1H-tetrazol-5-yl)-propyl}piperidine (HQL-79)–treated group (HQL-79 group, n⫽44) were treated orally with HQL-79 at 30 mg/kg body weight three times; immediately, day 1 and day 2 after MCA occlusion/reperfusion. Mice of the vehicle-treated group (control group, n⫽44) were treated orally with 0.9% saline solution at a volume similar to that used in the HQL-79 group. HQL-79 was suspended or dissolved in 0.5% methylcellulose for oral administration. Mice were maintained on a 12-h light/dark cycle and provided with food and water ad libitum. Ischemia was induced by the intraluminal vascular occlusion method as described previously (Komine-Kobayashi et al., 2004). Briefly, mice were anesthetized and the left MCA was occluded for 60 min followed by release of the occlusion. Regional cerebral blood flow (rCBF) was measured by laser Doppler flowmetry before, during, and after MCAO, as well as before euthanasia. An observer blinded to the study protocol scored the postural reflexes using a modified neurological scoring system described previously (Hara et al., 1996). In this system, score 0 represents no observ-
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able neurological deficits; 1, failure to extend the left forepaw on lifting the whole body by the tail; 2, circling to the contralateral side; and 3, loss of walking or righting reflex. At pre, 3, 6, 24 and 72 h and 7 days, blood samples were withdrawn from the tail vein for leukocyte count in peripheral blood by using Turk’s solution (Muto Pure Chemicals Co., Tokyo, Japan). We also established EGFP chimera mice by bone marrow transplantation of EGFP into C57BL/6 mice (n⫽16), using the method described previously (Tanaka et al., 2003; Komine-Kobayashi et al., 2006). Transient cerebral ischemia was also induced in these animals 6 weeks later, using the above-described protocol. The distribution of neuronal damage and infarct area was evaluated by Cresyl Violet staining. Briefly, coronal sections (20-m thick) at 400 m intervals were obtained and treated with Cresyl Violet. The infarct area (mm2) of each slice was measured digitally (NIH image 1.63), and the total volume of infarction in the brain was estimated by adding all infarct areas of each slice and multiplying the product by the distance between sections (400 m).
Immunohistochemistry Six animals of each group were anesthetized by i.p. injection of pentobarbital (50 mg/kg i.p.) at 72 h and 7 days after reperfusion. The brain was removed immediately and postfixed for 24 h in 4% paraformaldehyde in PBS at 4 °C before cryoprotection by bathing in 30% sucrose. It was then frozen, and 20 m-thick consecutive coronal sections were prepared on a cryostat (CM-1900, Leica, Wetzlar, Germany).
Fig. 1. Effect of HQL-79 treatment on stroke outcome. (A) Survival analysis during 7 days after reperfusion in the control and HQL-79 groups. (B) Neurological deficit scores in the control and HQL-79 groups. (C) Temporal profiles of peripheral leukocyte count of the vehicle and HQL-79 groups at several time points after reperfusion. The leukocyte count increased significantly at 3 and 6 h after reperfusion and decreased in a time-dependent manner after 24, 72 h and 7 days after reperfusion, but there was no significant difference between the two groups. Data are expressed as mean⫾SEM; n⫽5 per group. (D) Numbers of NeuN-positive cells at the indicated time points. Data are mean⫾SEM of six mice in each group. (E) Coronal sections of mice ischemic brain stained with Cresyl Violet staining. The infarct volume was compared between vehicle and HQL-79 groups at different time points after reperfusion. Data are mean⫾SEM; n⫽5 per group.
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Immunohistochemical staining was performed for nitrotyrosine (Upstate Biotechnology, Lake Placid, NY, USA), ionized calcium-binding adapter molecule 1 (Iba1; Wako Pure Chemicals, Osaka, Japan), NeuN (Chemicon, Temecula, CA, USA), HPGDS (raised in the Department of Molecular Behavioral Biology, Osaka Bioscience Institute, Osaka, Japan), iNOS (BD BioScience, CA, USA), cyclooxygenase-2 (COX-2; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and nuclear factor kappa B (NF-kB; Santa Cruz Biotechnology). Sections were washed in phosphate-buffered saline (PBS), incubated in 0.3% H2O2 in PBS for 30 min, and incubated overnight at 4 °C with 10% normal goat serum (Dako Corp., Carpentaria, CA, USA) in PBS and anti-Iba1 (dilution, 1:1000) antibody, anti-NeuN (dilution, 1:100) antibody, anti-iNOS (dilution, 1:300) antibody, anti-HPGDS antibody (dilution, 1:10,000) anti-nitrotyrosine (dilution, 1:100) antibody, anti-NF-kB antibody (dilution, 1:500) and anti-COX-2 antibody (dilution, 1:500). Immunoreactivity was visualized by the avidin– biotin complex method (Vectastatin; Vector Laboratories, Burlingame, CA, USA) as described previously (Tanaka et al., 2003).
Double-immunofluorescence staining In six animals of each group 72 h and 7 days after reperfusion, the brain was removed immediately after euthanasia and post-fixed for 24 h in 4% paraformaldehyde in PBS at 4 °C before cryoprotection by bathing in 30% sucrose. Free-floating sections of EGFP
bone marrow chimera mice and C57BL/6 mice were washed with PBS and incubated in a blocking solution (10% Block Ace [Yukijirushi, Sapporo, Japan] in PBS) for 1 h at room temperature, followed by incubation overnight at 4 °C with a rabbit anti-Iba1 antibody (dilution, 1:1000, Wako), anti-HPGDS antibody (dilution, 1:5000), anti-iNOS antibody (dilution, 1:200, BD BioScience), antiNeuN (dilution, 1:100; Chemicon, a marker of neurons), antiNF-kB (dilution, 1:100), anti-Gr-1 (dilution, 1:200, Pharmingen, San Diego, CA, USA, a marker of granulocytes), anti-nitrotyrosine antibody (dilution, 1:50; Upstate Biotechnology) or anti– glial fibrillary acidic protein (GFAP) antibody (dilution, 1:100, Dako). For double labeling, sections were incubated with two primary antibodies; Cy3-conjugated anti-rat IgG antibody (dilution, 1:500; Jackson Immunoresearch Laboratories, West Grove, PA, USA) followed by fluorescein isothiocyanate (FITC)– conjugated antirabbit IgG antibody (dilution, 1:500; Vector Laboratories) for 2 h at room temperature. The sections were washed with PBS and mounted on microslide glass with Vectorshield Mounting Medium (Vector Laboratories). Finally, the sections were examined with a fluorescence microscope (Eclipse TE300; Nikon, Tokyo, Japan).
Western blotting Six animals of each group were decapitated after 72 h and 7 days after reperfusion. Samples were taken from two regions; the isch-
Fig. 2. (A) Schematic representation of distribution of neuronal damage in mouse brain after reperfusion delineated by loss of microtubule associated protein 2 (MAP-2) staining. The shaded area represents the infarct zone (a). The three areas subjected to immunohistochemical analysis are illustrated. Staining with anti-HPGDS antibody in control (b– d) and HQL-79 group (e, f) mouse brain after MCAO and reperfusion. Shown are sham control (b), 72 h (c, e) and 7 days (d, f) in the Co area after reperfusion. Scale bar⫽200 m. (B) Numbers of HPGDS-positive cells at the indicated time points. Data are mean⫾SEM of six mice in each group. (C) Western blot analysis of HPGDS. Samples were prepared from the brain at 72 h (control group: lanes 1 and 2; HQL-79 group: lanes 3 and 4) and 7 days (control group: lanes 5 and 6; HQL-79 group: lanes 7 and 8) after reperfusion. A 26-kDa band corresponding to HPGDS protein was detected on the stroke side and its intensity increased in a time-dependent manner. A weaker band was noted in HQL-79 group relative to the control group. Representative data of five experiments with similar results. (D) Densitometric analysis. Values are expressed as percentages of the respective ␣-tubulin. C: contralateral region; S: stroke side. Data are mean⫾SEM; of six mice in each group.
M. Liu et al. / Neuroscience 163 (2009) 296 –307 emic region and the contralateral cortex. Protein extraction and Western blotting were performed as described previously (Ito et al., 1998). Aliquots containing 30 g of protein were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Protein bands were transferred onto polyvinylidene fluoride membrane (Trans-blot Semi-dry Transfer Cell, Bio-Rad, Hercules, CA, USA) with the use of an electrophoretic transfer system (Bio-Rad). After they were blocked with Block Ace for 1 h, the membranes were incubated overnight at 4 °C with anti-Iba1 antibody (dilution, 1:5000), anti-HPGDS antibody (dilution, 1:30,000), or anti-␣-tubulin antibody (dilution, 1:1000; Santa Cruz Biotechnology Inc.). After incubation with the appropriate horseradish peroxidase– conjugated secondary antibody (dilution, 1:20,000; Amersham Place, Little Chalfont, Buckinghamshire, UK) for 1 h at room temperature, immunoreactive bands were visualized in the linear range with enhanced chemiluminescence (ECL Western blotting system, Amersham). For quantitative evaluation, the immunoreactive bands were subjected to densitometric analysis.
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Cell count and statistical analysis The numbers of Iba1/EGFP-positive extrinsic monocytes/macrophages and Iba1-positive intrinsic microglia/macrophages were counted in three predefined areas (0.25 mm2, each) of Iba1stained coronal sections of EGFP chimera ischemic mice. Furthermore, the number of microglia/macrophages coexpressing iNOS and EGFP and coexpressing HPGDS and EGFP (n⫽5, each group), as well as the numbers of NeuN, Iba1-, NF-kB-, COX-2-, iNOS- and nitrotyrosine-positive cells, was counted by the same method under a light microscope (EclipseTS100, Nikon) at a magnification of ⫻100 and assessed independently by two investigators. Values presented in this study are expressed as mean⫾SEM. After acquisition of all data, the randomization code was broken and data were assigned to each group. ANOVA followed by post hoc Fisher protected least significant differences test was used to determine the statistical significance of differences in neurologic score and volumes of infarction between the two groups. A prob-
Fig. 3. (A) Western blot analysis of Iba1. Samples were prepared from the brain at 72 h (vehicle group: lanes 1 and 2; HQL-79 group: lanes 3 and 4) and 7 days (vehicle group: lanes 5 and 6; HQL-79 group: lanes 7 and 8) after reperfusion. A 17-kDa band corresponding to Iba1 protein was detected on the stroke side and its intensity increased in a time-dependent manner. A weaker band was noted in HQL-79 group than vehicle group. Representative data of five experiments with similar results. (B) Densitometric analysis. Values are expressed as percentages of the respective ␣-tubulin. C: contralateral region; S: stroke side. (C) Photomicrographs showing Iba1-staining in the Tr area of representative vehicle (a, c) and HQL-79 (b, d) groups at 72 h (a, b) and 7 days (c, d) after reperfusion. Scale bar⫽200 m. (D) Numbers of Iba1-positive cells at the indicated time points. Data are mean⫾SEM of six mice in each group. (E) Photomicrographs showing Iba1-staining in the Co area of representative control (a, c) and HQL-79 (b, d) mice at 72 h (a, b) and 7 days (c, d) after reperfusion. Scale bar⫽100 m. (F) Numbers of Iba1-positive cells at the indicated time points. Data are mean⫾SEM of six mice in each group.
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ability value less than 0.05 denoted the presence of a statistically significant difference.
RESULTS Physiological parameters All mice of this model showed infarct in the cortical and striatal areas, which was identified by NeuN staining. Measurements of various physiological parameters and rCBF revealed no significant differences between the HQL-79 and control groups during the whole process of ischemia and reperfusion. Effects of HQL-79 treatment on transient focal cerebral ischemia After ischemia/reperfusion injury, white-stained infarct area and severe neurological deficit were observed only in the operation group. Fig. 1A shows that the survival rate of HQL-79 group (20%) was diminished significantly at 7 days after reperfusion compared with the vehicle group. The neurological deficit scores are shown in Fig. 1B. The scores of the HQL-79 group recorded at several time points after reperfusion were significantly higher (P⬍0.0001) than
those of the vehicle group. Peripheral blood leukocyte count was not different in the two groups at both 72 h and 7 days after reperfusion (Fig. 1C). NeuN-positive cells were detected in the penumbra area at 72 h and 7 days after reperfusion. In the HQL-79 group, the staining and number for NeuN were weaker than the vehicle group (P⬍0.0001; Fig. 1D). At 72 h after reperfusion, the infarct size in the vehicle group (37.8⫾2.2 mm3) was significantly smaller (P⬍0.05) than in the HQL-79 group (45.8⫾1.4 mm3) (Fig. 1E). A similar trend was noted at 7 days after reperfusion (infarct size: vehicle: 45.2⫾4.9, HQL-79: 58.7⫾8.5 mm3, P⬍0.0001). Effect of HQL-79 on HPGDS expression For detailed analysis of the distribution of immunoreactive cells, we divided each ischemic lesion into three areas; the ischemic core (Co), transition area (Tr), and peri-infarct (Pe) areas, as shown schematically in Fig. 2A-a. Fig. 2A-b shows a few faintly stained HPGDS-positive cells with a well-ramified form in the cerebral cortex of sham-control mouse brain. In the HQL-79 group, HPGDS immunoreactivity significantly (P⬍0.0001) suppressed in the Co area at 72 h (Fig. 2A-d) and 7 days (Fig. 2A-e), than the corre-
Fig. 4. Photomicrographs showing Iba1/EGFP double immunofluorescence staining of control (a– c, g–i) and HQL-79 (d–f, j–i) groups at 72 h (a–f) and 7 days (g–i) after reperfusion in Pe (A), Tr (C) and Co (E). Scale bar⫽20 m. (B) Numbers of extrinsic (Ex) activated monocytes/macrophages and intrinsic (In) activated microglia/macrophages were counted in each parietal cortex including the Pe area (B), Tr area (D), and Co area (F). Data are mean⫾SEM of five mice in each group. * P⬍0.05, compared with the corresponding control group.
M. Liu et al. / Neuroscience 163 (2009) 296 –307
sponding site of control group (Fig. 2B). Immunoblots of HPGDS were clearly detected as a 26-kDa protein band (Fig. 2C). In the HQL-79 group, the intensity of the band decreased on the stroke side (P⬍0.0001) in a time-dependent manner compared with the control group (Fig. 2D). Effect of HQL-79 on microglial activation Immunoblots for Iba1 were detected in the ischemic lesion, which appeared as a 17-kDa protein band. In the control group, the intensity of the band increased on the stroke side (72 h, P⬍0.05; 7 days P⬍0.0001) in a time-dependent manner compared with HQL-79 group (Fig. 3A, 3B). At 72 h and 7 days after reperfusion, the numbers and activity of Iba1-positive microglia/macrophages in the Tr area were significantly higher in the control group (Figs. 3C-a, 3C-c, 3D) than in the HQL-79 group (Figs. 3C-b, 3C-d). Conversely, in the Co area, the number of Iba1 positive microglia/macrophage was higher in the HQL-79 group (Figs. 3E-b, 3E-d, 3F). Effect of HQL-79 on temporal profile of bone marrow– derived monocytes/macrophages and intrinsic microglia/macrophages after transient focal cerebral ischemia At 72 h and 7 days after reperfusion, Iba1-positive activated microglia/macrophages in the Tr area were signifi-
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cantly lower in number and more activated in the HQL-79 group than in the control group (Fig. 3C, 3D). Immunohistochemical staining for Iba1 was performed to determine the origin of microglia/macrophages, i.e. intrinsic microglia/ macrophages (red colored) or extrinsic monocytes/macrophages (yellow in color with coexpression of Iba1 and EGFP) (Fig. 4A–F). The remaining green fluorescent cells were identified as leukocytes by coexpression of Gr-1 and EGFP (data not shown). In the Pe area, the numbers of intrinsic microglia/macrophages were significantly lower in the HQL-79 group than in the control group at both 72 h and 7 days after perfusion (Fig. 4A, B). In the Tr area, the numbers of resident microglia/macrophages were significantly lower in the HQL-79 group than in the control group at both 72 h and 7 days after reperfusion while the number of Iba1/EGFP-positive extrinsic monocytes/macrophages was significantly higher in the HQL-79 group at 7 days after perfusion (Fig. 4C, D). Conversely, in the Co area, the numbers of extrinsic microglia/macrophages were significantly higher in the HQL-79 group at both 72 h and 7 days after reperfusion while the number of intrinsic microglia/ macrophage was lower in the HQL-79 group, compared with the control (Fig. 4E, F). In the Pe, Tr and Co areas, the numbers of intrinsic microglia/macrophages were significantly lower in the HQL-79 group than in the control group at both 72 h and 7
Fig. 4. (Continued).
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days after reperfusion. Conversely, in the Co area, the numbers of extrinsic microglia/macrophages were significantly higher in the HQL-79 group at both 72 h and 7 days after reperfusion. Effect of HQL-79 on HPGDS expression in EGFP mice after transient focal cerebral ischemia In the Co area, the number of bone marrow/blood-derived macrophages was significantly higher in the HQL-79 group than in the vehicle group at both 72 h (P⫽0.0005) and 7 days (P⬍0.0001) after reperfusion while the number of HPGDS-positive cells was significantly lower in the HQL-79 group at both 72 h (P⫽0.01) and 7 days (P⫽0.0008) after perfusion. However, the number of EGFP/HPGDS coexpressing cells was not significantly different in the two groups at both 72 h and 7 days after reperfusion (Fig. 5A, B). Effect of HQL-79 on iNOS expression after transient focal cerebral ischemia Immunostaining for iNOS was detected in microglia in the Co area at 72 h and 7 days after reperfusion. The immunoreactivity for iNOS was stronger in the HQL-79 group than the control group (P⬍0.0001; Fig. 6A-b, A-d, B). In EGFP chimera mice, HQL-79 significantly increased (P⬍0.0001)
the number of EGFP/iNOS-positive extrinsic microglia/macrophages in the ischemic Co area (Fig. 6C, D). Effect of HQL-79 on nitrotyrosine formation after transient focal cerebral ischemia Nitrotyrosine-positive cells were detected in the penumbra area at 72 h and 7 days after reperfusion. In the HQL-79 group, immunoreactivity for nitrotyrosine was stronger than the vehicle group (P⬍0.0001; Fig. 7A-b, A-d, B). Double immunofluorescence labeling was performed to identify nitrotyrosine-expressing cells, these cells were positive for astrocyte marker (GFAP) in the Tr area at 72 h and 7 days after reperfusion (Fig. 7C). No nitrotyrosine and microglia co-staining was noted at any time point after reperfusion (data not shown). Effects of HQL-79 on COX-2 expression after transient focal cerebral ischemia Immunostaining for COX-2 was scarce in the control group throughout the penumbra area at both 72 h and 7 days after reperfusion (Fig. 8A-a, 8A-c). In contrast, COX-2 immunoreactivity was more prominent throughout the test period in the HQL-79 group. Furthermore, the number and intensity of immunoreactivity of COX-2-positive cells were significantly higher in the
Fig. 4. (Continued).
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Fig. 5. (A) Photomicrographs showing HPGDS/EGFP double immunofluorescence staining of vehicle (a– c, g–i) and HQL-79 (d–f, j–l) groups at 72 h (a–f) and 7 days (g–i) after reperfusion in Co area. Scale bar⫽40 m. (B) Number of HPGDS and EGFP merged cells. The number of EGFP/HPGDS coexpressing cells was not significantly different between the two groups at both 72 h and 7 days after reperfusion.
HQL-79 group than the control group (72 h, P⫽0.0006; 7 days, P⬍0.05; Fig. 8A-b, 8A-d, 8B). Effect of HQL-79 on NF-kB expression after transient focal cerebral ischemia Double immunostaining studies showed no co-staining for NF-kB and NeuN (a neuronal marker) at any time point after reperfusion (data not shown). In the Tr area, NF-kB immunostaining was observed in the microglia at 72 h and 7 days after reperfusion. In the HQL-79 group, immunoreactivity of NF-kB was stronger than the control group (72 h, P⫽0.0002; 7 days, P⬍0.05; Figs. 8C-b, 8C-d, 8D).
DISCUSSION In the present study, we analyzed the role of HPGDS in brain ischemia/reperfusion injury using a specific inhibitor of HQL-79. The major finding of the present study was that HPGDS provided protection against progression and expansion of infarct volume after focal cerebral ischemia followed by reperfusion. To our knowledge, this is the first report that demonstrates the anti-inflammatory role of HPGDS in the adult mouse brain, after transient MCA ischemia/reperfusion.
Microglial cells are the main effectors of the innate response after CNS injury, including ischemia. Resident microglia and hematogenous macrophages play crucial roles in the pathogenetic cascade that follows cerebral ischemia but may functionally differ regarding their neuroprotective and cytotoxic properties. After ischemia, activation of resident microglial precedes and predominates over macrophage infiltration (Schilling et al., 2003). The majority of phagocytes in the infarct area are derived from local microglia, predominating over phagocytes of hematogenous origin (Schilling et al., 2005). Stoll et al. (2004) also reported that microglial cells are essential as scavenger cells in tissue repair and are of functional importance since insufficient removal of cell debris has been identified as one of the major causes of failure of tissue regeneration (Stoll et al., 2004). The present study showed that HQL-79 treatment significantly increased the migration of EGFPpositive bone marrow– derived extrinsic monocytes/macrophages into the core area, while it markedly decreased the number of intrinsic Iba1-positive microglia in the transient area at 72 h and 7 days after reperfusion. Murakami et al. (2003) reported that cell therapy using retroviral HPGDS cDNA transfer could potentially exert an antiinflammatory effect in acute gout (Murakami et al., 2003).
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Fig. 6. (A) Photomicrographs showing iNOS-staining in the Co area of representative control (a, c) and HQL-79 (b, d) group at 72 h (a, b) and 7 days (c, d) after reperfusion. Scale bar⫽200 m. (B) Numbers of iNOS-positive cells at the indicated time points. Data are mean⫾SEM of six mice in each group. (C) Photomicrographs showing iNOS/EGFP double immunofluorescence staining of control (a–f) and HQL-79 (g–l) mice at 72 h (a–f) and 7 days (g–l) after reperfusion. Scale bar⫽20 m. (D) Numbers of merged cells of EGFP and iNOS were counted in the ischemic Co area. Data are mean⫾SEM of six mice in each group. ** P⬍0.0001, compared with the corresponding control group.
In addition, Taniguchi et al. (2007) reported that PGD2 protects neonatal mouse (postnatal day 7) brain from hypoxic ischemic injury mainly via DP1 receptors by preventing endothelial cell degeneration. We reported previously iNOS immunostaining in endothelial cells at 3 and 24 h after reperfusion in the Pe and Co area, but such staining was noted in microglia at 72 h and 7 days after reperfusion in the ischemic Co area (Zhang et al., 2005). In the present study, animals were treated orally with HQL-79/vehicle at immediately, day 1 and day 2 after reperfusion, and thus we could not examine endothelial cells in detail. We reported previously marked induction of HPGDS after ischemic injury and that it was mainly produced by endogenous microglia (Liu et al., 2007). In another study, Mohri et al. (2003) also demonstrated the presence of high and specific expression of HPGDS in microglia, and showed many HPGDS-positive microglia that had engulfed apopto-
tic nuclei. Taking into consideration our results of the distribution and number of intrinsic and extrinsic microglia/ macrophages, it is possible that the decreased number of intrinsic microglia and increased number of extrinsic microglia/macrophages were caused by HQL-79 treatment, which subsequently resulted in exacerbation of ischemic brain injury. However, the number of EGFP/HPGDS coexpressing cells was not significantly different between the two groups at both 72 h and 7 days after reperfusion. In the present study, another interrelated mechanism that might account for the neuroprotective effect of HPGDS is the anti-inflammatory effect mediated by inhibition of iNOS activity and nitrotyrosine production. This conclusion is consistent with recent evidence that inhibition of iNOS gene provides protection against cerebral ischemia (Iadecola et al., 1997). The present study showed that immunoreactivity for iNOS was stronger in the HQL-79 group
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Fig. 7. (A) Photomicrographs showing nitrotyrosine staining in the Tr area of representative control (a, c) and HQL-79 (b, d) mice at 72 h (a, b) and 7 days (c, d) after reperfusion. Scale bar⫽200 m. (B) Numbers of nitrotyrosine-positive cells at the indicated time points. Data are mean⫾SEM of six mice in each group. (C) Double immunofluorescent staining of nitrotyrosine (green) and GFAP (red). Shown are the penumbra area of representative control mouse (a– c, g–i) and HQL-79 mouse (d–f, j–l) at 72 h (a–f) and 7 days (g–l) after reperfusion. Nitrotyrosine immunoreactivity (green) in glial-like cells. GFAP immunoreactivity (red) shows the distribution of astrocytes. Coexistence (yellow) of nitrotyrosine and GFAP in glial prolongations. Scale bar⫽20 m.
than the control group. In EGFP chimera mice, HQL-79 significantly increased the number of EGFP/iNOS-positive extrinsic microglia/macrophages in the Co area. In addition, in the HQL-79 group, nitrotyrosine-positive astrocytes were stronger than the control group. Many variables, such as suppression of iNOS expression, are known to play a protective role in several experimental models, such as iNOS null mice (Iadecola, 1997; Zhao et al., 2000) and administration of iNOS inhibitors (Nagayama et al., 1998). The appearance of peroxynitrate (ONOO–)-mediated nitrotyrosine paralleled NO synthesis, demonstrated by induction of iNOS, which produces high, potentially toxic levels of NO (Ischiropoulos and al-Mehci, 1995; Van der Vliet et al., 1994). Based on our results that HPGDS reduced nitrotyrosine production in astrocytes, the possible scenario behind HPGDS-induced reduction of the infarct volume includes suppression of microglial activation followed by suppression of iNOS and inhibition of peroxynitrite production. These findings suggest that one neuroprotective mechanism for HPGDS may involve inhibition of peroxyni-
trite production through reduction of iNOS activity. In this study, to determine whether introduction of HPGDS exerts proinflammatory or anti-inflammatory effects, we treated with HQL-79, a specific inhibitor of HPGDS. Our results showed that HQL-79-activated extrinsic microglia/macrophages accelerated cerebral ischemic injury by overexpression of iNOS. Together, our data provide strong evidence that HPGDS plays a crucial anti-inflammatory role in vivo. Activated NF-kB is translocated into the nucleus, where it initiates the transcription of several genes. This activation of NF-kB requires phosphorylation by IkB kinase (IKK) of IkB proteins. Previous studies reported that 15dPGJ2 inhibits IKK and IkB phosphorylation (Castrillo et al., 2000). NF-kB activates the transcription of many genes thought to be involved in the pathogenesis of cerebral ischemia, such as IL-1, TNF␣, iNOS, COX-2 and ICAM-1. These genes will then produce downstream proteins that exacerbate tissue damage. In the present study, we demonstrated a significant increase of NF-kB in the HQL-79 group. Moreover, we also showed that COX-2, iNOS and
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Fig. 8. (A) Photomicrographs showing COX-2 staining in the Tr area of representative control (a, c) and HQL-79 (b, d) mice at 72 h (a, b) and 7 days (c, d) after reperfusion. Scale bar⫽200 m. (B) Numbers of COX-2 positive cells at the indicated time points. Data are mean⫾SEM of six mice in each group (* P⬍0.05; ** P⫽0.0006, compared with the corresponding control group). (C) Photomicrographs showing NF-kB staining in the Tr area of representative control (a, c) and HQL-79 (b, d) mice at 72 h (a, b) and 7 days (c, d) after reperfusion. Scale bar⫽200 m. (D) Numbers of NF-kB-positive cells at the indicated time points. Data are mean⫾SEM of six mice in each group (* P⬍0.05; ** P⫽0.0002, compared with the corresponding control group).
nitrotyrosine production was subsequently increased in the HQL-79 group. Thus, HPGDS seems to be involved in the regulation of inflammatory responses to brain injury.
CONCLUSION In conclusion, the present study demonstrated that HPGDS protects against the lethal effects of cerebral infarction in a focal transient cerebral ischemia model. In this study, we showed that HPGDS prevents infiltration of extrinsic microglia/macrophages and has anti-inflammatory effects (which result in the reduction of NF-kB production) and that both effects are important for the neuroprotective effects of HPGDS in our ischemia/reperfusion model. Taken together, these data provide support to the paradoxical neuroprotective role of prostaglandins, and thus represent a novel therapeutic target in neurological diseases. Acknowledgments—This study was supported in part by a High Technology Research Center grant and a Grant-in-Aid for Exploratory Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. The authors acknowledge Dr. Kosuke Aritake (Department of Molecular Behavioral Biology,
Osaka Bioscience Institute, Osaka 565-0874, Japan) for critical reading of the manuscript and helpful suggestions.
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(Accepted 11 June 2009) (Available online 13 June 2009)
PROTECTIVE ROLE OF HEMATOPOIETIC PROSTAGLANDIN D SYNTHASE IN TRANSIENT FOCAL CEREBRAL ISCHEMIA IN MICE M. LIU,a N. EGUCHI,b Y. YAMASAKI,c Y. URADE,b N. HATTORId AND T. URABEd*
rophages in ischemic peri-area and penumbra (P<0.0001) at 72 h and 7 days after reperfusion, suggesting involvement of monocytes/macrophages in HQL-79-induced expansion of ischemic injury. Our results demonstrated that the neuroprotective effects of HPGDS in our model are mediated by suppression of activation and infiltration of inflammatory cells. © 2009 IBRO. Published by Elsevier Ltd. All rights reserved.
a Research Institute for Disease of Old Age, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan b Department of Molecular Behavioral Biology, Osaka Bioscience Institute, Osaka 565-0874, Japan c Hanno Research Center, Taiho Pharmaceutical Co. Ltd., Saitama 357-8257, Japan
Key words: PGD2, PGDS, HQL-79, microglia/macrophage, inflammation, ischemic stroke.
d
Department of Neurology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
There is increasing evidence that inflammation plays an important role in CNS ischemia. Experimentally and clinically, stroke is followed by acute and prolonged inflammatory response characterized by an extremely rapid (within hours) activation of microglia/macrophages (Kato et al., 1996; Lehrmann et al., 1997). Resident microglial cells and infiltrating hematogenous macrophages play an important role during the pathogenetic cascade following cerebral ischemia since they express a plethora of growth factors, chemokines and regulatory cytokines as well as free radicals and other toxic mediators (Raivich et al., 1999) that are involved in secondary infarct expansion (Del Zoppo et al., 2000; Hallenbeck, 2002). Hematopoietic prostaglandin D synthase (HPGDS) is a key enzyme in the production of prostaglandin D and its J metabolites. Previous studies showed that PGD2 is spontaneously converted to 15-deoxy-⌬12,14-PGJ2 (15d-PGJ2) by non-enzymatic dehydration (Shibata et al., 2002; Gilroy et al., 1999). Recent results indicate that prostaglandin D2 protects neonatal mouse brain from hypoxic ischemic injury (Taniguchi et al., 2007) and inhibits the expression of inducible nitric oxide synthase (iNOS) in rat vascular smooth muscle cells (Nagoshi et al., 1998). Moreover, Trivedi et al. (2006) demonstrated that HPGDS⫺/⫺ mice bearing a delayed type hypersensitivity reaction display an exaggerated inflammatory response that fails to resolve. Although, 15d-PGJ2 inhibits the production of pro-inflammatory cytokines and iNOS in activated monocytes (Rossi et al., 2000; Castrillo et al., 2000), to our knowledge no report has described the role of HPGDS in cerebral ischemia/reperfusion injury. We reported previously marked induction of HPGDS after ischemia/reperfusion injury, which was mainly produced by endogenous microglia (Liu et al., 2007). In the present study, we tested the hypothesis that HPGDS is neuroprotective in cerebral ischemia/reperfusion injury using middle cerebral artery (MCA) occlusion (MCAO)/reperfusion model of C57BL/6 mice and bone-marrow chimera mice (Komine-Kobayashi et al., 2006). These mice are known to express enhanced green fluorescent protein
Abstract—Cerebral ischemia/reperfusion injury is characterized by the development of inflammatory response, in which vascular macrophages and endogenous microglia are involved. Recent studies showed marked induction of hematopoietic prostaglandin D synthase (HPGDS) after ischemic/reperfusion injury and its localization in microglia, but the molecular mechanism(s) of HPGDS actions in cerebral ischemia is not clear. To clarify the role of HPGDS in cerebral ischemia, C57BL/6 mice and bone marrow chimera mice with cerebral ischemia/reperfusion injury were treated with (4-benzhydryloxy-(1) {3-(1H-tetrazol-5-yl)-propyl}piperidine (HQL-79), a specific inhibitor of HPGDS. The bone marrow chimera mice exhibit expression of enhanced green fluorescent protein (EGFP) in bone marrow/blood-derived monocytes/macrophages. Mice were subjected to ischemia/reperfusion and either treated with HQL-79 (nⴝ44) or vehicle (nⴝ44). Brain sections prepared at 72 h and 7 days after reperfusion were analyzed for neuronal nuclei (NeuN), HPGDS, ionized calcium-binding adapter molecule 1 (Iba1), inducible NO synthase (iNOS), nitrotyrosine, nuclear factor kappa B (NF-kB) and cyclooxygenase-2 (COX-2). The mortality rate (80%) and infarct size were larger in HQL-79- than vehicle-treated mice (58.7ⴞ8.5 versus 45.2ⴞ4.9 mm3; meanⴞSEM, P<0.0001) at 7 days after reperfusion. HQL-79 reduced NeuN expression in the transition area and Iba1 expression (P<0.0001) in the ischemic peri- and penumbra area, but increased COX-2 (P<0.05) and NF-kB expression (P<0.05) in ischemic penumbra and increased formation of nitrotyrosine (P<0.0001) and iNOS (P<0.0001) in the ischemic core area at 72 h and 7 days after reperfusion. In EGFP chimera mice, HQL-79 increased the migration of Iba1/EGFPpositive bone marrow-derived monocytes/macrophages, and simultaneously upregulated iNOS expression in the ischemic core area (P<0.0001), but increased intrinsic microglia/mac*Corresponding author. Tel: ⫹81-3-3813-3111; fax: ⫹81-3-5684-0476. E-mail address: [email protected] (T. Urabe). Abbreviations: Co, ischemic core; COX-2, cyclooxygenase-2; EGFP, enhanced green fluorescent protein; GFAP, glial fibrillary acidic protein; HPGDS, hematopoietic prostaglandin D synthase; HQL-79, 4-benzhydryloxy-(1) {3-(1H-tetrazol-5-yl)-propyl}piperidine; Iba1, ionized calcium-binding adapter molecule 1; IKK, IkB kinase; iNOS, inducible nitric oxide synthase; MCA, middle cerebral artery; MCAO, middle cerebral artery occlusion; NF-kB, nuclear factor kappa B; PBS, phosphate-buffered saline; Pe, ischemic peri-infarct area; rCBF, regional cerebral blood flow; Tr, ischemic transition area; 15d-PGJ2, 15-deoxy-⌬12,14-PGJ2.
0306-4522/09 $ - see front matter © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2009.06.027
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(EGFP) in bone marrow/blood-derived monocytes/macrophages.
EXPERIMENTAL PROCEDURES Experimental protocol The study protocol was approved by the Committee on Animal Experimental Guidelines of Juntendo University School of Medicine. Adult 8-week old male C57BL/6 mice (Charles River Japan Inc., Kanagawa, Japan), were divided at random into two groups. Mice of the 4-benzhydryloxy-(1) {3-(1H-tetrazol-5-yl)-propyl}piperidine (HQL-79)–treated group (HQL-79 group, n⫽44) were treated orally with HQL-79 at 30 mg/kg body weight three times; immediately, day 1 and day 2 after MCA occlusion/reperfusion. Mice of the vehicle-treated group (control group, n⫽44) were treated orally with 0.9% saline solution at a volume similar to that used in the HQL-79 group. HQL-79 was suspended or dissolved in 0.5% methylcellulose for oral administration. Mice were maintained on a 12-h light/dark cycle and provided with food and water ad libitum. Ischemia was induced by the intraluminal vascular occlusion method as described previously (Komine-Kobayashi et al., 2004). Briefly, mice were anesthetized and the left MCA was occluded for 60 min followed by release of the occlusion. Regional cerebral blood flow (rCBF) was measured by laser Doppler flowmetry before, during, and after MCAO, as well as before euthanasia. An observer blinded to the study protocol scored the postural reflexes using a modified neurological scoring system described previously (Hara et al., 1996). In this system, score 0 represents no observ-
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able neurological deficits; 1, failure to extend the left forepaw on lifting the whole body by the tail; 2, circling to the contralateral side; and 3, loss of walking or righting reflex. At pre, 3, 6, 24 and 72 h and 7 days, blood samples were withdrawn from the tail vein for leukocyte count in peripheral blood by using Turk’s solution (Muto Pure Chemicals Co., Tokyo, Japan). We also established EGFP chimera mice by bone marrow transplantation of EGFP into C57BL/6 mice (n⫽16), using the method described previously (Tanaka et al., 2003; Komine-Kobayashi et al., 2006). Transient cerebral ischemia was also induced in these animals 6 weeks later, using the above-described protocol. The distribution of neuronal damage and infarct area was evaluated by Cresyl Violet staining. Briefly, coronal sections (20-m thick) at 400 m intervals were obtained and treated with Cresyl Violet. The infarct area (mm2) of each slice was measured digitally (NIH image 1.63), and the total volume of infarction in the brain was estimated by adding all infarct areas of each slice and multiplying the product by the distance between sections (400 m).
Immunohistochemistry Six animals of each group were anesthetized by i.p. injection of pentobarbital (50 mg/kg i.p.) at 72 h and 7 days after reperfusion. The brain was removed immediately and postfixed for 24 h in 4% paraformaldehyde in PBS at 4 °C before cryoprotection by bathing in 30% sucrose. It was then frozen, and 20 m-thick consecutive coronal sections were prepared on a cryostat (CM-1900, Leica, Wetzlar, Germany).
Fig. 1. Effect of HQL-79 treatment on stroke outcome. (A) Survival analysis during 7 days after reperfusion in the control and HQL-79 groups. (B) Neurological deficit scores in the control and HQL-79 groups. (C) Temporal profiles of peripheral leukocyte count of the vehicle and HQL-79 groups at several time points after reperfusion. The leukocyte count increased significantly at 3 and 6 h after reperfusion and decreased in a time-dependent manner after 24, 72 h and 7 days after reperfusion, but there was no significant difference between the two groups. Data are expressed as mean⫾SEM; n⫽5 per group. (D) Numbers of NeuN-positive cells at the indicated time points. Data are mean⫾SEM of six mice in each group. (E) Coronal sections of mice ischemic brain stained with Cresyl Violet staining. The infarct volume was compared between vehicle and HQL-79 groups at different time points after reperfusion. Data are mean⫾SEM; n⫽5 per group.
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Immunohistochemical staining was performed for nitrotyrosine (Upstate Biotechnology, Lake Placid, NY, USA), ionized calcium-binding adapter molecule 1 (Iba1; Wako Pure Chemicals, Osaka, Japan), NeuN (Chemicon, Temecula, CA, USA), HPGDS (raised in the Department of Molecular Behavioral Biology, Osaka Bioscience Institute, Osaka, Japan), iNOS (BD BioScience, CA, USA), cyclooxygenase-2 (COX-2; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and nuclear factor kappa B (NF-kB; Santa Cruz Biotechnology). Sections were washed in phosphate-buffered saline (PBS), incubated in 0.3% H2O2 in PBS for 30 min, and incubated overnight at 4 °C with 10% normal goat serum (Dako Corp., Carpentaria, CA, USA) in PBS and anti-Iba1 (dilution, 1:1000) antibody, anti-NeuN (dilution, 1:100) antibody, anti-iNOS (dilution, 1:300) antibody, anti-HPGDS antibody (dilution, 1:10,000) anti-nitrotyrosine (dilution, 1:100) antibody, anti-NF-kB antibody (dilution, 1:500) and anti-COX-2 antibody (dilution, 1:500). Immunoreactivity was visualized by the avidin– biotin complex method (Vectastatin; Vector Laboratories, Burlingame, CA, USA) as described previously (Tanaka et al., 2003).
Double-immunofluorescence staining In six animals of each group 72 h and 7 days after reperfusion, the brain was removed immediately after euthanasia and post-fixed for 24 h in 4% paraformaldehyde in PBS at 4 °C before cryoprotection by bathing in 30% sucrose. Free-floating sections of EGFP
bone marrow chimera mice and C57BL/6 mice were washed with PBS and incubated in a blocking solution (10% Block Ace [Yukijirushi, Sapporo, Japan] in PBS) for 1 h at room temperature, followed by incubation overnight at 4 °C with a rabbit anti-Iba1 antibody (dilution, 1:1000, Wako), anti-HPGDS antibody (dilution, 1:5000), anti-iNOS antibody (dilution, 1:200, BD BioScience), antiNeuN (dilution, 1:100; Chemicon, a marker of neurons), antiNF-kB (dilution, 1:100), anti-Gr-1 (dilution, 1:200, Pharmingen, San Diego, CA, USA, a marker of granulocytes), anti-nitrotyrosine antibody (dilution, 1:50; Upstate Biotechnology) or anti– glial fibrillary acidic protein (GFAP) antibody (dilution, 1:100, Dako). For double labeling, sections were incubated with two primary antibodies; Cy3-conjugated anti-rat IgG antibody (dilution, 1:500; Jackson Immunoresearch Laboratories, West Grove, PA, USA) followed by fluorescein isothiocyanate (FITC)– conjugated antirabbit IgG antibody (dilution, 1:500; Vector Laboratories) for 2 h at room temperature. The sections were washed with PBS and mounted on microslide glass with Vectorshield Mounting Medium (Vector Laboratories). Finally, the sections were examined with a fluorescence microscope (Eclipse TE300; Nikon, Tokyo, Japan).
Western blotting Six animals of each group were decapitated after 72 h and 7 days after reperfusion. Samples were taken from two regions; the isch-
Fig. 2. (A) Schematic representation of distribution of neuronal damage in mouse brain after reperfusion delineated by loss of microtubule associated protein 2 (MAP-2) staining. The shaded area represents the infarct zone (a). The three areas subjected to immunohistochemical analysis are illustrated. Staining with anti-HPGDS antibody in control (b– d) and HQL-79 group (e, f) mouse brain after MCAO and reperfusion. Shown are sham control (b), 72 h (c, e) and 7 days (d, f) in the Co area after reperfusion. Scale bar⫽200 m. (B) Numbers of HPGDS-positive cells at the indicated time points. Data are mean⫾SEM of six mice in each group. (C) Western blot analysis of HPGDS. Samples were prepared from the brain at 72 h (control group: lanes 1 and 2; HQL-79 group: lanes 3 and 4) and 7 days (control group: lanes 5 and 6; HQL-79 group: lanes 7 and 8) after reperfusion. A 26-kDa band corresponding to HPGDS protein was detected on the stroke side and its intensity increased in a time-dependent manner. A weaker band was noted in HQL-79 group relative to the control group. Representative data of five experiments with similar results. (D) Densitometric analysis. Values are expressed as percentages of the respective ␣-tubulin. C: contralateral region; S: stroke side. Data are mean⫾SEM; of six mice in each group.
M. Liu et al. / Neuroscience 163 (2009) 296 –307 emic region and the contralateral cortex. Protein extraction and Western blotting were performed as described previously (Ito et al., 1998). Aliquots containing 30 g of protein were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Protein bands were transferred onto polyvinylidene fluoride membrane (Trans-blot Semi-dry Transfer Cell, Bio-Rad, Hercules, CA, USA) with the use of an electrophoretic transfer system (Bio-Rad). After they were blocked with Block Ace for 1 h, the membranes were incubated overnight at 4 °C with anti-Iba1 antibody (dilution, 1:5000), anti-HPGDS antibody (dilution, 1:30,000), or anti-␣-tubulin antibody (dilution, 1:1000; Santa Cruz Biotechnology Inc.). After incubation with the appropriate horseradish peroxidase– conjugated secondary antibody (dilution, 1:20,000; Amersham Place, Little Chalfont, Buckinghamshire, UK) for 1 h at room temperature, immunoreactive bands were visualized in the linear range with enhanced chemiluminescence (ECL Western blotting system, Amersham). For quantitative evaluation, the immunoreactive bands were subjected to densitometric analysis.
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Cell count and statistical analysis The numbers of Iba1/EGFP-positive extrinsic monocytes/macrophages and Iba1-positive intrinsic microglia/macrophages were counted in three predefined areas (0.25 mm2, each) of Iba1stained coronal sections of EGFP chimera ischemic mice. Furthermore, the number of microglia/macrophages coexpressing iNOS and EGFP and coexpressing HPGDS and EGFP (n⫽5, each group), as well as the numbers of NeuN, Iba1-, NF-kB-, COX-2-, iNOS- and nitrotyrosine-positive cells, was counted by the same method under a light microscope (EclipseTS100, Nikon) at a magnification of ⫻100 and assessed independently by two investigators. Values presented in this study are expressed as mean⫾SEM. After acquisition of all data, the randomization code was broken and data were assigned to each group. ANOVA followed by post hoc Fisher protected least significant differences test was used to determine the statistical significance of differences in neurologic score and volumes of infarction between the two groups. A prob-
Fig. 3. (A) Western blot analysis of Iba1. Samples were prepared from the brain at 72 h (vehicle group: lanes 1 and 2; HQL-79 group: lanes 3 and 4) and 7 days (vehicle group: lanes 5 and 6; HQL-79 group: lanes 7 and 8) after reperfusion. A 17-kDa band corresponding to Iba1 protein was detected on the stroke side and its intensity increased in a time-dependent manner. A weaker band was noted in HQL-79 group than vehicle group. Representative data of five experiments with similar results. (B) Densitometric analysis. Values are expressed as percentages of the respective ␣-tubulin. C: contralateral region; S: stroke side. (C) Photomicrographs showing Iba1-staining in the Tr area of representative vehicle (a, c) and HQL-79 (b, d) groups at 72 h (a, b) and 7 days (c, d) after reperfusion. Scale bar⫽200 m. (D) Numbers of Iba1-positive cells at the indicated time points. Data are mean⫾SEM of six mice in each group. (E) Photomicrographs showing Iba1-staining in the Co area of representative control (a, c) and HQL-79 (b, d) mice at 72 h (a, b) and 7 days (c, d) after reperfusion. Scale bar⫽100 m. (F) Numbers of Iba1-positive cells at the indicated time points. Data are mean⫾SEM of six mice in each group.
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ability value less than 0.05 denoted the presence of a statistically significant difference.
RESULTS Physiological parameters All mice of this model showed infarct in the cortical and striatal areas, which was identified by NeuN staining. Measurements of various physiological parameters and rCBF revealed no significant differences between the HQL-79 and control groups during the whole process of ischemia and reperfusion. Effects of HQL-79 treatment on transient focal cerebral ischemia After ischemia/reperfusion injury, white-stained infarct area and severe neurological deficit were observed only in the operation group. Fig. 1A shows that the survival rate of HQL-79 group (20%) was diminished significantly at 7 days after reperfusion compared with the vehicle group. The neurological deficit scores are shown in Fig. 1B. The scores of the HQL-79 group recorded at several time points after reperfusion were significantly higher (P⬍0.0001) than
those of the vehicle group. Peripheral blood leukocyte count was not different in the two groups at both 72 h and 7 days after reperfusion (Fig. 1C). NeuN-positive cells were detected in the penumbra area at 72 h and 7 days after reperfusion. In the HQL-79 group, the staining and number for NeuN were weaker than the vehicle group (P⬍0.0001; Fig. 1D). At 72 h after reperfusion, the infarct size in the vehicle group (37.8⫾2.2 mm3) was significantly smaller (P⬍0.05) than in the HQL-79 group (45.8⫾1.4 mm3) (Fig. 1E). A similar trend was noted at 7 days after reperfusion (infarct size: vehicle: 45.2⫾4.9, HQL-79: 58.7⫾8.5 mm3, P⬍0.0001). Effect of HQL-79 on HPGDS expression For detailed analysis of the distribution of immunoreactive cells, we divided each ischemic lesion into three areas; the ischemic core (Co), transition area (Tr), and peri-infarct (Pe) areas, as shown schematically in Fig. 2A-a. Fig. 2A-b shows a few faintly stained HPGDS-positive cells with a well-ramified form in the cerebral cortex of sham-control mouse brain. In the HQL-79 group, HPGDS immunoreactivity significantly (P⬍0.0001) suppressed in the Co area at 72 h (Fig. 2A-d) and 7 days (Fig. 2A-e), than the corre-
Fig. 4. Photomicrographs showing Iba1/EGFP double immunofluorescence staining of control (a– c, g–i) and HQL-79 (d–f, j–i) groups at 72 h (a–f) and 7 days (g–i) after reperfusion in Pe (A), Tr (C) and Co (E). Scale bar⫽20 m. (B) Numbers of extrinsic (Ex) activated monocytes/macrophages and intrinsic (In) activated microglia/macrophages were counted in each parietal cortex including the Pe area (B), Tr area (D), and Co area (F). Data are mean⫾SEM of five mice in each group. * P⬍0.05, compared with the corresponding control group.
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sponding site of control group (Fig. 2B). Immunoblots of HPGDS were clearly detected as a 26-kDa protein band (Fig. 2C). In the HQL-79 group, the intensity of the band decreased on the stroke side (P⬍0.0001) in a time-dependent manner compared with the control group (Fig. 2D). Effect of HQL-79 on microglial activation Immunoblots for Iba1 were detected in the ischemic lesion, which appeared as a 17-kDa protein band. In the control group, the intensity of the band increased on the stroke side (72 h, P⬍0.05; 7 days P⬍0.0001) in a time-dependent manner compared with HQL-79 group (Fig. 3A, 3B). At 72 h and 7 days after reperfusion, the numbers and activity of Iba1-positive microglia/macrophages in the Tr area were significantly higher in the control group (Figs. 3C-a, 3C-c, 3D) than in the HQL-79 group (Figs. 3C-b, 3C-d). Conversely, in the Co area, the number of Iba1 positive microglia/macrophage was higher in the HQL-79 group (Figs. 3E-b, 3E-d, 3F). Effect of HQL-79 on temporal profile of bone marrow– derived monocytes/macrophages and intrinsic microglia/macrophages after transient focal cerebral ischemia At 72 h and 7 days after reperfusion, Iba1-positive activated microglia/macrophages in the Tr area were signifi-
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cantly lower in number and more activated in the HQL-79 group than in the control group (Fig. 3C, 3D). Immunohistochemical staining for Iba1 was performed to determine the origin of microglia/macrophages, i.e. intrinsic microglia/ macrophages (red colored) or extrinsic monocytes/macrophages (yellow in color with coexpression of Iba1 and EGFP) (Fig. 4A–F). The remaining green fluorescent cells were identified as leukocytes by coexpression of Gr-1 and EGFP (data not shown). In the Pe area, the numbers of intrinsic microglia/macrophages were significantly lower in the HQL-79 group than in the control group at both 72 h and 7 days after perfusion (Fig. 4A, B). In the Tr area, the numbers of resident microglia/macrophages were significantly lower in the HQL-79 group than in the control group at both 72 h and 7 days after reperfusion while the number of Iba1/EGFP-positive extrinsic monocytes/macrophages was significantly higher in the HQL-79 group at 7 days after perfusion (Fig. 4C, D). Conversely, in the Co area, the numbers of extrinsic microglia/macrophages were significantly higher in the HQL-79 group at both 72 h and 7 days after reperfusion while the number of intrinsic microglia/ macrophage was lower in the HQL-79 group, compared with the control (Fig. 4E, F). In the Pe, Tr and Co areas, the numbers of intrinsic microglia/macrophages were significantly lower in the HQL-79 group than in the control group at both 72 h and 7
Fig. 4. (Continued).
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days after reperfusion. Conversely, in the Co area, the numbers of extrinsic microglia/macrophages were significantly higher in the HQL-79 group at both 72 h and 7 days after reperfusion. Effect of HQL-79 on HPGDS expression in EGFP mice after transient focal cerebral ischemia In the Co area, the number of bone marrow/blood-derived macrophages was significantly higher in the HQL-79 group than in the vehicle group at both 72 h (P⫽0.0005) and 7 days (P⬍0.0001) after reperfusion while the number of HPGDS-positive cells was significantly lower in the HQL-79 group at both 72 h (P⫽0.01) and 7 days (P⫽0.0008) after perfusion. However, the number of EGFP/HPGDS coexpressing cells was not significantly different in the two groups at both 72 h and 7 days after reperfusion (Fig. 5A, B). Effect of HQL-79 on iNOS expression after transient focal cerebral ischemia Immunostaining for iNOS was detected in microglia in the Co area at 72 h and 7 days after reperfusion. The immunoreactivity for iNOS was stronger in the HQL-79 group than the control group (P⬍0.0001; Fig. 6A-b, A-d, B). In EGFP chimera mice, HQL-79 significantly increased (P⬍0.0001)
the number of EGFP/iNOS-positive extrinsic microglia/macrophages in the ischemic Co area (Fig. 6C, D). Effect of HQL-79 on nitrotyrosine formation after transient focal cerebral ischemia Nitrotyrosine-positive cells were detected in the penumbra area at 72 h and 7 days after reperfusion. In the HQL-79 group, immunoreactivity for nitrotyrosine was stronger than the vehicle group (P⬍0.0001; Fig. 7A-b, A-d, B). Double immunofluorescence labeling was performed to identify nitrotyrosine-expressing cells, these cells were positive for astrocyte marker (GFAP) in the Tr area at 72 h and 7 days after reperfusion (Fig. 7C). No nitrotyrosine and microglia co-staining was noted at any time point after reperfusion (data not shown). Effects of HQL-79 on COX-2 expression after transient focal cerebral ischemia Immunostaining for COX-2 was scarce in the control group throughout the penumbra area at both 72 h and 7 days after reperfusion (Fig. 8A-a, 8A-c). In contrast, COX-2 immunoreactivity was more prominent throughout the test period in the HQL-79 group. Furthermore, the number and intensity of immunoreactivity of COX-2-positive cells were significantly higher in the
Fig. 4. (Continued).
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Fig. 5. (A) Photomicrographs showing HPGDS/EGFP double immunofluorescence staining of vehicle (a– c, g–i) and HQL-79 (d–f, j–l) groups at 72 h (a–f) and 7 days (g–i) after reperfusion in Co area. Scale bar⫽40 m. (B) Number of HPGDS and EGFP merged cells. The number of EGFP/HPGDS coexpressing cells was not significantly different between the two groups at both 72 h and 7 days after reperfusion.
HQL-79 group than the control group (72 h, P⫽0.0006; 7 days, P⬍0.05; Fig. 8A-b, 8A-d, 8B). Effect of HQL-79 on NF-kB expression after transient focal cerebral ischemia Double immunostaining studies showed no co-staining for NF-kB and NeuN (a neuronal marker) at any time point after reperfusion (data not shown). In the Tr area, NF-kB immunostaining was observed in the microglia at 72 h and 7 days after reperfusion. In the HQL-79 group, immunoreactivity of NF-kB was stronger than the control group (72 h, P⫽0.0002; 7 days, P⬍0.05; Figs. 8C-b, 8C-d, 8D).
DISCUSSION In the present study, we analyzed the role of HPGDS in brain ischemia/reperfusion injury using a specific inhibitor of HQL-79. The major finding of the present study was that HPGDS provided protection against progression and expansion of infarct volume after focal cerebral ischemia followed by reperfusion. To our knowledge, this is the first report that demonstrates the anti-inflammatory role of HPGDS in the adult mouse brain, after transient MCA ischemia/reperfusion.
Microglial cells are the main effectors of the innate response after CNS injury, including ischemia. Resident microglia and hematogenous macrophages play crucial roles in the pathogenetic cascade that follows cerebral ischemia but may functionally differ regarding their neuroprotective and cytotoxic properties. After ischemia, activation of resident microglial precedes and predominates over macrophage infiltration (Schilling et al., 2003). The majority of phagocytes in the infarct area are derived from local microglia, predominating over phagocytes of hematogenous origin (Schilling et al., 2005). Stoll et al. (2004) also reported that microglial cells are essential as scavenger cells in tissue repair and are of functional importance since insufficient removal of cell debris has been identified as one of the major causes of failure of tissue regeneration (Stoll et al., 2004). The present study showed that HQL-79 treatment significantly increased the migration of EGFPpositive bone marrow– derived extrinsic monocytes/macrophages into the core area, while it markedly decreased the number of intrinsic Iba1-positive microglia in the transient area at 72 h and 7 days after reperfusion. Murakami et al. (2003) reported that cell therapy using retroviral HPGDS cDNA transfer could potentially exert an antiinflammatory effect in acute gout (Murakami et al., 2003).
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Fig. 6. (A) Photomicrographs showing iNOS-staining in the Co area of representative control (a, c) and HQL-79 (b, d) group at 72 h (a, b) and 7 days (c, d) after reperfusion. Scale bar⫽200 m. (B) Numbers of iNOS-positive cells at the indicated time points. Data are mean⫾SEM of six mice in each group. (C) Photomicrographs showing iNOS/EGFP double immunofluorescence staining of control (a–f) and HQL-79 (g–l) mice at 72 h (a–f) and 7 days (g–l) after reperfusion. Scale bar⫽20 m. (D) Numbers of merged cells of EGFP and iNOS were counted in the ischemic Co area. Data are mean⫾SEM of six mice in each group. ** P⬍0.0001, compared with the corresponding control group.
In addition, Taniguchi et al. (2007) reported that PGD2 protects neonatal mouse (postnatal day 7) brain from hypoxic ischemic injury mainly via DP1 receptors by preventing endothelial cell degeneration. We reported previously iNOS immunostaining in endothelial cells at 3 and 24 h after reperfusion in the Pe and Co area, but such staining was noted in microglia at 72 h and 7 days after reperfusion in the ischemic Co area (Zhang et al., 2005). In the present study, animals were treated orally with HQL-79/vehicle at immediately, day 1 and day 2 after reperfusion, and thus we could not examine endothelial cells in detail. We reported previously marked induction of HPGDS after ischemic injury and that it was mainly produced by endogenous microglia (Liu et al., 2007). In another study, Mohri et al. (2003) also demonstrated the presence of high and specific expression of HPGDS in microglia, and showed many HPGDS-positive microglia that had engulfed apopto-
tic nuclei. Taking into consideration our results of the distribution and number of intrinsic and extrinsic microglia/ macrophages, it is possible that the decreased number of intrinsic microglia and increased number of extrinsic microglia/macrophages were caused by HQL-79 treatment, which subsequently resulted in exacerbation of ischemic brain injury. However, the number of EGFP/HPGDS coexpressing cells was not significantly different between the two groups at both 72 h and 7 days after reperfusion. In the present study, another interrelated mechanism that might account for the neuroprotective effect of HPGDS is the anti-inflammatory effect mediated by inhibition of iNOS activity and nitrotyrosine production. This conclusion is consistent with recent evidence that inhibition of iNOS gene provides protection against cerebral ischemia (Iadecola et al., 1997). The present study showed that immunoreactivity for iNOS was stronger in the HQL-79 group
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Fig. 7. (A) Photomicrographs showing nitrotyrosine staining in the Tr area of representative control (a, c) and HQL-79 (b, d) mice at 72 h (a, b) and 7 days (c, d) after reperfusion. Scale bar⫽200 m. (B) Numbers of nitrotyrosine-positive cells at the indicated time points. Data are mean⫾SEM of six mice in each group. (C) Double immunofluorescent staining of nitrotyrosine (green) and GFAP (red). Shown are the penumbra area of representative control mouse (a– c, g–i) and HQL-79 mouse (d–f, j–l) at 72 h (a–f) and 7 days (g–l) after reperfusion. Nitrotyrosine immunoreactivity (green) in glial-like cells. GFAP immunoreactivity (red) shows the distribution of astrocytes. Coexistence (yellow) of nitrotyrosine and GFAP in glial prolongations. Scale bar⫽20 m.
than the control group. In EGFP chimera mice, HQL-79 significantly increased the number of EGFP/iNOS-positive extrinsic microglia/macrophages in the Co area. In addition, in the HQL-79 group, nitrotyrosine-positive astrocytes were stronger than the control group. Many variables, such as suppression of iNOS expression, are known to play a protective role in several experimental models, such as iNOS null mice (Iadecola, 1997; Zhao et al., 2000) and administration of iNOS inhibitors (Nagayama et al., 1998). The appearance of peroxynitrate (ONOO–)-mediated nitrotyrosine paralleled NO synthesis, demonstrated by induction of iNOS, which produces high, potentially toxic levels of NO (Ischiropoulos and al-Mehci, 1995; Van der Vliet et al., 1994). Based on our results that HPGDS reduced nitrotyrosine production in astrocytes, the possible scenario behind HPGDS-induced reduction of the infarct volume includes suppression of microglial activation followed by suppression of iNOS and inhibition of peroxynitrite production. These findings suggest that one neuroprotective mechanism for HPGDS may involve inhibition of peroxyni-
trite production through reduction of iNOS activity. In this study, to determine whether introduction of HPGDS exerts proinflammatory or anti-inflammatory effects, we treated with HQL-79, a specific inhibitor of HPGDS. Our results showed that HQL-79-activated extrinsic microglia/macrophages accelerated cerebral ischemic injury by overexpression of iNOS. Together, our data provide strong evidence that HPGDS plays a crucial anti-inflammatory role in vivo. Activated NF-kB is translocated into the nucleus, where it initiates the transcription of several genes. This activation of NF-kB requires phosphorylation by IkB kinase (IKK) of IkB proteins. Previous studies reported that 15dPGJ2 inhibits IKK and IkB phosphorylation (Castrillo et al., 2000). NF-kB activates the transcription of many genes thought to be involved in the pathogenesis of cerebral ischemia, such as IL-1, TNF␣, iNOS, COX-2 and ICAM-1. These genes will then produce downstream proteins that exacerbate tissue damage. In the present study, we demonstrated a significant increase of NF-kB in the HQL-79 group. Moreover, we also showed that COX-2, iNOS and
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Fig. 8. (A) Photomicrographs showing COX-2 staining in the Tr area of representative control (a, c) and HQL-79 (b, d) mice at 72 h (a, b) and 7 days (c, d) after reperfusion. Scale bar⫽200 m. (B) Numbers of COX-2 positive cells at the indicated time points. Data are mean⫾SEM of six mice in each group (* P⬍0.05; ** P⫽0.0006, compared with the corresponding control group). (C) Photomicrographs showing NF-kB staining in the Tr area of representative control (a, c) and HQL-79 (b, d) mice at 72 h (a, b) and 7 days (c, d) after reperfusion. Scale bar⫽200 m. (D) Numbers of NF-kB-positive cells at the indicated time points. Data are mean⫾SEM of six mice in each group (* P⬍0.05; ** P⫽0.0002, compared with the corresponding control group).
nitrotyrosine production was subsequently increased in the HQL-79 group. Thus, HPGDS seems to be involved in the regulation of inflammatory responses to brain injury.
CONCLUSION In conclusion, the present study demonstrated that HPGDS protects against the lethal effects of cerebral infarction in a focal transient cerebral ischemia model. In this study, we showed that HPGDS prevents infiltration of extrinsic microglia/macrophages and has anti-inflammatory effects (which result in the reduction of NF-kB production) and that both effects are important for the neuroprotective effects of HPGDS in our ischemia/reperfusion model. Taken together, these data provide support to the paradoxical neuroprotective role of prostaglandins, and thus represent a novel therapeutic target in neurological diseases. Acknowledgments—This study was supported in part by a High Technology Research Center grant and a Grant-in-Aid for Exploratory Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. The authors acknowledge Dr. Kosuke Aritake (Department of Molecular Behavioral Biology,
Osaka Bioscience Institute, Osaka 565-0874, Japan) for critical reading of the manuscript and helpful suggestions.
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(Accepted 11 June 2009) (Available online 13 June 2009)