Neuroinflammatory response to lipopolysaccharide is exacerbated in mice genetically deficient in cyclooxygenase-2
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Cyclooxygenases (COX) -1 and -2 are key mediators of the inflammatory response in the central nervous system. Since COX-2 is inducible by inflammatory stimuli, it has been traditionally considered as the most appropriate target for anti-inflammatory drugs. However, the specific roles of COX-1 and COX-2 in modulating a neuroinflammatory response are unclear. Recently, we demonstrated that COX-1 deficient mice show decreased neuroinflammatory response and neuronal damage in response to lipopolysaccharide (LPS).
In this study, we investigated the role of COX-2 in the neuroinflammatory response to intracerebroventricular-injected LPS (5 μg), a model of direct activation of innate immunity, using COX-2 deficient (COX-2-/-) and wild type (COX-2+/+) mice, as well as COX-2+/+ mice pretreated for 6 weeks with celecoxib, a COX-2 selective inhibitor.
Twenty-four hours after LPS injection, COX-2-/- mice showed increased neuronal damage, glial cell activation, mRNA and protein expression of markers of inflammation and oxidative stress, such as cytokines, chemokines, iNOS and NADPH oxidase. Brain protein levels of IL-1β, NADPH oxidase subunit p67phox, and phosphorylated-signal transducer and activator of transcription 3 (STAT3) were higher in COX-2-/- and in celecoxib-treated mice, compared to COX-2+/+ mice. The increased neuroinflammatory response in COX-2-/- mice was likely mediated by the upregulation of STAT3 and suppressor of cytokine signaling 3 (SOCS3).
These results show that inhibiting COX-2 activity can exacerbate the inflammatory response to LPS, possibly by increasing glial cells activation and upregulating the STAT3 and SOCS3 pathways in the brain.
KeywordsGlial Fibrillary Acidic Protein Celecoxib NADPH Oxidase Neuroinflammatory Response Glial Cell Activation
analysis of variance
bovine serum albumin
central nervous system
enzyme linked immunosorbent assay
glial fibrillary acidic protein
inducible nitric oxide synthase
monocyte chemoattractant protein-1
macrophage inflammatory protein 1 alpha
- mPGES-1 or -2
microsomal prostaglandin E synthase-1 or -2
cytosolic prostaglandin E synthase
nuclear factor- κB
reactive oxygen species
suppressors of cytokine signaling 3
scavenger receptor A
signal transducer and activator of transcription 3
Tumor Necrosis Factor alpha.
Prostaglandin endoperoxide synthases or cyclooxygenases (COX-1 and COX-2) play a central role in the inflammatory cascade by converting arachidonic acid (AA), released from membrane phospholipids by a phospholipase A2 (PLA2), into prostaglandin endoperoxide H2, which in turn is converted to bioactive prostanoids by specific terminal synthases. The two COX isoforms share 60% homology in their amino acids sequence and have comparable kinetics; however they also show individual differences. COX-1 is normally constitutively expressed in most tissues and thought to be involved in homeostasis, whereas COX-2 is inducible upon inflammatory and other stimuli . However, in the central nervous system (CNS), COX-1 and COX-2 are both constitutively expressed and COX-2 is mainly detected in the perinuclear, dendritic and axonal domains of neurons, particularly in cortex, hippocampus, amygdala and dorsal horn of the spinal cord of both rodent and human CNS [2, 3, 4]. In the CNS, COX-2 has been implicated in important physiological functions such as synaptic transmission, neurotransmitter release, blood flow regulation, and sleep/wake cycle [5, 6, 7, 8, 9].
Both COX-1 and COX-2 have been shown to play important roles in an inflammatory response, their contribution being different depending on the type of insult, the time after insult, and the tissue examined [6, 10]. Because COX-2 is highly inducible by inflammatory stimuli it has been traditionally considered as the most appropriate target for anti-inflammatory drugs [2, 11]. However, the exact role of each COX isoform in neuroinflammation is unclear. While we have recently reported that genetic deletion or pharmacological inhibition of COX-1 significantly ameliorate the neuroinflammatory response and brain injury following lipopolysaccharide (LPS) treatment , the role of COX-2 in the neuroinflammatory process remains controversial. For instance, COX-2 deficient (COX-2-/-) mice have been reported to be resistant to the febrile response induced by peripheral injection of LPS . On the other hand, selective pharmacological inhibition of COX-2, but not of COX-1, increases the expression of several pro-inflammatory genes in the vascular associated cells and the parenchymal microglia after systemic injection of LPS .
In this study we examined the neuroinflammatory response of COX-2-/- and wild type (COX-2+/+) mice to intracerebroventricular (icv) injection of LPS, which is a model of direct activation of brain innate immunity [15, 16, 17, 18, 19].
LPS, a component of the outer cell wall of gram-negative bacteria, mediates its effect through the CD14 receptor, a glycosylphosphatidylinositol-linked membrane protein that is present on microglial cells. The LPS-CD14 complex, together with other adaptor proteins, binds to the toll-like receptor 4 (TLR4), which is present on microglia, but not on astrocytes, oligodendrocytes or cortical neurons . This initiates a bifurcated signal transduction cascade that leads to the transcription of inflammatory and immune response genes, primarily via nuclear factor- κB (NF-κB) activation but also through c-Fos/c-Jun and Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3)-dependent pathways . The signaling events ultimately lead to the production of free radicals generated by NADPH oxidase, myeloperoxidase and inducible nitric oxide synthase (iNOS) in combination with cytokines and chemokines [22, 23], which are mediators of the LPS-induced injury [15, 16, 24]. In this regard, previous data suggest that interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) can contribute to neuronal death in models of acute CNS injury as well as in chronic neurodegenerative disease .
In this study, we demonstrate that COX-2-/- mice are more susceptible than COX-2+/+ mice to LPS-induced neuronal injury and exhibit an increase in microglia and astrocyte activation, and increases in the expression of genes and proteins for inflammatory cytokines, chemokines, reactive oxygen species-generating enzymes, such as iNOS and NADPH oxidase, and in the expression of STAT3 and suppressor of cytokine signaling 3 (SOCS3) signaling molecules. COX-2+/+ mice chronically treated with celecoxib, a COX-2 selective inhibitor, also exhibit an increased neuroinflammatory response compared to untreated wild-type mice.
Materials and methods
Three-month-old male COX-2+/+ and COX-2-/- mice on a C57BL/6-129/Ola genetic background were used . Mice were received at our animal facility at 6 weeks of age from a NIEHS colony maintained by Taconic Farms (Germantown, NY) with heterozygous by heterozygous breedings for greater than 35 generations. In order to prevent the inclusion of strain or genetic background confounders between COX deficient and wild type mice, all of the mice used in this study were progeny derived from heterozygous by heterozygous mating and therefore all contained the same strain and genetic background [26, 27]. Mice were housed at 25°C in our animal facility with a 12 h light/dark cycle with free access to food and water. For celecoxib pretreatment, COX-2+/+ mice were given free access for six weeks to a diet containing 0 or 6000 ppm celecoxib, a COX-2 specific inhibitor, as previously described . Briefly, celecoxib (Celebrex™) capsules (400 mg; Pfizer Inc, New York, NY) were obtained from the NIH Division of Veterinary Medicine and were incorporated into feed by Research Diets, Inc. (New Brunswick, NJ). All procedures were performed under a NICHD approved animal protocol in accordance with NIH guidelines on the care and use of laboratory animals.
Mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg, i.p.) and positioned in a stereotaxic apparatus (Kopf Instruments, Tujunga, CA). Vehicle (sterile phosphate buffer, 5 μl) or LPS (E. coli serotype 055:B5 (Sigma); 5 μg in 5 μl of sterile saline) was administered into the cerebral lateral ventricle using a 10 μl syringe with a 33 gauge needle (World Precision Instruments, Sarasota, FL) and a syringe pump (Stoelting, Wood Dale, IL) at a rate of 1 μl/min. This dose of LPS has been shown by us and by others to produce significant neuroinflammation when measured at 24 h [12, 24, 29]. The coordinates for the sterotaxic injections were -2.3 mm dorsal/ventral, -1.0 mm lateral, and -0.5 mm anterior/posterior from the bregma . The needle was kept in this position for an additional 5 min after injection and then retrieved slowly from the brain.
Tissue preparation and histology
Twenty-four hours after LPS injection, mice were anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and then rapidly perfused transcardially with 0.9% saline solution containing 0.5% sodium nitrate and heparin (10 U/ml), followed by ice cold 4% paraformaldehyde in 0.1 M phosphate buffer. After transcardiac perfusions, brains were rapidly removed, postfixed for 4 h, and then cryoprotected in 30% sucrose at 4°C. Frozen brains were cut into 30 μm coronal sections using a cryostat and stored at -20°C.
Neurodegeneration was assessed using Fluoro-Jade B (FJB), as previously described . Briefly, mounted brain sections were dried for 4 h, rehydrated through graded concentrations of alcohol (100, 70%; 1 min each), and rinsed for 1 min in distilled water. Sections were dipped and shaken in potassium permanganate (0.06%) for 20 min, rinsed for 1 min in distilled water, dipped, and shaken in a solution containing 0.004% FJB (Histochem, Jefferson, AR), 0.1% acetic acid for 20 min. The slides were thereafter rinsed three times in distilled water (1 min each), dried, dipped in xylene, and coverslipped with Permount mounting medium (Fisher Scientific, Ottawa, Ontario) .
For immunohistochemistry, free-floating sections were rinsed three times in phosphate-buffered saline (PBS) (10 min each) and then pretreated with PBS containing 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity. After PBS wash, brain sections were incubated once in PBS with 0.3% Triton X-100 and once in PBS containing 0.5% BSA for 30 min with gentle shaking. The sections were incubated overnight at 4°C with anti-mouse scavenger receptor A (SRA) (1:100; Serotec, Raleigh, NC) in PBS containing 5% normal serum; followed by treatment with a biotinylated secondary antibody (Vector Laboratories, Burlingame, CA) for 1 h in PBS plus 5% normal serum at room temperature, and then with the Vector ABC kit (Vector Laboratories) for 1 h at room temperature. The sections were visualized with the 3,3-diaminobenzidine tetrachloride (DAB; Vector Laboratories, Burlingame, CA). Mounted brain sections were dried for 4 h, dehydrated through graded concentration of alcohol, cleared in xylene, and coverslipped with Permount mounting medium (Fisher Scientific).
RNA extraction and quantitative real-time PCR
Brain total RNA was extracted using RNeasy Lipid Tissue Midi Kit (Qiagen, Valencia, CA, USA) as directed by the manufacturer. Total RNA extraction and reverse transcription were performed as previously described, using the Applied Biosystems Assay-On-Demand Gene Expression protocol with an ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) [32, 33, 34]. Briefly, five micrograms of total RNA were reverse transcribed using a High Capacity cDNA Archive kit (Applied Biosystems). Quantitative PCR for glial fibrillary acidic protein (GFAP), SRA1, TNF-α, IL-1β, interleukin 6 (IL-6), CCL2/monocyte chemoattractant protein-1 (MCP-1), CCL3/macrophage inflammatory protein 1 alpha (MIP-1α), iNOS, the NADPH oxidase subunits gp91phox and p67phox, mPGES-1, cPLA2, COX-1, NF-κB-P65, STAT3, and SOCS3, was performed using specific Taqman® probes (Applied Biosystems). Data were analyzed using the comparative threshold cycle (ΔΔ Ct) method . Results were normalized with phosphoglycerate kinase 1 (Pgk1) as the endogenous control, and expressed as fold difference from the vehicle injected COX-2+/+ mice.
Western blot analyses were carried out as described previously  and nuclear proteins were prepared by using a compartmental protein extraction kit (Chemicon, Temecula) according to the manufacturer's protocol. Briefly, protein fractions were separated on Criterion gels (Bio-Rad), blotted onto a polyvinylidene difluoride membrane (Bio-Rad), and then immunoblotted with antibodies that recognize p67phox (1:500; BD Biosciences), phosphorylated STAT3 (p-STAT3 (Tyr 705), 1:500, Cell signaling, USA), STAT3 (1:1000 Cell signaling, USA), COX-1 (1:500 Cayman Chemicals, USA), and glyceraldehyde dehydrogenase (GAPDH, 1: 2000, Santa Cruz, CA, USA) to control for protein loading. Blotted proteins were detected and quantified using an Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NB, U.S.A.). For IL-1β measurement, a 500 μl aliquot of the crude brain homogenate was centrifuged at 10,000 × g for 20 min at 4°C, and the supernatant was immediately assayed using an ELISA-based kit (mouse IL-1β/IL-1F2 Quantikine ELISA kit, R&D Biosystems, Minneapolis, USA). TNF-α and MIP-1α were measured in the brain supernatant (Searchlight® Sample testing service, Pierce Biotechnology, Woburn, MA, USA). Results were expressed as ng/g protein.
Data were expressed as mean ± SEM and were analyzed with a two-way ANOVA. For Real-Time PCR results, the two-way ANOVA was performed on the log-transformed ΔΔCt. p values < 0.05 were considered statistically significant.
Only COX-2-/-mice show FluorojadeB positive neurons 24 h after LPS
LPS-induced glial cell activation is increased in COX-2-/-mice
Then we determined the immunoreactivity to SRA. In vehicle-treated COX-2+/+ and COX-2-/- mice, no SRA immunoreactivity was seen (Fig. 2C). Intense immunoreactive SRA-positive microglia with enhanced staining intensity, enlarged cell bodies, and thickening of processes were observed 24 h after LPS injection in the cortical/caudate putamen and hippocampal area of COX-2+/+mice. In LPS-injected COX-2-/- mice, SRA-positive cells were numerous with higher cells retaining an enlarged cell body with thickening of ramified processes (Fig. 2C).
Expression of cytokines and chemokines is increased in COX-2-/-mice after LPS
mRNA expression of mPGES-1 is increased in COX-2-/-mice after LPS
Expression of reactive oxygen species generating enzymes is increased in COX-2-/-mice after LPS
Phosphorylated STAT3 and the mRNA expression of STAT3 and SOCS3, but not of NF-κB, are increased in COX-2-/-mice after LPS
Pretreatment of COX-2+/+ mice with celecoxib for 6 weeks increases LPS-induced brain IL-1β level, NADPH oxidase subunit p67phox, and phosphorylated STAT3
In this study we demonstrate for the first time that genetic deletion of COX-2 enhanced the neuroinflammatory response and increased the susceptibility to neuronal damage induced by centrally injected LPS. We also showed that chronic treatment with a selective COX-2 inhibitor, celecoxib, also increases LPS-induced protein levels of IL-1β, a major proinflammatory cytokine, of phosphorylated STAT3, a transcription factor involved in the progression of the inflammatory cascade, and of NADPH oxidase subunit p67phox, a marker of oxidative stress. We have previously demonstrated that this chronic dosing paradigm of celecoxib (6000 ppm for 6 weeks) leads to a plasma concentration of 18.2 ± 5.8 μg/ml . Assuming 98% binding of celecoxib to plasma proteins and that only free celecoxib can cross the blood brain barrier , brain concentration of celecoxib is approximately 640 nM, well above the IC50 (39 nM) of celecoxib for COX-2 . These plasma concentrations are within the same order of magnitude of steady state concentrations (2–3 μg/ml) observed in humans after acute administration of 400–800 mg of celecoxib, doses clinically used for the treatment of rheumatoid arthritis and familial adenomatous polyposis .
In this study, FJB-positive neurons were only observed in the COX-2-/- mice, suggesting that COX-2 deletion increases the susceptibility to LPS-induced neurodegeneration. There is a conflicting view about the role of COX-2 in neurodegeneration and neurotoxicity . For instance, COX-2 inhibition is believed to be neuroprotective in models such as MPTP (1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine), quisquallic acid induced damage [41, 42], and centrally injected NMDA-induced neurotoxicity [43, 44]. However, in these studies, where the toxins directly damage neurons, COX-2-mediated cytotoxicity does not appear to be linked to the inflammatory response . On the other hand, pre-treatment with COX-2 inhibitors or genetic deletion of COX-2 has been shown to increase seizure activity and neuronal damage in response to kainate [28, 45], and to exacerbate endotoxin-induced ocular inflammation  and tissue damage in ConA- and acetaminophen-induced hepatotoxicity [47, 48]. Another study reported, in support of our observations, that selective pharmacological inhibition of COX-2 with NS-398 increases the transcription of inflammatory genes (mPGES-1, TLR2, CD14, MCP-1) in vascular associated brain cells and parenchymal microglia after systemic injection of LPS . While these conflicting data highlight the importance of investigating the distinct roles of COX-1 and COX-2 in physiology and pathology, our findings suggest that COX-2-derived products selectively mediate a protective effect in the development and/or the resolution of inflammation in the brain after endotoxin activation of the innate immune system. In this regard, a recent review emphasizes that COX-2 mediates neuroprotection via specific anti-inflammatory lipid mediators . Furthermore, Gilroy and colleagues demonstrated that selective COX-2 inhibitors, by blocking the production of PGE2 and PGD2, disturbed the resolution phase of inflammation, leading to delay in return to homeostasis .
COX-1 protein levels were not significantly changed by LPS in either COX-2-/- or celecoxib-treated mice compared to COX-2+/+ mice, indicating that the increased neuroinflammatory response was not due to an increased compensatory expression of COX-1 in response to LPS when COX-2 is either genetically abrogated or pharmacologically inhibited. Increases in microglial activation and in the induction of cytokines and chemokines in the COX-2-/- mice could contribute to the susceptibility to LPS-induced damage. Overexpression of chemokines, small pleiotropic chemoattractant cytokines that promote leukocytes activation and migration, has been recently implicated in many neurological disorders including multiple sclerosis, and Alzheimer's disease [51, 52]. The overexpression of chemokines observed in the COX-2-/- mice after LPS may increase the leukocytes and monocytes recruitment in the inflamed brain and cause neuronal damages, in the absence of a "switch off" mechanism. The increased expression of cytokines could be due to the incapacity of the tissue to resolve the inflammation, leading to a persistent activation of the inflammatory cascade. One possibility is that COX-2 deletion or inhibition leads to a reduction in anti-inflammatory mediators or neurotrophic factors, which would impair the brain ability to resolve the inflammation.
iNOS and NADPH oxidase may also contribute to microglia-mediated LPS induced neurotoxicity by increasing the production of extracellular reactive oxygen and nitrogen species, which, in turn, stimulate the microglial release of pro-inflammatory mediators that, like radical oxygen species, are toxic to neurons [53, 54]. In this regard, NADPH inhibitors suppress LPS-induced expression of iNOS, IL-6, IL-1β and TNF-α in glial cells in vitro  and NADPH oxidase has been shown to regulate COX-2 mediated PGE2 production in cultured microglia .
The JAK/STAT pathway is a key player in the intracellular response to cytokines. SOCS3 is a potent inhibitor of the JAK/STAT signaling cascade, negatively regulating signal transduction pathways mediated by a variety of cytokines. SOCS3 has been suggested to play a critical role in integrating the neuroimmunoendocrine circuits . Although NF-κB p65 expression were similar in COX-2+/+ and COX-2-/- mice after LPS, we found that the mRNA expression of STAT3 and the levels of phosphorylated STAT-3 were significantly higher in the COX-2-/- mice compared to wild type mice. SOCS3 was also upregulated in the COX-2-/- mice compared to COX-2+/+ mice after LPS. SOCS3 is a negative modulator of inflammatory cytokine signaling  and can be induced by inflammatory stimuli such as LPS, TNF-α and IL-6 [58, 59]. SOCS3 mRNA up-regulation in the COX-2 deficient mice can thus be viewed as the consequence of the higher cytokine production in these mice after LPS. In this regard, SOCS3 overexpression has been shown to lead to neuroblastoma cell death . Overall, our data indicate a dysregulation of the cytokine signaling pathway in the COX-2-/- mice, which may mediate the increased neuroinflammatory response.
While independent epidemiological studies indicate that non steroidal anti-inflammatory drugs (NSAIDs) administration prevents or delays the onset and risk of developing Alzheimer's disease [61, 62, 63], clinical trials using COX-2 selective inhibitors in patients with mild to severe cognitive impairment, have been unsuccessful to date [64, 65, 66, 67], with the exception of a small double blind, placebo-controlled study with indomethacin, a preferential COX-1 inhibitor . We have recently demonstrated that genetic deletion or pharmacological inhibition of COX-1 significantly attenuates glial cells activation and the neuroinflammatory response, oxidative stress and neuronal damage in response to icv injected LPS . Our results show that while COX-1 selective inhibition may be beneficial, selective inhibition of COX-2 appears not to be beneficial in neurodegenerative diseases with a marked inflammatory component and may explain the failure of selective COX-2 inhibitors to protect AD patients from cognitive decline in clinical trials [64, 65, 66, 67].
These findings altogether indicate that the two COX isoforms display opposite roles in the brain during the acute neuroinflammatory process and that COX-2 inhibition worsens the inflammatory response to LPS, suggesting a neuroprotective function of COX-2-derived products. In this regard, further investigations are warranted to identify which specific COX-2 products may mediate the neuroprotective effects and more research should be focused on COX-1 selective inhibitors for the treatment of neurological and neurodegenerative diseases with an inflammatory component.
This work was supported by the Intramural Research Programs of the National Institute on Aging and the National Institute of Environmental Health Sciences, National Institutes of Health. We thank Drs. Sang-Ho Choi and Christopher D. Toscano for useful experimental suggestions and discussion and Dr. Alan B. Zonderman for statistical help.
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