cyclooxygenase (cox)-1 activity precedes the cox-2 induction in aβ-induced neuroinflammation

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Cyclooxygenase (COX)-1 Activity Precedes the COX-2 Induction in Aβ-Induced Neuroinflammation Leila Dargahi & Shiva Nasiraei-Moghadam & Azadeh Abdi & Leila Khalaj & Fatemeh Moradi & Abolhassan Ahmadiani Received: 27 April 2010 / Accepted: 26 May 2010 # Springer Science+Business Media, LLC 2010 Abstract Two different isoforms of cyclooxygenases, COX-1 and COX-2, are constitutively expressed under normal physiological conditions of the central nervous system, and accumulating data indicate that both isoforms may be involved in different pathological conditions. However, the distinct role of COX-1 and COX-2 and the probable interaction between them in neuroinflammatory conditions associated with Alzheimer s disease are conflicting issues. The aim of this study was to elucidate the comparable role of each COX isoform in neuro- inflammatory response induced by β-amyloid peptide (Aβ). Using histological and biochemical methods, 13 days after stereotaxic injection of Aβ into the rat prefrontal cortex, hippocampal neuroinflammation and neuronal inju- ry were confirmed by increased expression of tumor necrosis factor-alpha (TNF-α) and COX-2, elevated levels of prostaglandin E2 (PGE2), astrogliosis, activation of caspase-3, and neuronal cell loss. Selective COX-1 or COX-2 inhibitors, SC560 and NS398, respectively, were chronically used to explore the role of COX-1 and COX-2. Treatment with either COX-1 or COX-2 selective inhibitor or their combination equally decreased the level of TNF-α, PGE2, and cleaved caspase-3 and attenuated astrogliosis and neuronal cell loss. Interestingly, treatment with COX-1 selective inhibitor or the combined COX inhibitors pre- vented the induction of COX-2. These results indicate that the activity of both isoforms is detrimental in neuro- inflammatory conditions associated with Aβ, but COX-1 activity is necessary for COX-2 induction and COX-2 activity seems to be the main source of PGE2 increment. Keywords β-Amyloid peptide . Neuroinflammation . Cyclooxygenase-1 . Cyclooxygenase-2 . Prostaglandin E2 Introduction Inflammation of the central nervous system (CNS) or neuroinflammation is the most important but poorly understood pathological feature of neurodegenerative dis- eases such as Alzheimers disease and is likely to contribute to the progressive neuronal cell loss in these conditions (Allan and Rothwell 2003; Heneka and O'Banion 2007). It has been well documented that cyclooxygenases (COX) or prostaglandin endoperoxide synthases play a central role in the inflammatory cascade (Morita 2002; Simmons et al. 2004). Two distinct isoforms of cyclo- oxygenase, COX-1 and COX-2, catalyze the first commit- ted step in the conversion of arachidonic acid into unstable intermediate prostaglandin endoperoxide G2/H2, which is rapidly converted to a series of biologically active prosta- glandins (PGD2, PGE2, PGF2α, and PGI2) and thrombox- ane A2 (TXA2) by various isomerases. COX-1 and COX-2 exhibit similar catalytic activity and have a comparable structure, but are products of different genes and differ in tissue distribution, transcriptional regulation, and biological functions (Vane et al. 1998; Chandrasekharan and Simmons 2004). In the central nervous system, both COX-1 and COX-2 are constitutively expressed under normal physiological L. Dargahi : S. Nasiraei-Moghadam : A. Abdi : L. Khalaj : F. Moradi : A. Ahmadiani Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran L. Dargahi : A. Abdi : L. Khalaj : A. Ahmadiani (*) Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran e-mail: [email protected] J Mol Neurosci DOI 10.1007/s12031-010-9401-6

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Cyclooxygenase (COX)-1 Activity Precedes the COX-2Induction in Aβ-Induced Neuroinflammation

Leila Dargahi & Shiva Nasiraei-Moghadam &

Azadeh Abdi & Leila Khalaj & Fatemeh Moradi &Abolhassan Ahmadiani

Received: 27 April 2010 /Accepted: 26 May 2010# Springer Science+Business Media, LLC 2010

Abstract Two different isoforms of cyclooxygenases,COX-1 and COX-2, are constitutively expressed undernormal physiological conditions of the central nervoussystem, and accumulating data indicate that both isoformsmay be involved in different pathological conditions.However, the distinct role of COX-1 and COX-2 and theprobable interaction between them in neuroinflammatoryconditions associated with Alzheimer’s disease areconflicting issues. The aim of this study was to elucidatethe comparable role of each COX isoform in neuro-inflammatory response induced by β-amyloid peptide(Aβ). Using histological and biochemical methods, 13 daysafter stereotaxic injection of Aβ into the rat prefrontalcortex, hippocampal neuroinflammation and neuronal inju-ry were confirmed by increased expression of tumornecrosis factor-alpha (TNF-α) and COX-2, elevated levelsof prostaglandin E2 (PGE2), astrogliosis, activation ofcaspase-3, and neuronal cell loss. Selective COX-1 orCOX-2 inhibitors, SC560 and NS398, respectively, werechronically used to explore the role of COX-1 and COX-2.Treatment with either COX-1 or COX-2 selective inhibitoror their combination equally decreased the level of TNF-α,PGE2, and cleaved caspase-3 and attenuated astrogliosisand neuronal cell loss. Interestingly, treatment with COX-1selective inhibitor or the combined COX inhibitors pre-

vented the induction of COX-2. These results indicate thatthe activity of both isoforms is detrimental in neuro-inflammatory conditions associated with Aβ, but COX-1activity is necessary for COX-2 induction and COX-2activity seems to be the main source of PGE2 increment.

Keywords β-Amyloid peptide . Neuroinflammation .

Cyclooxygenase-1 . Cyclooxygenase-2 . Prostaglandin E2


Inflammation of the central nervous system (CNS) orneuroinflammation is the most important but poorlyunderstood pathological feature of neurodegenerative dis-eases such as Alzheimer’s disease and is likely to contributeto the progressive neuronal cell loss in these conditions(Allan and Rothwell 2003; Heneka and O'Banion 2007).

It has been well documented that cyclooxygenases(COX) or prostaglandin endoperoxide synthases play acentral role in the inflammatory cascade (Morita 2002;Simmons et al. 2004). Two distinct isoforms of cyclo-oxygenase, COX-1 and COX-2, catalyze the first commit-ted step in the conversion of arachidonic acid into unstableintermediate prostaglandin endoperoxide G2/H2, which israpidly converted to a series of biologically active prosta-glandins (PGD2, PGE2, PGF2α, and PGI2) and thrombox-ane A2 (TXA2) by various isomerases. COX-1 and COX-2exhibit similar catalytic activity and have a comparablestructure, but are products of different genes and differ intissue distribution, transcriptional regulation, and biologicalfunctions (Vane et al. 1998; Chandrasekharan and Simmons2004).

In the central nervous system, both COX-1 and COX-2are constitutively expressed under normal physiological

L. Dargahi : S. Nasiraei-Moghadam :A. Abdi : L. Khalaj :F. Moradi :A. AhmadianiNeuroscience Research Center,Shahid Beheshti University of Medical Sciences,Tehran, Iran

L. Dargahi :A. Abdi : L. Khalaj :A. Ahmadiani (*)Department of Pharmacology, School of Medicine,Shahid Beheshti University of Medical Sciences,Tehran, Irane-mail: [email protected]

J Mol NeurosciDOI 10.1007/s12031-010-9401-6

conditions (Yasojima et al. 1999). Neuronal cells expressboth isoforms and high basal levels of COX-2, mainlydetected in the cell bodies and dendritic regions of neurons,in the cerebral cortex, hippocampus, and amygdala, areimplicated in normal neuronal functions including synaptictransmission and plasticity (Yamagata et al. 1993; Kaufmannet al. 1996; Yang and Chen 2008). Neuronal COX-2overexpression can be induced by excitotoxic and oxidativemechanisms as well as inflammatory mediators andreportedly has been observed in traumatic brain injury,cerebral ischemia, and seizures, as well as in severalprogressive neurodegenerative conditions, e.g., Alzheimer’sdisease, Parkinson’s disease, Huntington’s disease, etc.(Minghetti 2004; Liang et al. 2007; Strauss 2008). On theother hand, microglial cells primarily express COX-1 undernormal physiological conditions (Yermakova et al. 1999;Hoozemans et al. 2001; Maihofner et al. 2003), and thefocal accumulation of COX-1 expressing microglia hasbeen reported in several neuropathological disorders like inischemic and traumatic lesions as well as around the senileplaques of Alzheimer’s disease (Yermakova et al. 1999;Schwab et al. 2000, 2002; Hoozemans et al. 2001).Therefore, both COX isoforms may be involved in neuro-inflammation. In this regard, cyclooxygenases have beenthe subject of a lot of investigations, but the distinct role ofeach isoform and the probable interaction between them arestill unrevealed and remain to be elucidated especiallyunder different pathological insults leading to a commonfeature called neuroinflammation.

The role of cyclooxygenases in Alzheimer’s disease(AD) associated neuroinflammation and neurodegenerationis controversial. Clinical effectiveness of NSAIDs andCOX-1 preferential inhibitors like indomethacin, in reduc-ing the incidence and risk of AD and delaying theprogression of cognitive defects (Rogers et al. 1993;Weggen et al. 2007) in comparison with the failure ofCOX-2 selective inhibitors (Aisen et al. 2003; Klegeris andMcGeer 2005), has raised the idea that COX-1 may playthe major detrimental role in neuroinflammatory process ofAD. However, the neurotoxic role of COX-2 is stillcontroversial. Although it has been shown that COX-2overexpression can accelerate β-amyloid pathology inamyloid precursor protein (APP)-transgenic mice (Xianget al. 2002) and can enhance age-dependent cognitivedefects and neuronal apoptosis (Andreasson et al. 2001),some studies imply that COX-2 may play a neuroprotectiverole in CNS and its inhibition may aggravate the neuro-inflammatory response to central or systemic administrationof lipopolysaccharide (LPS; Blais et al. 2005; Aid et al.2008). Therefore, elucidating the exact role of each COXisoform may provide additional insights for the design of

new therapeutic approaches for the prevention or treatmentof AD.

Cumulative evidence suggests that Aβ peptide plays apivotal role as inducer of neuroinflammation in Alzheimer’sdisease. Aβ is a 39–43 residue peptide that is producedduring normal proteolytic processing of APP first throughβ-secretase and then through γ-secretase complex. Aβ1–42

is the main species thought to be involved in AD pathology,due to its self-aggregation properties and capacity to forminsoluble plaques. In addition to direct neurotoxicity, Aβpotentially promotes inflammatory response through theactivation of a complement pathway and activation ofmicroglia and astrocytes (Selkoe 2001; Estrada and Soto2007; Heneka and O'Banion 2007). Therefore, Aβ-inducedneuroinflammation can be an appropriate experimentalmodel to study the chronic neuroinflammation contributingto Alzheimer’s disease (Netland et al. 1998; Paris et al.2002; Richardson et al. 2002).

In the present study, we used the injection of Aβ1–42

solution into the rat brain to induce neuroinflammation andexamine the comparable role of COX-1 and COX-2through the pharmacological inhibition of each COXisoforms. Neuroinflammatory response of hippocampusand the potential effect of selective COX inhibitors wereevaluated by measuring some biochemical markers ofinflammation (e.g., tumor necrosis factor-alpha (TNF-α)and PGE2) and also histological markers of astrogliosis andneuronal apoptosis. To explore the probable connectionbetween two isoforms in AD-associated neuroinflammatorycondition, we measured the level of expression of COX-1and COX-2 in the presence of pharmacological inhibitionof each isoforms.

Experimental Procedures


Adult male Wistar rats weighing 250–280 g were obtainedfrom Pasture Institute (Tehran, Iran). Rats were housedthree per box and maintained at a constant temperature ona 12-h light–dark cycle, with food and water ad libitum.After at least 1 week of habituation in the facilities,animals were admitted to the experimental procedures. Allexperiments were carried in accordance with the NationalInstitutes of Health guidelines for the use of experimentalanimals and approved by the local ethical committee(Neuroscience Research Center, Shahid Beheshti Univer-sity of Medical Sciences), and efforts were made tominimize animal suffering and to reduce the number ofanimals used. Three to five rats were used for each data

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point and for each type of experiment (i.e., histological andbiochemical analyses).

Aβ Injection and Drug Administration

Aβ1–42 fragment (Sigma) was prepared as stock solution(0.1 mg/ml) in sterile 0.1 M phosphate-buffered saline(PBS; pH7.4), diluted to the concentration of 10 ng/μl andthen aliquoted (15 μl per vial), frozen on dry ice, and storedat −80°C until use. In experiments with Aβ injection, 3 μlof defrosted Aβ solution (10 ng/μl) was used for injectioninto the cortex of each cerebral hemisphere. Sterile 0.1 Mphosphate-buffered saline was injected into control animals.The animals were anesthetized with ketamine (80 mg/kg)and xylazine (5 mg/kg) and supplemented throughout thesurgery as required. They were placed in a stereotaxicframe and Aβ or vehicle was bilaterally injected in the deepfrontal cortex (3.2 mm anterior and ±2 mm lateral to thebregma and the depth of 3 mm) according to van der Steltet al. (2006). Injections were made over 3 min using a 10-μl Hamilton syringe and the needle was left in place for anadditional 3 min before it was slowly retracted. Afterinjection of Aβ, intracerebroventricular (icv) cannulationwas done for repeated injection of COX inhibitors or theirvehicles. A 23-gauge, stainless steel tubing guide cannulawas implanted just above the right lateral cerebroventricle(according to coordinates of Paxinos and Watson (2007)atlas—0.48 mm posterior and +1.5 mm lateral to thebregma, and its lower end about 0.3 mm above theventricle) and fixed on the rat skull by acrylic cement.

In experiments with COX inhibitors, SC560 (Sigma) andNS398 (Sigma) were used, respectively, as COX-1 andCOX-2 selective inhibitor. SC560 (10 μg/3 μl/rat, dissolvedin 90% dimethyl sulfoxide (DMSO)), NS398 (5 μg/3 μl/rat,dissolved in 10% DMSO), SC560 + NS398 (10+5 μg/3 μl/rat, dissolved in 90% DMSO), or vehicle (3 μl, 90%DMSO) was given intracerebroventricularly 1 h after Aβinjection and thereafter at the first, second, fourth, and tenthday from the operation. PBS-injected rats treated withvehicle (comparable with intact animals, data not shown)were considered as the control group. All animals werescarified on the 13th day from operation, based on previousstudy conducted by van der Stelt et al. (2006).

Tissue Preparation and Biochemical Analyses

Animals were euthanized by cervical translocation, and thebrains were removed from the skulls. The hippocampustissue was separated from each hemisphere and was snap-frozen in liquid nitrogen and kept at −80°C to time ofbiochemical analysis.

Western Blot Analysis

Western blot analysis was performed on hippocampihomogenates to determine TNF-α, COX-1, COX-2, andcleaved caspase-3 protein expression levels. Briefly, thewhole hippocampus was homogenized in ice-cold lysisbuffer (Tris–HCl 50 mM, NaCl 150 mM, Triton X-1000.1%, sodium deoxycholate 0.25%, SDS 0.1%, EDTA1 mM, and protease inhibitor cocktail 1%) by rapid passagethrough a 23-gauge syringe needle five to six times, and thetotal protein extract was then obtained by centrifugation for45 min at 13,000 rpm at 4°C and a further 15 min. Proteinconcentration was determined by the Bradford assay, andequivalent amounts (80 μg) of each sample were subjectedto SDS-PAGE electrophoresis. The proteins were trans-ferred onto polyvinylidene difluoride membranes (Milli-pore), for assessment of TNF-α, cleaved caspase-3, COX-2,and β-actin, and onto nitrocellulose membranes (Bio-Rad)for assessment of COX-1, by using Bio-Rad immunoblot-ting apparatus. Afterward, the membranes were blocked in2% skim milk (prepared in 0.1% Tween 20, Tris-bufferedsaline, TBS) for 75 min at room temperature and thenincubated at 4°C overnight with TNF-α rabbit monoclonalantibody (cell signaling) (1:1,000 v/v), COX-2 rabbitpolyclonal antibody (ABR-Affinity BioReagents)(1:10,000 v/v), COX-1 mouse monoclonal antibody(Abcam) (1:2,000 v/v), cleaved caspase-3 rabbit monoclo-nal antibody (cell signaling) (1:1,000 v/v), or β-actin rabbitmonoclonal antibody (cell signaling) (1:1,000 v/v). Themembranes were washed three times with 0.1% Tween 20and TBS and then incubated with horseradish peroxidase-conjugated gout anti-mouse or anti-rabbit secondaryantibodies (cell signaling) (1:10,000 v/v). The immuno-complexes were visualized by the ECL chemilumines-cence method (GE Healthcare, Amersham). The relativeexpression of the protein bands was quantified byscanning of the X-ray films and densitometric analysiswith ImageJ software.

Prostaglandin Assay

For PGE2 measurement, hippocampus tissues wereweighed and then homogenized by 500-μl cold methanolcontaining formic acid 96% (10 μl/ml), indomethacin(10 μg/ml), and PMSF (10 μM), allowed to sit at 4°C for15 min, and centrifuged at 13,000 rpm for 25 min at 4°C.The supernatants were diluted to 10% methanol by addingdistilled water, and purification was performed by solid-phase extraction technique as described previously (Yue etal. 2004). In short, samples pre-equilibrated at pH3.0 wereloaded onto C18 columns (Chromabond) and eluted with

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10 ml of ethyl acetate (Merck). Samples were concentratedby a nitrogen stream evaporator, and PGE2 was measuredby using enzyme immunoassay kits (Correlate-EIA™,assay designs), according to the manufacturer’s instruc-tions. Results were corrected according to the tissue weightand expressed relative to the value of the control group.

Tissue Preparation and Histological Analyses

The deeply anesthetized rats were transcardially perfusedusing a left ventricular cannula with 100 ml of PBS,followed by 200 ml of freshly prepared 4% phosphate-buffered paraformaldehyde (pH value, 7.4). The brainhemispheres were removed, postfixed in 4% paraformalde-hyde for 24 h, and subsequently embedded in paraffin.Hippocampal areas were delimited according to the atlas ofPaxinos and Watson (2007), −3.5 to –4.2 mm from thebregma, and brain tissue sections corresponding to thesecoordinates were subjected to histological assays. Coronalsections (4–5 μm thickness) were prepared serially using amicrotome rotary apparatus (Cut5062, Germany). Twosections were selected from each eight sections. The firstsection was assigned to Nissl staining and the secondsection was assigned to glial fibrillary acidic protein(GFAP) immunostaining. At each 90-μm interval, a 10-μm section was prepared for terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) staining.

Nissl Staining

Paraffin-embedded tissue sections were deparaffinized inxylene, rehydrated, and soaked in 0.1% cresyl violet acetate(Sigma) solution for 10 min. Sections were rapidlydifferentiated in 95% ethanol, then dehydrated in absolutealcohol (twice for 10 min each), cleared, and mounted withPermount mounting medium (Entellan®, MERK) andanalyzed under light microscopy.


TUNEL was performed on 10-μm-thick paraffin-embeddedsections using the In Situ Cell Death Detection Kit, POD(Roche Applied Science, Germany). Tissue sections weredeparaffinized in xylene, rehydrated, and immersed in 3%hydrogen peroxide to block the endogenous peroxidaseactivity. After rinsing with PBS, sections were treated withproteinase K solution at 37°C for 15 min to enhance thestaining, incubated for 60 min at 37°C with 50 μl ofTUNEL reaction mixture, and then incubated for 30 min at37°C with 50 μl of converter-POD. Sections were rinsed inPBS, then incubated for 10 min at 15–25°C with 50 μl ofdiaminobenzidine (DAB) substrate solution and rinsedagain with PBS. Counter staining was achieved with 0.5%

methyl green. For the positive control, the sections wereincubated in DNase solution at 15–25°C for 10 min, and forthe negative control, sections were omitted from theenzyme solution. Finally, the sections were dehydratedagain and coverslipped as described above and analyzedunder light microscopy.


Immunohistochemistry for GFAP was performed on 4–5-μm-thick paraffin-embedded sections according to theprotocol provided by GFAP mouse monoclonal antibody(Cell Signaling). In brief, sections were deparaffinized inxylene and rehydrated. Antigen retrieval was carried out bymicrowaving in citrate buffer (pH value 6) for 10 min. Thesections were quenched with 3% hydrogen peroxide (H2O2)in absolute methanol. Sections were treated with 5% normalgoat serum in PBS prior to the addition of the primaryantibody to block nonspecific binding sites. This wasfollowed by an overnight incubation with the primaryantibody (1:100) at 4°C. The sections were washed andthen incubated with a ready-to-use anti-mouse secondaryantibody from Dako (EnVision Plus®), and color reactionwas developed using DAB as the chromagen. The slideswere then counterstained with hematoxylin, dehydratedusing graded alcohols and xylene, and coverslipped asdescribed above. Sections serving as negative controls wereincubated with the primary antibody diluent, PBS, insteadof the primary antiserum.

Cell Counting

Morphometric analysis was performed by cell quantificationin the CA1 and CA3 subfields of the hippocampus. At leastfive histological sections, corresponding to the definedrostro-caudal level (−3.5 to −4.2 mm posterior to bregma),were processed for each animal. For Nissl staining, thenumber of survived pyramidal cells was counted in a 1-mmlength of the middle portion of hippocampal CA1 and CA3subfields under ×40 magnification of light microscopy asperformed previously (Xu et al. 2009). The percentage ofneuronal reduction was calculated in Aβ-injected rats andsubsequently in Aβ-injected, COX inhibitor-treated animals.The number of TUNEL-positive pyramidal cells and GFAPpositive astrocytes were counted, respectively, within threeadjacent ×100 and ×40 microscopic fields in CA1 and CA3areas of hippocampus and the average number of all sixfields in at least five sections for each animal was recorded.

Statistical Analysis

The average results for each group are presented as mean ±SEM with the exception of cell counts (TUNEL-positive

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neurons and GFAP-positive astrocytes) in which the dataare presented as mean ± SD. Statistical significancebetween groups was established with a one-way ANOVA(using SPSS16) followed by the Tukey HSD post hoc test.A P value of <0.05 was considered statistically significant.


Aβ-Induced Neuroinflammation: Increased the Expressionof TNF-α and COX-2 and Elevated the Level of PGE2

To ascertain the inflammatory response evoked by solubleand diffusible Aβ peptide, 13 days after the injection of Aβinto the rat cortex, the hippocampal expression of TNF-α,as a pro-inflammatory cytokine, and COX-1 and COX-2, asinflammatory enzymes, were determined quantitativelyusing Western blot analysis. As compared to the controlgroup, Aβ injection significantly increased the expressionof TNF-α (Fig. 1a) and COX-2 (Fig. 1b), but did not alterthe level of COX-1 protein (Fig. 1c). We also measured thehippocampus level of PGE2 as a major pro-inflammatoryand pathologic product of COX activity. Following Aβinjection, the hippocampus level of PGE2 was significantlyincreased as compared to control animals (Fig. 2).

Aβ-Induced Neuronal Damage and Astrogliosisin Hippocampus

To confirm the hippocampal neuronal cell death previouslyobserved in several studies of Aβ-induced neuroinflamma-tion and neurotoxicity (Harkany et al. 1999; Richardson etal. 2002), we performed Western blot and histologicalanalyses. Thirteen days after the injection of Aβ into thecortex, Western immunoblots showed a significant increasein hippocampus level of cleaved caspase-3, executionerenzyme in apoptotic cell death, as compared to controlanimals (Fig. 1d). Using Nissl staining, neuronal damagewas observed in the CA1 and CA3 regions of hippocampusin consecutive slices from the Aβ-injected versus controlanimals. In addition to localized neuronal cell loss, Aβcaused a widespread damage over the whole hippocampalregion, as can be seen in the reduced thickness ofhippocampus pyramidal cell layer (Fig. 3) and a significantreduction in the number of neuronal cells in CA1 and CA3regions (Table 1). In fact, significantly increased, albeitlimited, TUNEL-positive neurons were also found in thehippocampus of Aβ-injected versus control rat brains(Fig. 4).

To determine glial cell response to Aβ, we examined theexpression of GFAP as a specific marker for astrocytes.Sections adjacent to those used for Nissl and TUNELstaining were used to determine immunoreactivity to GFAP.

Aβ injection markedly increased immunoreactive GFAP-positive astrocytes in the hippocampus as compared tovehicle-injected animals (Fig. 5).

Both COX-1 and COX-2 Inhibition Reducedthe Aβ-Induced Expression of Inflammatory Mediatorsand Reversed the Neuronal Damage

To determine the distinct role of COX-1 and COX-2 inneuroinflammatory response to Aβ and neuronal apopto-sis, we used selective pharmacological inhibition of COX-1 and COX-2 by chronic intracerebroventricular injectionof SC560 and NS398, respectively. Western immunoblotsshowed that selective inhibition of either COX-1 or COX-2 significantly reduced the expression of TNF-α ascompared to Aβ-injected, vehicle-treated animals. Therewere no significant differences between the effects of thetwo inhibitors on the protein levels of TNF-α (Fig. 1a).The hippocampus level of PGE2 was significantly reducedand reached the normal value in either COX-1 or COX-2inhibitor-treated, Aβ-injected animals compared to Aβ-injected and vehicle-treated ones. It is of interest that inthis case there were also no significant differencesbetween the inhibitory effects of the two applied COXselective inhibitors (Fig. 2). Chronic treatment of Aβ-injected rats with combined selective COX-1 and COX-2inhibitors significantly reduced the hippocampal level ofTNF-α protein and PGE2, without any significant differ-ences with separated COX-1 or COX-2 inhibitor treatment(Figs. 1a and 2). The increased level of cleaved caspase-3due to Aβ injection was also significantly reduced by theanti-inflammatory effect of either COX-1 or COX-2inhibitor or their combination as compared to Aβ-injected and vehicle-treated animals (Fig. 1d). Biochem-ically confirmed, the protective effects of selectivepharmacological inhibition of COX-1 and COX-2 onAβ-induced hippocampal neuronal damage were alsoverified by histological evaluation. As shown in Fig. 3,the overall neuronal damage caused by Aβ injection wasprevented by chronic injection of either SC560 or NS398or their combination. Counting the neuronal cells in CA1and CA3 regions of consecutive hippocampal slices fromAβ-injected animals receiving chronic treatment witheither COX-1 or COX-2 inhibitor or their combinationrepresented a significant decrease in the percentage ofneuronal reduction as compared to the reduction observedin Aβ-injected, vehicle-treated animals (Table 1; mean ±SE, n=5 slices from three to five distinct brains in eachgroup). In addition, counting the TUNEL-positive neuronsand also the immunoreactive GFAP-positive astrocytes inthe hippocampus regions of brain slices from Aβ-injectedanimals chronically treated with either COX-1 or COX-2inhibitor or their combination showed a significant

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decrease as compared to Aβ-injected and vehicle-treatedones (Figs. 4 and 5), without any significant differencesbetween the three treatments.

Chronic icv injection of SC560 and NS398 or theircombination in the absence of Aβ injection did not causeany significant changes in the biochemical factors andhistopathological remarks assessed in this study as com-pared to vehicle-injected animals (data not shown).

Selective COX-1 Inhibition Completely Attenuatedthe Increased Expression of COX-2 in Response to Aβ

To elucidate the cause of equal efficacy of COX-1 andCOX-2 selective inhibition in attenuating the inflammatoryand neurotoxic response to Aβ, we analyzed the hippo-campus level of COX-1 and COX-2 protein in Aβ-injectedanimals receiving chronic treatment with either SC560 or


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Figure 1 Effects of Aβ and chronic treatment with COX-1 or COX-2inhibitors or their combination on the hippocampal levels of TNF-α,COX-2, COX-1, and cleaved caspase-3 proteins. The expression of aTNF-α, b COX-2, c COX-1, and d activation of caspase-3 weredetermined by Western blotting. The quantitative data obtained fromdensitometric analysis of scanned X-ray films were normalized to

corresponding immunoblots of β-actin as an internal standard andrepresented as relative density. All panels show the representativeimmunoblots of control, Aβ, Aβ + SC560, Aβ + NS398, and Aβ +(SC560 + NS398) groups from left to right. Data are mean ± SEM(n=3–5). ***P<0.001 versus control group, ##P<0.01, ###P<0.001versus Aβ-injected group

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NS398 or their combination. COX-2 selective inhibitionmodestly but significantly decreased the expression ofCOX-2 induction in response to Aβ although it was yetsignificantly higher than control animals. However, COX-1selective inhibition by SC560 or the combination of SC560and NS398 completely blocked the induction of COX-2 inresponse to Aβ (Fig. 1b). It is of interest that COX-1expression did not show any significant change in Aβ-injected, COX inhibitor-treated groups as compared to Aβ-injected, vehicle-treated group (Fig. 1c). In addition,chronic icv injection of SC560 and NS398 or theircombination in the absence of Aβ injection did not causeany significant changes in COX-1 or COX-2 expressionlevels (data not shown).


In the present study, we showed that chronic treatmentwith either COX-1 or COX-2 selective inhibitor equallyattenuated neuroinflammatory response to Aβ. In supportof inflammation and neurotoxicity by using biochemicaland histological analyses, we revealed that injection of Aβinto deep frontal cortex of rat-induced TNF-α expressionand astrocyte activation, increased COX-2 expression andproduction of PGE2, enhanced the cleaved caspase-3, andcaused neuronal damage and apoptosis in rat hippocam-pus. While we did not observe any significant increase inthe level of COX-1 protein, the inhibition of COX-1 aswell as selective COX-2 inhibition interestingly decreasedthe level of TNF-α and PGE2 to normal values, reducedastrogliosis, and prevented the activation of caspase-3 andneuronal cell death.

Data concerning the contribution of COX-2 to neuro-inflammation are controversial. Based on accumulated data,it has been deduced that the neurotoxic or neuroprotectiverole of COX-2 depends on the type of pathological insult,the cellular target of the stimulus, and whether inflamma-tion is a primary or secondary response (Choi et al. 2009).In this regard, COX-2 can mediate neuroprotection in thecase of primary neuroinflammation such that occurs inresponse to LPS that is able to directly induce microgliaand exert neurotoxicity secondary to this activation, withoutdirect toxic effect on neurons (Jeohn et al. 1998; Suzumuraet al. 2006; Aid et al. 2008). However, in conditions whenneuroinflammation is a secondary response to directneuronal injury such as ischemia and traumatic brain injury,





Figure 3 Representative photomicrographs of hippocampal CA1 (toppanels) and CA3 (bottom panels) subfields stained with cresyl violetacetate (Nissl staining) showing the effects of Aβ and chronictreatment with COX-1 or COX-2 inhibitors or their combination onthe hippocampus neuronal survival. From left to right: control, Aβ,

Aβ + SC560, Aβ + NS398, and Aβ + (SC560 + NS398) groups. Notethat the pyramidal cell layers in CA1 and CA3 areas in Aβ-injectedsections are markedly thinner than in the control. However, thedamage induced by Aβ was significantly less in the COX inhibitortreated rats. Bars=30 μm


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Figure 2 Effects of Aβ and chronic treatment with COX-1 or COX-2inhibitors or their combination on the hippocampal PGE2 levels. Dataare mean ± SEM (n=3–5). ***P<0.001 versus control group, ###P<0.001 versus Aβ-injected group

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COX-2 overexpression is involved in neurotoxicity (Li etal. 2008; Strauss 2008). In the case of neurodegenerativediseases like Alzheimer’s disease, neuroinflammation is acomplex phenomenon while Aβ peptides in addition todirect toxic effects on neurons that can interrupt intra-neuronal homeostasis and induce cell death (Mattson et al.1992; Ueda et al. 1994; Iwata et al. 2004; Malinin et al.2005; Sanz-Blasco et al. 2008) may also promote neuro-inflammation and neurodegeneration through the activationof microglial cells and astrocytes (Hu et al. 1998; Pan et al.2008). In the present study, the beneficial effect of COX-2selective inhibitor on alleviation of the Aβ-induced neuro-

inflammation and neuronal cell death implies that COX-2induction in response to Aβ is involved in neurotoxicity.

The data obtained from analysis of postmortem brains ofAD patients have revealed that neuronal COX-2 expressionincreases especially in the first stages of AD (Pasinetti andAisen 1998; Ho et al. 2001). In our experiment, 13 daysafter intracranial injection of Aβ, the level of COX-2protein significantly increased in the hippocampus, as it hasbeen reported in a similar study conducted by van der (2006). In vitro investigations show that Aβ can up-regulate COX-2 expression in neurons (Pasinetti and Aisen1998), but about the induction of microglial COX-2 in






L p





s (m




### ### ###

Figure 4 TUNEL staining of rat hippocampus. a Representativephotomicrographs of TUNEL-stained sections from control, Aβ, Aβ +SC560, Aβ + NS398, and Aβ + (SC560 + NS398) groups, respectively,from left to right. Bars=20 μm. b The number of TUNEL-positive

neurons was counted in three ×100 magnification fields of CA1 and CA3areas of hippocampus and the average number is represented as mean ±SD. ***P<0.001 versus control group, ###P<0.001 versus Aβ-injectedgroup

Table 1 Neuronal loss in hippocampus after Aβ injection and the protective effects of COX inhibitors

Group Percentage of neuronal reduction (mean ± SEM)


Control 0 0

Aβ 20.76±2.55*** 18.36±2.83***

Aβ + SC560 10.27±1.88## 8.71±2.43#

Aβ + NS398 12.16±2.05# 9.81±2.62#

Aβ + (SC560 + NS398) 8.9±1.79### 7.14±3.03##

The number of surviving pyramidal cells in a 1-mm length of CA1 and CA3 subfields of the hippocampus was counted at ×40 magnification andthe percentage of neuronal reduction was calculated [(average neuron number in control sections − neuron number in treated sections) / averageneuron number in control sections × 100]. Data are mean ± SEM

***P<0.001 versus control, # P<0.05, ## P<0.01, ### P<0.001 versus Aβ group

J Mol Neurosci

response to Aβ, there are some different reports. One studyindicates that Aβ fails to induce COX-2 in both human andrat microglia and another study shows that COX-2induction in microglia and production of inflammatorymediators including PGE2 in response to Aβ can aggravateneuronal damage (Hoozemans et al. 2002a; Pan et al.2008). However, it seems that neurons are the main sourceof COX-2 expression following Aβ injection, since micro-glia expressing COX-2 have not been also detected in ADbrains (Yermakova et al. 1999; Hoozemans et al. 2001).Increased production of glial inflammatory mediators suchas TNF-α and prostaglandins is the other stimulus that caninduce neuronal COX-2 expression (Fehrenbacher et al.2005; Pan et al. 2008).

Neuronal COX-2 overexpression can directly injureneurons by several mechanisms, including the induction ofcell cycle proteins, increased production of prostaglandins,and generation of reactive oxygen species (Nicol et al. 1992;Hoozemans et al. 2002b; Im et al. 2006). Prostaglandin E2 isthe major product of cyclooxygenase activity and its elevatedlevels have been observed in the CSF of AD patients (Ho etal. 2000; Combrinck et al. 2006). In addition to directneurotoxic and apoptogenic effects, PGE2 can impair thephagocytic activity of microglia and thereby increase thetoxicity of Aβ and also can potentiate the production ofinflammatory cytokines by astrocytes (Fiebich et al. 2001;

Takadera et al. 2004; Shie et al. 2005a, b; Kawano et al.2006). In the present study, we showed that chronictreatment with COX-2 selective inhibitor, NS398, preventedthe increment of TNF-α and PGE2, attenuated the cleavageand activation of caspase-3, and reduced the number ofTUNEL-positive neurons, and these are in support of theprotective role of COX-2 inhibition.

In a recent study, it has been shown that COX-2 deletionor inhibition may exacerbate the acute neuroinflammatoryresponse to LPS and proposed that COX-2 may exert aneuroprotective role in such acute neuroinflammatoryprocesses (Aid et al. 2008). It should be considered thatLPS induces COX-2 expression in microglia and endothe-lial cells while the neurons are the main source of COX-2overexpression in chronic neuroinflammatory responseevoked by Aβ (Akundi et al. 2005; Xia et al. 2006; Chunget al. 2010). Therefore, in addition to the hypothesis thatprolonged COX-2 expression in the brain interferes withintrinsic neuroprotective mechanisms and contributes to theestablishment of a vicious cycle in which cell death ratherthan survival pathways dominates (Strauss 2008), it seemsthat the type of cellular source of COX-2 overactivity isalso a determining factor for declaring the neurotoxic orneuroprotective role of COX-2.

In support of our results indicating the beneficial role ofCOX-2 inhibition in AD-associated neuroinflammation,












te N





D) ## ## ##


Figure 5 GFAP immunostaining of astrocytes in rat hippocampus. aRepresentative photomicrographs of GFAP-immunostained sectionsfrom control, Aβ, Aβ + SC560, Aβ + NS398, and Aβ + (SC560 +NS398) groups, respectively, from left to right. Bars=30 μm. b The

number of GFAP expressing astrocytes was counted in three ×40magnification fields of CA1 and CA3 areas of hippocampus and theaverage number is represented as mean ± SD. ***P<0.001 versuscontrol group, ##P<0.01 versus Aβ-injected group

J Mol Neurosci

some other studies indicated that COX-2 inhibition oralleviation may reduce neuroinflammation and oxidativestress and improve Aβ-mediated suppression of memoryand synaptic plasticity in some experimental models of AD(Melnikova et al. 2006; Kotilinek et al. 2008; Nivsarkar etal. 2008; Echeverria et al. 2009).

In addition to COX-2, recent evidences imply that COX-1, due to its predominant localization in microglia, plays amajor role in neuroinflammatory and neurodegenerativeprocesses (Choi et al. 2009), but the precise mechanism ofcontribution of COX-1 has not been clearly established.There are no indications that COX-1 expression levels inmicroglia are changed under neuropathological conditions(Hoozemans et al. 2008). However, the focal accumulationof microglia in several neuropathological disorders like ADmay result in a local increase in COX-1 activity (Yermakovaet al. 1999) and a significant COX-1-dependent PGE2production is also reported in some experimental models ofbrain injury such as global cerebral ischemia, NMDA-induced excitotoxicity, and in a MPTP model of Parkinson’sdisease (Candelario-Jalil et al. 2003; Teismann et al. 2003;Pepicelli et al. 2005).

In our study, although Aβ injection did not have any effecton COX-1 expression, the chronic inhibition of COX-1 bySC560 interestingly resulted in a protection comparable to itproduced by COX-2 selective inhibitor. Regarding thesignificant increase of COX-2 expression in response toAβ, it was anticipated that the enhanced PGE2 concentrationcould be due to COX-2 activity; however, COX-1 inhibitiondiminished the TNF-α and PGE2 levels and protectedneuronal cells from apoptosis. Such results have beenobtained from some other studies evaluating the role ofCOXs in neuroinflammatory response to icv injection of LPS.Although LPS like Aβ in our model does not induce COX-1in hippocampus tissue and just increases COX-2 proteinlevels, COX-1 deletion or preferential COX-1 inhibitionprovides sufficient protection against neuronal injury (Choi etal. 2008). Therefore, it can be predicted that there may be aninterplay between the two isoforms. We analyzed the level ofCOX-1 and COX-2 proteins in Aβ-injected rats that werechronically treated with either COX-1 or COX-2 inhibitorsand observed that COX-1 inhibition completely abrogated theCOX-2 induction in response to Aβ-induced neuroinflamma-tion. These data suggest that COX-1 activity is essential forCOX-2 induction in response to neuroinflammatory stimuli.Supporting this hypothesis, it has been reported that geneticdeletion or pharmacological inhibition of COX-1 decreasesthe expression of ROS generating enzymes includinginducible nitric oxide synthase, NADPH oxidase, andmyeloperoxidase as well as COX-2 in response to icvinjection of LPS (Choi et al. 2008). However, consideringthe fact that selective inhibition of COX-2 in this study

completely prevented the elevation of PGE2, it seems thatCOX-2 activity may be the main source of pathologicalincrease in PGE2 concentration.

It is of interest that the detrimental role of COX-1activity is not limited to microglia and should also beconsidered in neurons. Since it has been shown that COX-1selective inhibitors not only reduce Aβ1–42-induced PGsproduction in postmortem human microglia (Hoozemans etal. 2002a) but also inhibit Aβ induced cell death in murinecortical neurons or human neuroblastoma cells (Bate et al.2003; Ferrera and Arias 2005).

Treatment of Aβ-injected animals with the combinationof NS398 and SC560 (i.e., nonselective COX inhibition)resulted in a protection comparable to those produced byeach treatment alone. Considering the data obtained fromthree groups of treatment in which using SC560 resulted incomplete inhibition of COX-2 induction and PGE2 incre-ment, NS398 resulted in a modest decrease in COX-2induction but complete inhibition of PGE2 increment andSC560 + NS398 resulted in complete inhibition of COX-2induction and PGE2 increment, it can be concluded thatalthough either selective COX-1 or COX-2 inhibitionpotentially may exert similar protective effects, COX-1inhibition may be a more fundamental treatment in Aβ-induced neuroinflammation and nonselective inhibitionmay be more beneficial.

The results from other studies indicating the beneficialeffect of indomethacin and ibuprofen on suppression ofAβ-induced neuroinflammation and toxicity (Netland et al.1998; Richardson et al. 2002; Ferrera and Arias 2005)further confirm this hypothesis.

Taken together, our results suggest that both COX-1 andCOX-2 are involved in Aβ-induced neuroinflammation, butCOX-1 plays a preliminary and prominent role and isnecessary for pathological increment of COX-2 in responseto inflammatory and neurotoxic insults due to Aβ.Therefore, COX-1 selective or COX nonselective inhibitionmay provide more reliable strategy for anti-inflammatorytreatment of AD-like neurodegenerative diseases.

Acknowledgments The authors are grateful to T. Al-Tarihi, M.Motevalian, and F. Khodagholi for their valuable help and toNeuroscience Research Center of Shahid Beheshti University ofMedical Sciences for financial support.


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