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Research Report

Acetaminophen reduces mitochondrial dysfunction duringearly cerebral postischemic reperfusion in rats

Sunanda S. Baliga, Kathryn M. Jaques-Robinson, Norell M. Hadzimichalis,Roseli Golfetti, Gary F. Merrill⁎

Department of Cell Biology and Neuroscience, Rutgers University, 604 Allison Road, Piscataway, New Jersey 08854, USA

A R T I C L E I N F O

⁎ Corresponding author. Fax: +1 732 445 5870.E-mail address: merrill@biology.rutgers.eAbbreviations: 2VO/HT, Two-vessel occlu

Animal Care; ANOVA, Analysis of variancIntravenous; IR, Ischemia–reperfusion; MAPNational Institutes of Health; PaCO2, ArterialTTC, 2,3,5-triphenyltetrazolium chloride

0006-8993/$ – see front matter © 2010 Elsevidoi:10.1016/j.brainres.2010.01.013

A B S T R A C T

Article history:Accepted 6 January 2010Available online 14 January 2010

Acetaminophen, a popular analgesic and antipyretic, has been found to be effective againstneuronal cell death in in vivo and in vitro models of neurological disorders. Acute neuronaldeath has been attributed to loss of mitochondrial permeability transition coupled withmitochondrial dysfunction. The potential impact of acetaminophen on acute injury fromcerebral ischemia–reperfusion has not been studied. We investigated the effects ofacetaminophen on cerebral ischemia–reperfusion-induced injury using a transient globalforebrain ischemia model. Male Sprague–Dawley rats received 15mg/kg of acetaminophenintravenously during ischemia induced by hypovolemic hypotension and bilateral commoncarotid arterial occlusion, which was followed by reperfusion. Acetaminophen reduced tissuedamage, degree of mitochondrial swelling, and loss of mitochondrial membrane potential.Acetaminophen maintained mitochondrial cytochrome c content and reduced activation ofcaspase-9 and incidence of apoptosis. Our data show that acetaminophen reduces apoptosisvia a mitochondrial-mediated mechanism in an in vivo model of cerebral ischemia–reperfusion. These findings suggest a novel role for acetaminophen as a potential stroketherapeutic.

© 2010 Elsevier B.V. All rights reserved.

Keywords:MitochondriaAcetaminophenStrokeApoptosis

1. Introduction

Cerebral ischemia–reperfusion triggers a complex cascade ofbiochemical events including excitotoxicity, ionic imbalance,oxidative and nitrosative stresses and apoptotic-like celldeath mechanisms that lead to total breakdown of cellularintegrity and eventually cell death (Mohr, 2004). Nearly

du (G.F. Merrill).sion/hypotension; AAALAe; FACS, Fluorescence-a, Mean arterial pressure;blood carbon dioxide; PaO

er B.V. All rights reserved

13 years after its approval by the US Food and DrugAdministration, tissue plasminogen activator (tPA) remainsthe only effective medical therapy for acute ischemic stroke.However, only about 2% of patients with acute ischemicstroke benefit from this therapy due to its narrow therapeuticwindow (Kleindorfer et al., 2008). Over the last two decades,several neuroprotective agents have been investigated in

C, Association for Assessment and Accreditation of Laboratoryctivated cell sorting; HBSS, Hank's balanced salt solution; i.v.,Mg, Milligrams; MPT, Mitochondrial permeability transition; NIH,2, Arterial blood oxygen; Min, Minute; SEM, Standard error ofmean;

.

Table 1 – Comparison of hemodynamic and metabolicparameters between SO, CIR, and AIR groups.

Baseline Ischemia Reperfusion

Mean arterial pressure, MAP (mm Hg)SO 115.3±4.3 118.2±5.2 112.1±6.1CIR 110.7±5.1 61.6±2.9* 90.0±3.3AIR 109.3±5.1 61.3±4.3* 95.0±6.1

Heart rate (beats/min)SO 268.2±6.5 270.3±10 262.7±12CIR 266.5±7.6 271.7±17 252.8±10AIR 260.2±8.7 255.0±12 271.5±11

PaCO2 (mm Hg)SO 29.5±1.2 30.4±2.3 28.0±2.3CIR 26.0±1.4 30.3±1.9 26.8±1.5AIR 28.8±1.6 31.7±2.9 27.8±2.6

PaO2 (mm Hg)SO 92.6±5.4 90.2±3.2 89.8±4.6CIR 84.7±4.3 70.7±2.8* 88.3±6.2AIR 91.7±7.6 77.3±3.9* 88.0±5.3

pHSO 7.44±0.003 7.43±0.05 7.44±0.006CIR 7.43±0.007 7.39±0.01 7.38±0.008AIR 7.44±0.009 7.40±0.01 7.40±0.01

Body temperature (°C)SO 37.6±0.2 37.3±1.0 38.0±0.7CIR 37.0±0.3 37.1±0.3 36.7±0.2AIR 37.0±0.5 36.9±1.1 37.0±0.5

Physiological variablesdonot deviate betweengroups. Hemodynamicandmetabolic dataobtained fromSO,CIR andAIR groupsdonot differsignificantly from one another. Blood samples were taken at the endof baseline, ischemia, and reperfusion time points. Ischemia andreperfusion hemodynamic and blood gas values for group SO wereobtained 15min and 60min from the end of the baseline period,respectively. During ischemia, blood pressure was maintained at~55mmHg for both CIR and AIR animals. *P<0.05 as determinedby one-way ANOVA followed by Tukey's multiple comparisontest compared to baseline values from corresponding groups. Dataare±SEM. SO=sham-operated; CIR=control ischemia–reperfusion;AIR=acetaminophen ischemia–reperfusion.

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animal models of cerebral ischemia. While many of theseagents have been found to be neuroprotective in animalmodels, they have failed to be translated from the laboratoryto the emergency room (Cheng et al., 2004). Thus, there is ahuge unmet medical need to develop novel therapies foracute ischemic stroke.

Acetaminophen is a widely used over-the-counter med-ication generally used as an analgesic and antipyretic. It hasbeen shown to have under-appreciated antioxidant and anti-inflammatory properties. Acetaminophen has been demon-strated as an effective cardioprotective agent during myo-cardial ischemia–reperfusion injury (Golfetti et al., 2002;Hadzimichalis et al., 2007; Merrill et al., 2001; Merrill andGoldberg, 2001; Merrill et al., 2004; Zhu et al., 2006). Similarly,the drug has been shown to protect hippocampal neuronsand PC12 cultures from Aβ peptide-induced oxidative stressthrough reduction of lipid peroxidation and by loweringcytoplasmic levels of peroxides (Bisaglia et al., 2002). Thisstudy also showed that acetaminophen blunts neuronalapoptosis via reduction of the inflammatory transcriptionfactor NF-kappaB (Bisaglia et al., 2002). It has been shown toinhibit superoxide anion generation, lipid peroxidation andcell damage induced by quinolinic acid, a neurotoxicmetabolite implicated in the pathogenesis of neurodegener-ative disease, in the rat hippocampus (Maharaj et al., 2006).Acetaminophen has been shown to protect dopaminergicneurons in vitro from oxidative damage evoked by acuteexposure to 6-hydroxydopamine or excessive levels ofdopamine (Locke et al., 2008). Finally, recent studies haveshown that low-dose acetaminophen reduces inflammatoryprotein release from cultured brain endothelial cells exposedto oxidant stress and increases expression of the anti-apoptotic protein Bcl-2 in brain neurons (Tripathy andGrammas, 2009).

The goals of the present study were to explore whetheracetaminophen could have protective effects on mitochondrialdysfunction in an in vivo model of cerebral ischemia–reperfu-sion. To achieve these, we first investigated if acetaminophenhad any effect on tissue damage after ischemia–reperfusion.Acetaminophen indeed reduced the extent of tissue damageafter ischemia–reperfusion. We then investigated the effects ofacetaminophen onmitochondrial dysfunction by analyzing theoccurrenceofmitochondrial swelling, changes inmitochondrialmembrane potential, release of pro-apoptotic factor cyto-chrome c, activation of caspase-9 and incidence of apoptoticcell death after ischemia–reperfusion. Our results show thatacetaminophen reduces apoptotic incidence following cerebralischemia-reperfusion in vivo, likely by reducing mitochondrialdysfunction.

2. Results

2.1. Effects of acetaminophen on physiological parameters

The effects of acetaminophen on heart rate, mean arterialblood pressure, pH, blood oxygen (PaO2), and carbon dioxide(PaCO2) were measured in all groups. All respiratory andcardiovascular parameters remainedwithin baseline values inSO, AIR and CIR groups. In addition, the core body temperature

also did not differ significantly between groups, thus rulingout hypothermia-mediated effects on the results (Table 1).

2.2. Effectiveness of surgical procedure to induce injury

The areas of damage in AIR and CIR rat brains weresignificantly larger than those found in SO rats, thus provingthat our surgical procedurewas effective in producing cerebraldamage. Significantly smaller areas of tissue damage werenoted in group AIR when compared to group CIR (Fig. 1, n=4 ineach group). Group SO exhibited negligible or no damage andwas excluded from the analysis.

2.3. Effect of acetaminophen treatment on mitochondrialswelling

Group CIR brains had a higher occurrence of mitochondrialswelling compared to brains from group AIR. A significantdecrease in light absorbance was evident in group CIR,whereas group AIR exhibited higher light absorbance values(Fig. 2, n=4 in each group).

Fig. 1 – Acetaminophen significantly reduces tissue damagein ischemia-reperfused tissue. A: Representative brain slicesdemonstrating tissue damage in CIR rats compared to AIRbrains. Arrowheads indicate damaged areas. B: Volume oftissue damagewasmeasured and expressed as a percentageof the total volume of the brain slice. §P<0.05 as determinedby Student–reperfusion; AIR=acetaminophen ischemia–reperfusion.

Fig. 2 – Acetaminophen reduces mitochondrial swellingfollowing ischemia–reperfusion. A: Cortical tissue was submit-ted to subcellular fractionation as described in Experimentalprocedures. RepresentativeWestern blots show a lack ofmitochondrial-specific, voltage-dependent anion channel(VDAC) and cytosol-specific β-tubulin in the cytosolic (C) andmitochondrial (M) fractions respectively. B: Spectrophotometricanalysis of mitochondrial swelling comparing changes in lightabsorbance (absorbance at 540 nm, A540) between AIR and CIRrats expressed as a ratio to SO values (A540 control). *P<0.05 asdetermined by Student’s t test compared to CIR values.Error bars indicate SE. CIR=control ischemia–reperfusion;AIR=acetaminophen ischemia–reperfusion.

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2.4. Effect of acetaminophen treatment on mitochondrialmorphology

Quantitative analysis ofmitochondria in electronmicrographsconfirmed significantly smaller mitochondrial to cytosolicarea ratios in group AIR versus group CIR (Fig. 3, n=2 in eachgroup). Additionally, cristae in acetaminophen-treated mito-chondria were well-defined compared to CIR brains.

2.5. Effect of acetaminophen treatment on mitochondrialmembrane potential

Analysis of mitochondrial membrane potential revealed thatgroup AIR exhibited higher ratios of red fluorescence overgreen fluorescence compared to group CIR, representingsignificantly higher mitochondrial membrane potentialvalues (Fig. 4, n=4 in each group).

2.6. Effect of acetaminophen treatment on translocation ofcytochrome c

Cytosolic translocation of cytochrome c from the mitochon-dria was assessed using Western blot in cytosolic and

mitochondrial fractions of SO, CIR and AIR brains. Analysisof blot intensity revealed significantly higher levels ofcytochrome c in the cytosol of CIR and AIR rats compared toSO rats. Interestingly, cytosolic concentration of cytochrome cdid not differ between CIR and AIR rats (Fig. 5, n=4 in allgroups). In mitochondrial samples, a significant decrease incytochrome c content was evident in CIR rats compared to AIRand SO rats. No significant differences in mitochondrialcytochrome c content were observed between SO and AIRrats (Fig. 6, n=4 in all groups).

2.7. Effect of acetaminophen treatment on apoptosis

To determine the effect of acetaminophen on apoptotic celldeath, activated caspase-9 expression and apoptosis-mediat-ed DNA fragmentation were assayed in cortical tissue sectionsobtained from SO, CIR and AIR rats. A significant increase inactivated caspase-9 expression was noted in group CIR,compared to groups SO and AIR (Fig. 7, n=4 in all groups). Asimilar trend was noted with DNA fragmentation, where SOand AIR sections exhibited significantly lower incidence ofapoptosis-positive cells compared to CIR sections (Fig. 8, n=4in all groups).

Fig. 3 – Acetaminophenmaintains mitochondrial morphologyfollowing ischemia–reperfusion. A: Representative electronmicrographs of brain tissue from all groups showedwell-defined cristae in groupsSOandAIR,with loss of cristae ingroup CIR (arrows, image insets). B: Morphometric analysis ofthepercentage coverageof cytoplasmic areabymitochondria incortical tissue between groups AIR and CIR. *P<0.05 asdetermined by ANOVA followed by Tukey’s multiplecomparison test compared toSOvalues. §P<0.05 asdeterminedby ANOVA followed by Tukey’s multiple comparison testcompared to CIR values. Error bars indicate SE. SO=sham-operated; CIR = control ischemia-reperfusion; AIR=acetaminophen ischemia–reperfusion. Scale bars=1 µm.

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3. Discussion

In this study, we investigated the effects of acetaminophen onmitochondrial dysfunction in rats subjected to transientglobal cerebral ischemia–reperfusion. Our results show thatacetaminophen reduces tissue damage and mitochondrialdysfunction as measured by mitochondrial swelling andmorphology, changes in mitochondrial membrane potential,release of pro-apoptotic factor cytochrome c, activation of

caspase-9 and incidence of apoptotic cell death, followingcerebral ischemia–reperfusion.

Stroke is the third leading cause of death behind cardiovas-cular disease and cancer. It is also a leading cause of serious,long-termdisability (Rosamond et al., 2008). Current treatmentsfor prevention of stroke-related morbidity are limited tothrombolytic agents, such as tPA, and surgery. These therapieshave proven clinical utility, but can only be used in aminority ofpatients with acute stroke (Adams et al., 2007; Kleindorfer et al.,2008).Acetaminophen, beinganover-the-counter drug, is oneofthe most popular drugs for the treatment of pain and fever(Prescott, 2000). It hashighoral availability andwithamolecularmass of 152Da, can easily penetrate the blood–brain barrier(Bertolini et al., 2006). It has a wide margin of safety attherapeutic doses and needs no dose adjustment for elderlypatients (Prescott, 2000). Clinical studies testing the antipyreticproperty of acetaminophen in patients with acute ischemicstroke have demonstrated the safety of the drug in thispopulation (Dippel et al., 2001; Dippel et al., 2003).

Mitochondria are highly susceptible to cerebral insultsgiven the high metabolic rate of the brain and dependency onextracellular sources of nutrients (Szeto, 2006). Mitochondrialrespiration generates oxidant by-products that are quicklyquenched by endogenous antioxidants. A dramatic increase inoxygen during reperfusion sends the mitochondria intooverdrive, tipping the oxidant to antioxidant ratio in favor ofthe former. Oxidative stress is an important underlying factorin cell death induced by cerebral ischemia–reperfusion (Chan,2001; Sugawara and Chan, 2003), producing DNA lesions andinterfering with protein function (Slemmer et al., 2008).Changes in gene and protein expression are reported tooccur as early as 15 min from the onset of reperfusion (Cooperet al., 1977; Nowak et al., 1985; Zhang et al., 1993) and tissuedamage occurs within 20 min of reperfusion (Bahcekapiliet al., 2007). Additionally, an increase in Ca+2 permeabilityresults in the loss of mitochondrial membrane potential andeventual rupture of the mitochondrial membranes. Theseevents are generally regarded as the mitochondrial perme-ability transition (MPT) and precede apoptosis and necrosis(Tsujimoto et al., 2006).

We found that acetaminophen treatment preserved mito-chondrial integrity, morphology and function in our ratmodel.To exclude the possibility that this protection is due tohemodynamic effects, we continuously monitored arterialblood pressure in our experiments. Our recordings demon-strate that there was no difference between the AIR and CIRgroups in the vascular variables both before and after thehypotension and carotid occlusion (Table 1).

The mitochondrial morphometric analysis in our studyshows that acetaminophen prevented mitochondrial swellingduring cerebral ischemia–reperfusion, and provides evidencefor the mitochondrial-protective properties of acetaminophenat the cellular level. Swollenmitochondria are associated witha decrease in light absorbance (Tedeschi and Harris, 1955). Asignificant decrease in light absorbance by CIR brains suggeststhat reperfusion was severely detrimental to mitochondrialintegrity. Mitochondrial swelling plays a crucial role in cellinjury as it is affected by several processes critical to theischemia–reperfusion cascade including mitochondrial elec-tron transport, production of reactive oxygen species,

Fig. 4 – Acetaminophen preserves mitochondrial membrane potential following ischemia-reperfusion. A: Representative dotplot scatter analysis of cells obtained from SO, CIR and AIR brains reveals higher values for mitochondrial membrane potentialin SO and AIR brains, with a near complete abolishment of mitochondrial membrane potential in CIR brains. B: AIR brainsexhibited higher ratios of red fluorescence (N2) over green fluorescence (N4) compared to CIR brains. *P<0.05, as determined byANOVA followed by Tukey’s multiple comparison test, compared to CIR values. Error bars indicate SE. SO=sham-operated;CIR=control ischemia–reperfusion; AIR=acetaminophen ischemia–reperfusion.

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cytochrome c release, mechanical signaling pathways, andopening of MPT. Mitochondrial rupture occurs in the lastphase of mitochondrial swelling (Kaasik et al., 2007). Thusmorphometric and spectrophotometric analyses of mitochon-drial size between CIR and AIR animals allow a quantitativemeasure of the degree of cell injury sustained duringischemia–reperfusion.

In this study, loss of mitochondrial membrane potentialwas determined using the dye JC-1. JC-1 is a cationic dye thatexhibits potential-dependent accumulation and formation ofred fluorescent J-aggregates in mitochondria with highmembrane potential. In contrast, JC-1 exists as a monomerand produces green fluorescence in the cytoplasm andmitochondria with low membrane potential. Formation of J-aggregates in the mitochondria is indicated by a fluorescenceemission shift from green (535 nm) to red (590 nm). Mitochon-drial depolarization is indicated by a decrease in the red/greenfluorescence intensity ratio. We found that acetaminophensignificantly inhibited decreases in mitochondrial membranepotential.

There is combined necrotic and apoptotic cell death aftercerebral ischemia–reperfusion (Friedlander, 2003). Necroticcell death is more prevalent in the core of the lesion, where

hypoxia is most severe, and apoptosis occurs in thepenumbra, where collateral blood flow reduces the degreeof hypoxia (Friedlander, 2003). Morphological and biochem-ical evidence of apoptosis has been well documented inexperimental animal models of ischemic brain injury (Suga-wara et al., 2004). The most convincing morphologicalevidence of postischemic apoptosis was detected at earlyhours after the onset of an ischemic insult in the penumbraand during reperfusion (Charriaut-Marlangue et al., 1996;Fujimura et al., 1998; Li et al., 1995, 1997, 1998). Inhibition ofapoptosis provides an important therapeutic strategy forstroke (Nakka et al., 2008; Rami et al., 2008; Taoufik andProbert, 2008; Yuan, 2009).

In this study, the presence of cytochrome c in thecytoplasm and the mitochondria was assayed with Westernblot. Rats in group SO exhibited high cytochrome c content inthe mitochondria with traces of cytochrome c in the cyto-plasm. Group CIR exhibited a marked increase in cytosoliccytochrome c content accompanied by a subsequent decreaseof cytochrome c in the mitochondria. Interestingly, group AIRalso hadmarked increases in cytosolic cytochrome c; however,mitochondrial cytochrome c levels were significantly higherthan in CIR rats. While apoptosismight constitute the primary

Fig. 5 – Cytosolic cytochrome c content followingischemia–reperfusion. A: Representative Western blotsperformed on SO, CIR and AIR cytosolic fractions show a lack ofcytochrome c content in SO rats compared toCIR andAIR rats. B:Quantitative densitometric analysis of cytochrome c bandintensity revealedasignificant increase incytochrome c contentin CIR and AIR samples compared to SO. No significantdifferences in cytochrome c content were observed betweengroupsCIR andAIR. *P<0.05 as determined byANOVA followedby Tukey’s multiple comparison test compared tosham-operated controls. Error bars indicate SE. SO=sham-operated; CIR=control ischemia-reperfusion; AIR=acetaminophen ischemia–reperfusion.

Fig. 6 – Mitochondrial cytochrome c content followingischemia–reperfusion.A:RepresentativeWesternblotsobtainedfrom SO, CIR and AIR mitochondrial fractions show amarkeddecrease incytochrome ccontent inCIRsamplescompared toSOand AIR. B: Quantitative densitometric analysis ofcytochrome c band intensity revealed a significant decrease incytochrome c content inCIRmitochondriawhencompared toSOand AIRmitochondrial samples. No significant differences incytochrome c content were observed between SO and AIRmitochondrial samples. *P<0.05 as determined by ANOVAfollowed by Tukey’s multiple comparison test compared to SOvalues. §P<0.05 as determined by ANOVA followed by Tukey’smultiple comparison test compared to CIR values. Error barsindicate SE. SO=sham-operated; CIR = controlischemia–reperfusion; AIR=acetaminophenischemia–reperfusion; VDAC=voltage-dependent anionchannel.

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mode of cell death during early cerebral postischemicreperfusion, necrotic cell death mechanisms can also targetthe mitochondria during this time (Lobysheva et al., 2009).This could explain the discrepancy observed between CIR andAIR cytosolic cytochrome c expression since our study focusedsolely on the effects of acetaminophen on the mitochondrial-mediated apoptotic cell death cascade. We further investigat-ed the effect of acetaminophen on the apoptotic cascade byexamining the incidence of apoptosis-mediated DNA frag-mentation as well as the activation of caspase-9, an initiatorcaspase that propels the cell into apoptosis. When comparedto CIR rats, AIR tissue exhibited significantly lower incidencesof apoptotic DNA fragmentation and expression of activatedcaspase-9. These data suggest that acetaminophen has anti-apoptotic actions in the setting of cerebral ischemia–reperfusion.

While any of the multiple pathways involved in apoptoticcell death can be affected by acetaminophen, our data onmitochondrial dysfunction implicate the ‘intrinsic’ mitochon-drial pathway (Fig. 9). The intrinsic pathway involves activa-tion of pro-apoptotic members of the Bcl-2 family triggeringthe release of killer proteins from the mitochondrial inter-membrane space by MPT. These killer proteins lead to the

release of cytochrome c and activation of the initiator caspase-9 (Krajewska et al., 2004; Susin et al., 1999), and of caspase-3, a‘terminator’ caspase in the execution step of apoptosis (Joveret al., 2002). Anti-apoptotic Bcl-2 family members can atten-uate the release of killer proteins, influence mitochondrialintegrity and prevent apoptosis (Zhang et al., 2008). Cellsoverexpressing anti-apoptotic Bcl-2 exhibit a marked reduc-tion in apoptosis, which suggests that anti-apoptotic Bcl-2could independently affect the rate of apoptosis (Kang andReynolds, 2009). Similar studies employing neuronal cellsmodeling protein aggregation typical of Alzheimer's diseasehave also reported a significant reduction in apoptotic celldeath when these cells over expressed anti-apoptotic Bcl-2(Rohn et al., 2008). Recent in vitro data show that acetamino-phen increases expression of anti-apoptotic protein Bcl-2 inbrain endothelial cells and neuronal cells experiencingoxidative stress as a byproduct of inflammation (Tripathyand Grammas, 2009). Based on this data, we believe thatacetaminophen protects against apoptosis by increasing theexpression of anti-apoptotic Bcl-2 proteins (Fig. 9). This likelyprevents mitochondrial pore dysfunction, resulting in the

Fig. 7 – Acetaminophen attenuates activated caspase-9 expression following ischemia–reperfusion. A: Representative tissuesections obtained from SO, CIR and AIR rat brains revealing a marked increase in activated caspase-9 expression in CIR ratscompared to SO and AIR rats. Arrowheads indicate activated caspase-9 expression in cryosections of cortical tissue. B: Imageanalysis revealed a significant increase in caspase-9 expression in CIR tissue when compared to SO and AIR tissue.Acetaminophen treatment significantly reduced expression of activated caspase-9 compared to CIR tissue. *P<0.05 asdetermined by ANOVA followed by Tukey's multiple comparison test compared to SO values. §P<0.05 as determined byANOVA followed by Tukey's multiple comparison test compared to CIR values. Error bars indicate SE. SO=sham-operated;CIR=control ischemia–reperfusion; AIR=acetaminophen ischemia–reperfusion.

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stabilization of pro-apoptotic proteins, consequently mini-mizing their deleterious effects onMPT.We have not ruled outthe possibility that acetaminophen could prevent the inci-dence of the death receptor-mediated pathway of apoptosisand/or necrotic cell death mechanisms as the two modes ofcell death share characteristic features such as swollenmitochondria and rupture of plasma membranes (Galluzzi etal., 2009). Elucidating the exact mechanism of anti-apoptoticeffects of acetaminophen in vivo is the focus of ongoinginvestigation in our laboratory.

3.1. Conclusion

Here, for the first time, we show that acetaminophen reducescerebral tissue damage, mitochondrial dysfunction and theincidence of apoptosis during early postischemic reperfusion.Mitochondrial dysfunction is one of the pivotal events leadingto loss of neuronal function in neuropathological conditions.The prevention of mitochondrial rupture by some compoundshas resulted in improved brain function (Chu et al., 2007;Simpkins and Dykens, 2008; Yousuf et al., 2009), thusacetaminophen's ability to maintain mitochondrial function

soon after a cerebral insult shows promise for the drug as apotential stroke therapeutic.

4. Experimental procedures

4.1. Ethics statement

All animal housing conditions, surgical protocols and postop-erative care were reviewed and approved by the RutgersUniversity Institutional Animal Care and Use Committee andwere carried out in accordance with the National Institute ofHealth Guide for the Care and Use of Laboratory Animals (NIHPublications No. 80-23; revised 1996). Adequate measures weretaken tominimizepainordiscomfort to theanimals throughoutthe experimental procedure.

4.2. Materials

All chemicals were obtained from Sigma Aldrich, St. Louis,MO.Mitochondrialmembrane potential was determined usingMitoProbe JC-1 Assay Kit (Molecular Probes, Carlsbad, CA).

Fig. 8 – Acetaminophen reduces the incidence of apoptosis following ischemia–reperfusion. A: Representative tissue sectionsobtained from SO, CIR and AIR rat brains showed a higher incidence of apoptosis-mediated DNA fragmentation in CIR sampleswhen compared to SO andAIR samples. Arrowheads indicate apoptosis-positive cells in cryosections of cortical tissue. B: Imageanalysis showed CIR sections exhibiting significantly greater incidences of apoptosis-positive cells, with a concomitantdecrease in AIR sections. *P<0.05 as determined by ANOVA followed by Tukey's multiple comparison test compared to SOvalues. §P<0.05 as determined by ANOVA followed by Tukey's multiple comparison test compared to CIR values. Error barsindicate SE. SO=sham-operated; CIR=control ischemia–reperfusion; AIR=acetaminophen ischemia–reperfusion.

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4.3. Animal care and specifications

The experiments were performed on 78male Sprague–Dawleyrats weighing 350 to 400 g (Ace Animals, Boyertown, PA)housed two per cage in temperature and light-regulated (12 hlight:dark) AAALAC-accredited facilities. The animalswere fedstandard laboratory rat chow and water ad libitum.

4.4. Induction of cerebral ischemia–reperfusion

Global forebrain ischemia was induced using the bilateralcommon carotid artery (two-vessel) occlusion and hypovo-lemic hypotension (2VO/HT) model of Smith et al. (1984). Therats were anesthetized using ketamine/xylazine in an80:12 mg/kg i.p. ratio, with additional doses of ketamine at80 mg/kg, i.p. administered as necessary. Core body temper-ature wasmaintained thermostatically at 37 °C±0.5 °C with atemperature-controlled heating pad. Lidocaine was admin-istered to both inguinal areas and the ventral surface of theneck prior to surgery. The common carotid arteries wereisolated andmarked with 3-0 silk suture. The left jugular veinand both femoral arteries were cannulated for administeringdrugs and measuring hemodynamics, respectively. Heparin(250U/kg i.v.) was administered to prevent blood clotting.

Blood was withdrawn, via right femoral arterial access, into aheparinized syringe to a systemic mean arterial pressure(MAP) of ~55 mmHg and the carotid arteries were occludedusing bulldog clamps. To avoid changes in core body temper-ature during reperfusion, withdrawn blood was placed inheparinizedwater-jacketed vials andmaintained at 37 °C in awater bath for the duration of ischemia. At 15min, cerebralreperfusion was initiated by release of clamps from thecommon carotid arteries and reinfusion of the previouslywithdrawn heparinized blood to achieve normotension(MAP 100 mmHg). Anesthesia and temperature were main-tained for the duration of the experiment. At the end of thereperfusion period, animals were sacrificed by transcardialperfusion with ice-cold 0.1 M PBS until the blood wascompletely flushed out. At this time, animals were decapi-tated and brains extracted for analysis.

4.5. Measurement of physiological variables

Systemic mean arterial pressure was monitored using aphysiologic transducer (model BP-100; iWorx/CB Sciences,Dover, NH) in the left femoral artery. Arterial blood gases(PaO2, PaCO2, mmHg) were measured using a blood gasanalyzer (Radiometer America, Westlake, OH) at the following

Fig. 9 – Proposed mechanism of action of acetaminophen. Pro-apoptotic Bcl-2 proteins promote the formation of pores thatfacilitate the release of cytochrome c, one of the key events in the initiation of mitochondrial-mediated apoptosis.Acetaminophen upregulates the expression of the anti-apoptotic Bcl-2 protein that prevents this pore formation, thus limitingcytochrome c release and apoptosis. Dashed lines indicated alternativemodes of apoptotic cell death. See text for further detail.MPTP, mitochondrial permeability transition pore; FADD, Fas-activated death domain.

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time points: after 15min of steady-state conditions (baseline),15min of ischemia, and 45min of reperfusion (Fig. 1). Ratswere ventilated mechanically using a small animal respirator(model M683, Harvard Apparatus, Holliston, MA) to maintainblood pH at physiologic levels. Core body temperature wasmonitored with a rectal probe (model BAT-12, Physitemp,Clifton, NJ) sampled every 15min throughout the experiment.A data acquisition system (model 214; iWorx/CB Sciences,Dover, NH) in series with a personal computer runningLabscribe software (version 6.0, CB Sciences; Dover, NH) wasused to record monitored variables.

4.6. Experimental groups

Rats were randomly divided into three groups and treatedfollows:

1. Sham-operated (Group SO): Rats were instrumented andmaintained at steady-state conditions for the duration ofthe experiment.

2. Acetaminophen, ischemia–reperfusion (Group AIR): Acet-aminophen was administered at 15 mg/kg dissolved invehicle (0.9% NaCl) as an intravenous bolus of 0.2–0.3 mlfollowing induction of hypotension andprior to carotid arteryocclusion. Ratswere subjected to 15min of ischemia followedby 45min of reperfusion. We chose a duration of 45min forreperfusion based on our previously published reports wherea reperfusion period three times the duration of ischemiaproduced the greatest damage (Merrill et al., 2004).

3. Control, ischemia–reperfusion (Group CIR): Only the vehi-cle (0.9% NaCl) was administered as an intravenous bolusof 0.2–0.3 ml following induction of hypotension and prior

to carotid artery occlusion. Rats were subjected to 15min ofischemia followed by 45min of reperfusion.

4.7. Determination of tissue damage

Tissue damagewas assessed as previously described (Yang et al.,1998; Zhang, 2004) in all groups. In brief, extracted rat brainsweresliced into 2mm thick slices and incubated in a 0.1% solution ofTTC for 30min at 37 °C. The brain slices were transferred to 10%formalin after incubation. Acquisition of images was carried outwithin 24 hours of sacrifice by scanning stained brain sectionswithadigital color flatbedscanner (MC7420, Brother, Bridgewater,NJ). Colorless regions of the brain slices were defined as areas ofdamage,whereas regions that stained bright redwere consideredundamaged. Areas of tissue damage were manually determinedby outlining the margins of the white regions using imageanalysis software (ImageJ version 1.40, public domain softwaredeveloped at the NIH and available at http://rsb.info.nih.gov/ij/).In each coronal section, the area of damage (in square milli-meters) and the total area of the slice (in squaremillimeters)weretraced. Both areasweremultiplied by the slice thickness of 2mmto obtain the damaged tissue volume and total cerebral volumefor each slice. The volumes from all slices were added to obtainthe total volume of damaged area and total cerebral volume forthe brain. The percentage of the total volume of damage in thetotal supratentorial brain volume was then calculated to correctindividual difference in brain volumes (Fig. 2).

4.8. Mitochondrial morphometry

Mitochondrial morphometry was performed separately aspreviously described (Hirai et al., 2001) to compare structural

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changes inmitochondrial morphology between all groups. Ratswere transcardially perfused with 0.1 M PBS followed byTrump's Fixative (pH ~7.2) and the brains were extracted.Cerebral tissuewas sectioned into 1 mm3blocks and submergedin Trump's fixative. Blocks were postfixed with 1% osmiumtetroxide and subsequently dehydrated in graded ethanol.Samples were embedded in Epon-Araldite cocktail, sectionedwith a diamond knife ultramicrotome (model LKB-2088, LKB,Bromma, Sweden), and viewed with an electron microscope(model JEM-100CXII, JEOL USA, Peabody, MA) using standardprotocols (Bozzola et al., 1999).Mitochondriawere identifiedanddigital photomicrographs were acquired at a magnification of1 μm. Each mitochondrion was outlined and the total mito-chondrial and cytosolic areas were measured using imageanalysis software (Scion Image, public domain software devel-oped at the NIH and available at http://www.scioncorp.com/).Cytosolic area excluded the nucleus. The total mitochondrialareaswere expressed as a percentage of the total cytosolic areasand compared between all groups (Fig. 3).

4.9. Subcellular fractionation

Rats from groups SO, AIR and CIR were transcardially perfusedwith ice-cold 0.1 M PBS at the termination of reperfusion.Brains were extracted and the cerebrum excised, mincedfinely and placed in homogenization buffer (10 ml/g) contain-ing (in mM): 210.0 mannitol, 7.0 sucrose, and 5.0 4-morpholi-nopropanesulfonic acid, pH 7.4, 25 °C, with 1 mM PMSF andprotease inhibitor cocktail. Tissue was homogenized with 10–15 strokes using a Dounce Homogenizer (Fisher Scientific,Waltham, MA). Separation of mitochondrial and cytosolicfractions was obtained as previously described (Hadzimichaliset al., 2007). Briefly, the homogenatewas centrifuged at 1000×gfor 10 min at 4 °C and the resulting supernatant was centri-fuged at 7000×g for 10 min at 4 °C. The pellet from the secondcentrifugation represented the mitochondrial fraction andwas resuspended in 10 mM sodium phosphate buffer, pH 9.0.The supernatant represented the cytosolic fraction. Cytosolicand mitochondrial fractions were stored at −20 °C until use.

4.10. Measurement of cytosolic and mitochondrialcytochrome c content

Protein concentration in each sample was determined usingthe Bradford method. Cytosolic and mitochondrial proteins(10 µg) were resolved on a 12% SDS-polyacrylamide gel at 100 Vfor 2 h. Proteins were then transferred to an Immobilon-PPVDF membrane (Millipore, Billerica, MA) at 300 mA for 1.5 h.Membranes were blocked using 2% BSA and then incubatedwith primary rabbit polyclonal antibody against cytochrome c(1:1000, Santa Cruz Biotech, Santa Cruz, CA) overnight at 4 °C.Membranes were washed with TBS-T buffer and then incu-bated with secondary goat anti-rabbit HRP-conjugated anti-bodies against cytochrome c (1:6000, Bio-Rad, Hercules, CA) in2% nonfat milk for 45 min. Blots were developed with ECL+reagent (Amersham Biosciences, Piscataway, NJ) and exposed.Film was analyzed using NIH-developed software (ImageJversion 1.40, public domain software developed at the NIH andavailable at http://rsb.info.nih.gov/ij/). Membranes werestripped using mild stripping buffer (0.2 mM glycine,

0.003 mM SDS, 0.01% Tween-20) and blocked with 2% BSA for2 h. Then, the membranes were probed against β-tubulinusing primary rabbit polyclonal antibody (1:800, Santa CruzBiotech, Santa Cruz, CA) and voltage-dependent anion chan-nel using primary goat polyclonal antibody (VDAC, 1:800,Santa Cruz Biotech, Santa Cruz, CA) with secondary goat anti-rabbit (1:3000, Bio-Rad, Hercules, CA) and donkey anti-goat(1:3000, Santa Cruz Biotech, Santa Cruz, CA) HRP-conjugatedpolyclonal antibodies respectively. Blots were obtained asabove. The level of cytochrome c in mitochondrial andcytosolic samples in each treatment group was expressed asa ratio to β-tubulin (cytosolic samples) or VDAC (mitochon-drial samples). Experiments were repeated two to three times.

4.11. Measurement of mitochondrial swelling

Total protein concentration was measured in mitochondrialsuspensions from all groups using the Bradford assay.Mitochondrial suspensions were subject to spectrophotomet-ric analysis (540 nm) at 25 °C with a spectrometer (SpectraMaxplus384; Molecular Devices, Toronto, Canada) as previouslydescribed (Schinzel et al., 2005). Light absorbance values wereexpressed as a ratio to SO values.

4.12. Measurement of alterations in mitochondrialmembrane potential

Changes in mitochondrial membrane potential were mea-sured with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimida-zolylcarbocyanine iodide (JC-1) using Flow Activated CellSorting (FACS) in groups SO, AIR and CIR. Mitochondrial size,shape, and density do not influencemitochondrial membranepotential, making JC-1 a reliable means of comparing mito-chondrial membrane potential between samples (Reers et al.,1995). Cell suspensions for FACS analysis were generated aspreviously described (Campanella et al., 2002). Briefly, cerebraltissue was homogenized in HBSS using a Dounce Homogeniz-er (Fisher Scientific, Waltham, MA), passed through a 40 μmnylon mesh strainer, and centrifuged at 1000×g for 10 min.The resulting pellet was subjected to a Percoll density gradient(ρ=1.095 and ρ=1.03), overlayered with HBSS and centrifugedat 1000×g for 20min. Cells were collected from the interface ofthe two layers, washed with 10% FBS and centrifuged at 400×gfor 10 min. Cells were then treated with JC-1 according to themanufacturer's instructions and run through the flow cyt-ometer (Beckman-Coulter, Fullerton, CA). Excitation wave-length was set at 488 nm. Quantification of changes inmitochondrial membrane potential was performed as previ-ously described (Cossarizza et al., 1993; Mathur et al., 2000;Reers et al., 1991) and was obtained by calculating the ratio ofcells fluorescing red to cells fluorescing green (Fig. 4).

4.13. Evaluation of activated caspase-9 expression in braintissue

At the end of reperfusion, animals were transcardiallyperfused with ice-cold 0.1 M PBS followed by perfusion with4% paraformaldehyde in 0.1 M PBS (PFA). The brains wereextracted and the cerebrums were quickly excised from therest of the brain. Brain tissue was placed in 4% PFA overnight

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at 4 °C followed by 8 h incubations in 10, 20 and 30% sucroserespectively at 4 °C. Tissue was prepared for cryosectioning byincubation in 30% sucrose/50% OTC (v/v) for 1 h followed byincubation in 100% OTC at 4 °C for 1 h. The tissue was thenembedded in OTC over dry ice and 10 µm-thick sections wereobtained using a cryostat (model L-CM1900, Leica, St. Gallen,Switzerland). Slides were stored at −80 °C until use.

For immunohistochemical analysis, sections were rehy-drated and endogenous hydrogen peroxidases were quenchedusing quenching solution (0.3%H2O2 in PBS). Sectionswere thenblocked in 1% BSA followed by incubation with primarypolyclonal goat antibody against activated caspase-9 (1:50,Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 °C ina humidified chamber. Slideswere then incubated in secondarydonkey anti-goat antibody (1:50, Bio-Rad, Hercules, CA) anddeveloped using DAB solution with hematoxylin as a counterstain prior to mounting. Digital images of four randomlyselected fields from the cerebral cortex of each slide wereacquired using a microscope (Nikon Diaphot 3000). Histogramsof the fourdifferent fieldswereacquiredusingAdobePhotoshopSoftware (Adobe, San Jose, CA) and the mean values werecompared between groups SO, CIR and AIR. Values wereexpressed as a percentage compared to group SO. Alternateslides were stained with H&E for morphological analysis.

4.14. In situ detection of apoptotic DNA fragmentation

Apoptotic cell death was assayed in brain tissue sections aspreviously described (Wang et al., 2007) using the TACS·XL InSitu Apoptosis Detection kit (Trevigen Inc, Gaithersburg, MD).Briefly, slides were fixed by incubation in 3.7% bufferedformaldehyde and permeabilized by incubation in Cytonin™.Endogenous peroxidase activity was removed by immersingsections in quenching Solution (1:9 dilution 30% H2O2: metha-nol). BrdU was targeted using a highly specific antibody. Colordevelopment was achieved using DAB. Sections were thencounterstained using Methyl Green, mounted and digitalimages of four randomly selected fields from the cerebral cortexof each slide were acquired using a microscope (Nikon Diaphot3000). The number of apoptosis-positive cells was measuredusing the Adobe Photoshop Software (Adobe, San Jose, CA).Histograms of the four different fields were acquired and themean values were compared between groups SO, CIR and AIR.Values were expressed as a percentage compared to group SO.

4.15. Statistical analysis

Data are presented as means±SEM. Statistical analysisbetween two groups was assessed using two-tailed, unpairedStudent's t tests. Differences between three groups wereanalyzed using one-way analysis of variance (ANOVA) fol-lowed by Tukey's post-test for means comparison (GraphPadSoftware, La Jolla, CA). Significance was accepted at P<0.05.

Acknowledgments

We gratefully acknowledge the donation of animals andmaterials by Dr. Bonnie L. Firestein and Dr. David T. Denhardt.We thank Carole Lewandowski for her enthusiastic support of

this work. We would also like to acknowledge the ElectronMicroscopy Facility and EOHSI Facility at Rutgers Universityfor assisting in the acquisition and interpretation of electronmicrographs and flow cytometry data respectively.

Funding: This work was funded in part by Johnson &Johnson Corporate Office of Science and Technology/McNeilConsumer Specialty Products (to Gary F. Merrill).

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