Chronic paracetamol treatment increases alterations in cerebral vessels in cortical spreading depression model

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Microvascular Research xxx (2014) xxx–xxx

YMVRE-03425; No. of pages: 11; 4C:

Contents lists available at ScienceDirect

Microvascular Research

j ourna l homepage: www.e lsev ie r .com/ locate /ymvre

Chronic paracetamol treatment increases alterations in cerebral vesselsin cortical spreading depression model

OFWaranurin Yisarakun a, Weera Supornsilpchai b, Chattraporn Chantong a,

Anan Srikiatkhachorn c, Supang Maneesri-le Grand a,⁎a Department of Pathology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand 10330b Department of Physiology, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand 10330c Department of Physiology, Faculty of Medicine, Chulalongkorn Univ, Bangkok, Thailand 10330
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⁎ Corresponding author at: ElectronMicroscope Unit, Dof Medicine, Chulalongkorn University, Bangkok, Thailand

E-mail address: [email protected] (S. Ma

http://dx.doi.org/10.1016/j.mvr.2014.04.0120026-2862/© 2014 Published by Elsevier Inc.

Please cite this article as: Yisarakun, W., etdepression model, Microvasc. Res. (2014), h

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Article history:Accepted 30 April 2014Available online xxxx

Keywords:ParacetamolCortical spreading depressionCerebral microvesselUltrastructureCell adhesion moleculeMigraineBlood brain barrierVascular cell adhesion molecule-1Intercellular adhesion molecule-1Long-term treatment

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Recently, a number of non-beneficial effects of chronic treatment with paracetamol (APAP) have been reportedin several systems, including circulatory system. In this study, the effects of acute (1 hour) and chronic (30 days)APAP treatments on cerebral microvessels in a cortical spreading depression (CSD) migraine animalmodel wereinvestigated. Rats were divided into control, CSD only, and APAP treatment with or without CSD groups. A singledose (200 mg/kg body weight) or once-daily APAP treatment over 30 days was intraperitoneally injected intothe acute and chronic APAP treated groups, respectively. CSD was induced by topical application of potassiumchloride on the parietal cortex. Ultrastructural alterations and the expressions of cell adhesion molecules(ICAM-1 and VCAM-1) of the cerebralmicrovesselsweremonitored in all experimental groups. The results dem-onstrated that the induction of CSD caused ultrastructural alterations of the cerebral endothelial cells, as indicat-ed by increases inmicrovillous and pinocytic formations and swelling of the astrocytic foot plates. The expressionof ICAM-1 was significantly elevated in the CSD groups as compared with the control groups. Pretreatment withAPAP 1 hour prior to CSD activation attenuated the alterations induced byCSD. However, chronic APAP treatmentresulted in an enhancement of the ultrastructural alterations and the expressions of cell adhesion molecules inthe cerebral microvessels that were induced by CSD. Interestingly, the rats that received chronic APAP treatmentalone exhibited higher degrees of ultrastructural alterations and ICAM-1 expression than those in the controlgroup. Based on these results, we suggest that short-term treatment with APAP has no effect on cerebralmicrovessels and that chronic APAP treatment can alter cerebral microvasculature, especially when combinedwith CSD activation.

© 2014 Published by Elsevier Inc.

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Introduction

Paracetamol (acetaminophen, APAP) is one of the most populardrugs for the treatment of fever, pain, and headaches, including mi-graine. Given the low price of APAP, its minimal side effects and easyavailability without a prescription, the possibility exists that this drugis used for chronic treatment. Although the mechanism of action ofthis drug is still controversial, it is well accepted that APAP acts primar-ily in the central nervous system (CNS) (Brommet al., 1992;Woodbury,1965).

APAP has been recognized as a relatively safe drug; hepatotoxicity isone of the only reported toxic effects of a high-dose APAP. However, inthe last decade, several studies have gradually revealed the adverse ef-fects of this drug on several systems, including the respiratory system,the CNS, and the circulatory system (Curhan et al., 2002; Dedier et al.,

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epartment of Pathology, Faculty10330. Fax: +66 2 6524208.neesri-le Grand).

al., Chronic paracetamol treattp://dx.doi.org/10.1016/j.mv

2002; Forman et al., 2007; Gonzalez-Barcala et al., 2013; Sudano et al.,2010; Supornsilpchai et al., 2010a, 2010b). Several studies have demon-strated that long-term treatment with APAP, even at therapeutic doses,can induce the alteration in the circulatory system. The associations be-tween long-term treatment with this drug and increases in blood pres-sure and the risk of hypertension have been reported (Curhan et al.,2002; Dedier et al., 2002; Forman et al., 2007; Sudano et al., 2010).

It is well accepted that the mechanism underlying the toxic effect ofAPAP is mediated via its toxic metabolite, n-acetyl-p-benzoquinoneimine (NAPQI) (Dahlin et al., 1984; Huq, 2007). At low levels, nearlyall APAP is conjugated with glucuronic acid and sulfate in the liver be-fore being excreted in the urine, but a small fraction is metabolized bythe enzyme cytochrome P450 2E1 (CYP2E1), which results in the for-mation of NAPQI (Hansson et al., 1990; Haorah et al., 2005; Posadaset al., 2010). After its formation, NAPQI is always rapidly detoxified viaan interaction with glutathione (GSH) (Manyike et al., 2000; Posadaset al., 2010). NAPQI itself can directly damage cells by binding with cel-lular proteins (Kon et al., 2004). Thus, when NAPQI is present at highlevels, it can also cause the depletion of GSH, which leads to increases

tment increases alterations in cerebral vessels in cortical spreadingr.2014.04.012

http://dx.doi.org/10.1016/j.mvr.2014.04.012

mailto:[email protected]

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http://www.sciencedirect.com/science/journal/00262862


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in oxidative stress and ultimately induces more cellular damage(Jaeschke et al., 2003; James et al., 2003). Together, the availableevidence has shown that several cell types in the brain, including endo-thelial cells, astrocytes, pericytes, microglia, and neurons, express theenzyme CYP2E1 (Hansson et al., 1990; Haorah et al., 2005; Posadaset al., 2010). APAP can directly cross the blood brain barrier (BBB)(Kumpulainen et al., 2007) thus, after APAP reaches the cerebral circu-lation, it can be directly metabolized by this enzyme, which results inthe formation of NAPQI in the brain.

Among the disorders for which APAP is commonly used as a treat-ment drug, migraine (a neurovascular disorder) is of particular interestto our research groupdue to its pathogenesis,which is tightly associatedwith the activation of the trigeminovascular system (Edvinsson et al.,2012). The activation of this system can alter either the trigeminal neu-rons or cerebral vessels (Edvinsson et al., 2012; Just et al., 2006). Studiesin the migraine animal model have demonstrated that the integrity ofthe BBB is altered after cortical spreading depression (CSD) activation(Gursoy-Ozdemir et al., 2004; Moskowitz, 2007). Additionally, a studyof migraine patients has demonstrated that, 2 hours after migraine at-tack, soluble ICAM-1 levels have increased in the blood of the internaljugular vein (Sarchielli et al., 2006). Abnormalities in the cerebralvessels in migraine have been confirmed by findings that have demon-strated the association between migraine, particularly migraine withaura, and abnormalities in the cerebrovascular system (Tietjen andKhubchandani, 2009; Tietjen et al., 2012). Given the instability of the ce-rebral vessels in migraine patients, the addition of chronic APAP treat-ment might enhance the abnormalities in the cerebral microvessels ofthese patients.

In order to prove this hypothesis, this study aimed to investigatethe effects of chronic APAP treatment on the alteration of cerebralmicrovessels in the CSD migraine animal model. The expressions ofcell adhesion molecules (ICAM-1 and VCAM-1) were studied with im-munohistochemistry and western blot analysis, and ultrastructuralchanges in the cerebral microvessels (pinocytic vesicles and microvilli),including swelling of the astrocytic foot plate, were studied with elec-tron microscopy.

Materials and methods

Animals and drug treatment

Adult male Wistar rats weighing 200–250 grams were obtainedfrom the National Laboratory Animal Center, Mahidol University,Thailand. The rats were housed five per cage in a temperature- andhumidity-controlled room with a 12-hour dark/light cycle. Food anddrink were available ad libitum. The protocols of this study wereapproved by the Ethical Committee of the Faculty of Medicine,Chulalongkorn University, Thailand.

To investigate the effects of chronic APAP treatment on the alter-ation of cerebral microvessels, this study was divided into two experi-ments. The first experiment aimed to investigate the effect of acuteAPAP treatment on the alteration of cerebral microvessels in the CSDmigraine animal model. The rats were divided into the following fourgroups (n=10 in each group): control, CSD only, APAP-treatedwithoutCSD, and APAP-treated with CSD. A single dose of APAP (200 mg/kgbody weight (bw)) was intraperitoneally injected into the APAP-treated groups, and the same volume of 0.9% sterile salinewas adminis-tered to the non-APAP-treated groups. CSD was induced in the groupwith CSD activation at 1 hour after APAP or saline injection.

For the second experiment, which aimed to investigate the effect ofchronic APAP treatment on the alteration of cerebralmicrovessels in theCSD migraine animal model, the rats were again divided into fourgroups (n = 10 for each group): control, CSD only, APAP-treated with-out CSD, and APAP-treated with CSD. Rats in the APAP treated groupsreceived daily injections of APAP for 30 days, and 0.9% sterile salinewas injected daily into the non-APAP-treated groups. At 24 hours

Please cite this article as: Yisarakun, W., et al., Chronic paracetamol treadepression model, Microvasc. Res. (2014), http://dx.doi.org/10.1016/j.mv

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after the last injection of APAP or saline, the animals underwent surgery,and CSD was induced in the groups with CSD activation.

Surgical procedure and CSD induction

After the last injection of APAP or saline, all rats were anesthetized byan intraperitoneal injection of sodiumpentobarbital (60 mg/kg bw). Therats were firmly fixed to the head holder of a stereotaxic frame. The skinand soft tissue were removed before a craniotomy (2 mm in diameter)was made in the right parietal bone with a saline-cooled drill at 7 mmposterior and 1mm lateral to bregma. The duramaterwas then removedand solid KCl (3 mg) was applied to the surface of the parietal cortex inthe CSD induced groups, andNaCl (3mg)was applied in the non-CSD in-duced groups.

For the electron microscopic and immunohistochemical studies, therats from all experimental groups (n = 5 per group)were deeply anes-thetized with an excessive dose of sodium pentobarbital 2 hours afterthe application of KCl or NaCl and were then transcardially perfusedwith 250 ml of 0.1 M phosphate buffered saline (PBS) followed by250 ml of 4% paraformaldehyde in 0.1 M PBS, pH 7.4. The brain was re-moved, and a 3mm thick brain slicewas cut at 3mmanterior to bregmaand immersed in 4% paraformaldehyde for immunohistochemicalstudy. The frontal cortex ipsilateral to the area of KCl or NaCl applicationand 6 mm anterior to bregma was cut into small cubes (1 mm3), whichwere submerged in 3% glutaraldehyde for transmission electron micro-scopic study.

For the western blot analysis (n= 5 per group), 2 hours after KCl orNaCl application, all rats were euthanized with an excessive dose of so-dium pentobarbital followed by transcardial perfusion with normal sa-line before decapitation. The frontal cortex ipsilateral to the KClor NaCl application was rapidly dissected on ice at 3 mm anterior tobregma andmaintained at−80 °C until further processing (i.e., proteinextraction) as previous described by Deng et al. (2003).

Immunohistochemistry assay

Brain sliceswere processed and embedded in paraffin blocks. For thedetection of ICAM-1 and VCAM-1, 5-μm thick serial coronal sections ofthe brain were cut, and 1 of every 10 sections was collected onSuperfrost plus slides (Thermo Scientific, Portsmouth, New Hampshire,USA). In total, 4 sectionswere selected from each animal (n=5 for eachgroup). All slides were deparaffinized and then incubated with citratebuffer pH 6.0 (antigen retrieval solution, Dako, Glostrup, Denmark),3% H2O2 (endogenous peroxidase blocking), and 3% normal horseserum (PAN Biotech GmbH, Aidenbach, Germany) in PBS. ICAM-1 andVCAM-1 were labeled by incubating the sections with mouse anti-ICAM-1 (1:100 dilution; BD Bioscience Pharmingen, California, USA)and rabbit anti-VCAM-1 (1:100 dilution; Santa Cruz Biotechnology,California, USA) at 37 °C for 30 min. Immunoreactivities were detectedwith the ultraView Universal DAB Detection Kit (Ventana MedicalSystems, Inc., Arizona, USA). This entire process was conducted withan automatic slide staining machine (Benchmark XT, Ventana MedicalSystems, Inc., USA). Next, all slides were dehydrated in a graded seriesof ethanol, mounted, cover-slipped, and scanned with a slide scanner(Aperio ScanScope, Aperio, Vista, California, USA). To determinethe number of ICAM-1 and VCAM-1 immunoreactive microvessels, a1,000 × 600 μm grid was drawn in the area of lamina I-VI of the brainipsilateral to the site of KCl or NaCl application. The numbers of ICAM-1 or VCAM-1 immunopositive microvessels in eight randomly selectedareas were counted. The results are reported as the average numbersof ICAM-1 or VCAM-1 immunopositive vessels per 5 mm2.

Ultrastructural study

After overnight fixation with 3% glutaraldehyde, all 1 mm3 frontalcortex cubes were processed for transmission electron microscopic



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study according to procedures that have been described previously(Maneesri et al., 2010). Briefly, the specimens were post-fixed with 1%osmium tetroxide and dehydrated via a graded series of ethanol. Thecubes were then passed through propylene oxide and embedded inEpon (Epon 812; Electron Microscopy Sciences, Fort Washington,USA). Ultra-thin (70–90 nm thick) sections were prepared and stainedwith uranyl acetate and lead citrate. To investigate the ultrastructuralchanges in the cerebral microvessels, fifteen capillaries (diameter: 8–10 μm)per samplewere randomly selected under transmission electronmicroscopic examination (JEM 1210; JEOL, Tokyo, Japan), in total 75capillaries per group were examined. Indicators of ultrastructuralchanges, including the numbers of microvilli, the numbers of endotheli-al pinocytic vesicles, and astrocytic foot plate swelling, weremonitored.The numbers ofmicrovilli were counted and are reported as the averagenumbers ofmicrovilli per vessel. To count the numbers of pinocytic ves-icles, two electron micrographs that covered the area of the endothelialcells were taken from each capillary at a final magnification of 80,000×.The numbers of pinocytic vesicles were counted and are reported as theaverage numbers of pinocytic vesicles per μm2. To evaluate astrocyticfoot plate swelling, a photograph of every vessel was taken at a finalmagnification of 12,000×, and the degree of astrocytic foot plate swell-ing was scored based on the following criteria: normal — no astrocyticfoot plate swelling observed around the blood vessel, moderate — lessthan 50% of the astrocytic foot plates around the blood vessel wereswollen, and severe—more than 50% of the astrocytic foot plates aroundthe blood vessel were swollen.

Western blot analyses

The frozen frontal cortices were homogenized in ice-cold RIPA lysisbuffer (Cell Signaling Technology®, Massachusetts, USA) containing20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA,1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1mM b-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin and aprotease/phosphatase inhibitor cocktail (Cell Signaling Technology®,Massachusetts, USA). The samples were then centrifuged at 12,000g,4 °C for 15 min. The supernatants were collected, and the proteinconcentrations were measured using the Pierce™ BCA protein assay(Thermo Scientific, Illinois, USA). Ten and 60 μg/μl of the protein sam-ples were loaded on 7.5% SDS polyacrylamide gels for the detection ofVCAM-1 and ICAM-1, respectively. The proteins were then transferredonto nitrocellulose membranes using the Mini Trans-Blot® Electropho-resis Transfer Cell (Bio-Rad, California, USA). Next, themembraneswereincubated with 5% non-fat dry milk for 1 hour at room temperaturefollowed by incubation with anti-VCAM-1 (1:1,000 dilution; SantaCruz Biotechnology, California,, USA) or anti-ICAM-1 (1:1,000 dilution;BD Bioscience Pharmingen, California, USA) antibody at 4 °C overnightto detect the expressions of VCAM-1 and ICAM-1, respectively. Afterwashing, the membranes were incubated with anti-rabbit secondaryantibody conjugated with horseradish peroxidase (HRP) at a dilutionof 1:10,000 (Sigma, St. Louis, Missouri, USA) at room temperature for1 hour for the detection of VCAM-1. For the detection of ICAM-1, themembranes were incubated with anti-mouse secondary antibody con-jugated with HRP at a dilution of 1:5,000 (Sigma, St. Louis, Missouri,USA). Immunoreactive bands were visualized using a chemilumines-cence system (Amersham™ ECL™ Prime Western Blotting DetectionReagent, GE Healthcare Life Sciences, Buckinghamshire, UK). The inten-sities of the immunoreactive bands were analyzed with Image J soft-ware (National Institutes of Health, Bethesda, Maryland, USA). Theresults are reported as the ratios of the densities of VCAM-1 or ICAM-1to β-actin.

Statistical analysis

The results are presented as the means ± the S.E.M. The statisticalanalyses were performed using one-way ANOVA and Bonferroni’s test


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formultiple comparisons. P values less than 0.05were considered statis-tically significant.

Results

Neither acute nor chronic treatment 200 mg/kg bw APAP affectedthe rats’ body weights. The average body weights of the control and30-day APAP-treated groupswere 367±6 and 350±10 grams, respec-tively. Our results also indicated that 30 days of APAP treatment did notaffect the rats’ liver, as examination of the major functional enzymes ofthe liver (alanine aminotransferase; AST, aspartate aminotransferase;ALT, alkaline phosphatase; ALP) and the morphology of the liver re-vealed no significant differences between the APAP-treated animals ascompared with those in the control group (data not shown).

Effect of APAP treatment on the CSD-induced ultrastructural alterations ofcerebral endothelial cells

Our results revealed that the induction of CSD by the application ofKCl caused ultrastructural changes in the cerebral microvessels andthat the application of NaCl had no effect on these microvessels. Thenumbers of microvilli and pinocytic vesicles in the endothelial cells ob-tained from the CSD group were significantly higher than those ob-served in the control group (P b 0.05). Astrocytic foot plate swellingaround the cerebral vessels was clearly evident in the animals thatunderwent CSD activation. This finding correlates with the findings ofa previous study (Maneesri et al., 2010). The results obtained from theanimals that received APAP pretreatment revealed that pretreatmentwith this drug 1 hour prior to the induction of CSD attenuated the alter-ations of cerebral microvessels. The numbers of microvilli and pinocyticvesicles in the APAP pretreatment group (Figs. 1D and 2D) were signif-icantly lower than those observed in the CSD group (Figs. 1C and 2C)that did not receive treatment (Figs. 1A–D, 2A–D, and 3A and Table 1).Pretreatment with APAP also had a protective effect against the astro-cytic foot plate swelling induced by CSD. The proportion of astrocyticfoot plate swelling in the perivascular areas of the vessels obtainedfrom the APAP pretreated group (Fig. 1D)was lower than that observedin the CSD group (Fig. 1C). There was no significant difference observedin the ultrastructure (astrocytic foot plate swelling and formation ofmi-crovilli and pinocytic vesicle) between the cerebralmicrovessels obtain-ed from the acute APAP treatment group (Figs. 1B and 2B) as comparedwith the control group (Figs. 1A and 2A).

However, chronic APAP treatment (30 days) produced a differenteffect on the ultrastructural alterations induced by CSD. We found thatchronic APAP treatment caused a significant increase in the number ofmicrovilli (Fig. 1F) and pinocytic vesicles (Fig. 2F) as compared withthose from the control group (Figs. 1E and 2E) (P b 0.01). These dataare shown in Figs. 1E–H and 2E–H and Table 1. These alterations ofthe cerebral microvessels, including astrocytic foot plate swelling,were more severe when chronic APAP treatment was combined withCSD activation. The number of pinocytic vesicles in the endothelialcells obtained from the chronic APAP treatment with CSD activationgroup (Fig. 2H) was significant higher than that observed in the CSDgroup (Fig. 2G). The astrocytic foot plate swelling in the perivascularareas of the cerebralmicrovessels obtained from the chronic APAP treat-ment with CSD activation group (Fig. 1H) was also more severe thanthat observed in the CSD group (Fig. 1G). These data are shown inFigs. 1E–H and 3B.

Effect of APAP treatment on the CSD-induced expression of cell adhesionmolecules on cerebral microvessels

Using western blot analysis, the expressions of cell adhesion mole-cule proteins (ICAM-1 and VCAM-1) were evaluated. The results re-vealed that CSD activation increased the expression of cell adhesionmolecules in the cerebral cortex. The level of ICAM-1 expression



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Fig. 1. Effects of APAP treatment on CSD-induced ultrastructural changes in cerebral endothelial cells. Electronmicrographs showing the ultrastructures of cerebral microvessels obtainedfrom the control (A, E), APAP-treated without CSD (B, F), CSD (C, G), and APAP-treated with CSD (D, H) groups of 2 different experiments. The acute APAP treatment experiment (A–D);the chronic APAP treatment experiment (E–H). Microvillous formation (M); Swelling of the astrocytic foot plate (As). Scale bar = 1 μm.


Please cite this article as: Yisarakun, W., et al., Chronic paracetamol treatment increases alterations in cerebral vessels in cortical spreadingdepression model, Microvasc. Res. (2014), http://dx.doi.org/10.1016/j.mvr.2014.04.012

image of Fig.�1


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Fig. 2. Effects of APAP treatment on CSD-induced ultrastructural changes in cerebral endothelial cells. Electronmicrographs showing pinocytic formations in the endothelial cells obtainedfrom the control (A, E), APAP-treatedwithout CSD (B, F), CSD (C, G), and APAP-treatedwith CSD (D, H) groups from the 2 experiments. The acute APAP treatment experiment (A–D); thechronic APAP treatment experiment (E–H); Pinocytic vesicle (P); Microvillous formation (M). Scale bar = 200 nm.


Please cite this article as: Yisarakun, W., et al., Chronic paracetamol treatment increases alterations in cerebral vessels in cortical spreadingdepression model, Microvasc. Res. (2014), http://dx.doi.org/10.1016/j.mvr.2014.04.012

image of Fig.�2


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Fig. 3. Effects of APAP treatment on CSD-induced ultrastructural changes in astrocytes. Histograms showing the proportions of astrocytic foot plate swelling observed in the control, APAP-treated without CSD, CSD, and APAP-treated with CSD groups from the acute APAP treatment (A) and chronic APAP treatment (B) experiments.

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observed in the CSD group was significantly higher than that observedin the control group (P b 0.05). Pretreatment with APAP 1 hour priorto the induction of CSD attenuated the expression of ICAM-1 inducedby CSD as demonstrated in Fig. 4A. No differences in the expression ofVCAM-1 were observed in any experimental group as shown in Fig. 4C.

Interestingly, our results reveal that the expressions of cell adhesionmolecules in the rats that were chronically exposed to APAP treatment(30 days) were differentially affected by this drug. Compared withthose from the control group, long-term treatment with APAP eitheraloneor in combinationwith CSD significantly increased the ICAM-1 ex-pression (P b 0.05 and P b 0.001, respectively). Additionally, we foundthat the level of VCAM-1 protein expression in only the chronic APAP-treated with CSD groupwas significantly higher than that in the controlgroup (P b 0.01). These data are shown in Fig. 4B and D.

The immunohistochemical results correlate well with the results ob-tained from the western blot analyses. The numbers of ICAM-1- andVCAM-1-immunopositive vessels were significantly higher after CSDactivation. Pretreatment with APAP 1 hour prior to CSD activation de-creased the numbers of ICAM-1- and VCAM-1-immunopositive vesselsthat were induced by CSD (Figs. 5A–D and 6A–D and Table 2). However,following chronic APAP treatment, the induction of CSD caused a sig-nificant increase in the number of those immunopositive vesselscompared to the CSD group (P b 0.01 and P b 0.001, for ICAM-1 andVCAM-1, respectively). These data are shown in Figs. 5E–H and 6E–Hand Table 2.

Discussion

Our results revealed that acute and chronic treatment with APAPresulted in different responses of the cerebral microvessels to CSDactivation. We found that CSD activation alone induced increases in

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Table 1Effects of acute and chronic APAP treatment on CSD-induced ultrastructural changes in cerebr

Variables Control group APAP-trea

Acute APAP treatmentPinocytic vesicles (number per μm2) 16.89 ± 0.92 18.13 ± 0Microvilli (number per vessel) 1.20 ± 0.19 1.32 ± 0

Chronic APAP treatmentPinocytic vesicles (number per μm2) 17.16 ± 0.48 30.15 ± 2Microvilli (number per vessel) 0.86 ± 0.35 2.60 ± 0

⁎ P b 0.05 compared with the control group.⁎⁎ P b 0.01 compared with the control group.⁎⁎⁎ P b 0.001 compared with the control group.

# P b 0.05 compared with the CSD group.


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the cerebral microvessels. While acute APAP treatment attenuated thealteration of cerebral microvessels induced by CSD, the opposite re-sponse was observed in the rats that received chronic APAP treatment.Importantly, none of the alterations observed in the rats that receivedchronic APAP treatment were induced by hepatotoxicity because thethreemain liver enzymes and the liver morphologies of the rats that re-ceived the chronic APAP treatment were normal (data not shown).

It is known that CSD induction can initiate alterations in both neuraland vascular compartments (Lauritzen and Fabricius, 1995; Leão, 1944).In the vascular compartment, studies of both migraine patients and an-imal models have demonstrated that alterations in cerebral circulationoccur after the activation of the trigeminovascular system. In migrainepatients, increases in the expression of vascular adhesion moleculeshave been detected during migraine attacks (Sarchielli et al., 2006). Al-terations in the cerebral microvessels have been further confirmed byobservations of plasma protein extravasation from rat cerebral vesselsafter CSD activation (Gursoy-Ozdemir et al., 2004; Moskowitz, 2007).Several mechanisms, including increases in oxidative stress and neuro-genic inflammation caused by increases in vasoactive neurotransmitters(i.e., calcitonin gene related peptide (CGRP), substance P (SP), and nitricoxide (NO)) released from perivascular nerve terminals, are involved inthese hemodynamic changes (Brain and Grant, 2004; Busija et al.,2008). The results of the present study are in line with these observa-tions. A greater degree of ultrastructural alteration and increases inthe expressions of cell adhesion molecules (ICAM-1 and VCAM-1)were observed in animals that underwent CSD activation compared tothe control group.

Our results demonstrated that acute pretreatment with APAP priorto the induction of CSD attenuated the effect of CSD activation interms of alterations in cerebral microvessels. Our previous study that

al endothelial cells.

ted group CSD group APAP-treated with CSD group

.58 22.96 ± 1.26⁎ 19.12 ± 0.89

.32 2.54 ± 0.41⁎ 1.38 ± 0.21

.25⁎⁎ 23.35 ± 0.55 33.54 ± 1.82⁎⁎⁎#

.27⁎⁎ 2.80 ± 0.15⁎⁎ 2.98 ± 0.18⁎⁎⁎


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Fig. 4. Effects of APAP treatment on CSD-induced the expression of cell adhesion molecules in the cerebral cortex. The ICAM-1 (A, B) and VCAM-1 (C, D) protein levels were analyzed bywestern blotting and are shown for the control, APAP-treated without CSD, CSD, and APAP-treated with CSD groups from the acute APAP treatment (A, C) and chronic APAP treatment(B, D) experiments. Quantitative data are expressed as relative densities compared to β-actin. *P b 0.05 compared to the control group, **P b 0.01 compared to the control group,***P b 0.001 compared to the control group, ##P b 0.01 compared to the CSD group, δδδP b 0.001 compared to the APAP-treated without group.


employed the CSD animal model demonstrated that acute treatmentwith APAP attenuates the neuronal excitability induced by CSD(Supornsilpchai et al., 2010b). This effect might result in decreases inthe amounts of vasoactive neurotransmitters that are released fromperivascular neurons. Furthermore, several studies have demonstratedthat APAP inhibits the neuronal responses to SP activation and this ac-tion is associated with the ability of this drug to attenuate the synthesisof NO (Bujalska, 2004; Godfrey et al., 2007).


The anti-inflammatory effect of APAP has also been reported in pre-vious studies by Tripathy and Grammas (2009a, 2009b). These authorsdemonstrated that pretreatment with APAP increases both neuronaland endothelial cell survival and inhibits the oxidative stress-inducedexpression of pro-inflammatory cytokines (Tripathy and Grammas,2009a, 2009b). The recent study by Slosky et al. (2013) has demonstrat-ed that single treatment of APAP (500 mg/kg bw) in rats could increaseeither expression or activity of P-glycoprotein (P-gp) (Slosky et al.,


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Fig. 5. Effects of APAP treatment on CSD-induced ICAM-1 expression in the cerebral cortex. Photomicrographs showing ICAM-1-immunoreactive cells (arrows) in the cerebral corticesobtained from the control (A, E), APAP-treated without CSD (B, F), CSD (C, G), and APAP-treated with CSD (D, H) groups in the acute APAP treatment (A–D) and chronic APAP treatment(E–H) experiments. Scale bar = 200 μm.


2013)which is the transporter protein responsible for the efflux of a va-riety of compounds (including toxin and toxic metabolites) across theBBB (Lin and Yamazaki, 2003). Thus, in addition to its ability to decreaseneuronal excitation, the enhancing of P-gp functional expression, theanti-oxidant and anti-inflammatory effects of APAP may be the mecha-nisms that underlie the protective effect of APAP against the changes inthe cerebrovasculature that are induced by CSD activation.

Interestingly, our present results from the chronic APAP treatmentexperiment indicate that long-term treatment with this drug (30 days)may change the effects of APAP on cerebral microvessels from protectiveto threatening as demonstrated by the increased alteration of the


cerebral microvessels. This opposite effect of APAP that occurs whenthe drug is used as a long-term treatment has previously been reportedin our studies in the same animal model (Supornsilpchai et al., 2010a,2010b). The results demonstrated that, while acute APAP treatmentattenuates CSD-induced cortical neuron excitation, chronic APAP treat-ment results in the sensitization of these cortical neurons as demonstrat-ed by increases in the frequency of depolarizing shifts after CSDactivation (Supornsilpchai et al., 2010a, 2010b). The results indicatethat chronic APAP treatment might cause the increase in the release ofvasoactive neurotransmitters (i.e., CGRP, SP and NO) from perivascularneurons, which may induce neurogenic inflammation and ultimately


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Fig. 6. Effect of APAP treatment on CSD-induced the expression of VCAM-1 in the cerebral cortex. Photomicrographs showing VCAM-1-immunoreactive cells (arrows) in the cerebral cor-tices obtained from the control (A, E), APAP-treatedwithout CSD (B, F), CSD (C, G), and APAP-treatedwith CSD (D, H) groups in the acute APAP treatment (A–D) and chronic APAP treat-ment (E–H) experiments. Scale bar = 200 μm.


lead to the alteration of cerebral vessels. The present results have con-firmed this hypothesis. We demonstrated that chronic APAP treatmentinduced cerebral microvessel abnormalities and, when combined withCSD activation, those abnormalities were more severe.

Additionally, it is known that APAP easily crosses the BBB and can bemetabolized by the enzyme CYP2E1. This metabolic pathway results inthe formation of NAPQI in the brain (Hansson et al., 1990; Haorahet al., 2005; Posadas et al., 2010). NAPQI is a toxic substance that is al-ways rapidly captured by brain GSH. Because GSH is a major cellularanti-oxidant that neutralizes toxins and several types of free radicalsin the brain, the depletion of GSH in the brain likely occurs after chronic


APAP treatment. Increases in NAPQI levels together with depletions ofGSH can result in increases in oxidative stress, which can lead to a seriesof alterations in the brain that include damage to brain cells and alter-ations in the pro- and anti-inflammatory cytokine balance.We have re-cently demonstrated that chronic treatment with APAP enhances theexpression of pro-inflammatory cytokines in several brain regions in-cluding the cerebral cortex and hippocampus (Chantong et al., 2013;Maneesri-le Grand et al., 2011). We suggest that increments in pro-inflammatory cytokines in the brain are involved in the alteration of ce-rebral microvessels observed after chronic APAP treatment. Based onour results, the duration of treatment seems to be a key factor that is


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t2:1 Table 2t2:2 Effects of acute and chronic APAP treatment on CSD-induced expression of ICAM-1 and VCAM-1 in the cerebral cortex.

Variables Control group APAP-treated group CSD group APAP-treated with CSD groupt2:3

Acute APAP treatmentt2:4

ICAM-1 expression (positive vessels per 5 mm2) 0.00 ± 0.00 0.00 ± 0.00### 8.00 ± 2.00⁎⁎⁎ 1.50 ± 0.50##t2:5

VCAM-1 expression (positive vessels per 5 mm2) 0.00 ± 0.00 0.00 ± 0.00### 7.50 ± 0.50⁎⁎⁎ 0.00 ± 0.00###t2:6

t2:7Chronic APAP treatmentt2:8

ICAM-1 expression (positive vessels per 5 mm2) 0.00 ± 0.00 45.00 ± 1.00⁎⁎⁎## 8.50 ± 0.50 43.50 ± 4.5⁎⁎##t2:9

VCAM-1 expression (positive vessels per 5 mm2) 0.00 ± 0.00 30.00 ± 2.00⁎⁎⁎### 9.50 ± 0.50 35.00 ± 3.00⁎⁎⁎###t2:10

t2:11 ⁎⁎ P b 0.01 compared with the control group.t2:12 ⁎⁎⁎ P b 0.001 compared with the control group.t2:13 ## P b 0.01 compared with the CSD group.t2:14 ### P b 0.001 compared with the CSD group.


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responsible for the dual effects of this drug. However, the exact mecha-nisms underlying these effects require further investigation.

Our results are in line with recent data demonstrating that chronicor repeated treatment with APAP alters its therapeutic effects. Regard-ing the effects of APAP on the circulatory system, accumulating evidenceindicates that chronic APAP treatment, even at therapeutic doses, canaffect the circulatory system (Curhan et al., 2002; Dedier et al., 2002;Forman et al., 2007; Sudano et al., 2010). Studies on women and menwho frequently took APAP at a dosage of 500 mg/day found a nearly2-fold increase in the relative risk of incident hypertension comparedto nonusers (Curhan et al., 2002; Dedier et al., 2002; Forman et al.,2007). The effect of chronic APAP treatment on the circulatory systemhas recently been confirmed by Sudano et al. (2010). These authorsfound that collateral treatment with APAP at a therapeutic dose(1 gram/day) and standard cardiovascular therapy for two weeks in-duced increases in both systolic and diastolic blood pressure in patientswith coronary heart disease (Sudano et al., 2010). Additionally, a studyof mouse airways conducted by Nassini et al. (2010) found that singleand repeated treatment with APAP at therapeutic doses results in in-creases in the formation of NAPQI and increases in pro-inflammatorycytokine levels (i.e., monocyte chemotactic protein-1 (MCP-1), IL-1β,and TNF-α) (Nassini et al., 2010). These authors further demonstratedthat NAPQI has a key role in the series of alterations that are observedafter APAP treatment including increments in the amounts of neuropep-tides (SP and CGRP) released from the sensory nerve terminal, increasesin pro-inflammatory cytokine production and neurogenic inflammationand plasma protein extravasation in the mice airway (Nassini et al.,2010). These authors hypothesized that the inflammatory responsesto APAP treatment are limited to highly innervated tissues (Nassiniet al., 2010). Because cerebral microvessel tissue is highly innervatedby the perivascular neurons, this tissue may be highly susceptible toAPAP treatment.

Taken together, our results suggest that increases in NAPQI forma-tion, pro-inflammatory cytokine production and oxidative stressmay all be involved in the alterations of cerebral microvessels thatwere observed in the rats that received chronic APAP treatment.When combined with CSD activation, greater amounts of vasoactiveneurotransmitters are released from perivascular neurons due to thehyperexcitation of these cells. The cumulative effects of the neurogenicinflammation induced by these neurotransmitters on the cerebral ves-sels that occurred during chronic APAP treatment resulted in a greaterdegree of cerebrovascular damage in the rats that received chronicAPAP treatment and CSD activation.

In summary, the data obtained from this study indicate that theshort-term use of APAP still protected the cerebral microvessels againstCSD activation; however, long-term treatment with this drug at thesame dose caused damage to these vessels. When combined with CSDactivation, the damage to cerebral microvessels induced by long-termAPAP treatment was more severe. Thus, long-term treatment withAPAP for patients with CSD-related disorders, particularly migraineheadaches, might need to be carefully monitored.


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OFAcknowledgments

This study was supported by the National Research Council ofThailand (NRCT, GRB_BSS_48_55_30_07), Thailand Research Fund(RSA 5580034) and the Integrated Innovation Academic Center (IIAC)2012: Chulalongkorn University Centenary Academic DevelopmentProject.

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