Research Article Therapeutic Time Window for Edaravone Treatment of Traumatic Brain ...downloads.hindawi.com/journals/bmri/2013/379206.pdf · 2019. 7. 31. · In spite of the fact

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 379206 13 pageshttpdxdoiorg1011552013379206

Research ArticleTherapeutic Time Window for Edaravone Treatment ofTraumatic Brain Injury in Mice

Kazuyuki Miyamoto12 Hirokazu Ohtaki1 Kenji Dohi2

Tomomi Tsumuraya1 Dandan Song1 Keisuke Kiriyama1

Kazue Satoh1 Ai Shimizu1 Tohru Aruga2 and Seiji Shioda1

1 Department of Anatomy Showa University School of Medicine 1-5-8 Hatanodai Shinagawa-ku Tokyo 142-8555 Japan2Department of Emergency and Critical Care Medicine Showa University School of Medicine 1-5-8 Hatanodai Shinagawa-kuTokyo 142-8555 Japan

Correspondence should be addressed to Seiji Shioda shiodamedshowa-uacjp

Received 5 January 2013 Revised 8 March 2013 Accepted 11 March 2013

Academic Editor Norma Possa Marroni

Copyright copy 2013 Kazuyuki Miyamoto et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Traumatic brain injury (TBI) is a major cause of death and disability in young people No effective therapy is available to ameliorateits damaging effects Our aim was to investigate the optimal therapeutic time window of edaravone a free radical scavenger whichis currently used in Japan We also determined the temporal profile of reactive oxygen species (ROS) production oxidative stressand neuronal death Male C57Bl6 mice were subjected to a controlled cortical impact (CCI) Edaravone (30mgkg) or vehiclewas administered intravenously at 0 3 or 6 hours following CCIThe production of superoxide radicals (119874

2

∙minus) as a marker of ROSof nitrotyrosine (NT) as an indicator of oxidative stress and neuronal death were measured for 24 hours following CCI Superoxideradical production was clearly evident 3 hours after CCI with oxidative stress and neuronal cell death becoming apparent after 6hours Edaravone administration after CCI resulted in a significant reduction in the injury volume and oxidative stress particularlyat the 3-hour time point Moreover the greatest decrease in 119874

2

∙minus levels was observed when edaravone was administered 3 hoursfollowing CCI These findings suggest that edaravone could prove clinically useful to ameliorate the devastating effects of TBI

1 Introduction

In spite of the fact that traumatic brain injury (TBI) is amajor cause of death and disability particularly in youngpeople and given the huge socioeconomic costs of caringfor affected persons there is still no adequate treatmentavailable to ameliorate its damaging effects [1 2] The overallincidence of TBI in the United States is estimated to be 540cases per 100000 persons and the prevalence of long-termdisability is estimated to be between 32 and 53 million In2000 the economic impact of TBI in the United States wasestimated to be $92 billion in lifetimemedical costs and $512billion in lost productivity Falls and motor vehicle accidentsare the leading causes of TBI with most cases transferredimmediately to an emergency department [3] Given thatmoderate and severe TBIs are associated with neurologic and

functional impairments [4] further intensive care followinginitial treatment and diagnostic assessment are also usuallyrequired

Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) is aderivative of antipyrin and was approved as free radicalscavenger for the treatment of acute cerebral infarction inJapan [5] Edaravone was first reported to strongly scavengehydroxyl radicals (OHminus) produced by the Fenton reaction invitro and to decrease lipid and L-tyrosine oxidation [6] Theeffects of edaravone have been studied in relation to brainischemia in animals and humans and decreased brain edemainfarction endothelial damage and oxidative damage havebeen reported [6ndash11] Edaravone has also been used in otherneural injury models such as spinal cord injury [12] TBI[8 13 14] and brain hemorrhage [15] andwas found to reducelesion size and oxidative stress levels We have previously

2 BioMed Research International

reported that intravenous edaravone (30mgkg) treatmentimmediately after cortical impact suppressed traumatic neu-ral damage in rodents [13] and decreased hydroperoxide(ROO∙) and alkoxyl (RO∙) radical formation in both rodentsand patients with TBI [13 16] Edaravone treatment at 2hours and again at 12 hours also decreased neuronal lossin a dose-dependent fashion (075 15 or 3mgkg) in theCA3 layer of the hippocampus after TBI [8] while at 3mgkgiv it suppressed apoptotic neuronal cell death and oxidativedamage after TBI [14] In spite of these positive outcomes thetherapeutic time window of edaravone on TBI has not beenexamined in detail

It has been suggested that reactive oxygen species (ROS)generation is activated in the lesion area after TBI leading tothe initial production of superoxide (O2

∙minus) and nitric oxide(∙NO) radicalsThese ROS then react andmetabolize to formstronger oxidants in the form of peroxynitrite (ONOOminus)hydroxyl (∙OH) carbonate (CO3

∙minus) and nitrogen dioxide(∙NO2) radicals [17 18] which in turn react with proteins

lipids sugars and nucleotides and impair the normal phys-iological function of cells Although it is considered that O2

∙minus

does not have strong oxidative potential and that edaravonedoes not scavenge O2

∙minus in vitro [6] we previously reportedthatmice deficient inGp91phox (NOX2) a subunit of NADPHoxidase and a generator of O2

∙minus exhibited reduced lesion sizeand oxidative stress following TBI [19] Moreover knockoutmice lacking interleukin-1 a proinflammatory cytokine wereless susceptible to neuronal cell death than their wild-typelittermates and displayed less inducible nitric oxide synthasegene expression and reduced O2

∙minus and ONOOminus productionduring ischemia [20 21]

In the present study we investigated the therapeutic timewindow of edaravone on TBI and oxidative metabolite gen-eration in mice following a controlled cortical impact (CCI)We also evaluated the temporal profile of ROS productionoxidative stress and neuronal cell death in order to estimatethe relationship between the effect of edaravone on braindamage and the sequence of events leading to ROS generationfollowing CCI

2 Materials and Methods

21 Animals and CCI Model All experimental proceduresinvolving animals were approved by the Institutional AnimalCare and Use Committee of Showa University (00158)Young adult male C57BL6 mice (8ndash12 weeks of age 20ndash26 gper body weight) were anesthetized with 2 sevoflurane inN2OO2(7030) and positioned in a computer-guided

stereotaxic system (Leica Angle Two Leica MicrosystemsWetzlar Germany) incorporating an electromagnetic CCIdevice (Benchmarked Stereotaxic Impactor Leica Microsys-tems) Following a midline scalp incision a 4mm2 openingwas made in the skull 3mm lateral and 2mm caudal ofbregma thereby exposing the right parietotemporal cortex ACCI was carried out at a depth of 10mm from the duramaterat a stroke velocity of 37msecond using an impact devicewith a rounded tip of approximately 12mm in diameter(Figures 1(a) and 1(b)) [22]

After removing hemorrhaged blood resulting from theimpact the skull was covered with 4mm diameter artificialdura (GORE Preclude W L Gore amp Associates Newark NYUSA) and a 5mm diameter artificial bone plate made fromdental cement (GC Fuji I GC Corporation Tokyo Japan)The core body temperature of the mice was maintained at37∘C during the surgery

22 Experimental Design We performed 3 experiments asgiven in the following

Experiment 1 The animals were divided into 4 experimentalgroups to determine the possible therapeutic time windowof edaravone (30mgkg 119899 = 10 for each group) Group 1(Ed 0 h) edaravone was administered immediately (0 hour)after the CCI Group 2 (Ed 3 h) edaravone was administered3 hours following CCI Group 3 (Ed 6 h) edaravone wasadministered 6 hours following CCI Group 4 vehicle (Vh)this served as the control group in which normal saline wasgiven immediately (0 hour) following CCI (119899 = 9) Allanimals were sacrificed 24 hours following CCI (Figure 1(d))

Experiment 2 Mice (119899 = 3 in each group) were divided into 4groups They were sacrificed group 1 immediately (0) group2 3 hours group 3 6 hours and group 4 24 hours after CCIto investigate the temporal profiles of ROS oxidative stressand neuronal death in the brain (Figure 1(e))Experiment 3 Three groups were set (119899 = 3 in each group)to investigate the effect of edaravone treatment to ROS(Figure 1(f)) Group 1 (Ed 0 h) edaravone was administeredimmediately (0 hour) after the CCI and in situ detection ofO2∙minus was performed 4 hours after CCI Group 2 (Ed 3 h)

edaravone was administered 3 hours following CCI and insitu detection of O2

∙minus was performed 4 hours following CCIGroup 3 (Vh 3 h) vehicle was administered 3 hours after CCIand in situ detection of O

2

∙minus was also performed 4 hoursfollowing CCI as the control group

23 Administration of Edaravone Edaravone a free radicalscavenger was a gift fromMitsubishi Tanabe Pharma (OsakaJapan) Animals were placed in the supine position andanesthetized with sevoflurane administered by inhalationthrough a facemaskThe skin over the left clavicle was incisedto expose the left jugular vein Edaravone dissolved in salinewas slowly administered at a dosage of 30mgkg bodyweight(100ndash150120583L volume) into this vein

24 Tissue Preparation Under sodiumpentobarbital (50mgkg ip) anesthesia the animals were transcardially perfusedwith 09 NaCl followed by 2 paraformaldehyde (PFA) in50mM phosphate buffer (pH 72) The brain and skull werethen removed intact and postfixed in 2 PFA overnightafter which the skull was carefully removed and the brainwas immersed in 20 sucrose for 2 days for cytoprotectionThe brain was next frozen in liquid nitrogen-chilled 2-methylbutane and coronally cryosectioned at a thicknessof 50120583m from bregma to approximately 39mm caudal

BioMed Research International 3

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Figure 1 Establishment of TBI model and experimental protocol (a) Brain images following TBI The contusion was conducted over theright parietotemporal cortex 20mm caudal of bregma and 30mm lateral of the midline (b) High power images of the CCI device showing arounded tip of approximately 12mm in diameter (c) Coronal cryosections (thickness 50 120583m) from bregma to approximately 39mm caudalof bregma encompassing the injury regionwere used to determine the injury areaThe core-injury area was defined as the direct impact region12mm to 24mm caudal of bregma The peri-injury area was defined as being lt12mm and gt24mm caudal of bregma (d) The free radicalscavenger edaravone was injected intravenously into the jugular vein following CCI To determine the possible therapeutic time windowedaravone (30mgkg 119899 = 10 for each time point) was administered either immediately (0) or 3 or 6 hours following CCI As a vehicle-treated control group saline was administered immediately (0 hour) following CCI (119899 = 9) (e) Temporal profiles of ROS oxidative stressand neuronal death in the brain following CCI were determined immediately (0) and at 3 6 and 24 hours after CCI (119899 = 3) (f) Edaravonetreatment to ROS was investigated Edaravone was administered to group 1 immediately (0 hour) or group 2 3 hours after the CCI and in situdetection of O2

∙minus were performed 4 hours post-CCI As the control Group 3 vehicle was administered 3 hours post-CCI and in situ detectionof O2

∙minus was also performed 4 hours following CCI

of bregma thus ensuring coverage of the injured region(Figure 1(c)) The sections were then immediately immersedin PBS containing 01 Tween 20 (PBST) for subsequenthistological assessment

25 Fluoro-Jade B and Toluidine Blue Staining Fluoro-JadeB (FJB) staining was used to detect degenerating neuronsas previously reported with only minor modifications [2324] A series of sections was collected at 600 120583m intervalsfrom 0 to 36mm caudal of bregma (7 sections per mouse)and mounted on poly-L-lysine-coated glass slides After airdrying the slides were incubatedwith freshly prepared 006potassium permanganate for 15min and rinsed with distilledwater The sections were then immersed in 00005 FJB(Millipore Billerica MA USA) in a dark room for 30min

after which they were completely air-dried before beingimmersed in xylene and enclosed in malinol (Muto PureChemicals Tokyo Japan) A second series of adjacent sectionswas stained with toluidine blue (TB) [25] The sectionswere observed with a fluorescence microscope (Biozero 8100Keyence Osaka Japan)

26 Measurement of Lesion Volume FJB- and TB-stainedsections were used to semiquantify the injury area basedon sections that displayed FJB labeling or little or no TBstaining Some sections particularly those near the injurycore lacked part of the neocortex due to severe tissue damagethis area was also included in our calculations of lesionvolume The outlines of the affected regions were traced andthe areas were calculated using NIH Image software The


injury volume was then determined by summing these areasThis was performed by an investigator whowas blinded to theexperimental groups

We further defined core- and peri-injury areas Given thatthe diameter of the impact tip was around 12mm we definedthe core-injury area as encompassing the region 12mm to24mmcaudal of bregma with the peri-injury area surround-ing this (Figure 1(c))

27 Immunostaining of Nitrotyrosine (NT) Another seriesof sections at 600120583m intervals (6 sections per animal)was used to label for nitrotyrosine (NT) a peroxynitrite(ONOOminus) oxidative metabolite by free-floating immunohis-tochemistry After immersion in 03 H

2O2 the sections

were incubated in 5 normal horse serum and immersedovernight with a polyclonal affinity-purified rabbit anti-NTantibody (1 1000 Upstate Biotechnology Lake Placid NYUSA) The sections were then incubated with biotinylatedgoat anti-rabbit IgG (1 200 Santa Cruz Biotechnology SantaCruz CA USA) followed by an avidin-biotin complexsolution (Vector Burlingame CA USA) using diaminoben-zidine (Vector) as a chromogen The area of dark brownNT-immunopositive staining as well as that of the severelydamaged core-injury region as determined using NIH Imagesoftware and the injury volume calculated by summing theseareas

To determine the identity of the NT-positive cells wecolabeled for various cell markers After blocking the sec-tions were coincubated with anti-NT antibody and eithermonoclonal mouse anti-NeuN antibody (1 2000 a neuronalmarker Millipore) or monoclonal mouse anti-glial fibril-lary acidic protein (GFAP) antibody (1 2000 an astroglialmarker Sigma St Louis MO USA) followed by the sec-ondary antibodies Alexa 488-conjugated goat anti-rabbitIgG antibody (1 400) and Alexa 546-conjugated goat anti-mouse IgG antibody (1 800 Molecular Probes EugeneOR) Nuclei were stainedwith 46-diamidino-2-phenylindoledihydrochloride (DAPI 1 10000 Roche Mannheim Ger-many)

28 In Situ Detection of O2∙minus Hydroethidium (HEt) rapidly

penetrates into the brain parenchyma reacts with O2∙minus

and generates ethidium (Et) which can be detected at anemission wavelength of 510ndash550 nm [26 27] Mice wereanesthetized with 2 sevoflurane in N

2OO2(7030) and

were administered 10mgmL HEt solution (in 09 NaClcontaining 1 DMSO) into the left jugular vein Fifteenminutes after HEt infusion the animals were perfuse-fixedand their brains frozen and sectioned To identify the Et-positive cells some sections were also stained with primaryantibody for NeuN or GFAP All sections were nuclear-stained with DAPI Fluorescence was detected using an AxioImager optical sectioning microscope with an ApoTomeslider module

29 Statistical Analysis Data were expressed asmean plusmn SEMStatistical comparisons were made by Studentrsquos t-test for twogroups and by one-way ANOVA followed by Tukey-Kramer

tests for multiple group comparisons A value of 119875 lt 005was considered to indicate statistical significance

3 Results

31 Edaravone Has a Therapeutic Time Window of 6 Hoursfor the Treatment of TBI We commenced by investigatingthe effect of edaravone and the optimal therapeutic timewindow for its administration in our experimental TBImodel Animals were administered either vehicle (0 hours119899 = 9) or 30mgkg edaravone (0 3 or 6 hours after CCI)(119899 = 10) (Figure 1(d)) and the efficacy of this treatmentwas evaluated at 24 hours based on FJB (Figure 2) and TB(Figure 3) staining

One vehicle-treated mouse died during the experimentThe total injury volume calculated from FJB staining wassignificantly reduced in edaravone-treated animals (0 h 483plusmn 032mm3 119875 lt 001 3 h 313 plusmn 043mm3 119875 lt 00016 h 431 plusmn 050mm3 119875 lt 0001) compared with that in thevehicle-treated cohort (0 h 638 plusmn 034mm3) Interestinglythe total injury volume in mice treated with edaravone at3 hours was significantly less than that which occurred inthose treated at 0 hours (119875 lt 005) with the former animalsalso displaying the strongest neuroprotection observed acrossall groups No significant differences in injury volumes wererecorded between the 0- and 6-hour or the 3- and 6-hourtreatment groups (Figure 2)

To confirm these results we determined the injuryvolume based on TB staining and obtained similar results(Figure 3) The total injury volume detected by TB stainingwas significantly reduced in the edaravone-treated groups(0 h 478 plusmn 040mm3 119875 lt 005 3 h 309 plusmn 039mm3 119875 lt0001 6 h 375 plusmn 051mm3 119875 lt 001) compared with thevehicle-treated controls (0 h 614 plusmn 042mm3) with the bestresult again being obtained for the 3-hour treatment groupcompared with the 0-hour group (119875 lt 005)

To estimate which region of the affected tissue edaravonerescued from cell death we also analyzed the volume ofthe core- and peri-injury areas separately using FJB staining(Figure 2(d)) The animals treated with edaravone at 0hours had a significantly lower peri-injury volume comparedwith controls treated with vehicle at the same time point(208 plusmn 017mm3 versus 309 plusmn 021mm3 119875 lt 001) butno significant difference was observed for the core-injuryvolume (275plusmn 018mm3 versus 329plusmn 019mm3) In contrastedaravone treatment at both 3 and 6 hours significantlyreduced the TBI-induced volumes in both the core- (3 h 164plusmn 031mm3 P lt 0001 6 h 206 plusmn 016mm3 119875 lt 005) andperi- (3 h 149 plusmn 021mm3 P lt 0001 6 h 211 plusmn 036mm3119875 lt 0001) injury sites To be more specific concerningthe core injury area edaravone treatment at both 3 and 6hours rescued the frontal region to a greater extent thanthat seen for the 0-hour treatment (Figure 2(b)) Notablythe core-injury volume following edaravone administrationat 3 hours was significantly less than that measured inresponse to edaravone treatment at 0 hours The resultsfor TB staining were similar to those for FJB staining(Figure 3)


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lowastlowast

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Figure 2 Effect of edaravone on TBI following CCI (a) Representative core- (upper) and peri-injury (lower) images of FJB staining at 24hours following CCI Edaravone was injected 0 3 or 6 hours following CCI vehicle treatment occurred at 0 hours The inset shows a higherpower view of the boxed region in the main image (b) The TBI area was semiquantified for each experimental group Edaravone (119899 = 10)treatment at 3 and 6 hours led to a significant decrease in the core-injury area compared with the vehicle-treated control (119899 = 9) The injuryvolume was calculated by integration of the TBI areas and expressed as total (c) or core- and peri-injury (d) volumes Data are expressed asmean plusmn SEM Asterisk (lowast) indicates a significant difference between groups based on Tukey-Kramer tests (119875 values are described in the text)

32 Edaravone Treatment Suppresses Oxidative Stress follow-ing TBI Theprevious results revealed that edaravone reducesthe area affected by TBI and that it can exert a therapeuticeffect when administered up to 6 hours following injuryAs previous studies have reported that edaravone acts as aradical scavenger and reduces oxidative stress in a number ofdiseases [28ndash30] we therefore next investigated whether theeffect of edaravone in our model was due to the suppressionof oxidative stress To assess this we determined the NT-positive volume 24 hours after CCI in animals treated witheither vehicle (119899 = 9) at 0 hours or edaravone (119899 = 10) at 03 or 6 hours (Figure 1(d)) The total NT-positive volumes forthe edaravone-treated groups at 0 hours (391 plusmn 015mm3 P lt0001) 3 hours (228 plusmn 030mm3 P lt 0001) and 6 hours (315plusmn 045mm3 P lt 0001) were significantly less than those ofthe vehicle-treated control mice (614 plusmn 038mm3) (Figure 4)There was also a marked difference between the 0- and 3-hour edaravone treatment groups (391 plusmn 015mm3 versus

228 plusmn 030mm3 P lt 0001) Similar results were obtainedfor the core- and peri-injury volumes of the animals treatedwith edaravone at 3 hours (core 133 plusmn 016mm3 P lt 0001peri 095 plusmn 038mm3 P lt 0001) and 6 hours (core 183 plusmn023mm3 P lt 001 peri 132 plusmn 022mm3 P lt 001) comparedwith the vehicle-treated controls (core 329 plusmn 028mm3peri 285 plusmn 015mm3) However 0-hour edaravone treatmentonly produced a significant (P lt 005) decrease in the NT-positive peri-injury volume (core 281 plusmn 028mm3 peri 194plusmn 022mm3)Moreover the animals treatedwith edaravone at3 hours showed significantly greater neuroprotection at boththe core- and peri-injury sites versus the 0-hour treatmentgroup (Figure 4)

33 Neurodegeneration in the Core Injury Area Occurs at 6Hours following CCI and Spreads to Peri-Injury Area withTime We next determined the time course (Figure 1(e)) of


Core

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Figure 3 Effect of edaravone on TBI after CCI based on TB staining (a) Representative core- (upper) and peri-injury (lower) images of TBstaining 24 hours after CCI The region with little or no staining was defined as the injury area Edaravone (119899 = 10) was injected at 0 3 or6 hours and vehicle (119899 = 9) at 0 hours after CCI The total (b) or core- and peri-injury (c) areas were semiquantified in each experimentalgroup Data are expressed as mean plusmn SEM Asterisk (lowast) indicates a significant difference between the groups based on Tukey-Kramer tests

neurodegeneration following CCI (119899 = 3 for each timepoint) Only a few FJB-positive cells were present in the core-injury area 3 hours after CCI By 6 hours however diffuselyscattered FJB-positive cells were observed not only in the coreinjury area but also in the peri-injury area By 24 hours boththe number of FJB-positive cells and the size of the affectedarea had increased even further (Figure 5)The injury volumewas significantly greater at 6 (249 plusmn 030mm3 P lt 001) and24 hours (638 plusmn 034mm3 P lt 001) compared with 0 (119899 = 3010 plusmn 003mm3) and 3 (019 plusmn 005mm3) hours (Figure 5(c))

34 Oxidative Metabolites Increase 6 Hours after CCI inNeurons We subsequently investigated the temporal profile(Figure 1(e)) of oxidative stress by using NT immunos-taining after CCI (119899 = 3 for each time point) Minimalimmunoreactivity was observed immediately after CCI (0 h)However dark brown NT staining began to appear from 6hours following CCI in the neocortex around the epicenterof the impact site By 24 hours the NT-positive area hadexpanded and some of the tissue at the impact site had been

lost (Figure 6(a)) The volume of the NT-positive regionincreased significantly in a time-dependent manner afterCCI accounting for 018 plusmn 003mm3 038 plusmn 010mm3 and207plusmn 063mm3 (119875 lt 005 versus 0 hour) and 614plusmn 038mm3(119875 lt 001 versus 0 hour) at 0 3 6 and 24 hours after CCIrespectively (Figure 6(b))

Colabeling with cell markers at the core-injury siteindicated that the oxidative stress was occurring mainly inneurons (Figure 6(c))

35 ROS Increases in Neurons 3 Hours after CCI We nextdetermined the time course (Figure 1(e)) of ROS generationbased on the in situ detection of O2

∙minus using HEt at 0 3 6and 24 hours following CCI (119899 = 3 for each time point) Alow Et signal was initially observed in the core-impact regionHowever by 3 hours following CCI this signal had increasedmarkedly and continued to rise slightly up until the 24-hourtime point (Figure 7(a)) Colabeling for Et and various cellmarkers revealed that the affected cells were mainly NeuN-positive neurons (Figure 7(b))


Core

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(a)

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Figure 4 Effect of edaravone on oxidative stress followingCCI (a) Representative core- (upper) and peri-injury (lower) images ofNT stainingat 24 hours following CCI Edaravone (119899 = 10) was injected at 0 3 or 6 hours following CCI vehicle (119899 = 9) treatment occurred at 0 hoursThe inset shows a higher power view of the boxed region in the main image The injury volume was calculated by integration of the TBIareas (based on seven 50 120583m coronal sections at 500120583m intervals) and expressed as total (b) or core- and peri-injury (c) volumes Data areexpressed as mean plusmn SEM Asterisk (lowast) indicates a significant difference between groups based on Tukey-Kramer tests (119875 values are describedin the text)

36 Edaravone Suppresses the ROS Production Cycle followingCCI Finally we also investigated the effect of edaravonetreatment 3 hours following CCI on O2

∙minus generation atthe core-injury site using HEt injection Mice (119899 = 3in each group) were administered edaravone 0 or 3 hoursfollowing CCI or vehicle 3 hours following CCI with theEt signal evaluated 4 hours after injury as a measure ofO2∙minus generation As illustrated in Figure 7(c) vehicle-treated

brains showed a large number of affected cells and a strong Etsignal intensity Fewer cells were affected following edaravonetreatment at 0 hours but the intensity of the Et signal becamestronger thereafterHowever the 3-hour treatment resulted inonly a few affected cells and a very weak Et signal suggestingthat edaravone administered at this time point suppressedthe ROS and oxidative stress cycle and provided a greaterneuroprotective effect

4 Discussion

Edaravone is a free radical scavenger approved in Japan forthe treatment of stroke It could be a suitable candidatefor treating TBI as well given the results of several rodentstudies showing that this drug is able to decrease neuronal celldeath However the therapeutic time window of edaravoneon TBI has not been examined in detail In the presentstudy we have demonstrated that the intravenous injectionof edaravone (3mgkg) decreased TBI and reduced oxidativestress when administered after a delay of up to 6 hoursfollowing CCI We also determined the temporal profiles ofROS production oxidative stress and neuronal cell death inorder to understand the relationship between brain damageand the sequence of events underlying ROS generationfollowing CCI


0 h

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Figure 5 Detection of neuronal cell death by FJB labeling after CCI (a) Neuronal cell death as indicated by FJB labeling increases in atime-dependent manner after CCI (119899 = 3 at each time point) No FJB-positive cells were observed at 0 (also see inset) and 3 hours after CCIhowever by 6 and 24 hours (also see inset) FJB labeling and an increase in the area of cortical disruption produced by the contusion wereobserved (b) Seven 50120583m coronal sections at 500120583m intervals were used to semiquantify the area of FJB immunoreactivity together withthe area of cortical disruption produced by the TBI This analysis revealed a marked increase in the affected area at 6 and 24 hours Dataare expressed as mean plusmn SEM (119899 = 3) (c) A significant increase in the TBI volume was observed 6 and 24 hours following CCI Data areexpressed as mean plusmn SEM (119899 = 3) Asterisk (lowast) indicates a significant difference between groups based on Tukey-Kramer tests (119875 values aredescribed in the text)

We previously reported that the intravenous adminis-tration of edaravone (3mgkg) immediately after TBI sup-pressed cortical damage [13] Another study also demon-strated that edaravone injected 2 and 12 hours after TBIdecreased neuronal cell loss in the CA3 layer of the hip-pocampus in a dose-dependent fashion (075 15 or 3mgkg)[8] In the present study we examined the therapeutic

time window of intravenously injected edaravone (3mgkg)on TBI and showed that edaravone administered for upto 6 hours at least after CCI suppressed the lesion sizeComparisons of lesion sizes following edaravone treatmentat 0 3 and 6 hours after CCI demonstrated that edaravonetreatment at 3 hours provided the greatest neuroprotectiveeffect compared with the other treatment groups and that


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MergeDAPINT

20 120583m

20 120583m

(c)

Figure 6 Detection of oxidative stress by NT labeling following CCI (a) Very few NT-immunopositive cells were observed at 0 (also seeinset) and 3 hours following CCI however by 6 and 24 hours (also see inset) this number and the area of cortical disruption produced by thecontusion had increased markedly (b) Semiquantification of the NT-positive volume revealed a significant increase at 6 and 24 hours afterCCI Data are expressed as mean plusmn SD (119899 = 3) Asterisk (lowast) indicates a significant difference between groups based on Tukey-Kramer tests(119875 values are described in the text) (c) Multiple immunofluorescence staining of NT and cell markers The NT-positive staining overlappedwith that of the neuronal marker NeuN (green upper panel) but not with the astroglial marker GFAP (green lower panel)The sections werealso counterstained with the nuclear dye DAPI (blue)

a significant difference was observed compared with treat-ment at the 0-hour time point To estimate the extent ofthe neuroprotective effect we further compared the lesionsize by measuring both core- and peri-injury areas Edar-avone treatment 6 hours after CCI decreased the lesionsize significantly both in the peri- and core-injury sitescompared with control but the area was slightly larger thanthat seen in mice treated with edaravone 3 hours after CCI

Although mice treated with edaravone at the 0-hour timepoint showed a significantly decreased lesion size in theperi-injury site compared with vehicle-treated animals nostatistically significant differences with respect to the core-injury site size were seen Furthermore the size of the core-injury site in the 0 hour edaravone treatment group wassignificantly greater than that measured in mice treated 3hours after CCI In particular the lesion size in the frontal


Het negative 0 h 3 h

6 h 24 h

50 120583m

(a)

GFAP MergeEt DAPI

Et DAP MergeNeuN

50 120583m

50 120583m

(b)

50 120583m

Edaravone 3 hEdaravone 0 hVehicle 3 h

(c)

Figure 7 In situ detection of ROS as O2∙minus using Het (a) NoO2

∙minus (Et) signals (red) were detected in control animals Inmice subjected to TBIa low level of O2

∙minus was observed immediately after CCI By 3 hours this level had increasedmarkedly remaining high 6 and 24 hours after CCI(b) Multiple immunofluorescence staining of O2

∙minus and cell markers O2∙minus (Et) signals (red) strongly colocalized with the neuronal marker

NeuN (green upper panel) and to lesser extent with the astroglial marker GFAP (green lower panel) with nuclei labelled by DAPI (blue) (c)The effect of edaravone treatment on O2

∙minus production after CCI (119899 = 3 in each group) is shown As a control vehicle was administered 3hours post-CCI (vehicle 3 h) Edaravone was administered at 0 hours (edaravone 0 h) or 3 hours (edaravone 3 h) after CCI The productionof O2∙minus was evaluated based on the in situ detection of the Et signal (red) in the core-injury area 4 hours after CCI Nuclei (blue) were labeled

with DAPI

area was significantly different between these two groupsObservations made with respect to NT immunostaining asan indicator of oxidative stress also demonstrated a similartendency to that seen with lesion size

We subsequently examined the temporal profiles of ROSproduction oxidative stress and neuronal cell death inorder to understand the relationship between brain damageand the sequence of events giving rise to ROS generationfollowing CCI Superoxide detected by HEt was used forthe determination of ROS levels because O2

∙minus is initiallyincreased after injury [26] while the oxidative metabolitesONOOminus and ∙OH also contribute to increasing oxidativestress and damage in tissue [11] The O2

∙minus signal was initiallyobserved 3 hours following CCI and increased with time up

to 24 hours The oxidative stress detected by NT which isa metabolite of L-tyrosine oxidation by ONOOminus [31] wasobserved at 6 hours following CCI in the core-injury regionand increased for up to 24 hours with extension to the peri-injury area This was reflected in the rise in O2

∙minus productionand the concomitant increase in neuronal cell death detectedby FJB staining suggesting that excessive O2

∙minus productionafter CCImight result in the induction of oxidative stress andneurodegeneration in the brain

From the results of the temporal profiles of ROS pro-duction oxidative stress and neuronal cell death the effectsof treatment with edaravone were consistent with the timepoints which fall before during and after the production ofROS respectivelyWhile a precise explanation for this cannot


be given we suggest that it could have something to do withthe half-life of edaravone which is reported to be approx-imately one hour [29] Therefore the animals treated withedaravone immediately after (0 hour) CCI might actuallyhave had a decreased effective concentration of this drug atthe time of maximum ROS production By 6 hours followingCCI a weaker neuroprotective effect was observed due to thefact that significant oxidative damage and neuronal cell deathhad already occurred Nonetheless treatment at this timepoint still had a therapeutic effect given that the level of ROScontinued to increase for 24 hours These results suggest thatthere might be an optimal time for treatment with edaravoneto suppress the degree of injury An experimental study inwhichmice were exposed to hypobaric conditions for 3 hoursafter TBI supports our present results these animals showedexacerbated secondary traumatic injury severity becauseof a greatly heightened inflammatory response due to thehypobaric condition [32] We recently found that edaravoneimproved cerebral blood flow (CBF) after TBI [33] In thisway CBF in control animals in that study was significantlyreduced probably as a consequence of vasospasm in theipsilateral hemisphere of the brain 3 to 6 hours after CCI [33]Treatment with edaravone significantly ameliorated the CBFOther studies have reported that edaravone improves bloodcirculation in the heart [34] and lung [27] suggesting thatedaravone treatmentmay suppress traumatic ischemic injuryas observed in the present study by ameliorating circulation

This finding is important given that most TBIs areassociated with falls and motor vehicle accidents There-fore in most cases the onset of TBI is clear and patientsare transferred to an emergency department within a fewhours From a clinical perspective it is noteworthy thatdelayed treatment with edaravone in our study was moreefficacious than administration immediately after the CCIthe time delay between the onset of ROS production andneuronal cell death means probably that these cases wouldfall within a therapeutic time window for the treatment ofTBI At the present time edaravone (60mgday injectedintravenously in 2 divided doses) is used in Japan for thetreatment of stroke Therefore the dosage of edaravone inour study was approximately triple compared with clinicaluse Further study is required to determine the minimumdosage of edaravone necessary to suppress ROS productionand lesion size in this animal model and for this findingto be translated to the clinical setting whereby edaravoneis used to suppress the lesion size in clinical cases ofTBI

Althoughmany studies have reported that oxidative stresscontributes to TBI very few have linked this directly toROS production in the brain We have previously reportedthat both patients suffering a neurological emergency andanimals subjected to TBI display increased blood alkoxy-radical levels as detected by an electron spin resonance (ESR)spin trapping method [13 35] Based on the rapid elevationof intracellular Ca2+ and the impairment of CBF the sourceof the O2

∙minus is considered to be primarily the mitochondria[36] The intracellular O2

∙minus impairs mitochondrial functionand induces neuronal cell death Extracellular O2

∙minus mightbe produced by NADPH oxidase in microgliamacrophages

followingCCI [19] Based onour previous demonstration thatmice deficient in one of the subunits of NADPH oxidaseGp91phox display decreased O2

∙minus levels and that inhibitionof Gp91phox by apocynin reduces the severity of TBI invivo [19 36 37] it appears that this extracellular O2

∙minus

also contributes to the induction of neuronal cell death inresponse to injury Therefore both intra- and extracellularlyproduced O2

∙minus together with their associated metabolitesmay play important roles in the generation of oxidative stressfollowing CCI

Many clinical trials of antioxidant agents or radical scav-engers have been performed in cases of cerebral infarctionsubarachnoid hemorrhage and TBI [15 38 39] Howeverthe only agent which has been granted approval to date isedaravone [9 40] even though patients are treated withoutconcomitant monitoring of ROS or oxidative metabolitelevels Human TBI presents as a more complex and diversecondition than animal experimental models Therefore careneeds to be exercisedwhen determining the likely therapeutictime window of edaravone We suggest that edaravone andpossibly other radical scavengers or antioxidants should beadministered in response to ROS generation meaning thatthe bedside monitoring of ROS could shed more light on thepotential benefit of these agents Recently some groups havereported that Overhauser enhanced MRI which is a doubleresonance technique creates images of free radical distribu-tion in small animals by enhancing the water proton signalintensity by means of the Overhauser effect [41] Althoughnot available for bedside monitoring this technique couldbe adapted to directly monitor ROS levels in the brainWe have also previously reported that one particular ROSthe alkoxy radical is increased both in patients suffering aneuroemergency and in animals subjected to TBI and canbe monitored by ESR [13 35] These new techniques couldrepresent powerful tools for the diagnosis of ROS productionprior to oxidative stress thereby facilitating more effectivetreatment

5 Conclusion

In the present study we have demonstrated that edaravonesuppresses neuronal damage in mice subjected to a CCIwith the greatest effect observed when the drug is given3 hours after TBI This time window is consistent withthe increase in ROS produced in the cortex after CCI Wetherefore suggest that edaravone could prove clinically usefulto ameliorate the devastating effects of TBI To ensure optimalefficacy however it is critical that ROS levels are measuredconcomitantly

Conflict of Interests

No competing financial interests exist

Acknowledgments

The project was supported by a Grant-in-Aid for YoungScientists B from the JapaneseMinistry of EducationCulture


Sports Science and Technology (Kazuyuki Miyamoto) JSPSKAKENHI (23592683) Grant-in-Aid for Scientific Research(C) This work was also supported in part by the MEXT-Supported Program for the Strategic Research Foundation atPrivate Universities 2008ndash2012

References

[1] A W Brown E P Elovic S Kothari S R Flanagan and CKwasnica ldquoCongenital and acquired brain injury 1 Epidemiol-ogy pathophysiology prognostication innovative treatmentsand preventionrdquo Archives of Physical Medicine and Rehabilita-tion vol 89 no 3 pp S3ndashS8 2008

[2] E M Manno A A Rabinstein E F M Wijdicks et alldquoA prospective trial of elective extubation in brain injuredpatients meeting extubation criteria for ventilatory support AFeasibility Studyrdquo Critical Care vol 12 no 6 article R138 2008

[3] W Rutland-Brown J A Langlois K E Thomas and Y L XildquoIncidence of traumatic brain injury in the United States 2003rdquoJournal of Head Trauma Rehabilitation vol 21 no 6 pp 544ndash548 2006

[4] E Zaloshnja T Miller J A Langlois and A W SelassieldquoPrevalence of long-term disability from traumatic brain injuryin the civilian population of the United Statet 2005rdquo Journal ofHead Trauma Rehabilitation vol 23 no 6 pp 394ndash400 2008

[5] H YoshidaH Yanai YNamiki K Fukatsu-Sasaki N Furutaniand N Tada ldquoNeuroprotective effects of edaravone a novel freeradical scavenger in cerebrovascular injuryrdquoCNSDrug Reviewsvol 12 no 1 pp 9ndash20 2006

[6] T Watanabe S Yuki M Egawa and H Nishi ldquoProtectiveeffects of MCI-186 on cerebral ischemia possible involvementof free radical scavenging and antioxidant actionsrdquo Journal ofPharmacology and Experimental Therapeutics vol 268 no 3pp 1597ndash1604 1994

[7] Y Inokuchi S Imai Y Nakajima et al ldquoEdaravone a freeradical scavenger protects against retinal damage in vitro and invivordquo Journal of Pharmacology and Experimental Therapeuticsvol 329 no 2 pp 687ndash698 2009

[8] G H Wang Z L Jiang Y C Li et al ldquoFree-radical scavengeredaravone treatment confers neuroprotection against traumaticbrain injury in ratsrdquo Journal of Neurotrauma vol 28 pp 2123ndash2134 2011

[9] E Otomo H Tohgi K Kogure et al ldquoEffect of a novelfree radical scavenger edaravone (MCI-186) on acute braininfarction randomized placebo-controlled double-blind studyatmulticentersrdquoCerebrovascularDiseases vol 15 no 3 pp 222ndash229 2003

[10] P Sharma M Sinha R K Shukla R Garg R Verma and MK Singh ldquoA randomized controlled clinical trial to comparethe safety and efficacy of edaravone in acute ischemic strokerdquoAnnals of Indian Academy of Neurology vol 14 no 2 pp 103ndash106 2011

[11] R Tabrizchi ldquoEdaravoneMitsubishi-TokyordquoCurrentOpinion inInvestigational Drugs vol 1 no 3 pp 347ndash354 2000

[12] S Ohta Y Iwashita H Takada S Kuno and T NakamuraldquoNeuroprotection and enhanced recovery with edaravone afteracute spinal cord injury in ratsrdquo Spine vol 30 no 10 pp 1154ndash1158 2005

[13] K Dohi K Satoh T Nakamachi et al ldquoDoes edaravone (MCI-186) act as an antioxidant and a neuroprotector in experimentaltraumatic brain injuryrdquo Antioxidants and Redox Signaling vol9 no 2 pp 281ndash287 2007

[14] T Itoh T Satou S Nishida et al ldquoEdaravone protects againstapoptotic neuronal cell death and improves cerebral functionafter traumatic brain injury in ratsrdquo Neurochemical Researchvol 35 no 2 pp 348ndash355 2010

[15] AMunakata HOhkuma TNakanoN Shimamura K Asanoand M Naraoka ldquoEffect of a free radical scavenger edaravonein the treatment of patients with aneurysmal subarachnoidhemorrhagerdquo Neurosurgery vol 64 no 3 pp 423ndash428 2009

[16] K Dohi K Satoh Y Mihara et al ldquoAlkoxyl radical-scavengingactivity of edaravone in patients with traumatic brain injuryrdquoJournal of Neurotrauma vol 23 no 11 pp 1591ndash1599 2006

[17] E D Hall M R Detloff K Johnson and N C KupinaldquoPeroxynitrite-mediated protein nitration and lipid peroxida-tion in a mouse model of traumatic brain injuryrdquo Journal ofNeurotrauma vol 21 no 1 pp 9ndash20 2004

[18] A Lewen P Matz and P H Chan ldquoFree radical pathways inCNS injuryrdquo Journal of Neurotrauma vol 17 no 10 pp 871ndash8902000

[19] K Dohi H Ohtaki T Nakamachi et al ldquoGp91phox (NOX2)in classically activated microglia exacerbates traumatic braininjuryrdquo Journal of Neuroinflammation vol 7 no 41 2010

[20] H Mizushima C J I Zhou K Dohi et al ldquoReduced postis-chemic apoptosis in the hippocampus of mice deficient ininterleukin-1rdquo Journal of Comparative Neurology vol 448 no2 pp 203ndash216 2002

[21] H Ohtaki A Takaki L Yin et al ldquoSuppression of oxidativestress after transient focal ischemia in interleukin-1 knock outmicerdquo Acta Neurochirurgica no 86 pp 191ndash194 2003

[22] D L Brody C Mac Donald C C Kessens et al ldquoElectro-magnetic controlled cortical impact device for precise gradedexperimental traumatic brain injuryrdquo Journal of Neurotraumavol 24 no 4 pp 657ndash673 2007

[23] L C Schmued and K J Hopkins ldquoFluoro-Jade B a highaffinity fluorescent marker for the localization of neuronaldegenerationrdquo Brain Research vol 874 no 2 pp 123ndash130 2000

[24] H Ohtaki J H Ylostalo J E Foraker et al ldquoStemprogenitorcells from bone marrow decrease neuronal death in globalischemia by modulation of inflammatoryimmune responsesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 105 no 38 pp 14638ndash14643 2008

[25] H Inoue H Ohtaki T Nakamachi S Shioda and Y OkadaldquoAnion channel blockers attenuate delayed neuronal cell deathinduced by transient forebrain ischemiardquo Journal of Neuro-science Research vol 85 no 7 pp 1427ndash1435 2007

[26] H Ohtaki T Takeda K Dohi et al ldquoIncreased mitochondrialDNA oxidative damage after transient middle cerebral arteryocclusion in micerdquo Neuroscience Research vol 58 no 4 pp349ndash355 2007

[27] S Yamaguchi M H Hussein G A Daoud et al ldquoEdaravonea hydroxyl radical Scavenger ameliorates the severity of pul-monary hypertension in a porcine model of neonatal sepsisrdquoTohoku Journal of Experimental Medicine vol 223 no 4 pp235ndash241 2011

[28] T Koizumi H Tanaka S Sakaki and S Shimazaki ldquoThetherapeutic efficacy of edaravone in extensively burned ratsrdquoArchives of Surgery vol 141 no 10 pp 992ndash995 2006

[29] Y Sano T Motomura F Yamamoto M Fukuda T Mukaiand M Maeda ldquo1-(31015840-[125I]iodophenyl)-3-methy-2-pyrazolin-5-one preparation solution stability and biodistribution innormal micerdquo Chemical and Pharmaceutical Bulletin vol 58no 8 pp 1020ndash1025 2010


[30] A Sonoda N Nitta A Seko et al ldquoEdaravone prevents bowelinfarction after acute superior mesenteric artery thromboem-bolism using autologous fibrin clots in a rabbit modelrdquo BritishJournal of Radiology vol 82 no 981 pp 711ndash715 2009

[31] E Vandelle and M Delledonne ldquoPeroxynitrite formation andfunction in plantsrdquo Plant Science vol 181 pp 534ndash539 2011

[32] M D Goodman A T Makley N L Huber et al ldquoHypobarichypoxia exacerbates the neuroinflammatory response to trau-matic brain injuryrdquo Journal of Surgical Research vol 165 no 1pp 30ndash37 2011

[33] K Miyamoto H Ohtaki K Dohi et al ldquoEdaravone increasedregional cerebral blood flow after TBIrdquoActa Neurochirurgica Inpress

[34] J I Oyama S Satoh N Suematsu et al ldquoScavenging freeradicals improves endothelial dysfunction in human coronaryarteries in vivordquo Heart and Vessels vol 25 no 5 pp 379ndash3852010

[35] K Dohi K Satoh T Nakamachi et al ldquoNovel free radicalmonitoring in patients with neurological emergency diseasesrdquoActa Neurochirurgica vol 106 pp 315ndash319 2010

[36] M Bains and E D Hall ldquoAntioxidant therapies in traumaticbrain and spinal cord injuryrdquo Biochimica et Biophysica Acta vol1822 pp 675ndash684 2012

[37] W Lo T Bravo V Jadhav E Titova J H Zhang and J TangldquoNADPH oxidase inhibition improves neurological outcomesin surgically-induced brain injuryrdquo Neuroscience Letters vol414 no 3 pp 228ndash232 2007

[38] J Dawson K R Lees C JWeir et al ldquoBaseline serumurate and90-day functional outcomes following acute ischemic strokerdquoCerebrovascular Diseases vol 28 no 2 pp 202ndash203 2009

[39] C W P M Hukkelhoven E W Steyerberg E Farace J DF Habbema L F Marshall and A I R Maas ldquoRegionaldifferences in patient characteristics case management andoutcomes in traumatic brain injury experience from the tiri-lazad trialsrdquo Journal of Neurosurgery vol 97 no 3 pp 549ndash5572002

[40] T Nakase S Yoshioka and A Suzuki ldquoFree radical scavengeredaravone reduces the lesion size of lacunar infarction inhuman brain ischemic strokerdquo BMC Neurology vol 11 article39 2011

[41] H Utsumi K I Yamada K Ichikawa et al ldquoSimultane-ous molecular imaging of redox reactions monitored byOverhauser-enhancedMRI with 14N- and 15N-labeled nitroxylradicalsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 103 no 5 pp 1463ndash1468 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


reported that intravenous edaravone (30mgkg) treatmentimmediately after cortical impact suppressed traumatic neu-ral damage in rodents [13] and decreased hydroperoxide(ROO∙) and alkoxyl (RO∙) radical formation in both rodentsand patients with TBI [13 16] Edaravone treatment at 2hours and again at 12 hours also decreased neuronal lossin a dose-dependent fashion (075 15 or 3mgkg) in theCA3 layer of the hippocampus after TBI [8] while at 3mgkgiv it suppressed apoptotic neuronal cell death and oxidativedamage after TBI [14] In spite of these positive outcomes thetherapeutic time window of edaravone on TBI has not beenexamined in detail

It has been suggested that reactive oxygen species (ROS)generation is activated in the lesion area after TBI leading tothe initial production of superoxide (O2

∙minus) and nitric oxide(∙NO) radicalsThese ROS then react andmetabolize to formstronger oxidants in the form of peroxynitrite (ONOOminus)hydroxyl (∙OH) carbonate (CO3

∙minus) and nitrogen dioxide(∙NO2) radicals [17 18] which in turn react with proteins

lipids sugars and nucleotides and impair the normal phys-iological function of cells Although it is considered that O2

∙minus

does not have strong oxidative potential and that edaravonedoes not scavenge O2

∙minus in vitro [6] we previously reportedthatmice deficient inGp91phox (NOX2) a subunit of NADPHoxidase and a generator of O2

∙minus exhibited reduced lesion sizeand oxidative stress following TBI [19] Moreover knockoutmice lacking interleukin-1 a proinflammatory cytokine wereless susceptible to neuronal cell death than their wild-typelittermates and displayed less inducible nitric oxide synthasegene expression and reduced O2

∙minus and ONOOminus productionduring ischemia [20 21]

In the present study we investigated the therapeutic timewindow of edaravone on TBI and oxidative metabolite gen-eration in mice following a controlled cortical impact (CCI)We also evaluated the temporal profile of ROS productionoxidative stress and neuronal cell death in order to estimatethe relationship between the effect of edaravone on braindamage and the sequence of events leading to ROS generationfollowing CCI

2 Materials and Methods

21 Animals and CCI Model All experimental proceduresinvolving animals were approved by the Institutional AnimalCare and Use Committee of Showa University (00158)Young adult male C57BL6 mice (8ndash12 weeks of age 20ndash26 gper body weight) were anesthetized with 2 sevoflurane inN2OO2(7030) and positioned in a computer-guided

stereotaxic system (Leica Angle Two Leica MicrosystemsWetzlar Germany) incorporating an electromagnetic CCIdevice (Benchmarked Stereotaxic Impactor Leica Microsys-tems) Following a midline scalp incision a 4mm2 openingwas made in the skull 3mm lateral and 2mm caudal ofbregma thereby exposing the right parietotemporal cortex ACCI was carried out at a depth of 10mm from the duramaterat a stroke velocity of 37msecond using an impact devicewith a rounded tip of approximately 12mm in diameter(Figures 1(a) and 1(b)) [22]

After removing hemorrhaged blood resulting from theimpact the skull was covered with 4mm diameter artificialdura (GORE Preclude W L Gore amp Associates Newark NYUSA) and a 5mm diameter artificial bone plate made fromdental cement (GC Fuji I GC Corporation Tokyo Japan)The core body temperature of the mice was maintained at37∘C during the surgery

22 Experimental Design We performed 3 experiments asgiven in the following

Experiment 1 The animals were divided into 4 experimentalgroups to determine the possible therapeutic time windowof edaravone (30mgkg 119899 = 10 for each group) Group 1(Ed 0 h) edaravone was administered immediately (0 hour)after the CCI Group 2 (Ed 3 h) edaravone was administered3 hours following CCI Group 3 (Ed 6 h) edaravone wasadministered 6 hours following CCI Group 4 vehicle (Vh)this served as the control group in which normal saline wasgiven immediately (0 hour) following CCI (119899 = 9) Allanimals were sacrificed 24 hours following CCI (Figure 1(d))

Experiment 2 Mice (119899 = 3 in each group) were divided into 4groups They were sacrificed group 1 immediately (0) group2 3 hours group 3 6 hours and group 4 24 hours after CCIto investigate the temporal profiles of ROS oxidative stressand neuronal death in the brain (Figure 1(e))Experiment 3 Three groups were set (119899 = 3 in each group)to investigate the effect of edaravone treatment to ROS(Figure 1(f)) Group 1 (Ed 0 h) edaravone was administeredimmediately (0 hour) after the CCI and in situ detection ofO2∙minus was performed 4 hours after CCI Group 2 (Ed 3 h)

edaravone was administered 3 hours following CCI and insitu detection of O2

∙minus was performed 4 hours following CCIGroup 3 (Vh 3 h) vehicle was administered 3 hours after CCIand in situ detection of O

2

∙minus was also performed 4 hoursfollowing CCI as the control group

23 Administration of Edaravone Edaravone a free radicalscavenger was a gift fromMitsubishi Tanabe Pharma (OsakaJapan) Animals were placed in the supine position andanesthetized with sevoflurane administered by inhalationthrough a facemaskThe skin over the left clavicle was incisedto expose the left jugular vein Edaravone dissolved in salinewas slowly administered at a dosage of 30mgkg bodyweight(100ndash150120583L volume) into this vein

24 Tissue Preparation Under sodiumpentobarbital (50mgkg ip) anesthesia the animals were transcardially perfusedwith 09 NaCl followed by 2 paraformaldehyde (PFA) in50mM phosphate buffer (pH 72) The brain and skull werethen removed intact and postfixed in 2 PFA overnightafter which the skull was carefully removed and the brainwas immersed in 20 sucrose for 2 days for cytoprotectionThe brain was next frozen in liquid nitrogen-chilled 2-methylbutane and coronally cryosectioned at a thicknessof 50120583m from bregma to approximately 39mm caudal


0 3

minus2

minus4

Peri

Peri Core

(a) (b)


0

1

2

3

4


Impactor

Inju

ry ar

ea (m

m2)

(c)

CCI

Edaravone (Ed)

Vehicle (group 4)


Ed 6 h (group 3)

(3 mgkg iv)

(d)

CCI


0 h 3 h 6 h 24 h

(e)

Edaravone (Ed)

CCI

Ed 0 h (group 1)

Ed 3 h (group 2)



(f)












2O2 the sections






2OO2(7030) and




3 Results






Core

Peri

1 mm

1 mm 50 120583m


(a)

4

3

2

1

00 12 24 36


CorePeri Peri

Inju

ry ar

ea (m

m2)


lowast

lowast

lowast

lowast

lowastlowast

(b)

8

6

4

2

0


Total

Inju

ry v

olum

e (m

m3)

Vehicle 0 h 3 h 6 h

lowast

lowast

(c)

4

3

2

1

0


Core Peri

Inju

ry v

olum

e (m

m3)


lowastlowast

lowast

(d)






Core

Peri

1 mm

1 mm


(a)


0

2

6

4

8

Inju

ry v

olum

e (m

m3)

Vehicle 0 h 3 h 6 h

lowast

lowast

Total

(b)


1

3

2

4

0

Inju

ry v

olum

e (m

m3)


lowastlowast

lowast

Core Peri

(c)









Core

Peri


1 mm

1 mm 400 120583m

(a)

Vehicle


Total

0

2

4

6

8

NT-

posit

ive v

olum

e (m

m3)

0 h 3 h 6 h

lowast

lowast

(b)


Core Peri

2

4

0

1

3


lowast

lowast lowast

lowast

NT-

posit

ive v

olum

e (m

m3)

(c)





4 Discussion



0 h

3 h

100 120583m 500 120583m

6 h

24 h

(a)


6 h24 h

4

3

2

1

00 12 24


Impactor

36

Inju

ry a

rea (

mm

2)

(b)

lowast

lowast

lowast

8

6

4

2

00 3 6 24


Inju

ry v

olum

e (m

m3)

(c)





0 h

3 h

6 h

1 mm

24 h

400 120583m

(a)

lowast

lowast

lowast

8

6

4

2

00 3 6 24


T-po

sitiv

e vol

ume (

mm3)

(b)

GFAP

NT NeuN DAPI Merge

MergeDAPINT

20 120583m

20 120583m

(c)






6 h 24 h

50 120583m

(a)

GFAP MergeEt DAPI

Et DAP MergeNeuN

50 120583m

50 120583m

(b)

50 120583m


(c)







with DAPI


















∙minus




5 Conclusion




Acknowledgments




References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 3

minus2

minus4

Peri

Peri Core

(a) (b)


0

1

2

3

4


Impactor

Inju

ry ar

ea (m

m2)

(c)

CCI

Edaravone (Ed)

Vehicle (group 4)


Ed 6 h (group 3)

(3 mgkg iv)

(d)

CCI


0 h 3 h 6 h 24 h

(e)

Edaravone (Ed)

CCI

Ed 0 h (group 1)

Ed 3 h (group 2)



(f)












2O2 the sections






2OO2(7030) and




3 Results






Core

Peri

1 mm

1 mm 50 120583m


(a)

4

3

2

1

00 12 24 36


CorePeri Peri

Inju

ry ar

ea (m

m2)


lowast

lowast

lowast

lowast

lowastlowast

(b)

8

6

4

2

0


Total

Inju

ry v

olum

e (m

m3)

Vehicle 0 h 3 h 6 h

lowast

lowast

(c)

4

3

2

1

0


Core Peri

Inju

ry v

olum

e (m

m3)


lowastlowast

lowast

(d)






Core

Peri

1 mm

1 mm


(a)


0

2

6

4

8

Inju

ry v

olum

e (m

m3)

Vehicle 0 h 3 h 6 h

lowast

lowast

Total

(b)


1

3

2

4

0

Inju

ry v

olum

e (m

m3)


lowastlowast

lowast

Core Peri

(c)









Core

Peri


1 mm

1 mm 400 120583m

(a)

Vehicle


Total

0

2

4

6

8

NT-

posit

ive v

olum

e (m

m3)

0 h 3 h 6 h

lowast

lowast

(b)


Core Peri

2

4

0

1

3


lowast

lowast lowast

lowast

NT-

posit

ive v

olum

e (m

m3)

(c)





4 Discussion



0 h

3 h

100 120583m 500 120583m

6 h

24 h

(a)


6 h24 h

4

3

2

1

00 12 24


Impactor

36

Inju

ry a

rea (

mm

2)

(b)

lowast

lowast

lowast

8

6

4

2

00 3 6 24


Inju

ry v

olum

e (m

m3)

(c)





0 h

3 h

6 h

1 mm

24 h

400 120583m

(a)

lowast

lowast

lowast

8

6

4

2

00 3 6 24


T-po

sitiv

e vol

ume (

mm3)

(b)

GFAP

NT NeuN DAPI Merge

MergeDAPINT

20 120583m

20 120583m

(c)






6 h 24 h

50 120583m

(a)

GFAP MergeEt DAPI

Et DAP MergeNeuN

50 120583m

50 120583m

(b)

50 120583m


(c)







with DAPI


















∙minus




5 Conclusion




Acknowledgments




References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





2O2 the sections






2OO2(7030) and




3 Results






Core

Peri

1 mm

1 mm 50 120583m


(a)

4

3

2

1

00 12 24 36


CorePeri Peri

Inju

ry ar

ea (m

m2)


lowast

lowast

lowast

lowast

lowastlowast

(b)

8

6

4

2

0


Total

Inju

ry v

olum

e (m

m3)

Vehicle 0 h 3 h 6 h

lowast

lowast

(c)

4

3

2

1

0


Core Peri

Inju

ry v

olum

e (m

m3)


lowastlowast

lowast

(d)






Core

Peri

1 mm

1 mm


(a)


0

2

6

4

8

Inju

ry v

olum

e (m

m3)

Vehicle 0 h 3 h 6 h

lowast

lowast

Total

(b)


1

3

2

4

0

Inju

ry v

olum

e (m

m3)


lowastlowast

lowast

Core Peri

(c)









Core

Peri


1 mm

1 mm 400 120583m

(a)

Vehicle


Total

0

2

4

6

8

NT-

posit

ive v

olum

e (m

m3)

0 h 3 h 6 h

lowast

lowast

(b)


Core Peri

2

4

0

1

3


lowast

lowast lowast

lowast

NT-

posit

ive v

olum

e (m

m3)

(c)





4 Discussion



0 h

3 h

100 120583m 500 120583m

6 h

24 h

(a)


6 h24 h

4

3

2

1

00 12 24


Impactor

36

Inju

ry a

rea (

mm

2)

(b)

lowast

lowast

lowast

8

6

4

2

00 3 6 24


Inju

ry v

olum

e (m

m3)

(c)





0 h

3 h

6 h

1 mm

24 h

400 120583m

(a)

lowast

lowast

lowast

8

6

4

2

00 3 6 24


T-po

sitiv

e vol

ume (

mm3)

(b)

GFAP

NT NeuN DAPI Merge

MergeDAPINT

20 120583m

20 120583m

(c)






6 h 24 h

50 120583m

(a)

GFAP MergeEt DAPI

Et DAP MergeNeuN

50 120583m

50 120583m

(b)

50 120583m


(c)







with DAPI


















∙minus




5 Conclusion




Acknowledgments




References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Core

Peri

1 mm

1 mm 50 120583m


(a)

4

3

2

1

00 12 24 36


CorePeri Peri

Inju

ry ar

ea (m

m2)


lowast

lowast

lowast

lowast

lowastlowast

(b)

8

6

4

2

0


Total

Inju

ry v

olum

e (m

m3)

Vehicle 0 h 3 h 6 h

lowast

lowast

(c)

4

3

2

1

0


Core Peri

Inju

ry v

olum

e (m

m3)


lowastlowast

lowast

(d)






Core

Peri

1 mm

1 mm


(a)


0

2

6

4

8

Inju

ry v

olum

e (m

m3)

Vehicle 0 h 3 h 6 h

lowast

lowast

Total

(b)


1

3

2

4

0

Inju

ry v

olum

e (m

m3)


lowastlowast

lowast

Core Peri

(c)









Core

Peri


1 mm

1 mm 400 120583m

(a)

Vehicle


Total

0

2

4

6

8

NT-

posit

ive v

olum

e (m

m3)

0 h 3 h 6 h

lowast

lowast

(b)


Core Peri

2

4

0

1

3


lowast

lowast lowast

lowast

NT-

posit

ive v

olum

e (m

m3)

(c)





4 Discussion



0 h

3 h

100 120583m 500 120583m

6 h

24 h

(a)


6 h24 h

4

3

2

1

00 12 24


Impactor

36

Inju

ry a

rea (

mm

2)

(b)

lowast

lowast

lowast

8

6

4

2

00 3 6 24


Inju

ry v

olum

e (m

m3)

(c)





0 h

3 h

6 h

1 mm

24 h

400 120583m

(a)

lowast

lowast

lowast

8

6

4

2

00 3 6 24


T-po

sitiv

e vol

ume (

mm3)

(b)

GFAP

NT NeuN DAPI Merge

MergeDAPINT

20 120583m

20 120583m

(c)






6 h 24 h

50 120583m

(a)

GFAP MergeEt DAPI

Et DAP MergeNeuN

50 120583m

50 120583m

(b)

50 120583m


(c)







with DAPI


















∙minus




5 Conclusion




Acknowledgments




References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Core

Peri

1 mm

1 mm


(a)


0

2

6

4

8

Inju

ry v

olum

e (m

m3)

Vehicle 0 h 3 h 6 h

lowast

lowast

Total

(b)


1

3

2

4

0

Inju

ry v

olum

e (m

m3)


lowastlowast

lowast

Core Peri

(c)









Core

Peri


1 mm

1 mm 400 120583m

(a)

Vehicle


Total

0

2

4

6

8

NT-

posit

ive v

olum

e (m

m3)

0 h 3 h 6 h

lowast

lowast

(b)


Core Peri

2

4

0

1

3


lowast

lowast lowast

lowast

NT-

posit

ive v

olum

e (m

m3)

(c)





4 Discussion



0 h

3 h

100 120583m 500 120583m

6 h

24 h

(a)


6 h24 h

4

3

2

1

00 12 24


Impactor

36

Inju

ry a

rea (

mm

2)

(b)

lowast

lowast

lowast

8

6

4

2

00 3 6 24


Inju

ry v

olum

e (m

m3)

(c)





0 h

3 h

6 h

1 mm

24 h

400 120583m

(a)

lowast

lowast

lowast

8

6

4

2

00 3 6 24


T-po

sitiv

e vol

ume (

mm3)

(b)

GFAP

NT NeuN DAPI Merge

MergeDAPINT

20 120583m

20 120583m

(c)






6 h 24 h

50 120583m

(a)

GFAP MergeEt DAPI

Et DAP MergeNeuN

50 120583m

50 120583m

(b)

50 120583m


(c)







with DAPI


















∙minus




5 Conclusion




Acknowledgments




References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Core

Peri


1 mm

1 mm 400 120583m

(a)

Vehicle


Total

0

2

4

6

8

NT-

posit

ive v

olum

e (m

m3)

0 h 3 h 6 h

lowast

lowast

(b)


Core Peri

2

4

0

1

3


lowast

lowast lowast

lowast

NT-

posit

ive v

olum

e (m

m3)

(c)





4 Discussion



0 h

3 h

100 120583m 500 120583m

6 h

24 h

(a)


6 h24 h

4

3

2

1

00 12 24


Impactor

36

Inju

ry a

rea (

mm

2)

(b)

lowast

lowast

lowast

8

6

4

2

00 3 6 24


Inju

ry v

olum

e (m

m3)

(c)





0 h

3 h

6 h

1 mm

24 h

400 120583m

(a)

lowast

lowast

lowast

8

6

4

2

00 3 6 24


T-po

sitiv

e vol

ume (

mm3)

(b)

GFAP

NT NeuN DAPI Merge

MergeDAPINT

20 120583m

20 120583m

(c)






6 h 24 h

50 120583m

(a)

GFAP MergeEt DAPI

Et DAP MergeNeuN

50 120583m

50 120583m

(b)

50 120583m


(c)







with DAPI


















∙minus




5 Conclusion




Acknowledgments




References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 h

3 h

100 120583m 500 120583m

6 h

24 h

(a)


6 h24 h

4

3

2

1

00 12 24


Impactor

36

Inju

ry a

rea (

mm

2)

(b)

lowast

lowast

lowast

8

6

4

2

00 3 6 24


Inju

ry v

olum

e (m

m3)

(c)





0 h

3 h

6 h

1 mm

24 h

400 120583m

(a)

lowast

lowast

lowast

8

6

4

2

00 3 6 24


T-po

sitiv

e vol

ume (

mm3)

(b)

GFAP

NT NeuN DAPI Merge

MergeDAPINT

20 120583m

20 120583m

(c)






6 h 24 h

50 120583m

(a)

GFAP MergeEt DAPI

Et DAP MergeNeuN

50 120583m

50 120583m

(b)

50 120583m


(c)







with DAPI


















∙minus




5 Conclusion




Acknowledgments




References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 h

3 h

6 h

1 mm

24 h

400 120583m

(a)

lowast

lowast

lowast

8

6

4

2

00 3 6 24


T-po

sitiv

e vol

ume (

mm3)

(b)

GFAP

NT NeuN DAPI Merge

MergeDAPINT

20 120583m

20 120583m

(c)






6 h 24 h

50 120583m

(a)

GFAP MergeEt DAPI

Et DAP MergeNeuN

50 120583m

50 120583m

(b)

50 120583m


(c)







with DAPI


















∙minus




5 Conclusion




Acknowledgments




References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



6 h 24 h

50 120583m

(a)

GFAP MergeEt DAPI

Et DAP MergeNeuN

50 120583m

50 120583m

(b)

50 120583m


(c)







with DAPI


















∙minus




5 Conclusion




Acknowledgments




References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










∙minus




5 Conclusion




Acknowledgments




References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Therapeutic Time Window for Edaravone Treatment of Traumatic Brain ...downloads.hindawi.com/journals/bmri/2013/379206.pdf · 2019. 7. 31. · In spite of the fact