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b r a i n r e s e a r c h 1 5 2 7 ( 2 0 1 3 ) 2 4 6 – 2 5 4

0006-8993/$ - see frohttp://dx.doi.org/10.

Abbreviations: DI

PI, propidium iodinCorresponding aE-mail addresses:

Research Report

Role of histidine/histamine in carnosine-inducedneuroprotection during ischemic brain damage

Ok-Nam Baea,b, Arshad Majida,c,n

aDivision of Cerebrovascular Diseases and Department of Neurology and Ophthalmology, Michigan State University,East Lansing, MI, United StatesbCollege of Pharmacy and Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan,Republic of KoreacDepartment of Neurology and Manchester Academic Health Sciences Centre, Salford Royal Hospital, Stott Lane,Salford, England M6 8HD, United Kingdom

a r t i c l e i n f o

Article history:

Accepted 3 July 2013

Urgent need exists for new therapeutic options in ischemic stroke. We recently demonstrated

that carnosine, an endogenous dipeptide consisting of alanine and histidine, is robustly

Available online 11 July 2013

Keywords:

Carnosine

Neuroprotection

Ischemic stroke

Histidine

Histamine

nt matter & 2013 Publish1016/j.brainres.2013.07.00

V, days in vitro; LDH, l

de; pMCAO, permanentuthor at: Department ofmajidarshad@msn.com,

a b s t r a c t

neuroprotective in ischemic brain injury and has a wide clinically relevant therapeutic time

window. The precise mechanistic pathways that mediate this neuroprotective effect are not

known. Following in vivo administration, carnosine is hydrolyzed into histidine, a precursor of

histamine. It has been hypothesized that carnosine may exert its neuroprotective activities

through the histidine/histamine pathway. Herein, we investigated whether the neuroprotec-

tive effect of carnosine is mediated by the histidine/histamine pathway using in vitro primary

astrocytes and cortical neurons, and an in vivo rat model of ischemic stroke. In primary

astrocytes, carnosine significantly reduced ischemic cell death after oxygen–glucose depriva-

tion, and this effect was abolished by histamine receptor type I antagonist. However, histidine

or histamine did not exhibit a protective effect on ischemic astrocytic cell death. In primary

neuronal cultures, carnosine was found to be neuroprotective but histamine receptor

antagonists had no effect on the extent of neuroprotection. The in vivo effect of histidine

and carnosine was compared using a rat model of ischemic stroke; only carnosine exhibited

neuroprotection. Taken together, our data demonstrate that although the protective effects of

carnosine may be partially mediated by activity at the histamine type 1 receptor on astrocytes,

the histidine/histamine pathway does not appear to play a critical role in carnosine induced

neuroprotection.

& 2013 Published by Elsevier B.V.

ed by Elsevier B.V.4

actate dehydrogenase; NMDA, N-methyl-d-aspartate; OGD, oxygen–glucose deprivation;

middle cerebral artery occlusion; tPA, tissue plasminogen activatorNeurology, Salford Royal Hospital, Stott Lane, Salford, England M6 8HD, United Kingdom.arshad.majid@srft.nhs.uk (A. Majid).

1. Introduction

Tissue plasminogen activator (tPA) is the only approvedacute drug treatment for ischemic stroke. However, the use

of tPA is still limited to 3–5% of all stroke patients largely dueto its short therapeutic time window. Although therehave been vigorous efforts to develop new treatments forischemic stroke, all candidate agents have failed to show

b r a i n r e s e a r c h 1 5 2 7 ( 2 0 1 3 ) 2 4 6 – 2 5 4 247

efficacy in human clinical trials (Fisher, 2011; Green, 2008;Lo et al., 2003).

Recently, we have reported that carnosine, an endogenousdipeptide consisting of alanine and histidine, significantlyreduced the extent of brain injury after ischemic stroke (Baeet al., 2013). Intravenous administration of carnosine reducedbrain injury in both permanent- and transient-ischemic ratmodels. In addition, carnosine exhibited a wide clinicallyrelevant therapeutic time window, without side effects ortoxicity. Others have also reported beneficial cerebroprotec-tive effects of carnosine. Carnosine improved neurologicalfunction, decreased mortality, and enhanced functional out-come after global ischemia in gerbil and rat models (Dobrotaet al., 2005; Gallant et al., 2000), and attenuated brain damagein hypoxic neonatal brain (Zhang et al., 2011). Notably, amongseveral carnosine analogues, carnosine was found to be themost effective (Min et al., 2008; Rajanikant et al., 2007).

There have been several attempts to elucidate the mechan-isms that mediate the protective effects of carnosine. Carno-sine exhibits pleiotropic properties that are thought to beneuroprotective such as antioxidant, heavy metal chelatingand anti-excitotoxic activities (Guiotto et al., 2005; Hipkiss,2009; Hipkiss et al., 2013; Horning et al., 2000; Trombley et al.,2000; Green and Shuaib, 2006). However, to further developcarnosine as a therapeutic agent, a better and more completeunderstanding of the mechanisms that mediate neuroprotec-tion are needed.

Interestingly, several studies on carnosine bioactivity havefocused on the role of histidine and histamine, which aremetabolites of carnosine. Following in vivo administration,carnosine can be easily broken down into alanine and histi-dine by the action of carnosinases (Teufel et al., 2003).Histidine is a precursor of histamine, a bioactive amino acidin the nervous system. Mice deficient in histamine or thehistamine receptor exhibited impaired cognition and beha-vioral function (Dai et al., 2007; Dere et al., 2003). Publisheddata also suggested that histamine may reduce ischemic braininjury in cultured neuronal cells and animal models (Adachi,2005; Dai et al., 2006; Hamami et al., 2004; Lozada et al., 2005).Despite its presence in the brain, histamine is not transported

Fig. 1 – Carnosine and histidine levels in serum following intrave2000 mg/kg) was dissolved in saline, and administered intravenwas collected from catheterized femoral vein. The level of carnoacid analyzer. Rats in the control group were administered with

from plasma to brain, but instead is derived from histidine bythe action of histidine decarboxylase (Adachi et al., 2005, Prellet al., 1996). The synthesis of histaminemostly depends on theconcentration of histidine; and histidine loading has beenshown to increase the level of histamine in brain (Prell et al.,1996; Schwartz et al., 1972). Since treatment with carnosineincreases the availability of histidine (Flancbaum et al., 1990;Nagai et al., 2012), it has been hypothesized that the protectiveeffects of carnosine may be mediated through histidine/histamine signaling. However, the exact contribution of thehistaminergic pathway in carnosine-mediated neuroprotec-tion still remains unclear.

In the present study, we have investigated whether thehistidine/histamine pathway plays a key role in carnosine-mediated neuroprotection against ischemic brain injury.Primary cortical astrocytes and cortical neurons were usedto evaluate neurotoxicity following exposure to NMDA oroxygen–glucose deprivation. In vivo activity was examinedusing the rat permanent middle cerebral artery occlusionmodel. Our data provide new insights into the mechanismsthat underlie carnosine neuroprotection. We have demon-strated that although the neuroprotective activity of carno-sine may be partially mediated through H1 receptors onastrocytes, it is not, however, dependent on conversion ofcarnosine to histidine and histamine.

2. Results

2.1. The level of carnosine and histidine after in vivocarnosine administration

Following in vivo administration, carnosine can be hydrolyzedinto alanine and histidine by carnosinases (Teufel et al., 2003).We measured both carnosine and histidine levels after intrave-nous administration of carnosine to examine if administrationof carnosine affects histidine levels. Intravenous administrationof carnosine resulted in increased levels of carnosine in serumin a dose-dependent manner. Subsequently, carnosine concen-trations in serum fell rapidly (Fig. 1A). Of note, the serum levels

nous administration of carnosine. (A) and (B) Carnosine (0 toously. At each time points after carnosine treatment, bloodsine (A) or histidine (B) in serum was analyzed using aminosaline as vehicle. n¼7.

Table 1 – Kinetic parameters in carnosine and histidine.

T1/2 (h) V (L) K (1/h) Cl (L/h)

Carnosine 0.3170.09 65.1722.7 2.4570.75 145.1719.4Histidine 2.1271.70 2979.672383.2 0.4570.23 1534.97966.1

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of histidine increased according to the dose carnosine that wasadministered (Fig. 1B). Interestingly, the clearance of histidinewas found to be slower compared to carnosine (Fig. 1 andTable 1). Based on these findings, we had hypothesized that theprotective effect of carnosine may be mediated through theactivity of histidine/histamine pathway.

2.2. Carnosine neuroprotection against astrocytic injury

To elucidate the role of the histaminergic pathway, weexamined the neuroprotective effect of carnosine in primaryastrocytes. Primary cortical astrocytes were exposed to oxy-gen–glucose deprivation (OGD) for 6 h, and subsequent cellinjury and death were examined by the extent of LDH releaseand PI staining. As expected, OGD resulted in astrocytic celldeath, and pretreatment with carnosine (0 to 100 μM) for30 min reduced OGD-induced cytotoxicity (Fig. 2A and B).To examine the involvement of the histamine pathway incarnosine-mediated protection, we used specific antagonistsfor each of the three histamine receptors, H1, H2 and H3.Astrocytes were pretreated with pyrilamine, cimetidine, orthioperamide (100 μM each) for 30 min prior to carnosine, toblock H1, H2 or H3 histamine receptors, respectively (Fiorettiet al., 2009). Protective activity of carnosine against OGD-induced astrocytic death was only significantly attenuated bypyrilamine, suggesting that H1 receptors may partially med-iate the neuroprotective activity of carnosine in astrocytes(Fig. 2C and D). Exposure of astrocytes to histamine receptorantagonists alone did not affect cell viability or vulnerabilityto OGD-induced injury (Fig. 2E).

2.3. The effects of histidine and histamine on ischemicastrocytic death

To further characterize the role of the histaminergic pathwayin carnosine-mediated protection, the effects of carnosine,histidine, and histamine (0 to 100 μM) were compared inOGD-induced astrocytic death. While carnosine reducedOGD-cytotoxicity, significant protection was not observed inhistidine- or histamine-pretreated astrocytes (Fig. 3A). Block-ade of histamine receptors did not influence the lack ofactivity of histidine or histamine (Fig. 3B and C).

2.4. The effect of carnosine on OGD- or NMDA-inducedprimary neuronal death

We also determined the effect of carnosine in primarycortical neurons and whether the histaminergic pathway isinvolved. OGD or excitotoxic exposure to NMDA (25 μM) wereused to induce neuronal damage. Pretreatment with carno-sine (0 to 100 μM) significantly inhibited OGD-induced neuro-nal death (Fig. 4A). However, histamine receptor antagonistshad no effect (Fig. 4B). Carnosine was also protective against

NMDA-induced excitotoxicity (Fig. 4C). The blockade of his-tamine receptors did not modulate carnosine-mediated neu-roprotection against NMDA stimulation (Fig. 4D).

2.5. The influence of carnosine or histidine in rat focalischemia

Next, we examined the role of the histidine/histamine path-way in a rat model of acute stroke. Carnosine (1000 mg/kg) orhistidine (1000 mg/kg) was administered intravenously at30 min prior to ischemia onset which was induced by perma-nent middle cerebral artery occlusion (pMCAO). IntraluminalpMCAO induced reproducible infarcts in saline-treated rats(47.0777.63% of the hemisphere). Carnosine administrationsignificantly reduced infarct volumes (26.2775.63% of thehemisphere) by 44.2% compared to saline-treated rats. Nota-bly, histidine had no effect on infarct volume (50.2976.94% ofinfarcted hemisphere), suggesting that carnosine but nothistidine is protective against ischemic brain damage (Fig. 5).

3. Discussion

We investigated whether carnosine-induced neuroprotectionis mediated by the histaminergic signaling pathway.Although our data suggest that carnosine may exert itsneuroprotective activity in part through H1 histamine recep-tors in astrocytes; histidine and histamine by themselves didnot influence astrocytic injury induced by OGD. In primaryneurons, we demonstrated a profound neuroprotective effectwith carnosine which was not affected by blockade ofhistamine receptors. Consistent with our in vitro findings,histidine had no effect on brain damage after pMCAO, whilecarnosine significantly reduced brain infarction. Takentogether, we have demonstrated that metabolism of carno-sine to histidine and histamine does not appear to be asignificant pathway although carnosine may partially exertits neuroprotective through H1 receptors.

Supporting our view, several other reports have shownthat the carnosine–histidine–histamine pathway dose notcontribute to the protective effect of carnosine. In a recentpaper, carnosine protection against ischemic damage waspreserved in mice lacking histidine decarboxylase, whichconverts histidine to histamine (Shen et al., 2010). Fu et al.(2008) demonstrated that the attenuated neurotoxicity ofamyloid beta by carnosine was not mediated by the histi-dine–histamine pathway in rat PC12 cells. Conversely, Shenet al. (2007) showed that NMDA-induced neurotoxicity indifferentiated rat PC12 was attenuated by carnosine throughhistidine–histamine pathway. It is likely that the difference inthe findings of Shen et al. and our findings may be due to thedifferent types of cells used.

Fig. 2 – Involvement of histamine receptors in carnosine cerebroprotection against oxygen–glucose deprivation (OGD)-inducedcell death in primary astrocytes. (A) and (B) Pretreatment with carnosine (1 to 100 μM) for 30 min significantly reducedastrocytic death induced by 6 h OGD, determined by the extent of lactate dehydrogenase (LDH) leakage (A) or staining withpropidium iodide (PI) (B). (C) and (D) To block histamine receptors, astrocytes were pretreated with pyrilamine (PY; 100 μM),cimetidine (CIM; 100 μM), or thioperamide (THIO; 100 μM) for 30 min prior to carnosine, against H1, H2 or H3 histaminereceptor, respectively. LDH leakage (C) or PI staining (D) was examined for astrocytic cytotoxicity. (E) Effects of H receptorantagonists on astrocytic viability. npo0.05 and nnpo0.01 vs. OGD-exposed group without carnosine. #po0.05 vs. OGD-exposed group with carnosine pretreatment. (A) n¼4, (B) n¼4, (C) n¼3, (D) n¼4, (E) n¼3. All values are means7SEM andanalyzed by ANOVA tests ((A) and (B)) or Student′s t-test ((C) to (E)).

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The potential of carnosine as a new multimodal endogen-ous molecule has been recognized in recent reviews, leadingto significant interest in possible pharmaceutical and nutri-tional applications of carnosine and/or its derivatives in

various diseases (Barski et al., 2013; Bellia et al., 2011, 2012;Hipkiss, 2009; Hipkiss et al., 2013). Carnosine has anti-inflam-matory, anti-cancer, and immuno-modulating activities. Inneurological disease models, carnosine exhibited significant

Fig. 3 – Effects of carnosine, histidine, or histamine on astrocytic cell death induced by OGD. (A) Primary astrocytes werepretreated with carnosine (0 to 100 μM), histidine (0 to 100 μM) or histamine (0 to 100 μM) for 30 min, and ischemic cell deathwas induced by OGD for 6 h. Astrocytic death was determined by PI staining at 24 h after OGD exposure. (B) and (C) Astrocyteswere pre-treated with histamine receptor antagonists, pyrilamine (PY; 100 μM), cimetidine (CIM; 100 μM), or thioperamide(THIO; 100 μM), 30 min prior to histidine (100 μM; (B)) or histamine (100 μM; (C)), and the cells were exposed to OGD stimulationfor 6 h. npo0.05 vs. control group. (A) n¼3–4, (B) n¼3, (B) n¼3. All values are means7SEM and analyzed by Student′s t-test.

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protective activities against neurodegenerative diseases suchas Alzheimer′s disease (Corona et al., 2011), Parkinson′sdisease (Boldyrev et al., 2008) and dementia (Ma et al., 2012),improving survival and learning ability. In ischemic braininjury, we and other researchers have shown that histologicaland behavioral functional damage were reduced by carnosinetreatment. The beneficial effects of carnosine have beenattributed to modulation of intracellular signaling, extracellu-lar environments, including anti-oxidative (Bellia et al., 2011;Guiotto et al., 2005; Kohen et al., 1988) and anti-nitosative(Calabrese et al., 2005), metal-chelating (Trombley et al., 2000),mitochondrial preserving (Bae et al., 2013), and anti-excitotoxic(Shen et al., 2010) activities.

Based on its biological functions, there has also been wideinterest in developing carnosine derivatives to increase ther-apeutic actions as well as to improve its resistance to carno-sinase, which hydrolyzes carnosine to histidine and alanine.Our findings show that the beneficial effects of carnosine maynot rely on hydrolysis to histidine/histamine. This suggeststhat carnosine derivatives which exhibit resistance to carno-sinases might be promising candidates for further evaluation(Vistoli et al., 2012).

In summary, we have demonstrated that although theneuroprotective activity of carnosine is partially mediatedthrough H1 activity on astrocytes, it is not, however, dependenton conversion of carnosine to histidine and histamine. Webelieve that our findings provide important mechanistic cluesto better understand carnosine-mediated neuroprotection.

4. Experimental procedures

4.1. Primary astrocytes/neurons

Primary cortical neuronal and astrocytic cultures were estab-lished as previously described (Bae et al., 2012). Cell culturemedia and reagents including Neurobasal A, B27, DMEM,glutamine, and penicillin/streptomycin were purchased fromInvitrogen (Carlsbad, CA).

4.1.1. Primary astrocytic culturesPrimary cultures of astrocytes were prepared using cerebralcortices isolated from C57BL/6 newborn mice at postnatal day1 and dissociated in dissection media (81.8 mM Na2SO4,

Fig. 4 – The role of histamine receptors in carnosine neuroprotection in primary cortical neurons. (A) and (B) Neurons werepre-treated with carnosine (0 to 100 μM) 30 min prior to OGD (2 h)-induced cell death in primary cortical neurons (A). Neuronswere pre-treated with antagonists against histamine receptors 30 min prior to carnosine treatment (B). Neuronal cell deathwas determined by LDH leakage at 24 h after OGD stimulation. (C) and (D) Effect of carnosine (C) or influence of histaminereceptors in carnosine neuroprotection (D) was examined in NMDA-induced neuronal cell death. npo0.05 and nnpo0.01 vs.control group. (A) n¼3, (B) n¼3–4, (C) n¼4, (D) n¼3–4. All values are means7SEM and analyzed by ANOVA tests ((A) and (C)) ort-test ((B) and (D)).

Fig. 5 – Protective effect of carnosine or histidine against brain damage in ischemic rat models. Thirty minutes after singleintravenous administration of saline, carnosine (1000 mg/kg) or histidine (1000 mg/kg) to rats, brain ischemia was initiated bypermanent middle cerebral artery occlusion (pMCAO). Brain damage was examined by TTC staining at 24 h after pMCAO, andanalyzed by ImageJ program. npo0.05 vs. saline-treated group. n¼10. All values are means7SEM and analyzed by Student′st-test. Representative photos are shown.

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30 mM K2SO4, 5.8 mM MgCl2, 0.252 mM CaCl2, 1.5 mM HEPES,20mM glucose, and 0.001% phenol red, pH 7.6) containing4 mM L-cysteine, 10 U/ml papain (Worthington), and 1000 U/ml

DNase (Roche) for 30min at 37 1C. Dissociated cells werewashed and triturated with a pipette, and the cells were platedin flasks coated with poly-D-lysine using Dulbecco′s modified

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Eagle′s medium (DMEM) supplemented with 10% fetal bovineserum and penicillin/streptomycin. Seven days after plating,the flasks were shaken for 4 h at 37 1C to dislodge contami-nated cells such as oligodendroglial and microglial cells thatwere loosely attached to the astrocyte monolayer. On daysin vitro (DIV) 14, astrocytes were detached by trypsinizationand plated on poly-D-lysine precoated wells. Astrocytes wereused between DIV 18-21, when they reach maximal sensitivityto OGD-induced cytotoxicity. These cultures contained 490%astrocytes as revealed by immunohistochemistry against glialfibrillary acidic protein (GFAP).

4.1.2. Primary neuronal culturesCortical neuronal cultures were established using similarprocedures as astrocytes. Cerebral cortices were isolated fromnewborn C57BL/6J mice at post-natal day 0 (Bae et al., 2013).Dissociated cells were washed with Neurobasal A and tritu-rated with a pipette, and plated onto poly-D-lysine-precoatedplates (Jeon et al., 2012). Three days after plating, 50% of themedium was changed and subsequently replaced every threedays with Neurobasal A supplemented with 2% B27. Neuronalcultures were maintained in a CO2 incubator at 37 1C in 5%CO2, and used between DIV 7 and 11. These cultures con-tained 490% neurons as revealed by NeuN/beta tubulin-immunohistochemistry.

4.2. In vitro ischemic insults

The protective effect of carnosine against neuronal cytotoxi-city was measured after oxygen–glucose deprivation (OGD)-or N-methyl-d-aspartate (NMDA)-stimulation (Kim et al.,2012; Panickar et al., 2008). Primary astrocytes were exposedto OGD to simulate the ischemic state. On DIV 18-21, theculture medium was replaced by glucose-free Earle′sbalanced salt solution (EBSS), and the cells were placed inan anaerobic chamber (Billups-Rothenberg Inc, Del Mar, CA)saturated with 5% CO2 and 95% N2 for 6 h. OGD was termi-nated by switching back to normal culture conditions. Controlcells were incubated in EBSS with glucose in a normoxicincubator for the same period. OGD in neurons was per-formed using the same procedure as astrocytes, except thecells were used on DIV 9 and OGD was applied for only 2 h.Cytotoxicity was determined at 24 h after OGD. For NMDAtoxicity studies, primary neuronal cells were treated withNMDA-containing media and incubated at 37 1C for 20 min onDIV 9. Exposure to NMDA was terminated by replacementwith the original media collected before NMDA treatment.NMDA-induced cytotoxicity was measured at 24 h afterNMDA exposure. To examine carnosine protection, cells werepretreated with carnosine for 30 min prior to ischemicinsults. To block histamine receptors, pyrilamine, cimetidineor thioperamide (Sigma) specific antagonists for H1, H2, or H3histamine receptors, respectively, were added 30 min beforecarnosine treatment.

4.3. Determination of cytotoxicity

OGD- or NMDA-induced cytotoxicity was determined usingpropidium iodide (PI)-staining or lactate dehydrogenase (LDH)assay. Briefly, at 24 h following OGD- or NMDA-exposure,

cells were stained with 5 μg/mL PI at 37 1C for 30 min, andexamined by fluorescent microplate reader (Ascent, ThermoLab systems, Franklin, MA). To detect the loss of membraneintegrity, a typical marker of cytotoxicity, the extent of LDHrelease was measured in conditioned media using the Cyto-tox 96R Non-Radioactive Cytotoxicity Assay (Promega Cor-poration). Cell viability in sister cells treated with 100 μMNMDA, which induces near complete neuronal death, wasused as the total cell death (100%).

4.4. In vivo rat studies

In vivo animal experiments were conducted using adult maleSprague-Dawley rats weighing 250 to 300 g (Harlan) andperformed in accordance with the NIH Policy and AnimalWelfare Act under the approval by Institutional Animal Careand Use Committee (IACUC) at Michigan State University.Treatment groups were allocated in a randomized fashionusing a Researcher Randomizer Program (http://www.randomizer.org/). Investigators were blind to treatment duringsurgeries and outcome evaluations.

4.4.1. Permanent middle cerebral artery occlusion (pMCAO) inratsPermanent focal cerebral ischemia was induced using asilicone-coated intraluminal filament (Doccol Co.), as pre-viously described (Bae et al., 2013). General anesthesia wasinduced by isoflurane inhalation, and rats were maintainedunder anesthesia throughout the surgical period. Rectaltemperature was maintained at 37 1C and the cerebral bloodflow (CBF) was monitored with laser Doppler (Perimed, NorthRoyalton, OH). The left common carotid artery (CCA) and theexternal carotid artery (ECA) were exposed and ligated by asuture. The occipital artery of the ECA was coagulated. Theinternal carotid artery (ICA) was exposed and the pterygopa-latine artery was ligated. Ischemia was produced by advan-cing a silicone-coated 4–0 monofilament nylon suture. Themonofilament was inserted into the CCA and advanced intothe ICA to the origin of the MCA. Rats were excluded from thestudy if the CBF was not decreased below 30% of baselineafter occlusion. Saline or carnosine (1000 mg/kg B W) or his-tidine (1000 mg/kg BW) were administered over 3 min intothe lateral tail vein at 30 min prior to the occlusion of themiddle cerebral artery.

4.4.2. Calculation of infarct volumeAt 24 h after onset of ischemia, rats were euthanized byisoflurane overdose, and decapitated. Brains were rapidlyremoved and cut into 2 mm sections, stained with 2% triphe-nyltetrazolium chloride (TTC), and fixed in 4% paraformalde-hyde (Saleem et al., 2008). Each section was scanned to adigital image, and analyzed using the NIH ImageJ software.The infarct volume for each section was determined andedema correction was performed by measurement of theinfarcted and control hemisphere.

4.4.3. Serum kinetic studiesMale SD rats were randomly divided into five groups (n¼7rats/group) and given a single intravenous bolus injection ofsaline or 100, 500, 1000, or 2000 mg/kg B W carnosine. Blood

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samples were drawn from femoral vein catheter beforeadministration and at 15 min, 1 h, 3 h, 6 h, 12 h, and 24 hpost-administration of carnosine. The concentration of car-nosine or histidine in serum at each time point was mea-sured using high-performance liquid chromatography (HPLC)on a Hitachi Model L-8800 Amino Acid Analyzer as previouslydescribed (Min et al., 2008).

4.5. Statistics

We calculated the means and standard errors of means (SEM)for all treatment groups. The data were subjected to Studentt-test or one-way ANOVA followed by Duncan′s test todetermine the significant differences between treatmentgroups. Statistical analysis was performed using SPSS software(Chicago, IL). In all cases, a p value of o0.05 was consideredsignificant.

Acknowledgment

This study was supported by the AHA grant to Arshad MajidMD and by Basic Science Research Program through theNational Research Foundation of Korea (NRF) to Ok-NamBae (2012R1A1A3013240). We thank Kelsey Serfozo andDr. Gary Stein for their technical assistance.

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