European Journal of Pharmacology 638 (2010) 65–71

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European Journal of Pharmacology

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Neuropharmacology and Analgesia

Pharmacological evaluation of glutamate transporter 1 (GLT-1) mediatedneuroprotection following cerebral ischemia/reperfusion injury☆

Rajkumar Verma a, Vikas Mishra a, Dinakar Sasmal b, Ram Raghubir a,⁎a Division of Pharmacology, Central Drug Research Institute (CDRI), P.O. Box 173, Lucknow (U.P.) 226001 (CSIR), Indiab Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi, 835215, India

☆ CDRI communication no: 7819.⁎ Corresponding author. Division of Pharmacology, C

P.O. Box 173, Lucknow (U.P.) 226001, India. Tel.: +991E-mail address: raghubirr@yahoo.com (R. Raghubir)

0014-2999/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.ejphar.2010.04.021

a b s t r a c t

a r t i c l e i n f o

Article history:Received 5 November 2009Received in revised form 12 March 2010Accepted 1 April 2010Available online 24 April 2010

Keywords:Glutamate transporter-1CeftriaxoneCerebral ischemiaGlutamine synthetaseDihydrokainateNeuroprotection

Recently glutamate transporters have emerged as a potential therapeutic target in a wide range of acute andchronic neurological disorders, owing to their novel mode of action. The modulation of GLT-1, a majorglutamate transporter has been shown to exert neuroprotection in various models of ischemic injury andmotoneuron degeneration. Therefore, an attempt was made to explore its neuroprotective potential incerebral ischemia/reperfusion injury using ceftriaxone, a GLT-1 modulator. Pre-treatment with ceftriaxone(100 mg/kg. i.v) for five days resulted in a significant reduction (Pb0.01) in neurological deficit as well ascerebral infarct volume after 1 h of ischemia followed by 24 h of reperfusion injury. It also caused asignificant (Pb0.05) upregulation of GLT-1 mRNA, protein and glutamine synthetase (GS) activity.Furthermore, inhibition of ceftriaxone-mediated increased glutamine synthetase activity by dihydrokainate(DHK), a GLT-1 specific inhibitor, confirms the specific effect of ceftriaxone on GLT-1 activity. In addition,ceftriaxone also induced a significant (Pb0.01) increase in [3H]-glutamate uptake, mediated by GLT-1 in glialenriched preparation, as evidenced by use of DHK and DL-threo-beta-benzyloxyaspartate (DL-TBOA). Thus,the present study provides overwhelming evidence that modulation of GLT-1 protein expression and activityconfers neuroprotection in cerebral ischemia/reperfusion injury.

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© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Glutamate induced excitotoxicity and ionic imbalance are themajorearly events of ischemic brain stress, which affect neuronal, non-neuronal and vascular elements in the affected brain territory. Ischemia-induced energy shortage results in excessive release of glutamate fromneurons and subsequent decreased uptake of glutamate by astrocytescauses an unwarranted exposure of neurons to glutamate leading toglutamate-mediated excitotoxicity (Anderson et al., 2003).

In order to protect neurons from glutamate excitotoxicity, neuronsand/or astrocytes must remove excessive glutamate from the extracel-lular space. Astrocytic glutamate transporters (GluTs) play a dominantrole in extracellular glutamate removal (Rothstein et al., 1996). Theuptake of glutamate is mainly achieved by a family of five subtypes ofsodium dependent transporters. Among these Excitatory Amino AcidTransporter 1 (EAAT 1) or Glutamate Aspartate Transporter (GLAST) andExcitatoryAminoAcidTransporter2 (EAAT2)orGlutamate transporter 1(GLT-1) play a major role, the latter may account for more than 90% ofglutamate uptake in adult forebrain (Danbolt, 2001). The dysfunction ofglutamate transporters has been implicated in the pathogenesis of

various debilitative neurological disorders of diverse etiology, includingamyotrophic lateral sclerosis, Alzheimer's disease, several forms ofepilepsy, ischemia/stroke and traumatic brain injury (Lee et al., 2008).Thus drugs and agents that increase glutamate transport activity, wouldact as powerful tool for enhancing the clearance of glutamate in suchpathological conditions. Several in vitroand in vivo studies have shownanupregulation and increased biological activity of EAAT 1 and 2 by severaldrugs/molecules such as MS-153, 522, dibutyryl cyclic (dbc)AMP andpituitary adenylate cyclase-activating polypeptide (PACAP), citicoline,arundic acid (ONO-2506) and beta-lactam antibiotics (ceftriaxone)(Beschorner et al., 2007a,b).

Glutamine synthetase (GS) is also considered a critical enzyme for theregulation and scavenging of extracellular glutamate in the brain. Thepost-ischemic increase in astrocytic GS activity suggests that the capacityto take up glutamate and to convert glutamate to glutamine is enhancedafter ischemia (Hoshi et al., 2006). It seems that there should be aneffective glutamate–glutamine cycle regulation between glutamateuptake and glutamine synthesis in astrocytes.We havemade an attemptto analyze this by usingGLT-1 specific inhibitor of glutamate uptakeDHKto establish a link betweenGLT-1 upregulation and enhancedGS activity.

After the pioneer work of Rothstein et al. (2005) in relation to GLT-1mediated neuroprotection by beta-lactam antibiotics in an animalmodel of amyotrophic lateral sclerosis disease, several other groupsinstantly studied and tried to establish the possible mode of action ofbeta-lactam antibiotics, specially ceftriaxone (Rothstein et al., 2005 and

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Lipski et al., 2007). At the outset, efforts were made to establish thedefinite role of GLT-1 in neuroprotection using in vivo model of focalcerebral ischemia. Hence, our approach was initially to establish GLT-1as a therapeutic target for neuroprotection and additionally to correlateits increased activity with glutamine synthetase activity.

2. Materials and methods

2.1. Animals

Male Sprague–Dawley albino rats, weighing 230–250 g, wereprocured from National Laboratory Animal Centre (NALC) of CentralDrugResearch Institute Lucknow, India andwereused in all experiments.They were allowed free access to food andwater andmaintained at 12 hday/night cycle. The approved standard procedures and the institutionalanimal ethical committee guidelines were followed, throughout theexperiments.

2.2. Experimental protocol

Animals were anaesthetized with chloral hydrate (300 mg/kg i.p.)and placed in a supine position over an operation table with heatingdevice to maintain a constant body temperature of 37±0.5 °C. Focalcerebral ischemia was induced by middle cerebral artery occlusion(MCAO) using modification of the intraluminal technique (Longa et al.,1989). Briefly, the left common carotid artery (CCA) was exposedthrough a midline incision in the neck region. The neck muscles wereseparated further to expose external carotid artery (ECA) and theninternal carotid artery (ICA). A 3.0 cm length 3-0 monofilament nylonsuture (Ethicon, Johnsons & Johnsons Ltd., Mumbai, India) wasintroduced into the ECA lumen through a small nick and gentlyadvanced from ECA to the ICA lumen until resistance was felt, to blockmiddle cerebral artery circulation. Recirculation/reperfusion wasallowed by removing the monofilament at predecided time points. Insham-operated animals, all the procedures except the insertion of thenylon filament were carried out. For behavioral and brain infarctionvolume study a total no. of 43 animalswere randomly divided in severalgroup as sham (n=6), vehicle (normal saline) control (n=14),ceftriaxone pre (n=12) and post-treatment (n=11). In addition, fourmore groups, consisting of 6 animals each, were used for glutaminesynthetase activity, GLT-1 protein and mRNA study (sham-operatedanimals; vehicle treated; ceftriaxone pre-treated and ceftriaxone post-treated). The doses of ceftriaxone and DHK were selected according tothe previous report (Chu et al., 2007) with some modification. A dailydose of 100 mg/kg i.v. was administered for five days in pre-treatmentgroup for behavioral, cerebral infarction, glutamine synthetase activity,PCR and western blot studies, whereas a single dose of 100 mg/kg i.v.was given 2 h after the reperfusion in post-treatment groups. The effectof DHK on glutamine synthetase activity in vehicle and ceftriaxone pre-treated group was also studied in two other groups comprising DHK+veh (n=5) and DHK+CTX (n=6). A dose of 100 μg/kg, i.c.v. was given1 h before the MCA occlusion using Hamilton syringe. The DHK wasprepared at the concentration of 2 mg/ml solution in artificial CSF. Thisamounts to a volume of approximately 10–12 μl depending upon thebodyweight of animals andwas given slowly over a period of nearly 5–6 min in the left ventricle of the brain according to the coordinates of ratbrain atlas using stereotaxic instrument [steolting] (Paxinos andWatson, 1986). The glutamate uptake activity was carried out in sixrats per group and each experimentwas performed at least in triplicate.DHK and DL-TBOA were purchased from Tocris and all other chemicalswere purchased from Sigma unless otherwise specified.

2.3. Assay of glutamine synthetase activity

Theglutamine synthetase activitywasmeasured in cytosolic fractionof tissue homogenates. It was based on the formation of 7-glutamylhy-

droxamate from glutamate and NH2OH. The reaction mixture contain-ing 100 mM imidazole buffer, pH 7.2, 10 mM ATP, 125 mM NH2OH,50 mM glutamate, 25 mM β mercaptoethanol and 20 mM MgCl2 wasequilibrated at 37 °C with 100 μl of tissue supernatant in a total volumeof 500 μl. After 30 min at 37 °C, 750 μl of stop mix containing 370 mMFeC13, 670 mMHCl and 200 mM trichloroacetic acid was added to stopthe reaction. The mixture was then centrifuged at 5000 g for 10 min atroom temperature. The absorbance of supernatant was measuredspectrophotometrically (Lambda 35, Perkin Elmer, US) at 535 nmagainst a reagent blank (Meister, 1985).

2.4. Preparation of glial enriched fraction from rat brain homogenatesand glutamate uptake assay

Glutamateuptakewasmeasured asdescribedbyXuet al. (2003)withminor modification. Briefly, the animals pre-treated with ceftriaxone(100 mg/kg, i.v. daily for 5 days) or salinewere decapitated after 5th day.The brains were then removed carefully. Brain samples were preparedfromboth cerebral hemisphere andwerehomogenizedbriefly in ice-coldbuffer containing 320 mM sucrose, 4 mM Tris, pH 7.4, 1 mM EDTA, and10 mM glucose using a Teflon homogenizer. Homogenates werecentrifuged (500 g for 10 min; 4 °C), and the supernatant thus obtainedwere spunat 10,000 g for 10 minat 4 °C. The glial enriched fractionswerefurther prepared by centrifugation on a discontinuous sucrose-Ficollgradient at 81,500g for 2 h using ultracentrifuge (Henn and Hemberger,1971). The pellets enriched in glial fraction were re-suspended in10 foldvolume of initial brain tissue weight in Krebs–Ringer–HEPES (KRH)medium, containing (in mM): 120 NaCl, 4.7 KCl, 2.2 CaCl2, 25 HEPES, 1.2MgSO4, 1.2 KH2PO4, and 10 glucose, pH 7.4. This suspension was furtherwashed twice with KRH buffer to remove excess endogenous glutamateand re-suspended in the samebuffer so as to give a protein concentrationof 2 mg/ml, which was determined by Lowry method (Lowry et al.,1951). Glutamate uptake in glial enriched preparation was measured infollowing sets comprising [3H]-Glutamate only, DHK (300 µM)+[3H]-glutamate and DL-TBOA (100μM)+[3H]-Glutamate in both ceftraixoneand saline or vehicle group. The glial fractions were treated with vehicle,DHK and DL-TBOA, 30 min prior to experiment. The assay was initiatedby adding [3H]-glutamate (20 nM, 29.0 Ci/mmol; GEHealthcare, UK) in afinal volume of 500 μl of KRH medium. After incubation at roomtemperature for 30 min, the uptake was terminated by adding chilledKRH buffer followed by filtration on glass-fiber filters (Whatman, UK).Nonspecific uptake was determined with sodium-free buffer that wasprepared by replacing NaCl with choline chloride.

2.5. Neurobehavioral assessment

The neurobehavioral assessment was performed according tomethod developed by Longa et al. (1989). Briefly, the neurobehavioralfindings were scored on five point scale with 10 grading scores: a scoreof 0 indicated no neurologic deficit, a score of 1 (failure to extendopposite forepaw fully) a mild focal neurologic deficit, a score of 2(contralateral circling) amoderate focal neurological deficit, and a scoreof 3 (falling to the opposite side to the damage when hanged on net) asevere focal deficit; rats with a score of 4 did not walk spontaneouslyand had a depressed level of consciousness. The scores of neurobeha-vioralfindings obtained after testingon each scalewere summedupanddenoted as neurological deficit score (Longa et al., 1989). Theneurological deficit was scored twice, initially after full recovery fromanesthesia during the reperfusion period i.e. about 3–4 h and later aftercompletion of 24 h of reperfusion. The animals showing no sign ofneurological deficit at both time points were excluded from the study.

2.6. Measurement of the cerebral infarct area and volume

After predetermined time point of ischemia/reperfusion, the brainswere quickly removed and sliced into coronal sections at 2 mm

Fig. 1. Neurological deficit scores in rats subjected to 1 h ischemia followed by 24 h ofreperfusion in vehicle, pre- and post-ceftriaxone treated groups (**Pb0.01 vs. veh).

Fig. 2. TTC stained brain slices showing the infarction area (A) inMCAOvs. ceftriaxone pre(100 mg/kg i.p. daily×5 days) and post (2 h post-reperfusion single dose) treated groups.Histogram (B) showing infarction volume in animals subjected to MCAO and ceftriaxonepre- and post-treated groups (**Pb0.01 vs. veh).

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intervals. Each slice was immersed in a 1.0% solution of 2, 3, 5–triphenyltetrazolium chloride (TTC) for 30 min at 37 °C and then fixedin 10% buffered formaldehyde solution. The stained brain slices weredigitally photographed and the infarct area, outlined in white, wasmeasured on posterior surface of each section using Biovis Image Plussoftware version 1.5. The percentage infarct area was calculated bysubtracting the outlinedwhite area from total area of the hemisphere ofaffected side of the brain divided by hundred. Infarct volume, expressedinmm3, was calculated by a linear integration of the infarct area of eachslice multiplied by thickness of brain section (Bederson et al., 1986).

2.7. RNA isolation and PCR studies

Total RNAwas isolated from rat brains collected at various ischemia/reperfusion time points in sham, vehicle or ceftriaxone treated groupusing the Tri Reagent (sigma) according to the manufacturer'sinstructions. Total RNA (2 μg) was subsequently transcribed intocDNA using omniscript RT kit. cDNA was amplified separately withspecific primer for GLT-1, GAPDH and IL-1β using Taq PCR core Kit(Qiagen USA). The PCR products were resolved in 1.2% agarose gelcontaining EtBr 5 μg/ml and the intensity of each band was analyzed byusing spot densitometry analysis software of Alphamager ™ 2200.

2.8. Western blot analysis

The sham operated as well as rats subjected to 1 h of ischemiafollowed by different time points of reperfusion, were subjected toperfusion with saline and the brain was rapidly removed. The differentbrain parts namely cortex, striatum and hippocampus were gentlyseparated. The brain parts were then homogenized in ten volumes ofice-cold lysate buffer (200 mMHEPES (pH 7.5), 250 mM sucrose, 1 mMdithiothreitol, 1.5 mM, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1×protease inhibitor cocktail) using a Teflon homogenizer. The homo-genates were then spun at 800g for 10 min. The 800g supernatant wasagain spun at 20,000 g for 20 min. The resultant pelletswere used as thecytoplasmic membrane protein, enriched in GLT-1. The proteinconcentration of each sample was determined spectrophotometrically(Lowry et al., 1951).

An aliquot of 20 µg of protein was subjected to 10% or 12% sodiumdodecylsulfate polyacrylamide gel electrophoresis. The separatedproteins were transferred on to nitrocellulose membrane. For immuno-blotting, the following primary antibodies were used: mouse monoclo-nal anti-GLT-1; anti-glutamine synthetase (BD biosciences) and rabbitpolyclonal anti-actin; anti-IL-1β (Santa Cruz Biotechnology USA)antibodies. The secondary antibodies used were HRP conjugated anti-IgG. The immunoreactive bands were visualized by enhanced chemilu-minescence (ECL) detection (GEHealthcare UK). The band intensitywasmeasured using spot densitometry analysis by Alphamager ™ 2200software.

2.9. Statistical analysis

The comparisons among different groups were made using theone-way analysis of variance (ANOVA) followed by Newman–Keulsmultiple comparison test. The comparison between identical timepoints of ischemia/reperfusion was made using Student's t-test andrepresented as mean±S.E.M. A P value of b0.05, b0.01 and b0.001were considered statistically significance.

3. Results

3.1. Neurobehavioral assessment

The neurological deficit scores of each experimental animal wererecorded at 1/24 h of ischemia/reperfusion injury in sham, vehicle andceftriaxone treated groups. The pre-treatment with ceftriaxone

(100 mg/kg i.v. daily×5 days) significantly (Pb0.01) reduced theneurological deficit in comparison to vehicle treated group (Fig. 1).

3.2. Effect of ceftriaxone treatment on cerebral infarct volume

The ischemia/reperfusion of 1/24 h produced marked infarct as isevidenced in the serial coronal brain section (Fig. 2A). The ceftriaxonepre-treatment reduced infarct volumeby58% as compared to thevehiclereperfusion group, at 24 h post-ischemia/reperfusion (Fig. 2B). Themean infarct volume in ceftriaxone pre-treated group was 119.2±53.5 mm3 vs. 240.2±43.8 mm3 in the vehicle reperfusion group(Pb0.01) and with post-treatment it was reduced to 197.1±46.21 mm3 in comparison to vehicle.

3.3. Effect of ceftriaxone treatment on GLT-1 mRNA and protein expression

The effect of ischemia/reperfusion stress onGLT-1mRNAandproteinexpression was determined after 1 h of ischemia followed by different

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timepoints of reperfusion in shamandvehicle control groups. Therewasa significant reduction in GLT-1 gene expression in the cortex andstriatum with increasing reperfusion time point, but a slight increasingpattern was observed in hippocampus (Fig. 3A). The protein expressionchanges were not well correlated with mRNA expression. A moderateincrease was found in the striatum up to 12 h, but it returned near thebasal level at 24 h post-reperfusion. However, in the cortex therewas aninitial increase followed by downward trend reaching to basal level, butGLT-1 did not show any sign of alterations in hippocampus (Fig. 3B).

The effect of both pre- and post-ceftriaxone treatment was alsoinvestigated, where a significant (Pb0.05) increase was observed in thecortex in comparison to vehicle in both pre- and post-treated groups(Fig. 4A). The changes in the striatum were insignificant. A similarpattern of expression was found in protein expression in both pre- andpost-treated ceftriaxone groups in the cortex region while in thestriatum, a similar pattern of expression was found only in the pre-treatment group (Fig. 4B). Effortswere alsomade to investigatewhetherthe neuroprotective effect of ceftriaxone may be partly mediated by itsanti-inflammatory action, we also analyzed mRNA and protein expres-sion changes in IL-1β, it was found that ceftriaxone treatment has noeffect on IL-1β in both brains nuclei except a significant effect on itsprotein level in striatal region following post-treatment (Fig. 5A and B).

Fig. 3.Differential expression of GLT-1 mRNA (A) and protein (B) in different brain regions afrelative differences in mRNA and protein expression in comparison to sham after normaliz

3.4. Effect of ceftriaxone treatment on GS activity and expression

GS is a key glutamate-metabolizing enzyme, which convertsglutamate into relatively non-toxic glutamine following its uptake bynon-neuronal cells specially astrocytes. We found a significant increase(Pb0.05) in GS activity in the cortex, but without any significant changein striatal region in the brain of animals pre-treated with ceftriaxone(Fig. 6A). Further the changes in GS activity were well correlated withincreased activity in GLT-1 protein because inhibition of GLT-1 with itsspecific blocker dihydrokainate (100 µg/kg i.c.v.) 1 h prior to ischemiaresults in significant (Pb0.05) drop in GS activity in the pre-treatmentgroup (Fig. 6B and C). The effect of ceftriaxone treatment on GS proteinwas also analyzed to examine, whether ceftriaxone has any effect on itsexpression. There was in general no significant change observed in GSprotein level in both control and ceftriaxone treated groups (Fig. 7).

3.5. Effect of ceftriaxone treatment on glutamate uptake

In order to validate our glial enriched preparation, we conducted anumber of experiments where, we first measured the uptake of [3H]-glutamate in the presence of various concentrations (0–240 µM) ofunlabeled glutamate. The results of this study showed that the uptake of

ter 1h of ischemia followed by 0, 3, 6, 12 and 24 h of reperfusion. Data are represented asation with β-actin (*Pb0.05 and **Pb0.01 vs. sham).

Fig. 4. Effect of ceftriaxone treatment onGLT-1mRNA (A) and protein expression (B) in thecortex and striatal region of the brain after 1 h of ischemia followed by 24 h of reperfusion(*Pb0.05vs. veh).β-actinwasusedas the loadingcontrol forwesternblot (B) (lowerpanel).

Fig. 5. Effect of ceftriaxone treatment on IL-1βmRNA (A) and protein expression (B) inthe cortex and striatal regions of the brain after 1 h of ischemia followed by 24 h ofreperfusion (*Pb0.05 vs. veh). β-actin was used as the loading control for western blot(B) (lower panel).

Fig. 6. Glutamate synthetase activity in (A) ceftriaxone (CTX) pre- and post-treatedanimals following 1/24 h of ischemia/reperfusion injury. Effect of GLT-1 specific inhibitorDHKonglutamine synthetase activity in the cortex (B) and striatum(C). (*Pb0.05 vs. veh).

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labeled glutamate was decreasing in linear fashion with increasing thedose of unlabeled glutamate (Fig. 8A). Further we validated GLT-1dependency of [3H]-glutamate uptake by using specific inhibitor of GLT-1, DHK at different doses (150, 300 and 600 μM), where we found that

Fig. 7. Western blot analysis for glutamine synthetase (GS) protein expression in the(A) cortex and (B) striatum in sham, vehicle and ceftriaxone pre- and post-treated groupsafter 1 h of ischemia followed 24 h of reperfusion.

Fig. 8. Uptake study of [3H]-glutamate in glial enrich preparations (A) in the presence ofvarious concentrations of unlabeled glutamate, (B) in the presence of various concentra-tions of DHK, (C) in vehicle and ceftriaxone treated group and (D) in ceftriaxone treatedgroup in the presence of saline, DHK and DL-TBOA. (**Pb0.01 and ***Pb0.001 vs. vehiclecontrol or saline only group).

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DHK was able to reduce the uptake in a dose dependent manner(Fig. 8B). The ceftriaxone treatment showed a significant increase(Pb0.01) in [3H]-glutamate uptake in comparison to vehicle (Fig. 8C).Additionally, we also used DL-TBOA, a complete inhibitor of glutamatetransport, besides DHK in order to assess the level of glutamate uptakeinhibition (Fig. 8D).

4. Discussion

Glutamate excitotoxicity is implicated in cerebral stroke and variousother CNS disorders, including Huntington disease, Alzheimer's disease,amyotrophic lateral sclerosis and epilepsy. Interestingly under thesepathological conditions the expression of GLT-1 is down regulated andleads to imbalance of glutamate homeostasis, which contributes toexcitotoxicity (Lee et al., 2008; Sheldon and Robinson, 2007). Therefore,the modulation of glutamate homeostasis seems to offer a potentialstrategy for the treatment of excitotoxic injury,whichmaybeachievedbyaffecting or blockade of glutamate release, glutamate-receptor andglutamate-transporter activators. The recent report indicates thatglutamate release inhibitors and glutamate-receptor antagonists werenot successful in clinical situation owing to their significant adverseeffects (Tanaka, 2005). However, glutamate transporter's mechanismappears as an attractive therapeutic target in cerebral stroke due to twomain reasons; (i) glutamate transporters can uptake glutamate releasedby calcium-dependent and calcium independentmechanisms and (ii) themodulation of glutamate-transporter activity subtly modulates glutama-tergic synaptic transmission (Tanaka, 2005). Therefore, treatment aimedat returning glutamate transporters to normal levels of expression andfunction may be beneficial in stroke situations; anticonvulsants andantipsychotics appear to work in part by reversing pathologicalalterations to glutamate transporters and restoring glutamatergichomeostasis (Sheldon and Robinson, 2007).

Therefore, in thepresent study,wehave investigated the role ofGLT-1protein in focal cerebral ischemia model of rat to assess its potential incerebral stroke. The results reveal a down regulation of GLT-1 proteinfollowing ischemia/reperfusion injury, which is in agreement with theobservations ofmany otherworkers (Chu et al., 2007; Lipski et al., 2007).Hence, it seems that upregulation of this proteinmight be able to provideneuroprotection in the above model. In our study, we found thatceftriaxone pre-treatment upregulates GLT-1 and reduces cerebralinfarct ashas beendemonstrated inTTC stainedbrain sections.Moreover,ceftriaxonepost-treatment also resulted inupregulation ofGLT-1protein

in the cortex, without any significant effect on cerebral infarct. Thisparadoxical effect of ceftriaxone may be due to glutamate transporter'sability to operate in both directions depending upon the extent ofischemic insults as well as ionic gradient across the plasma membrane(Mitani and Tanaka, 2003). Therefore, ceftriaxone post-treatmentupregulates GLT-1, but the reversed operation of GLT-1 might have ledto the release of glutamate into the extracellular space, which tends toattenuate the protective effect of ceftriaxone on cerebral damagefollowing the ischemic insult (Mitani and Tanaka, 2003). AlthoughGLT-1 upregulation by ceftriaxone post-treatment did not offer protec-tion due to increase in extracellular excitatory amino acid levels undercondition of transporter reversal. However, these adverse effects seem tobe more pronounced in striatum, which represents the ischemic coreregion and any neuroprotective mechanism will thus be ineffective(Popp et al., 2009). Further the area surrounding this core region consistsof potential salvageable tissue known as the ischemic penumbra(frontoparietal cortex), which provides opportunity for neuroprotectivetherapy (Weinstein et al., 2004). It seems that glutamate release viareversed GLT-1 is not significant in the ischemic penumbra asdihydrokainate (DHK), a non-transportable selective inhibitor of GLT-1transporter, didnot reduceexcitatoryaminoacid release inmild ischemicareas, suggesting a diminished transporter reversal role in the penumbra(Feustel et al., 2004).Moreover, thenormal functionofGLT-1, rather thanthe reversed operation of GLT-1 transporter, seems to dominate in theischemic penumbra. Therefore, treatment aimed at returning glutamatetransporters to basal level of expression and function may be beneficial(Sheldon and Robinson, 2007).

The sensory motor dysfunction is commonly observed in ratssubjected to focal cerebral ischemia. There is a significant motor deficitbehavioral recovery following pre-treatment, but not with ceftriaxonepost-treatment which may be due to GLT-1 protein upregulation. It iswell established that sensory motor dysfunction after transient focalcerebral ischemia in rats occurs because of consistent synaptictransmission failure within the motor cortex (Bolay and Dalkara,1998). Thus, an improvement in neurological score reflects recoveryof motor deficit (Bederson et al., 1986).

Glutaminesynthetasehas been recognized to beoneof the importantenzyme controlling synaptic glutamate availability. It has been proposedearlier that glial cells take up released glutamate and convert it intoglutamine by the action of glutamine synthetase (Hertz, 1979; Shankand Aprison, 1981). Glutamine further diffuses out of the glia andreaches into neurons, where it is reconverted into glutamate, therebyreplenishing transmitter content in the appropriate presynaptic term-inals. The loss of glutamine synthetase would thus be expected toincrease the level of brain glutamate. The exactmechanism of loss is notknown, but oxidative stress during ischemia/reperfusion injury mayaffect the enzyme activity (Oliver et al., 1990). It has been reported thatmild ischemia/reperfusion injury upregulates glutamine synthetase upto 48 h and has been proposed as a compensatory neuroprotectivemechanism. Therefore, it can be expected that increased glutaminesynthetase activity can buffer the excess glutamate. Our studydemonstrates that ceftriaxone treatment upregulates the glutaminesynthetase activity, which is reflected by increased glutamate-trans-porter activity. Ceftriaxonedoesnothave its direct effect onGSactivity aspre-treatment with DHK diminished its activity. These results furtherprovide evidence that GS activity can be used as a tool for glutamateuptake. Ceftriaxone has been reported for its upregulating effect on GLT-1 activity in various models of cell lines and primary culture of glial cells(Rothstein et al., 2005). In order to find out the effect on glutamateuptake by GLT-1, we prepared glial enriched membrane preparationfrom the brain of ceftriaxone pre-treated animals and validated for [3H]-glutamate uptake activity. Our results of uptake activity in thesepreparations showed a dramatic increase in uptake of [3H]-glutamate,which further confirms our in vivo observations. Interestingly theinhibition of this uptake by pre-treatment of DHK suggests theinvolvement of GLT-1 (Danbolt, 2001).

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Cerebral ischemia/reperfusion injury leads to upregulation of anumber of inflammatory genes e.g. Il-6, IL-8, TNF-α, Fas-L, andCox-2 etc.However, these inflammatorymediators appear to play a facilitatory rolein glutamate excitotoxicity, by inhibiting glutamate transporter'sfunction on astrocytes (Chu et al., 2007). Further, in our study it seemsthat ceftriaxonemayhave slight down regulating effects on Il-1βproteinin striatal region, a vulnerable area for ischemia/reperfusion damage.Thus, it appears that ceftriaxone might have some anti-inflammatoryaction which needs further investigation. In a recent study, Lee et al.(2008) found that ceftriaxone significantly elevated EAAT2 transcriptioninprimary human fetal astrocytes through the nuclear factor-κB (NF-κB)signaling pathway. The ceftriaxone promotes nuclear translocation ofp65 to activateNF-κB. Therefore, it seems that specificNF-κBbinding siteat −272 position of the EAAT 2 promoter may be responsible forceftriaxone-mediated EAAT 2 induction (Lee et al., 2008).

These findings thus strongly suggest that ceftriaxone has a modula-tory effect on glutamate transport and has the potential to ameliorateneuronal excitotoxicity following cerebral ischemia. Further, we havealso provided the evidence that ceftriaxone showed its protective effectby upregulation and increase in activity of GLT-1. Thus, our study furtherstrengthens the concept that upregulation of GLT-1 might be a potentialtherapeutic target for neuroprotection following cerebral ischemicinjury.

Acknowledgement

Rajkumar Verma and Vikas Mishra acknowledge the fellowshipfrom CSIR, India.

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