Neuroprotection afforded by prior citicoline administration in experimental brain ischemia: effects on glutamate transport

www.elsevier.com/locate/ynbdi Neurobiology of Disease 18 (2005) 336 – 345

Neuroprotection afforded by prior citicoline administration in experimental brain ischemia: effects on glutamate transport Olivia Hurtado,a Marı´a A. Moro,a Antonio Ca´rdenas,b Vero´nica Sa´nchez,a Paz Ferna´ndez-Tome´,b Juan C. Leza,a Pedro Lorenzo,a Julio J. Secades,c Rafael Lozano,c Antoni Da´valos,d Jose´ Castillo,e and Ignacio Lizasoaina,* a

Departamento de Farmacologı´a, Facultad de Medicina, Universidad Complutense de Madrid (UCM), 28040 Madrid, Spain Instituto de Farmacologı´a y Toxicologı´a, UCM-CSIC, Madrid, Spain c Departamento Me´dico, Grupo Ferrer SA, Barcelona, Spain d Servicio de Neurologı´a, Hospital Doctor Josep Trueta, Girona, Spain e Servicio de Neurologı´a, Hospital Clı´nico Universitario de Santiago, Santiago de Compostela, Spain b

Received 29 March 2004; revised 23 July 2004; accepted 13 October 2004 Available online 15 December 2004

Background and purpose: Cytidine-5V-diphosphocholine (citicoline or CDP-choline), an intermediate in the biosynthesis of phosphatidylcholine, has shown beneficial effects in a number of CNS injury models including cerebral ischemia. Citicoline is the only neuroprotectant that has proved efficacy in patients with moderate to severe stroke. However, the precise mechanism by which citicoline is neuroprotective is not fully known. The present study was designed to search for mechanisms of citicoline neuroprotective properties using in vivo and in vitro models of brain ischemia. Methods: Focal brain ischemia was produced in male adult Fischer rats by occluding both the common carotid and middle cerebral arteries. Brain glutamate levels were determined at fixed intervals after occlusion. Animals were then sacrificed, and infarct volume and brain ATP levels were measured. As in vitro model of ischemia, rat cultured cortical neurones or astrocytes, isolated or in co-culture, were exposed to oxygen–glucose deprivation (OGD) either in the absence or in the presence of citicoline (1–100 MM). Viability was studied by measuring LDH release. Glutamate release and uptake, and ATP levels were also determined. Results: Citicoline (0.5, 1 and 2 g/kg i.p. administered 1 h before the occlusion) produced a reduction of the infarct size measured at striatum (18, 27 and 42% inhibition, respectively, n = 8, P b 0.05 vs. ischemia), effect that correlated with the inhibition caused by citicoline on ischemia-induced increase in glutamate concentrations after the onset of the ischemia. Citicoline also inhibited ischemia-induced decrease in cortical and striatal ATP levels. Incubation of cultured rat cortical neurones with citicoline (10 and 100 MM) prevented OGDinduced LDH and glutamate release and caused a recovery in ATP levels after OGD, confirming our previous results. In addition, citicoline (100 MM) caused an increase in glutamate uptake and in EAAT2 glutamate transporter membrane expression in cultured rat astrocytes. Conclusions: Our present findings show novel mechanisms

* Corresponding author. Fax: +34 91 3941478. E-mail address: [email protected] (I. Lizasoain). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2004.10.006

for the neuroprotective effects of citicoline, which cooperate to decrease brain glutamate release after ischemia. D 2004 Elsevier Inc. All rights reserved. Keywords: Astrocytes; ATP; Cerebral infarction; Middle cerebral artery occlusion; Neurones; OGD; Stroke

Introduction Cytidine-5V-diphosphocholine (citicoline or CDP-choline) is a compound normally present in all cells throughout the body and an intermediate in the biosynthesis of phosphatidylcholine (PtdCho). It has been shown that citicoline produces neuroprotective effects in a variety of CNS injury models including cerebral ischemia. At the experimental level, it has been reported to decrease infarct volume and oedema, and/or to improve neurological deficits, either alone or in combination with other agents (Alkan et al., 2001; Andersen et al., 1999; Aronowski et al., 1996; Grieb et al., 2001; Kakihana et al., 1988; Onal et al., 1997; Schabitz et al., 1996, 1999; Shuaib et al., 2000). In humans, citicoline is the only neuroprotectant that has shown positive results in all randomized, double-blind trials and has demonstrated efficacy in a metaanalysis with an overall safety similar to placebo (Da´valos et al., 2002). The effects proposed to explain the neuroprotective actions of citicoline have been thoroughly reviewed (Adibhatla and Hatcher, 2002; Adibhatla et al., 2002; D’Orlando and Sandage, 1995; Secades, 2002) and include prevention of fatty acids release (Dorman et al., 1983), stimulation of PtdCho synthesis, preservation of cardiolipin and sphingomyelin levels (Rao et al., 2000), increase of glutathione synthesis and glutathione reductase

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activity (Adibhatla et al., 2001), restoration of Na+/K+-ATPase activity (Plataras et al., 2000; Rigoulet et al., 1979) and antiapoptotic effects (Alvarez et al., 1999; Krupinski et al., 2002). However, its precise mechanism of action is not fully known. Glutamate, a major excitatory neurotransmitter, has long been recognised to play key roles in the pathophysiology of ischemia, due to its excessive accumulation in the extracellular space and the subsequent activation of its receptors, mainly the N-methyl-daspartate (NMDA) type of glutamate receptor (Castillo et al., 1996; Choi and Rothman, 1990). Inhibition of glutamate actions has demonstrated to be a very powerful strategy to decrease brain damage after experimental ischemia and, indeed, the larger part of efforts to reduce ischemia-induced brain injury has primarily focused on attenuating excitotoxicity with several neuroprotective drugs that block glutamate receptors or inhibit glutamate release induced by brain ischemia (Lees, 2000). However, although some trials are still ongoing, the results from several completed trials have been disappointing, mainly due to severe adverse effects (Goldberg, 2002). Due to its actions on membrane stability, one possibility is that citicoline might be affecting the increased accumulation of extracellular glutamate caused by ischemia, either inhibiting its release or increasing its uptake. Therefore, we have used both in vivo and in vitro models of experimental ischemia in order to determine whether the neuroprotective effect caused by citicoline affects ischemia-induced extracellular glutamate accumulation.

Materials and methods In vivo cerebral ischemia Experimental groups Citicoline was administered by intra-peritoneal (i.p.) injection. Five groups were used for determinations of glutamate levels and infarct area: (1) sham-operated animals (SHAM; n = 8); (2) permanent middle cerebral artery occlusion (pMCAO) 1 h after an i.p. injection of saline (pMCAO; n = 8); (3) pMCAO 1 h after an i.p. injection of 0.5 g/kg citicoline (pMCAO + CDPCh0.5; n = 8); (4) pMCAO 1 h after an i.p. injection of 1 g/kg citicoline (pMCAO+CDPCh1; n = 8) and (5) pMCAO 1 h after an i.p. injection of 2 g/kg citicoline (pMCAO+CDPCh2; n = 8). In addition, four groups were used in order to determine brain ATP levels (sham-operated animals; SHAM; SHAM + CDPCh2, pMCAO and pMCAO + CDPCh2; n = 6 in each group). The time of administration of citicoline was chosen according to our previous data showing a neuroprotective effect of citicoline (Ca´rdenas et al., 2002). Permanent middle cerebral artery occlusion (pMCAO) in rats Experiments were performed on male Fischer rats weighing 250–300 g. Rats were anaesthetised with 2.5% halothane in a mixture of 70% nitrogen/30% oxygen. Permanent focal cerebral ischemia was induced by ligature of the left common carotid artery (CCA) and occlusion of the ipsilateral distal middle cerebral artery (MCA) as described previously (De Cristo´bal et al., 2001; Hurtado et al., 2003). Briefly, for the CCA ligature, a midline ventral cervical incision was made, and the CCA was isolated and permanently occluded with a silk ligature. For the MCA occlusion, a 1-cm incision perpendicular to the line connecting the lateral

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canthus of the left eye and the external auditory canal was made to expose and retract the temporalis muscle. A 2-mm burr hole was drilled and the MCA was exposed by cutting and retracting the dura. The MCA was elevated and cauterised. Rats in which the MCA was exposed but not occluded served as sham-operated controls (SHAM). Following surgery, subjects were returned to their cages and allowed free access to water and food. The body temperature of animals was monitored throughout the experiment and was maintained at 37.5 F 0.58C using a heating pad. All procedures conformed to the Committee of Animal Care at the Universidad Complutense of Madrid according with E.U. rules (DC86/609/CEE). Brain microdialysis Rats were anaesthetised with 2.5% halothane in a mixture of 70% nitrogen/30% oxygen and secured in a Kopf stereotaxic frame with the tooth bar at 3.3 mm below the interaural zero. The dialysis probe (3.5 mm  240 Am; Cuprophan) was implanted in the left striatum according to the following coordinates: +1.0 mm anteroposterior and +3.0 mm mediolateral to the bregma and 4.3 mm dorsoventral from the surface of the brain (Paxinos and Watson, 1986). Probes were secured to the skull as described (Hurtado et al., 2003). The correct placement of the probes was verified by dye perfusion on test animals prior to proceeding with experimental groups. The day after probe implantation, probes were perfused with artificial CSF (KCl: 2.5 mM; NaCl: 125 mM; MgCl2d 6H2O: 1.18 mM; CaCl2d 2H2O: 1.26 mM; NaH2PO4d H2O: 0.5 mM; Na2HPO4d 2H2O: 5 mM) at a rate of 1 Al/min. After a 60-min resting period, samples were collected every 30 min. Three basal samples were taken prior to pMCAO to achieve steady baseline concentration of glutamate. These samples were averaged and all subsequent values were expressed as a percentage of these basal preischemic levels. The in vitro probe recovery of a solution containing 1 ng/Al of glutamate was 20.5 F 1.5% (n = 6). Dialysate samples were collected at the times indicated during 24 h, and stored at 408C until glutamate determination as described (Hurtado et al., 2003). Glutamate levels are expressed in Amol/l. Infarct area determination The brains were removed 48 h after pMCAO, and series of 2 mm of coronal brain slices were obtained (Brain Matrix, WPI, UK) and stained in 1% TTC (2,3,5-triphenyl-tetrazolium chloride, Merck) in 0.1 M phosphate buffer, and infarct size was determined as described (Hurtado et al., 2003; Mackensen et al., 2001). Infarct volumes were measured by sampling stained sections with a digital camera (Nikon Coolpix 990, Nikon Corporation, Tokyo, Japan), and the image of each section was analysed by an image analyser (Scion Image for Windows 2000, Scion Corporation, Frederick, MD). The digitised image was displayed on a video monitor. With the observer blinded to the experimental conditions, the contralateral hemisphere perimeter was overlapped on the ipsilateral hemisphere to exclude edema, and infarct borders in cortex, subcortex and striatum were delineated using an operator-controlled cursor. The area of infarct, which was not stained, was determined by counting pixels contained within the outlined regions of interest and expressed in square millimeters. Infarct volumes (in mm3) were integrated from the infarct areas over the extent of the infarct calculated as an orthogonal projection.

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In vitro experiments Primary culture of rat cortical neurones All experimental protocols adhered to the guidelines of the Animal Welfare Committee of the Universidad Complutense (following DC 86/609/EU). Primary cultures of rat cortical neurones were performed as described (Romera et al., 2004). Studies were performed at in vitro days 9–10, time at which the cultures consisted of 94 F 6% neurones, as determined by flow cytometry (Romera et al., 2004). Astrocyte culture Primary astrocyte cultures were prepared from neonatal (P0) Wistar rat cortex, as previously described (McCarthy and de Vellis, 1980; Romera et al., 2004). Cells present in the culture were shown to be astrocytes (94 F 5%) after characterisation by flow cytometry as described above using a primary specific anti-GFAP antibody (Chemicon, Temecula, CA; 1:100 dilution). Neuronal–astrocyte co-culture Neuronal–astrocytic co-cultures were prepared as described (Romera et al., 2004). Briefly, after 10 days in culture, astrocytes were plated in culture plate inserts (1-Am membrane pore size, 4.2 cm2 of effective membrane growth area, Falcon, Beckton Dickinson Labware, Franklin Lakes, NJ). One day after, neurons plated in six-well companion plates (Falcon, Beckton Dickinson Labware) had their medium replaced, and astrocytes-containing inserts were transferred to these wells. Co-cultures were transferred to the normoxic incubator and maintained for 1 week, the time at which astrocytes showed a process-bearing morphology, indicative of differentiation (Swanson et al., 1997).

For determination of cellular ATP levels, cells were collected after exposure to a bsubmaximalQ OGD of 30 min, a time at which glutamate release had not yet begun, to avoid loss of ATP due to cell lysis or to consumption by exocytosis, allowing comparisons between intact control and OGD-exposed cortical neurons. The absence of cell lysis was determined by LDH release. ATP concentrations were measured as indicated below. Assessment of cell viability As a marker of necrotic tissue damage, lactate dehydrogenase (LDH) activity released from damaged cells was determined as described (Koh and Choi, 1987; Romera et al., 2004). LDH release is expressed as percentage of total cell LDH and is plotted as percentage of LDH release induced by OGD. Basal LDH release was 6 F 1% (n = 12). [3H]Glutamate uptake by neurones or astrocytes [3H]Glutamate uptake by neuronal or astrocytic cultures, either isolated of from co-cultures, was determined as described (De Cristo´bal et al., 2002). Briefly, cultures were washed in control solution and incubated in control solution containing 3 AM glutamate and 8 ACi/ml of [3H]glutamate for 90 s, a time in which glutamate uptake was found to proceed linearly with time. In a parallel set of experiments, cultures were incubated during the same time in a solution of the same composition but in which Na+ was equiosmotically substituted by choline. At the end of the incubation, solution was collected and cells were lysed by addition of perchloric acid (0.3 M). [3H]glutamate uptake was calculated by subtracting the uptake in the absence of Na+ from the uptake in its presence and expressed as percentage of total [3H]glutamate. Determinations

Exposure of cultures to oxygen–glucose deprivation (OGD) Oxygen–glucose deprivation was performed as described (De Cristo´bal et al., 2002; Romera et al., 2004). Culture medium was replaced by a solution containing (in mM) NaCl (130), KCl (5.4), CaCl2 (1.8), NaHCO3 (26), MgCl2 (0.8), NaH2PO4 (1.18) and 2% HS bubbled with 95% N2/5% CO2 for OGD cells (OGD solution). OGD cells were transferred to an anaerobic chamber (Forma Scientific, Hucoa Erloss, Spain) containing a gas mixture of 95% N2/5% CO2 and humidified at 378C, and maintained at a constant pressure of 0.15 bar. Time of exposure to OGD was 145 min. In some experiments, citicoline (1–100 AM) was included 30 min before and during OGD. OGD was terminated by replacing the exposure medium with oxygenated MEM containing 0.6% glucose, 0.029% glutamine, 50 I.U./ml penicillin, 50 Ag/ml streptomycin, 10% HS (reperfusion medium) and returned to the normoxic incubator. Control cultures in a solution identical to OGD solution but containing glucose (33 mM; control solution) were kept in the normoxic incubator for the same time period as the OGD, and then incubation solution was replaced with reperfusion buffer and cultures were returned to the normoxic incubator until the end of the experiment. Culture medium and cells were collected at times indicated for LDH determination. For glutamate determination, samples of incubation solution were collected at the end of the OGD period, a time at which LDH efflux was not significantly different from control values, thus allowing to study glutamate release caused by OGD and excluding the efflux of this excitatory amino acid due to damaged membranes. Samples were collected from cultures of either pure neurones or neuronal–astrocytic co-cultures.

HPLC determination of glutamate concentration Analysis of glutamate was performed by HPLC with fluorimetric detection (Perkin Elmer Binary LC Pump 250 and Fluorescence Detector LC 240) following pre-column derivatisation with the o-phtalaldialdehyde procedure (Lindroth and Mopper, 1979). Derivatives were separated isocratically on a reverse phase column (4.6  150 mm, 5 Am particle diameter, Nucleosil 100-C18) using a mobile phase consisting of sodium acetate buffer (0.05 M pH 6.5), 20% methanol and 2% tetrahydrofuran. The area of each peak was determined with a Perkin Elmer Nelson Model 1020 integrator (Phoenix 8088 ROM BIOS Version 2.52 software), and compared with the peak area of the corresponding external standard to determine glutamate concentration. Glutamate release is expressed as percentage of net release induced by OGD for in vitro experiments. Glutamate levels in microdialysate are expressed in percent of pre-ischemic values. Basal release for in vitro experiments was below the detection limit of our assay (0.17 Amol/l). ATP determination For determination of ATP levels from SHAM, SHAM + citicoline, pMCAO and pMCAO + citicoline groups, rats were sacrificed 30 min after MCAO or SHAM operation and immediately immersed into liquid nitrogen; once frozen, brains were taken out and stored at 808C until ATP determination. From frozen brain samples, ipsilateral cortices and striata were dissected. From cultures, cells were lysed immediately after the experiment. ATP levels determined with a firefly luciferin-

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luciferase assay-based commercial kit (ATP Bioluminescence Assay Kit HSII, Roche, Barcelona, Spain). Data are expressed as percentage of sham/control levels. Basal ATP levels are expressed in Amol/g. Biotinylation Biotinylation of cell surface proteins was performed as described by Davis et al. (1998), Qian et al. (1997) and modified by Duan et al. (1999). After 30-min incubation with or without citicoline (100 AM), cultures were washed with balanced salt solution (BSS) for 6 min and rinsed twice with PBS/Ca–Mg containing (mM): NaCl (138), KCl (2.7), CaCl2 (0.1), KH2PO4 (1.5), MgCl2 (1.0), NaH2PO4 (9.6), pH 7.3. Cultures were then incubated in sulfo-NHS-biotin solution (Pierce, Rockford, IL) (1 mg/ml in with PBS/Ca–Mg) for 20 min at 48C. Biotinylation was terminated by washing twice in a quenching solution of PBS/Ca–Mg in which there was an equimolar substitution of 100 mM glycine for NaCl (to maintain 300 mOsm). This was followed by an additional 45-min incubation in the quenching solution at 48C. Quenching solution was removed, and the cells were lysed with 100 Al/well of radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors (100 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM iodoacetamide, 1 Ag/ml leupeptin, 5 Ag/ml aprotinin and 250 AM phenylmethylsulfonyl fluoride) for 1 h at 48C with vigorous shaking. The lysates were centrifuged at 14,000  g for 15 min at 48C. Three hundred microliters of the supernatant were taken for Western analysis (see below) as the whole cell fraction. The rest of the supernatant (600 Al) was incubated with 300 Al avidin bead suspension for 1 h at room temperature with gentle shaking. The avidin–lysate solution was then centrifuged for 15 min at 14,000  g, and the supernatant was taken for Western analysis as the intracellular fraction. The pellet was washed four times with 1 ml RIPA buffer and resuspended in 300 Al Laemmli buffer (62.4 mM Tris–HCl, pH 7.2, 2% SDS, 20% glycerol and 5% 2-mercaptoethanol) for 1 h with gentle shaking at room temperature. After centrifugation for 15 min at 14,000  g, the supernatant was taken for Western analysis as the biotinylated (plasma membrane) fraction. Western blot analysis of glutamate transporters in cultured astrocytes or neurones EAAT1, EAAT2 and EAAT3 glutamate transporters protein levels were determined in homogenates of astrocytes or neurones with or without exposure to citicoline (100 AM) for 25 min: cells were homogenised at 48C in 5 volumes of buffer containing 320 mM sucrose, 1 mM dl-dithiothreitol, 10 Ag/ml leupeptin, 10 Ag/ ml soybean trypsin inhibitor, 2 Ag/ml aprotinin, 0.2% Nonidetk (Roche), and 50 mM Tris brought to pH 7.0 at 208C with HCl (homogenisation buffer). Homogenate containing 10 Ag of protein was loaded, and the proteins were size-separated in 10% SDS-polyacrylamide gel electrophoresis (90 mA). The proteins were blotted onto a PVDF membrane (Millipore, Madrid, Spain) and incubated with the following specific primary antibodies: polyclonal antibodies against EAAT1/GLAST, EAAT2/GLT-1 (Santa Cruz Biotechnology, Santa Cruz, CA; 1:500) or monoclonal antibody against EAAT3/EAAC1 (Chemicon; 1:500 dilution) glutamate transporters. Proteins recognised by the antibody were revealed by ECLk-kit following manufacturer’s instructions (Amersham Pharmacia Biotech, Piscat-

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away, NJ). h-actin was used as loading control for total and intracellular proteins. To verify equal loading of the lanes corresponding to membrane fraction, membrane extracts were assayed for 5V-nucleotidase activity with a commercial kit (Molecular Probes, Invitrogen, Barcelona, Spain); differences between extracts were lower than 10%. Blots were also stained with Ponceau S and processed to confirm equal loading of protein (Moore and Viselli, 2000). Chemicals and statistical analyses Unless otherwise stated, chemicals were from Sigma (Madrid, Spain). Results are expressed as mean F standard error of the mean (SEM) of the indicated number of experiments; statistical analysis involved one-way analysis of variance (ANOVA, or the Kruskal–Wallis test when the data were not normally distributed) followed by individual comparisons of means (Student–Newman– Keuls, or Dunn’s method when the data were not normally distributed).

Results Effect of citicoline on infarct volume after pMCAO Cortical infarct volume measured at 48 h after pMCAO showed a reduction in the groups treated with 1–2 g citicoline before the occlusion (Fig. 1; n = 8, P b 0.05) when compared with the nontreated group. In contrast, when the striatal infarct volume was measured, the effect of citicoline was observed even at the lowest dose tested (Fig. 1; n = 8, P b 0.05). Effect of citicoline on brain extracellular glutamate concentrations after pMCAO As we have previously shown (Hurtado et al., 2003), pMCAO caused an increase in brain extracellular glutamate concentration (basal value: 2.3 F 0.4 Amol/l, n = 12; Fig. 2A). The onset of glutamate increase began immediately after occlusion, and was decreased after 24 h (Fig. 2A). The increase caused by pMCAO in brain glutamate levels was inhibited by previous treatment with citicoline (2 g/kg; Fig. 2A; n = 4, P b 0.05). Citicoline (2 g/kg) did not affect brain extracellular glutamate concentrations in sham animals (data not shown, n = 4, P N 0.05). Effect of citicoline on brain ATP levels after pMCAO Citicoline (2 g/kg) increased basal cortical and striatal ATP levels in sham-operated animals when measured 15 min after its administration (Fig. 2B; sham values: 5.4 F 0.5 and 5.5 F 0.7 Amol/g, respectively, n = 8, 100%). Occlusion of MCA caused a reduction of ATP levels in both tissues when compared with the sham group (Fig. 2B). Previous treatment with citicoline (2 g/kg) partially prevented ATP reduction induced by the ischemic insult (Fig. 2B; P b 0.05, n = 6). Effects of citicoline on rat cortical neurones Exposure of rat cortical neurones to OGD caused neuronal death, as shown by an increase in LDH efflux to the medium at 3 and 24 h after OGD compared with control (basal LDH release was

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Effects of citicoline on rat cortical astrocytes Exposure of rat cultured astrocytes to OGD did not affect either LDH release, extracellular glutamate concentrations or ATP levels at the times studied (n = 6–12, P N 0.05, data not shown). Citicoline also failed to affect ATP levels in these cells (control value: 2.8 F 0.2 Amol/g; citicoline 1, 10 and 100 AM: 1.9 F 0.6, 2.6 F 0.2, 2.0 F 0.5 Amol/g, respectively; P N 0.05, n = 4). In contrast, citicoline (100 AM) increased glutamate uptake in astrocytes (control cultures: 3.5 F 0.3 ACi/mg protein, n = 7, P b 0.05, Fig. 4A). In order to elucidate the mechanisms of citicoline-induced glutamate uptake by astrocytes, expression of the glial glutamate transporters EAAT1 and EAAT2 was performed by Western blot analysis. Our results show that total cellular levels of EAAT2 were unaffected by 100 AM citicoline (Fig. 4B). However, citicoline caused a significant increase in EAAT2 levels in the biotinylated (cell surface) fraction of this transporter. Our assay was not sensitive enough to detect EAAT1 in our samples (data not shown).

Fig. 1. Effect of citicoline (CDPCh 0.5, 1 and 2 g/kg) on infarct volume measured at cortex and striatum after pMCAO. Data are mean F SEM, n = 8. (See Materials and methods for details). *P b 0.05 vs. pMCAO. Photomicrographs of representative animals.

6 F 1%; Fig. 3A). Citicoline (10 and 100 AM) inhibited OGDinduced neuronal death, as shown by a reduction in OGD-induced LDH release (Fig. 3A; P b 0.05, n = 12). In these cells, OGD also caused an increase in extracellular glutamate concentration (0.91 F 0.18 nmol/106 cells, n = 12) when compared with the control group (below detection limit, see Materials and methods; Fig. 3B). The incubation of cells with citicoline (10 and 100 AM) before and during the OGD period caused an inhibition of OGD-induced glutamate release (Fig. 3B, P b 0.05, n = 7). In addition, the exposure of cortical neurons to a bsubmaximalQ OGD caused a decrease in ATP levels compared with control cells (Fig. 3C, P b 0.05, n = 4; control value: 4.5 F 0.5 Amol/g). Citicoline caused a recovery in ATP levels after OGD (Fig. 3C, P b 0.05, n = 4). Citicoline also increased ATP levels in control neurones (120 F 12%, 139 F 10% and 146 F 8% of control levels at 1, 10 and 100 AM citicoline, respectively, n = 4, P b 0.05). Incubation with citicoline (1–100 AM) for 30 min did not affect glutamate uptake in rat cortical neurones (control cultures: 1.03 F 0.20 ACi/mg protein; citicoline 1, 10 and 100 AM: 1.06 F 0.10, 0.82 F 0.20, 1.26 F 0.10 ACi/mg, respectively; P N 0.05, n = 8).

Fig. 2. Effect of citicoline (CDPCh 2 g/kg) on (A) brain glutamate levels after pMCAO (data are mean F SEM, n = 8, *P b 0.05 vs. pMCAO; see Materials and methods for details) and (B) cortical and striatal ATP levels after pMCAO (data are mean F SEM, n = 6–8, #P b 0.05 vs. SHAM; *P b 0.05 vs. pMCAO).

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Citicoline (100 AM) did not affect [3H]glutamate uptake when studied in neurones from co-cultures, either in control or OGDexposed cells (control: 0.75 F 0.20, 0.77 F 0.07, 0.82 F 0.08 and 0.75 F 0.13 ACi/mg in control, citicoline, OGD and OGD + citicoline, respectively; P N 0.05, n = 8). These results were in agreement with the levels of the neuronal EAAT3 glutamate transporter, which was unaffected either by OGD or citicoline (100 AM) at the times studied (data not shown). In contrast, citicoline (100 AM) did increase glutamate uptake in control astrocytes which had been co-cultured with neurones (control cultures: 5.9 F 0.4 ACi/mg protein, n = 8, P b 0.05, Fig. 5A). OGD also caused an increase in glutamate uptake when compared with the control group, an effect that was not further changed by citicoline (Fig. 5A). As opposed to undifferentiated astrocytes, there was a detectable membrane expression of the glial transporter EAAT2 in control astrocytes from co-cultures, which was further enhanced by citicoline (100 AM, Figs. 5B,C). Accordingly, exposure to OGD increased membrane EAAT2 expression, an effect that was not affected by citicoline (100 AM, Figs. 5B,C).

Fig. 3. Effect of citicoline on cultured cortical neurones. Effect of Citicoline (CDPCh: 1–100 AM) on (A) LDH release at 3 h (5) or 24 h (n) after oxygen–glucose deprivation (OGD). LDH is expressed as the percentage of total LDH and plotted as the percentage of LDH release after OGD (67 F 6% of total LDH; n = 12). Data are mean F SEM; n = 12; *P b 0.05 vs. OGD. (B) Glutamate release after OGD (0.91 F 0.18 nmol/106 cells, n = 12). Data are mean F SEM; n = 7–12; *P b 0.05 vs. OGD. (C) ATP levels after OGD (control value = 4.5 F 0.5 Amol/g protein). Data are mean F SEM; n = 4; *P b 0.05 vs. control.

Effect of citicoline on neuronal–astrocytic co-cultures As astrocytes differentiate in the presence of neurones, glutamate uptake and release as well as glutamate transporters were studied in neuronal–astrocytic co-cultures, both in control conditions and after exposure to OGD.

Fig. 4. Effect of citicoline on cultured astrocytes. Effect of Citicoline (CDPCh: 1–100 AM) on (A) [3H] glutamate uptake (control uptake: 3.5 F 0.3 ACi/mg protein; n = 7), *P b 0.05 vs. control; and (B) EAAT2 glutamate transporter expression (CDPCh:100 AM). Lower panel shows densitometric analysis of bands. Data are mean F SEM; n = 4; *P b 0.05 vs. nontreated group. Apart from h-actin, equal loading of proteins was verified by checking 5V-nucleotidase activity and by staining of membrane with Ponceau S (see Materials and methods for further details).

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Fig. 5. Effect of citicoline on neuronal-astrocytic co-cultures. Effect of Citicoline (CDPCh, 100 AM) on (A) [3H] astrocytic glutamate uptake (control uptake: 5.9 F 0.4 ACi/mg protein, n = 8), *P b 0.05 vs. control; (B) EAAT2 glutamate transporter expression (CDPCh:100 AM). Lower panel shows densitometric analysis of bands. Data are mean F SEM; n = 4; *P b 0.05 vs. nontreated group. (C) EAAT2 glutamate transporter expression in biotinylated fractions of control and OGD-exposed astrocytes from co-cultures, in the presence or absence of 100 AM citicoline (CDPCh). Data are mean F SEM; n = 4; *P b 0.05 vs. control. Apart from h-actin, equal loading of proteins in membrane fractions was verified by checking 5V-nucleotidase activity and by staining of membrane with Ponceau S (see Materials and methods for further details).

Exposure of co-cultures to OGD caused an increase in extracellular glutamate (0.60 F 0.06 nmol/106 neurones, n = 10), which was decreased in cells pre-incubated with 100 AM citicoline (below detection limit, P b 0.05, n = 10).

Discussion Previous evidences have demonstrated a neuroprotective effect of citicoline in experimental brain ischemia, as indicated by a reduction in infarct volume and/or neurological deficits after MCAO. We have now confirmed the neuroprotective effect of citicoline after MCAO and studied further the mechanisms implicated. Our results indicate that citicoline decreases extracellular glutamate accumulation after ischemia by a dual mechanism

involving both a decreased neuronal glutamate efflux and an increased astrocytic glutamate uptake. We first tested the effect of several doses of citicoline on infarct outcome. Previous studies have demonstrated that citicoline, either alone (Schabitz et al., 1996; Shuaib et al., 2000) or in combination with other drugs (Andersen et al., 1999; Onal et al., 1997; Schabitz et al., 1999; Shuaib et al., 2000), decrease infarct volume after MCAO in rat. Confirming such evidences, our results show that intraperitoneal administration of citicoline prior to MCAO is effective in improving stroke outcome, as demonstrated by a reduction in infarct volume, an effect that was more evident when determined in striatum. It is well known that glutamate plays a predominant role in the pathogenesis of ischemic brain injury (Castillo et al., 1996, 1997; Choi and Rothman, 1990). We and others have previously shown that glutamate increases in brain after cerebral ischemia in this permanent MCAO model (Baker et al., 1995; De Cristo´bal et al., 2001; Hurtado et al., 2003). Therefore, we tested the correlation between the neuroprotective effect of citicoline and glutamate levels in the dialysate. According to our hypothesis, this compound prevented ischemia-induced increase in brain glutamate levels. It has been shown that, during ischaemia, the striatum is exposed to higher cumulative concentrations of glutamate than the cortex (Osuga and Hakim, 1994). Our findings showing that the neuroprotective effects of citicoline are more potent in the striatum than in the cortex may be related to the fact that citicoline targets the increase in extracellular glutamate after ischaemia. High extracellular glutamate concentrations may result from an increased release and/or from a decreased uptake. Regarding ischemia-induced glutamate release, it has been shown to be largely due to reversed operation of neuronal glutamate transporters (Jabaudon et al., 2000; Rossi et al., 2000), a fact that results from a severe depletion in ATP levels caused by ischemia (Madl and Burgesser, 1993). Therefore, we have studied whether the effect of citicoline on ischemia-induced glutamate increase is related to an effect on brain ATP levels. Indeed, our data indicate that the ATP loss induced by the ischemic insult is inhibited by previous administration of citicoline and that this drug is able by itself to increase the levels of ATP even when it is administered to sham-operated animals. Our results using an in vitro model of cerebral ischemia by exposing rat cortical neurones to OGD confirm our data from the in vivo model. Indeed, OGD-induced neuronal LDH release was decreased by citicoline, an effect concomitant to the decrease in both OGD-induced glutamate elevation and ATP fall. However, although the concentration of 1 AM was able to prevent partly OGD-induced ATP fall, it failed to affect both glutamate and LDH release. Since it has been described that ATP concentrations need to fall below a threshold for glutamate transporter reversal to take place (Madl and Burgesser, 1993), it is likely that only the higher concentrations of citicoline are able to rise above such level. Taken together, in vivo and in vitro data strongly support the hypothesis that citicoline-induced effect on neuronal ATP levels account, at least in part, for the decrease in extracellular glutamate and the subsequent neuroprotection after ischemia, very likely due to a reduced reversal of the neuronal transporters. Several mechanisms might explain the effect of citicoline on ATP levels both in control and ischaemia, either in vivo or in vitro. In this context, it has been demonstrated that citicoline prevents the loss of cardiolipin, which is an exclusive inner mitochondrial phospholipid and it is essential for mitochondrial electron trans-

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port (Hoch, 1992). Furthermore, citicoline restores Na+/K+ATPase activity in vivo (Rigoulet et al., 1979) and has a direct stimulatory effect in vitro (Plataras et al., 2000). Although the inhibition of phospholipase A2 has been recently shown (Adibhatla and Hatcher, 2003), it is not known the precise mechanism by which citicoline produces those effects, and further studies are required to clarify this point. Of note, our results are in agreement with previous data in the literature indicating that strategies that prevent ATP fall are neuroprotective (Bickler and Buck, 1998; Ca´rdenas et al., 2000; De Cristo´bal et al., 2001, 2002; Galeffi et al., 2000; Hurtado et al., 2003; Kass and Lipton, 1982; Moro et al., 2000; Riepe et al., 1997). Interestingly, astrocytes play an important role in the maintenance of extracellular glutamate concentrations by regulating its uptake. Indeed, glutamate transporters in neurones and glia remove glutamate from extracellular space thereby helping to terminate glutamatergic synaptic transmission and to prevent the extracellular glutamate concentration from rising to neurotoxic values (Maragakis and Rothstein, 2001; Takahashi et al., 1997). Therefore, we studied whether astrocytes might be also a target for citicoline at the glutamate uptake level by using pure astrocytic cultures. Indeed, our results show that citicoline causes an increase in glutamate uptake in these cells, which was not observed in neurones. Thus, a plausible hypothesis is that citicoline increases glutamate uptake by regulating glutamate transporters. Of the five high-affinity, sodium-dependent glutamate transporters currently known (Seal and Amara, 1999), two of them, EAAT1 and EAAT2 are localised primarily in astrocytes. Several studies have now shown that EAAT2 deletion using either antisense or gene deletion strategies is related to larger increases in extracellular glutamate, neuronal damage and brain oedema after experimental brain ischemia (Hamann et al., 2002; Namura et al., 2002; Rao et al., 2001; Rothstein et al., 1996). Therefore, we examined the expression of this transporter by Western blotting finding that citicoline increased remarkably its membrane levels. These findings strongly suggest that citicoline induces the translocation of this transporter from the cytosol to the membrane, where it is functional and helps to decrease extracellular glutamate concentrations. In these experiments, we used polygonal, bundifferentiatedQ astrocytes, in which the basal membrane expression of EAAT2 transporter is very low (Swanson et al., 1997). Previously reported actions of citicoline on cell membrane (Adibhatla and Hatcher, 2002; Adibhatla et al., 2002; D’Orlando and Sandage, 1995; Secades, 2002) might play a role in its effect on astrocytic membrane. In the conditions used in the present study, astrocytes were resistant to ischemia since exposure to OGD at those times that decreased neuronal survival did not affect viability, extracellular glutamate concentration or cellular ATP levels when studied in cultured astrocytes, in agreement with previous evidences indicating that ATP depletion and necrosis occur in OGD-exposed neurones, but not in OGD-exposed astrocytes (Almeida et al., 2002). These results indicate that the mechanism of action of citicoline on astrocytes is different from the one that we describe in neurones. It is likely that more severe ischaemic conditions are able to affect glutamate release and viability in astrocytes, as it has been previously described (review in Chen and Swanson, 2003). We next examined the effects of citicoline on neuronal– astrocytic co-cultures. This preparation allows the intercellular communication between the two cell populations by means of

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soluble factors that may diffuse through the insert pores. Then, the two populations may be separated for different studies. In coculture, due to the presence of neurones, the astrocytes transform into a stellate, process-bearing phenotype (Swanson et al., 1997). Control glutamate uptake in these astrocytes was higher than in nondifferentiated astrocytes, in agreement with the presence of EAAT2 in their plasma membrane and with previous studies (Swanson et al., 1997). The incubation with citicoline increased even further both glutamate uptake and EAAT2 plasma membrane expression. Interestingly, exposure to OGD caused a remarkable increase in both uptake and EAAT2 membrane expression, which was not further enhanced in the presence of citicoline. The effect of OGD may be due to an increased extracellular concentration of glutamate from neurones, as this excitatory amino acid has been demonstrated to be a major regulator of glutamate transporter expression in astrocytes (Duan et al., 1999). The neuroprotective effect of citicoline may then result from a dual action on both neurones and astrocytes: increased ATP levels in neurones, either from an increased production or from decreased consumption, may delay the reversal of neuronal glutamate transporters that leads to glutamate release and subsequent excitotoxicity; in addition, an increased capacity of astrocytes to remove excess extracellular glutamate would contribute to decrease ischaemic damage; although citicoline failed to increase further glutamate uptake in these cells, at the beginning of the ischaemic insult, citicoline-treated astrocytes already possess an increased glutamate transporter capacity, and are able to remove glutamate more efficiently than the nontreated cells. This is strongly supported by the fact that, although OGD-induced glutamate release in co-cultures is of the same magnitude than in neuronal cultures, the inhibitory effect of a comparable concentration of citicoline (100 AM) is much more pronounced. In summary, our results show that citicoline exhibits a remarkable and specific protection that occurs concomitantly with an inhibition of ischemia-induced neuronal glutamate release and an increase in astrocytic glutamate uptake. These results may possess important therapeutic implications in the management of patients at risk of ischemic events, since we demonstrated that early neurological progression of patients with acute ischemic stroke is associated with high concentrations of glutamate in blood and cerebrospinal fluid (Castillo et al., 1997).

Acknowledgments This work was partly supported by grants from SAF200204487-C02-01 (IL), FIS-PI03/0314 (MAM), and CAM08.5/ 0001.1/2003 (PL). OH is recipient of a postdoctoral fellowship funded by Comunidad de Madrid.

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