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Intern. J. Neuroscience, 114:183–196, 2004Copyright Taylor & Francis Inc.ISSN: 0020-7454 / 1543-5245 onlineDOI: 10.1080/00207450490249329

THE EFFECTS OF CITICOLINEAND LAMOTRIGINE ALONE ANDIN COMBINATION FOLLOWING

PERMANENT MIDDLE CEREBRALARTERY OCCLUSION IN RATS

SÜREYYA ATAUSM. ZÜLKÜF ÖNALDepartment of NeurologyAkdeniz University Medical FacultyAntalya, Turkey

SADI S. OZDEMDepartment of PharmacologyAkdeniz University Medical FacultyAntalya, Turkey

KENNETH W. LOCKEInterneuron Pharmaceuticals, Inc.Lexington, Massachusetts, USA

SEVIN BALKANDepartment of NeurologyAkdeniz University Medical FacultyAntalya, Turkey

Received 28 May 2003.This study is supported by Akdeniz University, Unit of Research Projects. We would like

to thank Taha Karaman and Evren Tercan for their valuable statistics consultation and ErolNizamoglu for his technical support. Citicoline was provided by Interneuron Pharmaceuticals Inc.,Lexington, Massachusetts, USA.

Address correspondence to Dr. Mehmet Zülküf Önal, Akdeniz University Medical Faculty,Department of Neurology, 07070, Antalya, Turkey. E-mail: mzonal@akdeniz.edu.tr

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The neuroprotective efficacies of citicoline and lamotrigine, alone andin combination, were investigated in experimental permanent focal is-chemia. Seven groups of adult male rats underwent focal cerebral is-chemia and were given the following treatments: placebo (P), low andhigh doses of citicoline (C250 and C500, 250 and 500 mg/kg/day i.p.,respectively), low and high doses of lamotrigine (L50 and L100, 50 and100 mg/kg/day p.o., respectively), and combination regimes of both drugsin low (C250 + L50) and high doses (C500 + L100). Citicoline, but notlamotrigine, exerted neuroprotective efficacy during this acute ischemicstroke model. The citicoline and lamotrigine combination did not pro-vide a significant additive neuroprotective effect.

Keywords cerebral ischemia, citicoline, lamotrigine, neuroprotection, rat

Neuroprotective agents and strategies have been studied for years,and appear to be effective in a variety of pre-clinical stroke models.Neuroprotective agents act on different steps of the ischemic cas-cade initiated after vessel occlusion, leading to irreversible cell death(Önal & Fisher, 1997). In animal models, many of these agentsreduce the volume of cerebral infarction when given either beforeor shortly after the ischemic insult (Minematsu & Fisher, 1993;Phillis, Song, & Regan, 1997; Schabitz et al., 1999; Wiard et al.,1995; Önal & Fisher, 1996; Schabitz & Fisher, 2001; Zivin, 1998).However, at present, conclusive evidence of clinical efficacyhas yet to be demonstrated in clinical trials; almost none of thedrugs has proven conclusively to be effective in humans. This isexactly related to issues of drug dosage, time window, sex differ-ences, and, in particular, the stroke target population (Finkelstein etal., 1999).

Most of the neuroprotective agents are not well tolerated in hu-mans, especially at higher doses, due to significant side effects (Alberts,1994). Some studies obviously demonstrate that subpopulations ofpatients can benefit from the neuroprotective approach (Schabitzet al., 1999). The exploration of the effectiveness of combinationtherapies for stroke seems rational because cerebral ischemiatriggers a multitude of pathophysiologic and biochemical eventsthat affect the evaluation of focal ischemia differently. Therefore,low dose combinations of agents acting through different mecha-nisms were evaluated for potential synergistic and/or additive activ-ity, which means increased neuroprotective effects with relatively

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Citicoline and Lamotrigine in Ischemia 185

fewer side effects (Önal & Fisher, 1997; Önal & Fisher, 1996; Schabitzet al., 1999; Lyden & Lonzo, 1994; Önal et al., 1997).

Citicoline (cytidine 5'-diphosphate choline, CDP-choline) was selectedas an agent that has antioxidant and membrane reparative activity,while lamotrigine (3,5-diamino-6-(2,3-dichlorophenyl)-1,2,4-triazine)was selected as an antagonist of glutamate neurotoxicity. Citicolineis a key intermediary in the biosynthesis of phosphatidylcholine, animportant component of cell membranes (Schabitz et al., 1999).Antiepileptic agent lamotrigine stabilizes presynaptic neuronal mem-branes by inhibiting voltage-sensitive sodium channels, and by re-ducing excessive glutamate release after global and focal cerebralischemia (Rataud et al., 1994; Stuart & Meldrum, 1995; Calabresiet al., 2000). Both agents reduce the size of the cerebral infarct andimprove neurological outcome in animal models of focal cerebralischemia, and have been used in several clinical studies of acuteischemic stroke (Schabitz et al., 1999; Wiard, Dickerson, Beek, Norton,& Cooper, 1995; Önal, Li, Tatlisumak, Locke, Sandage, & Fisher,1997; Rataud, Debemot, Mary, Pratt, & Stutzmann, 1994; Calabresiet al., 2000; Aronowski, Strong, & Grotta, 1996; Schabitz et al.,1996; Shuaib et al., 1995; Crumrine, Bergstrand, Cooper, Faison, &Cooper, 1997; Bacher and Zornow, 1997; Clarke, Warach, Pettignew,Gammans, & Sabounjian, 1997; Smith & Meldrum, 1995; Balkanet al., 1997).

In the present study, we investigated two drugs separately and incombination treatment. The effects of low and high dose citicolineand lamotrigine versus placebo on cerebral infarct volume and neu-rological outcome were assessed using the intraluminal suture oc-clusion model of the middle cerebral artery (MCA) in rats.

MATERIAL AND METHODS

Seven groups of adult, normotensive, male Wistar rats (n = 7 pergroup), weighing 280 to 350 g, underwent focal cerebral ischemia bythe intraluminal suture occlusion method. Animals were maintainedon a 12-h light/12-h dark cycle, and had free access to food and waterbefore and after the surgical procedures. All experimental proce-dures, including surgery, daily drug administration, daily monitoring,

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digitizing, and evaluation of postmortem brain slices were conductedin a blinded, randomized manner. The procedures were approved bythe official ethic committee of our university.

The animals were anesthetized intramuscularly with xylazine (2%;15 mg/kg) and ketamine HCI (100 mg/kg). Physiological measure-ments were obtained during the surgical procedure and kept in anormal range.

Focal brain ischemia was induced by the intraluminal suture MCAocclusion (MCAO) model, which has been previously described indetail (Minematsu et al., 1992; Koizumi et al., 1986; Zea et al., 1989).After a midline ventral neck incision, the right common carotid artery(CCA) and the right external carotid artery (ECA) were exposed, andthe proximal CCA was ligated with a 3-0 silk suture, and the ECAwas ligated just after its bifurcation with a 6-0 silk suture. A 4-0 nylonsuture 40 mm in length, whose tip had been rounded by heating neara flame, was inserted through the incision and gently advanced intothe internal carotid artery. The suture was inserted approximately 17mm from the bifurcation to occlude unilaterally the origin of MCA.

The animals were allowed to recover from anesthesia at roomtemperature and to eat and drink freely. Once daily, at 24-h inter-vals, animals were weighed and neurological evaluations were per-formed, using a six-point scale; 0 = no neurological deficit, 1 =failure to extend left forepaw fully, 2 = circling to the left, 3 =falling to left, 4 = no spontaneous walking with a depressed level ofconsciousness, and 5 = death.

Animals were randomly and blindly assigned to one of the follow-ing treatment groups: placebo (P), low dose citicoline (C250 250 mg/kg/day, i.p.), high dose citicoline (C500 = 500 mg/kg/day, i.p.), lowdose lamotrigine (L50 50 mg/kg/day, p.o.), high dose lamotrigine(L100 = 100 mg/kg/day, p.o.), or the combination of both drugs inlow doses (C250 + L50) or high doses (C500 + L100). The treatmentprotocol was as follows: the first treatment (either saline or citicoline)was given intraperitoneally 30 min after MCAO, and the secondtreatment (either saline or lamotrigine) was given orally via a naso-gastric tube 90 min after MCAO. The respective treatments wereadministered at 24-h intervals for an additional 3 days or for as longas the animal survived during the follow-up period. Injections wereperformed by an investigator blinded to the treatment assigned.

After completing the final treatment and neurological examina-

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Citicoline and Lamotrigine in Ischemia 187

tion on the third day, the rats were re-anesthetized with a high doseof ketamine HCl (250 mg/kg) and decapitated. The brains were thenremoved quickly and placed in a deep freezer (–20°C) for 30 minand then sectioned coronally into six 2-mm thick slices (from ros-tral to caudal). The brain slices were incubated for 30 min in a 2%solution of 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma®) at37°C and fixed by immersion in a 10% buffered formalin solution(Sigma®) for 24 h. These slices were photographed, and the imageswere stored on a computer. The unstained area was defined as thearea of infarct. The cortical and subcortical infarct areas and totalhemispheric areas were calculated separately using an image pro-cessing software package (UTHSCSA®, Image Tool for Windows,Version 2.0 Texas, USA). A corrected infarct volume was calcu-lated to compensate for the effect of brain edema. The correctedinfarct area for each slice was calculated according to the followingequation: Corrected Infarct Area = Left Hemisphere Area – (RightHemisphere Area – Infarct Area). Total cortical and subcortical,uncorrected and corrected infarct volumes were calculated by mul-tiplying the infarcted area by the slice thickness and summing thevolumes of the six slices. Brain edema was calculated by subtract-ing the corrected from the uncorrected infarct volume.

The brains of animals that died prematurely between the seconddose and the last dose of treatment regime were included in thestudy. They were harvested within 60 min of death and evaluated ina manner similar to those which were killed electively on the thirdday of the experiment.

The randomization code was broken after all data were acquired.For the determination of statistical significance of parametric vari-ables, ANOVA with subsequent post-hoc Bonferroni’s multiple com-parison tests were used. Kruskal-Wallis, Chi-square, and Fisher-exacttests were performed for non-parametric variables where appropriate.Values are presented as mean ± standard error of mean (s.e.m.). Forall tests, values of p < .05 were considered statistically significant.

RESULTS

Body weights at baseline and 3 days after infarction revealed nosignificant differences between the seven treatment groups, although

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total body weights declined from day 1 to day 3 (data not shown).Physiological parameters compared before and after surgical proce-dures also revealed no significant difference (data not shown).

The premature mortality rate, after 24 h and MCAO, was 57.1%in the P group, 42.9% in both the L50 and C250 groups, 28.6% inthe L100 group, 14.3% in both the C500 and C500 + L100 groups,and 0% in the C250 + L50 group. There was no significant dif-ference in the mortality rate between the groups (p in Chi-squaretest and p = .07 in Fisher-exact test between C250 + L50 andP groups).

The mean daily neurological scores were not significantly differ-ent between the 7 treatment groups. However, there was a trendtoward a lower mean neurological score in the high dose citicolineand lamotrigine groups (Figure 1).

The mean corrected total, cortical, and subcortical infarct vol-umes for each treatment group are shown in Table 1 and Figure 2.The C250 group had significantly smaller mean corrected total inf-arct volume compared to the P group (p < .05), and significantlylarger subcortical infarct volume compared to the L100 + C500

FIGURE 1. Mean daily neurological scores of the seven treatment groups (n = 7 foreach group). For animals dying prematurely, a score of 5 was assigned from the day ofdeath to day 3. P = placebo, L50 = lamotrigine 50 mg/kg/day, p.o., L100 = lamotrigine100 mg/kg/day, p.o., C250 = citicoline 250 mg/kg/day, i.p., C500 = citicoline 500 mg/kg/day, i.p. The Kruskal-Wallis test revealed no significance. p < .05 was consideredstatistically significant.

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Citicoline and Lamotrigine in Ischemia 189

group (p < .05). Mean corrected total, cortical, and subcortical in-farct volumes of the C500 group were significantly smaller than theP (p < .001, .001, and .01, respectively) and the L50 groups(p < .01, .01, and .001, respectively). Also, the C500 group hadsignificantly smaller mean cortical infarct volume than the L100group (p < .05). The mean corrected total, cortical, and subcorticalinfarct volumes of the L50 and the L100 groups were not signifi-cantly different from the P group. The L50 + C250 low dose com-bination group had significantly smaller mean corrected total andcortical infarct volumes than the P group (p < .01, for both). The

TABLE 1. Infarct volume analysis data

Group TIV DF CIV DF SIV DF

1. P 388.0 ± 30 4,5,6,7 286.6 ± 30 5,6,7 159.7 ± 12 5,72. L50 365.7 ± 37 5,6,7 256.0 ± 27 5,7 174.5 ± 12 3,5,73. L100 307.0 ± 22 7 245.5 ± 12 5,7 123.8 ± 8 24. C250 262.4 ± 24 1 204.8 ± 16 – 135.2 ± 9 75. C500 208.4 ± 21 1,2 155.2 ± 11 1,2,3 101.6 ± 6 1,26. L50 + C250 239.4 ± 23 1,2 174.4 ± 13 1 129.9 ± 15 –7. L100 + C500 189.8 ± 15 1,2,3 130.3 ± 6 1,2,3 85.6 ± 6 1,2,4

P = placebo, L50 = lamotrigine 50 mg/kg/day, p.o., L100 = lamotrigine 100 mg/kg/day, p.o., C250 =citicoline 250 mg/kg/day, i.p., C500 = citicoline 500 mg/kg/day, i.p., n = 7 for each group. TIV = totalinfarct volume (corrected), CIV = cortical infarct volume, SIV = subcortical infarct volume. Volumevalues are in mm3. TIV, CIV, and SIV values are presented as mean ± s.e.m. p < .05 were consideredstatistically significant. DF = different from (ANOVA with post-hoc Bonferroni’s multiple comparisontests).

FIGURE 2. Mean corrected total, cortical, and subcortical infarct volumes of the seventreatment groups (n = 7 for each group). *, π, and γ: p < .05 compared to correspond-ing values in P group (ANOVA with post-hoc Bonferroni’s multiple comparison tests).P = placebo, L50 = lamotrigine 50 mg/kg/day, p.o., L100 = lamotrigine 100 mg/kg/day,p.o., C250 = citicoline 250 mg/kg/day, i.p., C500 = citicoline 500 mg/kg/day, i.p.

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mean corrected total infarct volume in this group was also signifi-cantly smaller than the L50 group (p < .05). The L100 + C500 highdose combination group had significantly smaller mean correctedtotal and cortical infarct volumes compared to the P (p < .001, forboth), L50 (p < .001, for both), and L100 groups (p < .05 and .01,respectively). The mean subcortical infarct volume in the L100 + C500 high dose combination group was significantly smaller com-pared to those in the P (p < .001), L50 (p < .001), and C250 (p <.05) groups.

DISCUSSION

The concept of neuroprotection depends on the pathophysiologicmechanisms underlying acute ischemic stroke. Many molecules, suchas free radical scavengers, calcium channel blockers, growth fac-tors, statins, lipid peroxidation inhibitors, excitatory amino acid an-tagonists, and others have been studied for many years (Finkelsteinet al., 1999). Here, we applied combination therapy to investigatethe possible advantage of intervening the steps of the pathophysi-ological process of the ischemic cascade. This has been referred toas the “cocktail” approach that has been used in the clinical man-agement of AIDS and cancer patients (Finkelstein et al., 1999).

The low dose administration of citicoline provided a significantreduction in the corrected mean total infarct volume, and also atrend to reduce the cortical, and the subcortical infarct volume com-pared to the placebo administration. The high dose citicoline admin-istration brought an additional benefit that reduced the correctedmean total, the cortical and the subcortical infarct volumes com-pared to the placebo administration. Based on these findings it seemedthat citicoline treatment alone produced a dose-related, significantreduction in the mean corrected total infarct volume.

Neuroprotective activity of citicoline is shown by Schabitz et al.(1996) as reducing the infarct volume in an animal model of stroke.Citicoline has beneficial effects on brain dysfunction induced bycerebral ischemia (Kakihana et al., 1988). It is employed in Japanand Europe to treat head injury and acute stroke (Bazan, 1970).Cerebral ischemia induces free fatty acid (FFA) contents, decreased

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Citicoline and Lamotrigine in Ischemia 191

phospholipid content, and uncoupling of mitochondrial oxidativephosphorylation (Bazan, 1970; Yatsu & Moss, 1971; Majeswska,Strosznajder, & Lazarewicz, 1978, Kuwashima et al., 1976). Addi-tionally, FFAs inhibit oxidative phosphorylation of mitochondondrialpreparations in vitro (Kuwashima et al., 1976). Citicoline stimulatesthe synthesis of phosphatidylcholine and restores the membraneintegrity (Schabitz et al., 1996). The exogenous administration ofciticoline has been shown in animal models to reduce cell mem-brane breakdown, leading to increased synthesis of phosphatidyl-choline and decreased levels of FFAs (Trovarelli et al., 1981; Dormanet al., 1982). Neuroprotective effect of citicoline probably occursbecause it inhibits the release of FFAs and resynthesis of phospho-lipids (Hayaishi et al., 1961; Bazan, 1970; Yatsu & Moss, 1971;Majeswska et al., 1978).

In agreement with our findings, the use of citicoline treatmenthas been shown to be beneficial in several animal models of is-chemia or hypoxia, including recent studies with reversible focalocclusion and in an intracerebral hemorrhage model (Önal et al.,1997; Aronowski et al., 1996; Kakihan, Fukuda, Suno, & Nagaoka,1988; Dorman et al., 1982; Weiss, 1995; Yamamoto, Shimizu, &Okamiya, 1990; De Medio et al., 1983; Mykita, Golly, Dreyfus,Freysz, & Massarelli, 1986; Clark et al., 1998). Additionally, inclinical studies, treatment with citicoline improved cognitive andbehavioral function in patients with memory deficits (Spiers, Myers,Hochanadel, Lieberman, & Wurtman, 1996; Alvarez et al., 1997),and produced partly beneficial effects in a clinical stroke trial in theparticular subgroup of stroke population, revealed in a diffusion-weighted magnetic resonance imaging study (Schabitz & Fisher,2001; Clarke et al., 1997). A randomized efficacy trial of citicolinein patients with acute ischemic stroke, unfortunately, failed. How-ever, that study revealed partly a beneficial effect in the post hocanalyses of subgroup of patients (Clarke et al., 1999). In our study,we confirmed the potential neuroprotective efficacy of citicolineduring an acute ischemic stroke model.

The excitatory amino acid glutamate is thought to have a crucialrole in the development of neurological damage once cerebral perfu-sion is reduced to a level at which metabolic activity is compromisedbut may potentially still recover (Astrup et al., 1981). Experimental

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and clinical studies for investigating the basic pathophysiologicalmechanisms of ischemic brain injury revealed that the overactivationof excitatory amino acid receptors by endogenous glutamate is themajor phase of ischemic neuronal damage (Scatton, 1994). In cere-bral tissue, elevated glutamate levels are thought to be an importantfirst step to cause neuronal damage. Due to an ischemic insult andglutamate receptor, antagonists are protective in animal models offocal and global cerebral ischemia (Finkelstein et al., 1999; Crumrineet al., 1997; Bacher & Zornow, 1997; Clarke et al., 1997; Dyker &Lees, 1998).

Lamotrigine has been found to inhibit veratrine-induced aminoacid release in rat cerebral cortex (Leach, Baxter, & Critchley, 1991)which suggested that it acts at voltage-sensitive sodium channels tostabilize neuronal membranes, and to inhibit glutamate release (Rataudet al., 1994; Leach et al., 1991). Accordingly, lamotrigine was shownto suppress the amplitude of glutamatergic excitatory postsynapticpotentials, consistent with a presynaptic mode of action (Wang, Huang,Hsu, Tsai, & Gean, 1996; Calabresi et al., 1999). It was also re-ported that lamotrigine might reduce postsynaptic sensitivity to glu-tamate released from corticostriatal terminals (Calabresi et al., 1999).Accordingly, lamotrigine and its derivatives have been shown to beneuroprotective in several experimental ischemia studies (Leach etal, 1986; Gilland, Puka-Sundvall, Andine, Bona, & Hagberg, 1994;Smith, Lekieffre, Sowinski, & Meldrum, 1993). Wiard and colleagues(1995) demonstrated protection by lamotrigine from the behavioraldeficits and histological damage caused by the ligation of the com-mon carotid arteries in a gerbil model of global cerebral ischemia.Shuaib and colleagues (1995) found a significant recovery in behav-ioral testing and neuronal protection in animals treated with lamo-trigine either before or after the insult. Crumrine et al. (1997) inves-tigated the neuroprotective effect of lamotrigine in hippocampal CA1neurons of rats suffering cardiac arrest, and found more than a 50%reduction of the damage to the CA1 cell population at doses similarto those used to treat epilepsy. Smith and Meldrum (1995) andRataud and colleagues (1994) found a significant neuroprotectiveeffect of lamotrigine in rats subjected to focal cerebral ischemiawhen given shortly after the onset of the stroke. Balkan and col-leagues (1997) showed that lamotrigine caused a marked reduction

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Citicoline and Lamotrigine in Ischemia 193

in brain nitrite and cGMP levels during focal cerebral ischemia inrats. However, in the present study, neither low nor high doses oflamotrigine exerted a significant neuroprotective effect in experi-mental ischemic stroke, although they provided a small but nonsig-nificant trend to get additional benefit as in the reduction of infarctvolume in each of the infarct areas. Except for the mean subcorticalinfarct volume, there were no significant differences between theinfarct volumes of low and high dose lamotrigine groups. However,it is possible that the measures, such as dose increment or timing ofapplication, might cause alterations in possible neuroprotective ef-fects of this drug and need to be investigated.

Disruption of the ischemic cascade of events at multiple levels islikely to be more effective than disruptive at any point. With theuse of multiple neuroprotective therapies, each agent or approachcould be given or applied, either simultaneously or in rapid succes-sion, allowing each agent to work on different ischemic injury mecha-nisms (Finkelstein et al., 1999; Önal et al., 1997; Fisher et al., 1995).Moreover, multiple drug therapy may enable the use of lower dosesof the individual agents in the mixture; thus, potentially reducinglimiting side effects.

In the present study, we used a similar dosing paradigm to evalu-ate potential additive effects between citicoline and lamotrigine. TheC250 group had a significantly reduced mean corrected total infarctvolume compared to the P group. A similar reduction in total infarctvolume was also observed in the L50 + C250 group. However, acombination of low doses of citicoline and lamotrigine did not pro-vide an additional beneficial effect on this parameter, as there wasno significant difference between the mean total infarct volumes ofthese two groups. However, a low dose combination seemed to pro-vide a beneficial effect with respect to cortical infarct volume, as itwas significantly lower than placebo in L50 + C250, but not inC250 group. Although the low dose combination caused a morepronounced reduction in mean subcortical infarct volume when com-pared to low dose citicoline alone, none of them was found to besignificantly different from the placebo. The high dose combinationof lamotrigine and citicoline significantly reduced infarct volumesafter permanent focal cerebral ischemia in rats. However, it didnot provide a significant difference from the use of the high dose

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administration of citicoline alone in the comparison of each mea-surement, although it provided small but nonsignificant decreases ininfarct volumes.

The findings of the present study suggested that citicoline, butnot lamotrigine, exerted a dose-dependent neuroprotective efficacyduring this acute ischemic stroke model, and the reductions in inf-arct volumes observed in combination groups were mainly due tociticoline component. Therefore, it did not provide additional sup-port for the use of multiple drugs acting through different mecha-nisms in this stroke model. However, the possible additive and/orsynergistic effects in vivo and in vitro should be investigated infuture leading studies.

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