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Neuroscience Letters 564 (2014) 11–15

Contents lists available at ScienceDirect

Neuroscience Letters

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europrotective effect of the 3�5�-pregnanolone glutamatereatment in the model of focal cerebral ischemia in immature rats

enka Kleteckovaa,c, Grygoriy Tsenovb, Hana Kubovab, Ales Stuchlika, Karel Valesa,∗

Department of Neurophysiology of Memory, Institute of Physiology, v.v.i. Academy of Sciences of the Czech Republic, Videnska 1083, 14220 Prague 4,zech RepublicDepartment of Developmental Epileptology, Institute of Physiology, v.v.i. Academy of Sciences of the Czech Republic, Videnska 1083, 14220 Prague 4,zech Republic2nd Faculty of Medicine, Charles University, Prague, Czech Republic, V Uvalu 84, 150 06 Prague 5—Motol, Czech Republic

i g h l i g h t s

3�5�-Pregnanolone glutamate is a use-dependent antagonist of NMDA receptors.We demonstrated that PG has lack of neurotoxicity effect in 12-day-old rats.We showed that PG has neuroprotective effect in ET-1 induced model of ischemia in 12-day-old rats.

r t i c l e i n f o

rticle history:eceived 28 November 2013eceived in revised form 20 January 2014ccepted 29 January 2014

eywords:�5�-Pregnanolone glutamateMDA receptors

mmature ratsocal cerebral ischemiandothelin-1

a b s t r a c t

The perinatal hypoxic-ischemic insult frequently leads to mortality, morbidity and plays a key role inthe later pathological consequences. The ischemic insult causes a massive release of glutamate and sub-sequent excitotoxic damage. The neuroactive steroid 3�5�-pregnanolone glutamate (PG) is a NMDAreceptor antagonist acting via use-dependent mechanism and can be used as a neuroprotective agentthat may alleviate glutamatergic excitotoxicity in the brain.

First, a possible neurotoxic effect of the PG, a novel use-dependent NMDA antagonist, was studiedin immature rats. In addition, to compare this effect with a well-described non-competitive NMDAantagonist, the MK-801 (positive control) was used. Animals at postnatal day 12 (P12) were injectedintraperitoneally with PG in a doses 1 or 10 mg/kg or with MK-801 in a dose 1 mg/kg. Effect of PG treatmenton the immature brain was evaluated on Fluoro Jade B (FJB) stained sections. Second, a neuroprotectiveeffect of the PG was studied in the model of focal cerebral ischemia in P12. Focal cerebral ischemia wasinduced by the infusion of the endothelin-1 (ET-1) into the right dorsal hippocampus. PG at the doses1 or 10 mg/kg was administrated intraperitoneally 5 min after the end of ET-1 infusion. To evaluate the

neuroprotective effect after the PG treatment FJB staining was used. Our results demonstrate a lack of theneurotoxicity of the PG in intact P12. In the second part of the study in the model of the focal ischemia wedetected significantly lower occurrence of FJB-positive cells in the afflicted hippocampus in PG treatedgroups, while animals without PG treatment exhibited massive neurodegeneration. The neuroprotectivepotential of the PG can serve in the development of therapeutic strategies for brain damage induced bythe glutamate excitotoxicity.

ntroduction

Perinatal insults such as stroke are an important cause of neu-

ological morbidity in infants and children, with the incidence oforbidity in approximately eight cases out of 100,000 per year [9].

n children and newborns, stroke is often unrecognized because of

∗ Corresponding author. Tel.: +420 24106 2713/+420 29644 2713;ax: +420 24106 2488.

E-mail addresses: vales@biomed.cas.cz, vales@fgu.cas.cz (K. Vales).

304-3940/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.neulet.2014.01.057

© 2014 Elsevier Ireland Ltd. All rights reserved.

variation in evaluation and diagnosis. Generally, a perinatal strokeoccurs in approximately one in 4000 term births [23] and can resultin death or long-term neurological consequences including cogni-tive and motor disabilities.

Glutamate excitotoxicity has emerged as an important mecha-nism of injury in the adult brain. The pathophysiological processesas well as neuroprotection related to deregulated glutamate

neurotransmission are relatively well described in adults, butinformation concerning early postnatal period is largely lack-ing. Glutamate acts on various membrane receptors includingNMDA, AMPA and kainate receptors [36]. The activity of NMDA

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nd AMPA receptors are enhanced in the immature brain to pro-ote activity-dependent plasticity [12]. Ischemia disrupts synaptic

unction, leads to accumulation of extracellular glutamate and sub-equent excitotoxic brain damage. Taken together excitotoxicity isn attractive target for neuroprotective efforts in early postnataleriod. The model of the focal cerebral ischemia induced by infusionf a vasoconstrictive peptide endothelin-1 (ET-1) into the dorsalippocampus of immature rats was used. It is considered to be theeproducible model of human stroke [8]. It causes properly describeschemic area and is well feasible in immature rats. Intracerebralnfusion of the ET-1 leads to glutamate excitotoxicity, followedy the development of the ischemic lesions and ischemia-inducedeizures in immature rats [7,37].

Functional inhibition of NMDA receptors can be achievedhrough the actions at different recognition sites. Most agents thatompletely block NMDA receptors cause undesirable side effectsuch as memory impairment, psychotomimetic effects, ataxia andisruption of motor coordination [5]. Against this the ion channellockers with moderate affinity and low-affinity negative uncom-etitive modulators show a much better profile than high affinityhannel blockers [29].

Many experimental studies have shown therapeutic poten-ial of neurosteroids. We found that the neuroactive steroid�5�-pregnanolone glutamate (PG), a synthetic analog ofaturally-occurring 3�5�-pregnanolone sulfate, inhibits prefer-ntially tonically activated NMDA receptors included in thexcitotoxic action of glutamate. Previous results showed that thection of derivatives of pregnanolone is independent of the cellembrane potential opposite ion channel blockers of NMDA recep-

ors like a MK-801. PG binding to its inhibitory binding site isonditioned by the activation of NMDA receptor by agonist. There-ore, PG is a use-dependent allosteric inhibitor of NMDA receptors14,31]. Application of PG has no behavioral side effects and it canenetrate through the blood–brain barrier [32]. It can be assumed,hat drugs possessing neuroprotective properties with minimalide effects, i.e. with more favorable benefit/risk ratio are promisingor future therapies.

The aims of the study were:

1) To analyze the potential neurotoxic effect of PG administra-tion in P12 in comparison with MK-801 (positive control ofneurotoxicity).

2) To evaluate neuroprotective effect of PG in the model of focalcerebral ischemia induced by intrahippocampal infusion of ET-1 in P12.

aterial and methods

nimals

Experiments were performed in immature male albino Wistarats bred by Institute of Physiology of the Academy of Sciences ofhe Czech Republic (CZ 11760353), and the day of birth was defineds day 0. Rats were housed in a controlled environment (tempera-ure 22 ± 1 ◦C, humidity 50–60%, lights on 6:00 am–6:00 pm) withree access to food and water. Experiments were approved by thenimal Care and Use Committee of the Institute of Physiology of

he Academy of Sciences of the Czech Republic and by The Cen-ral Committee of the Academy of Sciences of the Czech Republicnumber approval 095/2010). The Institute of Physiology possessesIH Statement of Compliance with Standards for Human Care and

se of Laboratory Animals no. A5820-01 valid till 1/31/2014. Ani-al care and experimental procedures are conducted in accordanceith the guidelines of the European Community Council directives

6/609/EEC. In the first experiment a total of 13 animals were used:

Letters 564 (2014) 11–15

4 animals for 1 mg/kg of MK-801, 3 animals for 1 mg/kg of PG and6 animals for 10 mg/kg of PG. In the second experiment a total of48 animals were used: 6–8 animals were tested in each of the sixgroups.

Drugs

Neuroactive steroid PG were synthesized by the Department ofNeuroprotectives (Institute of Organic Chemistry and BiochemistryASCR v.v.i., Prague). PG was dissolved in a 1 ml of the 88 mM �-cyclodextrin (CDX); (No. C4767, Sigma-Aldrich, St. Louis, MO, USA)with addition of 3 ml saline and the final pH was adjusted to 7.4value. CDX is a well-established solubility enhancer of poorly sol-uble steroid substances. Freshly prepared solutions in the dosesof 1 and 10 mg/kg were sonificated for 30 min and stored at 4 ◦Covernight. The following day, the solutions were sonificated for30 min before administration. Solutions were used only once. Focalischemia was induced by the intrahippocampal infusion of ET-1(No. E7764, Sigma-Aldrich, St. Louis, MO, USA) dissolved in 10 mMphosphate buffer (No. 79382, Sigma-Aldrich, St. Louis, MO, USA) ina concentration 40 pmol and a total volume of 1 �l. ET-1 solutionhad been fractioned on a small volume aliquots (20 �l) and frozenat −20 ◦C. Each aliquot was used only once.

Controls received intrahippocampal infusion of a correspondingvolume of only phosphate buffer solution (PBS) or in combinationwith systemic administration of CDX. In addition, separate groupsof animals with only ET-1 infusion and with ET-1 in combinationwith CDX were done.

Experiment 1: Analysis of neurotoxicity effect of PG

Intact P12 animals were injected intraperitoneally with PG in adose of 1 or 10 mg/kg. Additional group of animals received MK-801 in a dose of 1 mg/kg as a positive control for neurotoxic effects[11]. Corresponding volume of the vehicle was intraperitoneallyadministrated to the control animals. All pups were returned totheir dams for necessary care and 24 h after application the animalswere anesthetized with urethane (No. U2500, Sigma-Aldrich, St.Louis, MO, USA) in a dose of 2 g/kg and transcardially perfused with4% paraformaldehyde as described before [6]. Degenerating cellswere detected using visual inspection of Fluoro Jade B (FJB) stainedsection.

Experiment 2: Analysis of neuroprotective effect of PG in themodel of the focal cerebral ischemia induced by intrahippocampalinfusion of ET-1

Surgical preparation of P12 animals was performed under1.5–2% isoflurane anesthesia (No. B306, Abbot Laboratories, Queen-borought, UK). Animals were placed into a stereotaxic apparatus,the skin on the head was carefully cut up and a cannula (No.C315IA/SP, Plastics One Inc., Roanoke, USA) for drug applicationwas implanted into the right dorsal hippocampus (AP = 3.7; L = 3.0;H = 3.5 mm relative to bregma). Coordinates were recalculated foreach animal according to Paxinos et al. [30].

The infusion was done by pump (kds No. 789200W, WPI, USA)with a constant flow rate of 0.25 �l/min and 1 min after the end ofthe infusion, the skin was glued by organic glue (No. 70330–11000,Collodium, Penta, Czech Republic) and anesthesia was terminated.Five minutes after the end of the ET-1 infusion PG in a dose of1 or 10 mg/kg was administrated intraperitoneally. Animals werevideo-monitored for 2.5 h for observation of the behavioral man-

ifestation of epileptic seizures. At the end of monitoring, animalswere returned to their dams for care. During all the experiments,rats were kept at the temperature of the nest (34 ± 0.5 ◦C). 24 hafter application, rats were anesthetized with urethane (No. U2500,

L. Kleteckova et al. / Neuroscience Letters 564 (2014) 11–15 13

Fig. 1. Fluoro Jade B—positive cells in the hippocampus 24 h after infusion of ET-1 (40 pmol; 1 �l) alone or in combination with PG. On the left side, infusion of ET-1 (braincode ETPG11, total damage score is 11.42) with intraperitoneal administration of CDX. FJB-positive cells are present in all hippocampal subfield and in small number also ind damaF us. CAt amic n

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dose of 10 mg/kg was 0.46 ± 0.29 (Fig. 2). The extensive hippocam-pal damage was evident (total score 10.25 ± 1.45) in animals withET-1 infusion followed by the systemic administration of corre-sponding volume of CDX. Ischemic injury in ET-1 and ET-1 + CDX

Fig. 2. Total neuronal damage score in experimental groups (data showed asmean ± S.E.M.). On axe Y are values of total neuronal damage (score) in theafflicted hippocampus in units are on axe Y; on X—individual bars represent exper-imental groups: PBS—intrahippocampal application of 10 mM PBS in total volume1 �l; (n = 5). PBS + CDX—intrahippocampal infusion of PBS followed by intraperi-toneally administration of CDX; (n = 6). ET-1—intrahippocampal application ofendothelin-1 (40 pmol; 1 �l); (n = 11). ET-1 + CDX—intrahippocampal infusion ofET-1 and systemic injection of CDX; (n = 6). ET-1 + PG1—intrahippocampal appli-

orsal thalamic nuclei. On the right side, infusion of ET-1 (brain code ETPG14, totalJB-positive cells is present only in restricted area of CA3 subfield of the hippocamphalamic nucleus, RSG—retrospenial granular cortex, VP-ventral posterolateral thal

igma-Aldrich, St. Louis, MO, USA) in a dose of 2 g/kg and transcar-ially perfused as described above.

istology

Brains were cryoprotected in gradual concentrations of sucrose,rozen and sectioned in the coronal plane (50 �m, 1-in-5 series).ll sections were collected in, and stored at −20 ◦C in the cryopro-

ective solution until used. One series of sections was FJB stained35] and used to analyze distribution of degenerating neurons inhe hippocampus as described before [37]. For regional determina-ion of the severity of injury, hippocampal subfield CA1 and CA3,he dentate granule cell layer and the hilus were evaluated sepa-ately. Severity of the damage was scored from 0 to 4 according toxtension of damaged subfield (0: 0–5% of the area is damaged; 1:–25%; 2: 26–50%; 3: 51–75%; 4: >75%). Average score was calcu-

ated for each hippocampal subfield and then summarized for eachnimal. Total score therefore ranged from 0 to 16. The percentagef animals with FJB-positive cells located in other brain areas wasalculated in each group.

tatistic

Statistical analyses for comparison between different groupsf animals were performed using nonparametric Mann Whitneytest. All parameters were analyzed using SigmaStat (SPSS Inc.,

hicago, IL, USA). The level of statistical significance was acceptedt p < 0.05.

esults

No signs of neurotoxic effects were detected in animals injectedith PG. FJB-positive cells were not observed in any of the P12 ani-als receiving PG in a dose of 1 mg/kg or 10 mg/kg in any brain

tructure. In contrast the animals injected with MK-801 (1 mg/kg)t P12 sparse FJB-positive cells were observed 24 h later in thenteroventral, anterodorsal and mediodorsal nuclei of the thala-us, in several cortical regions (the prelimbic, infralimbic, cingular

nd retrosplenial cortices), in the subiculum, and in the mamillaryucleus in all injected animal.

In P12 animals after focal cerebral ischemia, the neuroactive

teroid PG provided clear neuroprotective effect. Intrahippocam-al infusion of the ET-1 led to the development of lesion after 24 haverage damage score 11.56 ± 0.66), whereas the intensity of FJB-ositive cells distribution was significantly (p < 0.05) reduced in

ge score is 0.83) with intraperitoneal application of PG 1 mg/kg. Limited number of1, CA3—fields of hippocampus, DG—dentate gyrus, Hb—habenula, LD—laterodorsalucleus.

PG-treated groups (Fig. 1). Moreover, the neurodegeneration hada tendency to be more reduced if higher dose (10 mg/kg) of the PGwas used. The total score range in animals treated with the PG in adose of 1 mg/kg was 0.74 ± 0.31, while in animals with the PG in a

cation of ET-1 followed by intraperitoneal administration of PG 1 mg/kg; (n = 12).ET-1 + PG10—intrahippocampal application of ET-1 and intraperitoneal administra-tion of PG 10 mg/kg; (n = 8). Asterisk—significant difference (p < 0.001) as comparedto: PBS, PBS + CDX, ET-1 + PG1 and ET-1 + PG10 groups; cross-significant difference(p < 0.05) as compared to PBS group.

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roups was observed in all evaluated areas of an afflicted dorsalippocampus. Also in these groups isolated FJB-positive neurons inhe dorsal thalamic nuclei were found in 87.5% and 80% of animals,espectively (Fig. 2). Infusion of PG a dose of 1 mg/kg reduced thencidence of thalamic damage to 50% (p = 0.044) and PG in a dosef 10 mg/kg only to 12.5% (p < 0.001). Only negligible numbers ofJB-positive cells were observed in the area close to cannula afterntrahippocampal injection of PBS in a total volume of 1 �l as well asfter infusion of PBS in combination with systemic administrationf corresponding volume of CDX.

iscussion

Searching for novel drugs potentially useful for therapy of CNSamage belongs to the most investigated topic in contemporaryharmacology and neuroscience. The aim of the present study waso evaluate neuroprotective properties of neurosteroid PG in themmature brain.

The first finding is a lack of neurotoxicity of PG in intact P12.pplication of MK-801, a noncompetitive NMDA antagonist, dur-

ng the first two weeks after birth in developing brain leads toidespread apoptotic neurodegeneration [12], which is consistentith our findings. The similar apoptotic damage was also observed

fter systemic administration of other NMDA antagonists [11,33].n the other hand, memantine, a low affinity uncompetitiveMDA antagonist, protects hippocampal neurons against gluta-ate excitotoxic injury and its administration did not show serious

ehavioral side effects [4]. In addition, specific NR2B subunit antag-nists displayed absence of the signs of serious neurotoxicity [21].hese facts and our results confirm that administration of someinds of NMDA antagonists did not lead to extensive apoptotic neu-odegeneration and potentially have better benefit/risk ratio in themmature brain.

The second finding of this study is the neuroprotective effect ofG in the model of focal ischemia in P12 rats. This age correspondsith the human early developmental stages in the first year of life.oreover, the vascular network in this age has higher numbers

f anastomosis, which can contribute to more enormous ischemicnjury [10]. This fact can also worsen the lesion development, espe-ially due to a high concentration of extracellular glutamate duringhe reperfusion. In addition, ET-1 has been shown to inhibit gluta-

ate uptake by a non-vascular mechanism, which also contributeo a more extensive brain injury [18].

Some animal models of ischemia are difficult applicable inmmature rats. We did not use widely practiced middle cerebralrtery occlusion (MCAO), because a variable range and locationf ischemic injury could occur in this model [25,34]. Currentlylso exist the variant MCAO via ET-1 infusion. In this model issed higher concentration of ET-1, which can also lead to dis-nity and fragmentation of ischemic damage [34]. A localizedtroke induced by the ET-1, in the rat model of focal cerebralschemia, can be used to study ischemia-induced consequencesacute as well as long-term: neuronal damage, memory deficit,pileptogenesis, local metabolic changes, etc.). Our previous studies24,37] demonstrated that unilateral intrahippocampal injectionf ET-1 in immature animals leads to the development of elec-rographic seizures with behavioral manifestations and induce

well-nigh limited morphological damage of an afflicted hip-ocampus. In addition, in humans and in animal models, theippocampus appears to be particularly vulnerable to perinataltroke [1,3]. Therefore, we selected intrahippocampal application

f ET-1.

Previously we demonstrated a therapeutic effect of PG in theilateral NMDA-induced lesions of hippocampus [32]. A neuropro-ective effect of PG observed in our model of the focal ischemia

Letters 564 (2014) 11–15

coincided with several reports concerning neuroprotectivepotential of other neuroactive steroids, for example 3�-ol-5�-pregnan-20-one hemisuccinate (PHS) [15]. Intravenousadministration of PHS in adult rats 30 min after middle cerebralartery occlusion reduced the infarct size. In another study, PHSexpressively reduced consequences of reversible spinal cordischemia in a rabbit [17]. The same author has reported neuropro-tective properties of PHS in the rabbit small clot embolic strokemodel [15].

In the other models of ischemia, naturally-occurring neuros-teroids were also tested. For example, dehydroepiandrosteronsulphate (DHEAS) was tested in bilateral occlusion of the com-mon carotid arteries [20]. The same authors also demonstrated theneuroprotective effect of dehydroepiandrosterone (DHEA) in the4-vessel occlusion model. Single administration of DHEA causedreduction of neuronal death and improvement of spatial learningdeficit [19].

The administration of memantine in immature rats led tothe reduction of glutamate level and neuroprotection in bilateralcarotid ligation with subsequent hypoxia [2]. In another study, aneuroprotective effect of memantine was also demonstrated in therabbit small clot embolic stroke model [16]. Administration of MK-801 had a neuroprotective effect in the global cerebral ischemiacombined with hypotension [22] and the ischemic spinal cordinjury [13]. In addition, it has been confirmed in MCAO model viaET-1 infusion [28]. Nevertheless, it should be emphasized, that thetherapeutic application of MK-801 is difficult due to serious behav-ioral side effects observed after its administration.

Neuroactive steroids exert very complex and often biphasiceffects. The influence of neurosteroids is associated with mod-ulation of GABAA and NMDA receptors [26,27]. The crucial roleof NMDA receptors in glutamatergic excitotoxicity leads to risinginterest about interaction of neurosteroids and NMDA receptors[14]. Nonetheless, the neurochemical and pharmacological mech-anisms are not fully understood and maybe involve multipleneurotransmitter receptor systems. Further studies aimed at ana-lyzing the regional, cellular and molecular activities are requiredand planned.

Taken together, our results demonstrate that early administra-tion of PG leads to a significant reduction of neurodegenerativechanges in the model of the focal cerebral ischemia. Importantly, incomparison with literary data, administration of PG shows minimalneurotoxicity in the immature brain. The present results suggestthat use-dependent NMDA receptor antagonists, possessing neu-roprotective properties and having minimal side effects can bepromising drugs for future therapies.

Conclusions

We demonstrated the neuroprotective effect of PG in ischemicimmature brain. Well-timed systemic administration of PG signif-icantly reduced range of the neuronal damage in ET-1 inducedmodel of the focal cerebral ischemia. Importantly, as a potentialtherapeutic agent, this drug does not show direct neurotoxic effectin the immature brain. In the future we would like to focus on theimportance of PG and other synthetic neuroactive steroids in theischemic damage of the brain and in other pathophysiological pro-cesses influenced by glutamate excitotoxicity in adult and as wellas immature rats.

Conflict of interest statement

There is no conflict of interests.

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cknowledgments

We gratefully acknowledge the expert technical help by Mrs.lanka Cejkova and Mrs. Michaela Fialova. We are appreciative tor. Ladislav Vyklicky, Dr. Hana Chodounska and Dr. Eva Kudova

or scientific inspiration and for steroid synthesis. We would alsoike to express our gratitude to Dr. Jaroslava Folbergrova for criti-al reading of the manuscript and her inspiring comments. Last butot least, we want to express thank to Ms. Thuy Hua for languageheck. This study was supported by GACR grants P304/11/P386304/14/20613S P304/12/G069, P303/12/1464 TACR-TE01020028nd GAUK 604412, institutional support RVO: 67985823.

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