Neuroscience Letters 564 (2014) 11–15
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Neuroprotective effect of the 3␣5-pregnanolone glutamate treatment in the model of focal cerebral ischemia in immature rats Lenka Kleteckova a,c , Grygoriy Tsenov b , Hana Kubova b , Ales Stuchlik a , Karel Vales a,∗ a Department of Neurophysiology of Memory, Institute of Physiology, v.v.i. Academy of Sciences of the Czech Republic, Videnska 1083, 14220 Prague 4, Czech Republic b Department of Developmental Epileptology, Institute of Physiology, v.v.i. Academy of Sciences of the Czech Republic, Videnska 1083, 14220 Prague 4, Czech Republic c 2nd Faculty of Medicine, Charles University, Prague, Czech Republic, V Uvalu 84, 150 06 Prague 5—Motol, Czech Republic
h 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.
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Article history: Received 28 November 2013 Received in revised form 20 January 2014 Accepted 29 January 2014 Keywords: 3␣5-Pregnanolone glutamate NMDA receptors Immature rats Focal cerebral ischemia Endothelin-1
a b s t r a c t The perinatal hypoxic-ischemic insult frequently leads to mortality, morbidity and plays a key role in the later pathological consequences. The ischemic insult causes a massive release of glutamate and subsequent excitotoxic damage. The neuroactive steroid 3␣5-pregnanolone glutamate (PG) is a NMDA receptor antagonist acting via use-dependent mechanism and can be used as a neuroprotective agent that may alleviate glutamatergic excitotoxicity in the brain. First, a possible neurotoxic effect of the PG, a novel use-dependent NMDA antagonist, was studied in immature rats. In addition, to compare this effect with a well-described non-competitive NMDA antagonist, the MK-801 (positive control) was used. Animals at postnatal day 12 (P12) were injected intraperitoneally with PG in a doses 1 or 10 mg/kg or with MK-801 in a dose 1 mg/kg. Effect of PG treatment on the immature brain was evaluated on Fluoro Jade B (FJB) stained sections. Second, a neuroprotective effect of the PG was studied in the model of focal cerebral ischemia in P12. Focal cerebral ischemia was induced by the infusion of the endothelin-1 (ET-1) into the right dorsal hippocampus. PG at the doses 1 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 the neurotoxicity of the PG in intact P12. In the second part of the study in the model of the focal ischemia we detected significantly lower occurrence of FJB-positive cells in the afflicted hippocampus in PG treated groups, while animals without PG treatment exhibited massive neurodegeneration. The neuroprotective potential of the PG can serve in the development of therapeutic strategies for brain damage induced by the glutamate excitotoxicity. © 2014 Elsevier Ireland Ltd. All rights reserved.
Introduction Perinatal insults such as stroke are an important cause of neurological morbidity in infants and children, with the incidence of morbidity in approximately eight cases out of 100,000 per year [9]. In children and newborns, stroke is often unrecognized because of
∗ Corresponding author. Tel.: +420 24106 2713/+420 29644 2713; fax: +420 24106 2488. E-mail addresses: [email protected], [email protected] (K. Vales). 0304-3940/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2014.01.057
variation in evaluation and diagnosis. Generally, a perinatal stroke occurs in approximately one in 4000 term births [23] and can result in death or long-term neurological consequences including cognitive and motor disabilities. Glutamate excitotoxicity has emerged as an important mechanism of injury in the adult brain. The pathophysiological processes as well as neuroprotection related to deregulated glutamate neurotransmission are relatively well described in adults, but information concerning early postnatal period is largely lacking. Glutamate acts on various membrane receptors including NMDA, AMPA and kainate receptors [36]. The activity of NMDA
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and AMPA receptors are enhanced in the immature brain to promote activity-dependent plasticity [12]. Ischemia disrupts synaptic function, leads to accumulation of extracellular glutamate and subsequent excitotoxic brain damage. Taken together excitotoxicity is an attractive target for neuroprotective efforts in early postnatal period. The model of the focal cerebral ischemia induced by infusion of a vasoconstrictive peptide endothelin-1 (ET-1) into the dorsal hippocampus of immature rats was used. It is considered to be the reproducible model of human stroke [8]. It causes properly describe ischemic area and is well feasible in immature rats. Intracerebral infusion of the ET-1 leads to glutamate excitotoxicity, followed by the development of the ischemic lesions and ischemia-induced seizures in immature rats [7,37]. Functional inhibition of NMDA receptors can be achieved through the actions at different recognition sites. Most agents that completely block NMDA receptors cause undesirable side effects such as memory impairment, psychotomimetic effects, ataxia and disruption of motor coordination [5]. Against this the ion channel blockers with moderate affinity and low-affinity negative uncompetitive modulators show a much better profile than high affinity channel blockers [29]. Many experimental studies have shown therapeutic potential of neurosteroids. We found that the neuroactive steroid 3␣5-pregnanolone glutamate (PG), a synthetic analog of naturally-occurring 3␣5-pregnanolone sulfate, inhibits preferentially tonically activated NMDA receptors included in the excitotoxic action of glutamate. Previous results showed that the action of derivatives of pregnanolone is independent of the cell membrane potential opposite ion channel blockers of NMDA receptors like a MK-801. PG binding to its inhibitory binding site is conditioned by the activation of NMDA receptor by agonist. Therefore, PG is a use-dependent allosteric inhibitor of NMDA receptors [14,31]. Application of PG has no behavioral side effects and it can penetrate through the blood–brain barrier [32]. It can be assumed, that drugs possessing neuroprotective properties with minimal side effects, i.e. with more favorable benefit/risk ratio are promising for future therapies. The aims of the study were: (1) To analyze the potential neurotoxic effect of PG administration in P12 in comparison with MK-801 (positive control of neurotoxicity). (2) To evaluate neuroprotective effect of PG in the model of focal cerebral ischemia induced by intrahippocampal infusion of ET1 in P12. Material and methods Animals Experiments were performed in immature male albino Wistar rats bred by Institute of Physiology of the Academy of Sciences of the Czech Republic (CZ 11760353), and the day of birth was defined as day 0. Rats were housed in a controlled environment (temperature 22 ± 1 ◦ C, humidity 50–60%, lights on 6:00 am–6:00 pm) with free access to food and water. Experiments were approved by the Animal Care and Use Committee of the Institute of Physiology of the Academy of Sciences of the Czech Republic and by The Central Committee of the Academy of Sciences of the Czech Republic (number approval 095/2010). The Institute of Physiology possesses NIH Statement of Compliance with Standards for Human Care and Use of Laboratory Animals no. A5820-01 valid till 1/31/2014. Animal care and experimental procedures are conducted in accordance with the guidelines of the European Community Council directives 86/609/EEC. In the first experiment a total of 13 animals were used:
4 animals for 1 mg/kg of MK-801, 3 animals for 1 mg/kg of PG and 6 animals for 10 mg/kg of PG. In the second experiment a total of 48 animals were used: 6–8 animals were tested in each of the six groups. Drugs Neuroactive steroid PG were synthesized by the Department of Neuroprotectives (Institute of Organic Chemistry and Biochemistry ASCR 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 7.4 value. CDX is a well-established solubility enhancer of poorly soluble steroid substances. Freshly prepared solutions in the doses of 1 and 10 mg/kg were sonificated for 30 min and stored at 4 ◦ C overnight. The following day, the solutions were sonificated for 30 min before administration. Solutions were used only once. Focal ischemia was induced by the intrahippocampal infusion of ET-1 (No. E7764, Sigma-Aldrich, St. Louis, MO, USA) dissolved in 10 mM phosphate buffer (No. 79382, Sigma-Aldrich, St. Louis, MO, USA) in a concentration 40 pmol and a total volume of 1 l. ET-1 solution had been fractioned on a small volume aliquots (20 l) and frozen at −20 ◦ C. Each aliquot was used only once. Controls received intrahippocampal infusion of a corresponding volume of only phosphate buffer solution (PBS) or in combination with systemic administration of CDX. In addition, separate groups of animals with only ET-1 infusion and with ET-1 in combination with CDX were done. Experiment 1: Analysis of neurotoxicity effect of PG Intact P12 animals were injected intraperitoneally with PG in a dose of 1 or 10 mg/kg. Additional group of animals received MK801 in a dose of 1 mg/kg as a positive control for neurotoxic effects [11]. Corresponding volume of the vehicle was intraperitoneally administrated to the control animals. All pups were returned to their dams for necessary care and 24 h after application the animals were anesthetized with urethane (No. U2500, Sigma-Aldrich, St. Louis, MO, USA) in a dose of 2 g/kg and transcardially perfused with 4% paraformaldehyde as described before [6]. Degenerating cells were detected using visual inspection of Fluoro Jade B (FJB) stained section. Experiment 2: Analysis of neuroprotective effect of PG in the model of the focal cerebral ischemia induced by intrahippocampal infusion of ET-1 Surgical preparation of P12 animals was performed under 1.5–2% isoflurane anesthesia (No. B306, Abbot Laboratories, Queenborought, 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 application was implanted into the right dorsal hippocampus (AP = 3.7; L = 3.0; H = 3.5 mm relative to bregma). Coordinates were recalculated for each 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 of the 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 of 1 or 10 mg/kg was administrated intraperitoneally. Animals were video-monitored for 2.5 h for observation of the behavioral manifestation of epileptic seizures. At the end of monitoring, animals were returned to their dams for care. During all the experiments, rats were kept at the temperature of the nest (34 ± 0.5 ◦ C). 24 h after application, rats were anesthetized with urethane (No. U2500,
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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 (brain code 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 in dorsal thalamic nuclei. On the right side, infusion of ET-1 (brain code ETPG14, total damage score is 0.83) with intraperitoneal application of PG 1 mg/kg. Limited number of FJB-positive cells is present only in restricted area of CA3 subfield of the hippocampus. CA1, CA3—fields of hippocampus, DG—dentate gyrus, Hb—habenula, LD—laterodorsal thalamic nucleus, RSG—retrospenial granular cortex, VP-ventral posterolateral thalamic nucleus.
Sigma-Aldrich, St. Louis, MO, USA) in a dose of 2 g/kg and transcardially perfused as described above. Histology Brains were cryoprotected in gradual concentrations of sucrose, frozen and sectioned in the coronal plane (50 m, 1-in-5 series). All sections were collected in, and stored at −20 ◦ C in the cryoprotective solution until used. One series of sections was FJB stained [35] and used to analyze distribution of degenerating neurons in the hippocampus as described before [37]. For regional determination of the severity of injury, hippocampal subfield CA1 and CA3, the dentate granule cell layer and the hilus were evaluated separately. Severity of the damage was scored from 0 to 4 according to extension of damaged subfield (0: 0–5% of the area is damaged; 1: 6–25%; 2: 26–50%; 3: 51–75%; 4: >75%). Average score was calculated for each hippocampal subfield and then summarized for each animal. Total score therefore ranged from 0 to 16. The percentage of animals with FJB-positive cells located in other brain areas was calculated in each group.
PG-treated groups (Fig. 1). Moreover, the neurodegeneration had a tendency to be more reduced if higher dose (10 mg/kg) of the PG was used. The total score range in animals treated with the PG in a dose of 1 mg/kg was 0.74 ± 0.31, while in animals with the PG in a dose of 10 mg/kg was 0.46 ± 0.29 (Fig. 2). The extensive hippocampal damage was evident (total score 10.25 ± 1.45) in animals with ET-1 infusion followed by the systemic administration of corresponding volume of CDX. Ischemic injury in ET-1 and ET-1 + CDX
Statistic Statistical analyses for comparison between different groups of animals were performed using nonparametric Mann Whitney U test. All parameters were analyzed using SigmaStat (SPSS Inc., Chicago, IL, USA). The level of statistical significance was accepted at p < 0.05. Results No signs of neurotoxic effects were detected in animals injected with PG. FJB-positive cells were not observed in any of the P12 animals receiving PG in a dose of 1 mg/kg or 10 mg/kg in any brain structure. In contrast the animals injected with MK-801 (1 mg/kg) at P12 sparse FJB-positive cells were observed 24 h later in the anteroventral, anterodorsal and mediodorsal nuclei of the thalamus, in several cortical regions (the prelimbic, infralimbic, cingular and retrosplenial cortices), in the subiculum, and in the mamillary nucleus in all injected animal. In P12 animals after focal cerebral ischemia, the neuroactive steroid PG provided clear neuroprotective effect. Intrahippocampal infusion of the ET-1 led to the development of lesion after 24 h (average damage score 11.56 ± 0.66), whereas the intensity of FJBpositive cells distribution was significantly (p < 0.05) reduced in
Fig. 2. Total neuronal damage score in experimental groups (data showed as mean ± S.E.M.). On axe Y are values of total neuronal damage (score) in the afflicted hippocampus in units are on axe Y; on X—individual bars represent experimental groups: PBS—intrahippocampal application of 10 mM PBS in total volume 1 l; (n = 5). PBS + CDX—intrahippocampal infusion of PBS followed by intraperitoneally administration of CDX; (n = 6). ET-1—intrahippocampal application of endothelin-1 (40 pmol; 1 l); (n = 11). ET-1 + CDX—intrahippocampal infusion of ET-1 and systemic injection of CDX; (n = 6). ET-1 + PG1—intrahippocampal application of ET-1 followed by intraperitoneal administration of PG 1 mg/kg; (n = 12). ET-1 + PG10—intrahippocampal application of ET-1 and intraperitoneal administration of PG 10 mg/kg; (n = 8). Asterisk—significant difference (p < 0.001) as compared to: 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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groups was observed in all evaluated areas of an afflicted dorsal hippocampus. Also in these groups isolated FJB-positive neurons in the dorsal thalamic nuclei were found in 87.5% and 80% of animals, respectively (Fig. 2). Infusion of PG a dose of 1 mg/kg reduced the incidence of thalamic damage to 50% (p = 0.044) and PG in a dose of 10 mg/kg only to 12.5% (p < 0.001). Only negligible numbers of FJB-positive cells were observed in the area close to cannula after intrahippocampal injection of PBS in a total volume of 1 l as well as after infusion of PBS in combination with systemic administration of corresponding volume of CDX.
Discussion Searching for novel drugs potentially useful for therapy of CNS damage belongs to the most investigated topic in contemporary pharmacology and neuroscience. The aim of the present study was to evaluate neuroprotective properties of neurosteroid PG in the immature brain. The first finding is a lack of neurotoxicity of PG in intact P12. Application of MK-801, a noncompetitive NMDA antagonist, during the first two weeks after birth in developing brain leads to widespread apoptotic neurodegeneration [12], which is consistent with our findings. The similar apoptotic damage was also observed after systemic administration of other NMDA antagonists [11,33]. On the other hand, memantine, a low affinity uncompetitive NMDA antagonist, protects hippocampal neurons against glutamate excitotoxic injury and its administration did not show serious behavioral side effects [4]. In addition, specific NR2B subunit antagonists displayed absence of the signs of serious neurotoxicity [21]. These facts and our results confirm that administration of some kinds of NMDA antagonists did not lead to extensive apoptotic neurodegeneration and potentially have better benefit/risk ratio in the immature brain. The second finding of this study is the neuroprotective effect of PG in the model of focal ischemia in P12 rats. This age corresponds with the human early developmental stages in the first year of life. Moreover, the vascular network in this age has higher numbers of anastomosis, which can contribute to more enormous ischemic injury [10]. This fact can also worsen the lesion development, especially due to a high concentration of extracellular glutamate during the reperfusion. In addition, ET-1 has been shown to inhibit glutamate uptake by a non-vascular mechanism, which also contribute to a more extensive brain injury [18]. Some animal models of ischemia are difficult applicable in immature rats. We did not use widely practiced middle cerebral artery occlusion (MCAO), because a variable range and location of ischemic injury could occur in this model [25,34]. Currently also exist the variant MCAO via ET-1 infusion. In this model is used higher concentration of ET-1, which can also lead to disunity and fragmentation of ischemic damage [34]. A localized stroke induced by the ET-1, in the rat model of focal cerebral ischemia, can be used to study ischemia-induced consequences (acute as well as long-term: neuronal damage, memory deficit, epileptogenesis, local metabolic changes, etc.). Our previous studies [24,37] demonstrated that unilateral intrahippocampal injection of ET-1 in immature animals leads to the development of electrographic seizures with behavioral manifestations and induce a well-nigh limited morphological damage of an afflicted hippocampus. In addition, in humans and in animal models, the hippocampus appears to be particularly vulnerable to perinatal stroke [1,3]. Therefore, we selected intrahippocampal application of ET-1. Previously we demonstrated a therapeutic effect of PG in the bilateral NMDA-induced lesions of hippocampus [32]. A neuroprotective effect of PG observed in our model of the focal ischemia
coincided with several reports concerning neuroprotective potential of other neuroactive steroids, for example 3␣-ol5-pregnan-20-one hemisuccinate (PHS) [15]. Intravenous administration of PHS in adult rats 30 min after middle cerebral artery occlusion reduced the infarct size. In another study, PHS expressively reduced consequences of reversible spinal cord ischemia in a rabbit [17]. The same author has reported neuroprotective properties of PHS in the rabbit small clot embolic stroke model [15]. In the other models of ischemia, naturally-occurring neurosteroids were also tested. For example, dehydroepiandrosteron sulphate (DHEAS) was tested in bilateral occlusion of the common carotid arteries [20]. The same authors also demonstrated the neuroprotective effect of dehydroepiandrosterone (DHEA) in the 4-vessel occlusion model. Single administration of DHEA caused reduction of neuronal death and improvement of spatial learning deficit [19]. The administration of memantine in immature rats led to the reduction of glutamate level and neuroprotection in bilateral carotid ligation with subsequent hypoxia [2]. In another study, a neuroprotective effect of memantine was also demonstrated in the rabbit small clot embolic stroke model [16]. Administration of MK801 had a neuroprotective effect in the global cerebral ischemia combined with hypotension [22] and the ischemic spinal cord injury [13]. In addition, it has been confirmed in MCAO model via ET-1 infusion [28]. Nevertheless, it should be emphasized, that the therapeutic application of MK-801 is difficult due to serious behavioral side effects observed after its administration. Neuroactive steroids exert very complex and often biphasic effects. The influence of neurosteroids is associated with modulation of GABAA and NMDA receptors [26,27]. The crucial role of NMDA receptors in glutamatergic excitotoxicity leads to rising interest about interaction of neurosteroids and NMDA receptors [14]. Nonetheless, the neurochemical and pharmacological mechanisms are not fully understood and maybe involve multiple neurotransmitter receptor systems. Further studies aimed at analyzing the regional, cellular and molecular activities are required and planned. Taken together, our results demonstrate that early administration of PG leads to a significant reduction of neurodegenerative changes in the model of the focal cerebral ischemia. Importantly, in comparison with literary data, administration of PG shows minimal neurotoxicity in the immature brain. The present results suggest that use-dependent NMDA receptor antagonists, possessing neuroprotective properties and having minimal side effects can be promising drugs for future therapies.
Conclusions We demonstrated the neuroprotective effect of PG in ischemic immature brain. Well-timed systemic administration of PG significantly reduced range of the neuronal damage in ET-1 induced model of the focal cerebral ischemia. Importantly, as a potential therapeutic agent, this drug does not show direct neurotoxic effect in the immature brain. In the future we would like to focus on the importance of PG and other synthetic neuroactive steroids in the ischemic damage of the brain and in other pathophysiological processes influenced by glutamate excitotoxicity in adult and as well as immature rats.
Conflict of interest statement There is no conflict of interests.
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Acknowledgments We gratefully acknowledge the expert technical help by Mrs. Blanka Cejkova and Mrs. Michaela Fialova. We are appreciative to Dr. Ladislav Vyklicky, Dr. Hana Chodounska and Dr. Eva Kudova for scientific inspiration and for steroid synthesis. We would also like to express our gratitude to Dr. Jaroslava Folbergrova for critical reading of the manuscript and her inspiring comments. Last but not least, we want to express thank to Ms. Thuy Hua for language check. This study was supported by GACR grants P304/11/P386 P304/14/20613S P304/12/G069, P303/12/1464 TACR-TE01020028 and GAUK 604412, institutional support RVO: 67985823. References [1] Y. Ben-Ari, Effects of anoxia and aglycemia on the adult and immature hippocampus, Biology of the Neonate 62 (1992) 225–230. [2] H.S. Chen, J.W. Pellegrini, S.K. Aggarwal, S.Z. Lei, S. Warach, F.E. Jensen, S.A. Lipton, Open-channel block of N-methyl-d-aspartate (NMDA) responses by memantine: therapeutic advantage against NMDA receptor-mediated neurotoxicity, Journal of Neuroscience: Official Journal of the Society for Neuroscience 12 (1992) 4427–4436. [3] M.R. Del Bigio, L.E. Becker, Microglial aggregation in the dentate gyrus: a marker of mild hypoxic-ischaemic brain insult in human infants, Neuropathology and Applied Neurobiology 20 (1994) 144–151. [4] S.S. Deshpande, C.D. Smith, M.G. Filbert, Assessment of primary neuronal culture as a model for soman-induced neurotoxicity and effectiveness of memantine as a neuroprotective drug, Archives of Toxicology 69 (1995) 384–390. [5] R. Dingledine, K. Borges, D. Bowie, S.F. Traynelis, The glutamate receptor ion channels, Pharmacological Reviews 51 (1999) 7–61. [6] R. Druga, P. Mares, J. Otahal, H. Kubova, Degenerative neuronal changes in the rat thalamus induced by status epilepticus at different developmental stages, Epilepsy Research 63 (2005) 43–65. [7] K. Fuxe, B. Bjelke, B. Andbjer, H. Grahn, R. Rimondini, L.F. Agnati, Endothelin-1 induced lesions of the frontoparietal cortex of the rat. A possible model of focal cortical ischemia, NeuroReport 8 (1997) 2623–2629. [8] G. Gilmour, S.D. Iversen, M.F. O’Neill, D.M. Bannerman, The effects of intracortical endothelin-1 injections on skilled forelimb use: implications for modelling recovery of function after stroke, Behavioural Brain Research 150 (2004) 171–183. [9] M. Giroud, M. Lemesle, J.B. Gouyon, J.L. Nivelon, C. Milan, R. Dumas, Cerebrovascular disease in children under 16 years of age in the city of Dijon, France: a study of incidence and clinical features from 1985 to 1993, Journal of Clinical Epidemiology 48 (1995) 1343–1348. [10] I. Grivas, H. Michaloudi, C. Batzios, M. Chiotelli, C. Papatheodoropoulos, G. Kostopoulos, G.C. Papadopoulos, Vascular network of the rat hippocampus is not homogeneous along the septotemporal axis, Brain Research 971 (2003) 245–249. [11] C. Ikonomidou, F. Bosch, M. Miksa, P. Bittigau, J. Vockler, K. Dikranian, T.I. Tenkova, V. Stefovska, L. Turski, J.W. Olney, Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain, Science 283 (1999) 70–74. [12] M.V. Johnston, Excitotoxicity in perinatal brain injury, Brain Pathology 15 (2005) 234–240. [13] H. Kocaeli, E. Korfali, H. Ozturk, N. Kahveci, S. Yilmazlar, MK-801 improves neurological and histological outcomes after spinal cord ischemia induced by transient aortic cross-clipping in rats, Surgical Neurology 64 (Suppl 2) (2005) S22–S26, discussion S27. [14] M. Korinek, V. Kapras, V. Vyklicky, E. Adamusova, J. Borovska, K. Vales, A. Stuchlik, M. Horak, H. Chodounska, L. Vyklicky Jr., Neurosteroid modulation of N-methyl-d-aspartate receptors: molecular mechanism and behavioral effects, Steroids 76 (2011) 1409–1418. [15] P.A. Lapchak, 3alpha-OL-5-beta-pregnan-20-one hemisuccinate, a steroidal low-affinity NMDA receptor antagonist improves clinical rating scores in a rabbit multiple infarct ischemia model: synergism with tissue plasminogen activator, Experimental Neurology 197 (2006) 531–537.
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Neuroprotective effect of the 3␣5-pregnanolone glutamate treatment in the model of focal cerebral ischemia in immature rats Lenka Kleteckova a,c , Grygoriy Tsenov b , Hana Kubova b , Ales Stuchlik a , Karel Vales a,∗ a Department of Neurophysiology of Memory, Institute of Physiology, v.v.i. Academy of Sciences of the Czech Republic, Videnska 1083, 14220 Prague 4, Czech Republic b Department of Developmental Epileptology, Institute of Physiology, v.v.i. Academy of Sciences of the Czech Republic, Videnska 1083, 14220 Prague 4, Czech Republic c 2nd Faculty of Medicine, Charles University, Prague, Czech Republic, V Uvalu 84, 150 06 Prague 5—Motol, Czech Republic
h 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.
a r t i c l e
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Article history: Received 28 November 2013 Received in revised form 20 January 2014 Accepted 29 January 2014 Keywords: 3␣5-Pregnanolone glutamate NMDA receptors Immature rats Focal cerebral ischemia Endothelin-1
a b s t r a c t The perinatal hypoxic-ischemic insult frequently leads to mortality, morbidity and plays a key role in the later pathological consequences. The ischemic insult causes a massive release of glutamate and subsequent excitotoxic damage. The neuroactive steroid 3␣5-pregnanolone glutamate (PG) is a NMDA receptor antagonist acting via use-dependent mechanism and can be used as a neuroprotective agent that may alleviate glutamatergic excitotoxicity in the brain. First, a possible neurotoxic effect of the PG, a novel use-dependent NMDA antagonist, was studied in immature rats. In addition, to compare this effect with a well-described non-competitive NMDA antagonist, the MK-801 (positive control) was used. Animals at postnatal day 12 (P12) were injected intraperitoneally with PG in a doses 1 or 10 mg/kg or with MK-801 in a dose 1 mg/kg. Effect of PG treatment on the immature brain was evaluated on Fluoro Jade B (FJB) stained sections. Second, a neuroprotective effect of the PG was studied in the model of focal cerebral ischemia in P12. Focal cerebral ischemia was induced by the infusion of the endothelin-1 (ET-1) into the right dorsal hippocampus. PG at the doses 1 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 the neurotoxicity of the PG in intact P12. In the second part of the study in the model of the focal ischemia we detected significantly lower occurrence of FJB-positive cells in the afflicted hippocampus in PG treated groups, while animals without PG treatment exhibited massive neurodegeneration. The neuroprotective potential of the PG can serve in the development of therapeutic strategies for brain damage induced by the glutamate excitotoxicity. © 2014 Elsevier Ireland Ltd. All rights reserved.
Introduction Perinatal insults such as stroke are an important cause of neurological morbidity in infants and children, with the incidence of morbidity in approximately eight cases out of 100,000 per year [9]. In children and newborns, stroke is often unrecognized because of
∗ Corresponding author. Tel.: +420 24106 2713/+420 29644 2713; fax: +420 24106 2488. E-mail addresses: [email protected], [email protected] (K. Vales). 0304-3940/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2014.01.057
variation in evaluation and diagnosis. Generally, a perinatal stroke occurs in approximately one in 4000 term births [23] and can result in death or long-term neurological consequences including cognitive and motor disabilities. Glutamate excitotoxicity has emerged as an important mechanism of injury in the adult brain. The pathophysiological processes as well as neuroprotection related to deregulated glutamate neurotransmission are relatively well described in adults, but information concerning early postnatal period is largely lacking. Glutamate acts on various membrane receptors including NMDA, AMPA and kainate receptors [36]. The activity of NMDA
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and AMPA receptors are enhanced in the immature brain to promote activity-dependent plasticity [12]. Ischemia disrupts synaptic function, leads to accumulation of extracellular glutamate and subsequent excitotoxic brain damage. Taken together excitotoxicity is an attractive target for neuroprotective efforts in early postnatal period. The model of the focal cerebral ischemia induced by infusion of a vasoconstrictive peptide endothelin-1 (ET-1) into the dorsal hippocampus of immature rats was used. It is considered to be the reproducible model of human stroke [8]. It causes properly describe ischemic area and is well feasible in immature rats. Intracerebral infusion of the ET-1 leads to glutamate excitotoxicity, followed by the development of the ischemic lesions and ischemia-induced seizures in immature rats [7,37]. Functional inhibition of NMDA receptors can be achieved through the actions at different recognition sites. Most agents that completely block NMDA receptors cause undesirable side effects such as memory impairment, psychotomimetic effects, ataxia and disruption of motor coordination [5]. Against this the ion channel blockers with moderate affinity and low-affinity negative uncompetitive modulators show a much better profile than high affinity channel blockers [29]. Many experimental studies have shown therapeutic potential of neurosteroids. We found that the neuroactive steroid 3␣5-pregnanolone glutamate (PG), a synthetic analog of naturally-occurring 3␣5-pregnanolone sulfate, inhibits preferentially tonically activated NMDA receptors included in the excitotoxic action of glutamate. Previous results showed that the action of derivatives of pregnanolone is independent of the cell membrane potential opposite ion channel blockers of NMDA receptors like a MK-801. PG binding to its inhibitory binding site is conditioned by the activation of NMDA receptor by agonist. Therefore, PG is a use-dependent allosteric inhibitor of NMDA receptors [14,31]. Application of PG has no behavioral side effects and it can penetrate through the blood–brain barrier [32]. It can be assumed, that drugs possessing neuroprotective properties with minimal side effects, i.e. with more favorable benefit/risk ratio are promising for future therapies. The aims of the study were: (1) To analyze the potential neurotoxic effect of PG administration in P12 in comparison with MK-801 (positive control of neurotoxicity). (2) To evaluate neuroprotective effect of PG in the model of focal cerebral ischemia induced by intrahippocampal infusion of ET1 in P12. Material and methods Animals Experiments were performed in immature male albino Wistar rats bred by Institute of Physiology of the Academy of Sciences of the Czech Republic (CZ 11760353), and the day of birth was defined as day 0. Rats were housed in a controlled environment (temperature 22 ± 1 ◦ C, humidity 50–60%, lights on 6:00 am–6:00 pm) with free access to food and water. Experiments were approved by the Animal Care and Use Committee of the Institute of Physiology of the Academy of Sciences of the Czech Republic and by The Central Committee of the Academy of Sciences of the Czech Republic (number approval 095/2010). The Institute of Physiology possesses NIH Statement of Compliance with Standards for Human Care and Use of Laboratory Animals no. A5820-01 valid till 1/31/2014. Animal care and experimental procedures are conducted in accordance with the guidelines of the European Community Council directives 86/609/EEC. In the first experiment a total of 13 animals were used:
4 animals for 1 mg/kg of MK-801, 3 animals for 1 mg/kg of PG and 6 animals for 10 mg/kg of PG. In the second experiment a total of 48 animals were used: 6–8 animals were tested in each of the six groups. Drugs Neuroactive steroid PG were synthesized by the Department of Neuroprotectives (Institute of Organic Chemistry and Biochemistry ASCR 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 7.4 value. CDX is a well-established solubility enhancer of poorly soluble steroid substances. Freshly prepared solutions in the doses of 1 and 10 mg/kg were sonificated for 30 min and stored at 4 ◦ C overnight. The following day, the solutions were sonificated for 30 min before administration. Solutions were used only once. Focal ischemia was induced by the intrahippocampal infusion of ET-1 (No. E7764, Sigma-Aldrich, St. Louis, MO, USA) dissolved in 10 mM phosphate buffer (No. 79382, Sigma-Aldrich, St. Louis, MO, USA) in a concentration 40 pmol and a total volume of 1 l. ET-1 solution had been fractioned on a small volume aliquots (20 l) and frozen at −20 ◦ C. Each aliquot was used only once. Controls received intrahippocampal infusion of a corresponding volume of only phosphate buffer solution (PBS) or in combination with systemic administration of CDX. In addition, separate groups of animals with only ET-1 infusion and with ET-1 in combination with CDX were done. Experiment 1: Analysis of neurotoxicity effect of PG Intact P12 animals were injected intraperitoneally with PG in a dose of 1 or 10 mg/kg. Additional group of animals received MK801 in a dose of 1 mg/kg as a positive control for neurotoxic effects [11]. Corresponding volume of the vehicle was intraperitoneally administrated to the control animals. All pups were returned to their dams for necessary care and 24 h after application the animals were anesthetized with urethane (No. U2500, Sigma-Aldrich, St. Louis, MO, USA) in a dose of 2 g/kg and transcardially perfused with 4% paraformaldehyde as described before [6]. Degenerating cells were detected using visual inspection of Fluoro Jade B (FJB) stained section. Experiment 2: Analysis of neuroprotective effect of PG in the model of the focal cerebral ischemia induced by intrahippocampal infusion of ET-1 Surgical preparation of P12 animals was performed under 1.5–2% isoflurane anesthesia (No. B306, Abbot Laboratories, Queenborought, 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 application was implanted into the right dorsal hippocampus (AP = 3.7; L = 3.0; H = 3.5 mm relative to bregma). Coordinates were recalculated for each 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 of the 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 of 1 or 10 mg/kg was administrated intraperitoneally. Animals were video-monitored for 2.5 h for observation of the behavioral manifestation of epileptic seizures. At the end of monitoring, animals were returned to their dams for care. During all the experiments, rats were kept at the temperature of the nest (34 ± 0.5 ◦ C). 24 h after application, rats were anesthetized with urethane (No. U2500,
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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 (brain code 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 in dorsal thalamic nuclei. On the right side, infusion of ET-1 (brain code ETPG14, total damage score is 0.83) with intraperitoneal application of PG 1 mg/kg. Limited number of FJB-positive cells is present only in restricted area of CA3 subfield of the hippocampus. CA1, CA3—fields of hippocampus, DG—dentate gyrus, Hb—habenula, LD—laterodorsal thalamic nucleus, RSG—retrospenial granular cortex, VP-ventral posterolateral thalamic nucleus.
Sigma-Aldrich, St. Louis, MO, USA) in a dose of 2 g/kg and transcardially perfused as described above. Histology Brains were cryoprotected in gradual concentrations of sucrose, frozen and sectioned in the coronal plane (50 m, 1-in-5 series). All sections were collected in, and stored at −20 ◦ C in the cryoprotective solution until used. One series of sections was FJB stained [35] and used to analyze distribution of degenerating neurons in the hippocampus as described before [37]. For regional determination of the severity of injury, hippocampal subfield CA1 and CA3, the dentate granule cell layer and the hilus were evaluated separately. Severity of the damage was scored from 0 to 4 according to extension of damaged subfield (0: 0–5% of the area is damaged; 1: 6–25%; 2: 26–50%; 3: 51–75%; 4: >75%). Average score was calculated for each hippocampal subfield and then summarized for each animal. Total score therefore ranged from 0 to 16. The percentage of animals with FJB-positive cells located in other brain areas was calculated in each group.
PG-treated groups (Fig. 1). Moreover, the neurodegeneration had a tendency to be more reduced if higher dose (10 mg/kg) of the PG was used. The total score range in animals treated with the PG in a dose of 1 mg/kg was 0.74 ± 0.31, while in animals with the PG in a dose of 10 mg/kg was 0.46 ± 0.29 (Fig. 2). The extensive hippocampal damage was evident (total score 10.25 ± 1.45) in animals with ET-1 infusion followed by the systemic administration of corresponding volume of CDX. Ischemic injury in ET-1 and ET-1 + CDX
Statistic Statistical analyses for comparison between different groups of animals were performed using nonparametric Mann Whitney U test. All parameters were analyzed using SigmaStat (SPSS Inc., Chicago, IL, USA). The level of statistical significance was accepted at p < 0.05. Results No signs of neurotoxic effects were detected in animals injected with PG. FJB-positive cells were not observed in any of the P12 animals receiving PG in a dose of 1 mg/kg or 10 mg/kg in any brain structure. In contrast the animals injected with MK-801 (1 mg/kg) at P12 sparse FJB-positive cells were observed 24 h later in the anteroventral, anterodorsal and mediodorsal nuclei of the thalamus, in several cortical regions (the prelimbic, infralimbic, cingular and retrosplenial cortices), in the subiculum, and in the mamillary nucleus in all injected animal. In P12 animals after focal cerebral ischemia, the neuroactive steroid PG provided clear neuroprotective effect. Intrahippocampal infusion of the ET-1 led to the development of lesion after 24 h (average damage score 11.56 ± 0.66), whereas the intensity of FJBpositive cells distribution was significantly (p < 0.05) reduced in
Fig. 2. Total neuronal damage score in experimental groups (data showed as mean ± S.E.M.). On axe Y are values of total neuronal damage (score) in the afflicted hippocampus in units are on axe Y; on X—individual bars represent experimental groups: PBS—intrahippocampal application of 10 mM PBS in total volume 1 l; (n = 5). PBS + CDX—intrahippocampal infusion of PBS followed by intraperitoneally administration of CDX; (n = 6). ET-1—intrahippocampal application of endothelin-1 (40 pmol; 1 l); (n = 11). ET-1 + CDX—intrahippocampal infusion of ET-1 and systemic injection of CDX; (n = 6). ET-1 + PG1—intrahippocampal application of ET-1 followed by intraperitoneal administration of PG 1 mg/kg; (n = 12). ET-1 + PG10—intrahippocampal application of ET-1 and intraperitoneal administration of PG 10 mg/kg; (n = 8). Asterisk—significant difference (p < 0.001) as compared to: 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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groups was observed in all evaluated areas of an afflicted dorsal hippocampus. Also in these groups isolated FJB-positive neurons in the dorsal thalamic nuclei were found in 87.5% and 80% of animals, respectively (Fig. 2). Infusion of PG a dose of 1 mg/kg reduced the incidence of thalamic damage to 50% (p = 0.044) and PG in a dose of 10 mg/kg only to 12.5% (p < 0.001). Only negligible numbers of FJB-positive cells were observed in the area close to cannula after intrahippocampal injection of PBS in a total volume of 1 l as well as after infusion of PBS in combination with systemic administration of corresponding volume of CDX.
Discussion Searching for novel drugs potentially useful for therapy of CNS damage belongs to the most investigated topic in contemporary pharmacology and neuroscience. The aim of the present study was to evaluate neuroprotective properties of neurosteroid PG in the immature brain. The first finding is a lack of neurotoxicity of PG in intact P12. Application of MK-801, a noncompetitive NMDA antagonist, during the first two weeks after birth in developing brain leads to widespread apoptotic neurodegeneration [12], which is consistent with our findings. The similar apoptotic damage was also observed after systemic administration of other NMDA antagonists [11,33]. On the other hand, memantine, a low affinity uncompetitive NMDA antagonist, protects hippocampal neurons against glutamate excitotoxic injury and its administration did not show serious behavioral side effects [4]. In addition, specific NR2B subunit antagonists displayed absence of the signs of serious neurotoxicity [21]. These facts and our results confirm that administration of some kinds of NMDA antagonists did not lead to extensive apoptotic neurodegeneration and potentially have better benefit/risk ratio in the immature brain. The second finding of this study is the neuroprotective effect of PG in the model of focal ischemia in P12 rats. This age corresponds with the human early developmental stages in the first year of life. Moreover, the vascular network in this age has higher numbers of anastomosis, which can contribute to more enormous ischemic injury [10]. This fact can also worsen the lesion development, especially due to a high concentration of extracellular glutamate during the reperfusion. In addition, ET-1 has been shown to inhibit glutamate uptake by a non-vascular mechanism, which also contribute to a more extensive brain injury [18]. Some animal models of ischemia are difficult applicable in immature rats. We did not use widely practiced middle cerebral artery occlusion (MCAO), because a variable range and location of ischemic injury could occur in this model [25,34]. Currently also exist the variant MCAO via ET-1 infusion. In this model is used higher concentration of ET-1, which can also lead to disunity and fragmentation of ischemic damage [34]. A localized stroke induced by the ET-1, in the rat model of focal cerebral ischemia, can be used to study ischemia-induced consequences (acute as well as long-term: neuronal damage, memory deficit, epileptogenesis, local metabolic changes, etc.). Our previous studies [24,37] demonstrated that unilateral intrahippocampal injection of ET-1 in immature animals leads to the development of electrographic seizures with behavioral manifestations and induce a well-nigh limited morphological damage of an afflicted hippocampus. In addition, in humans and in animal models, the hippocampus appears to be particularly vulnerable to perinatal stroke [1,3]. Therefore, we selected intrahippocampal application of ET-1. Previously we demonstrated a therapeutic effect of PG in the bilateral NMDA-induced lesions of hippocampus [32]. A neuroprotective effect of PG observed in our model of the focal ischemia
coincided with several reports concerning neuroprotective potential of other neuroactive steroids, for example 3␣-ol5-pregnan-20-one hemisuccinate (PHS) [15]. Intravenous administration of PHS in adult rats 30 min after middle cerebral artery occlusion reduced the infarct size. In another study, PHS expressively reduced consequences of reversible spinal cord ischemia in a rabbit [17]. The same author has reported neuroprotective properties of PHS in the rabbit small clot embolic stroke model [15]. In the other models of ischemia, naturally-occurring neurosteroids were also tested. For example, dehydroepiandrosteron sulphate (DHEAS) was tested in bilateral occlusion of the common carotid arteries [20]. The same authors also demonstrated the neuroprotective effect of dehydroepiandrosterone (DHEA) in the 4-vessel occlusion model. Single administration of DHEA caused reduction of neuronal death and improvement of spatial learning deficit [19]. The administration of memantine in immature rats led to the reduction of glutamate level and neuroprotection in bilateral carotid ligation with subsequent hypoxia [2]. In another study, a neuroprotective effect of memantine was also demonstrated in the rabbit small clot embolic stroke model [16]. Administration of MK801 had a neuroprotective effect in the global cerebral ischemia combined with hypotension [22] and the ischemic spinal cord injury [13]. In addition, it has been confirmed in MCAO model via ET-1 infusion [28]. Nevertheless, it should be emphasized, that the therapeutic application of MK-801 is difficult due to serious behavioral side effects observed after its administration. Neuroactive steroids exert very complex and often biphasic effects. The influence of neurosteroids is associated with modulation of GABAA and NMDA receptors [26,27]. The crucial role of NMDA receptors in glutamatergic excitotoxicity leads to rising interest about interaction of neurosteroids and NMDA receptors [14]. Nonetheless, the neurochemical and pharmacological mechanisms are not fully understood and maybe involve multiple neurotransmitter receptor systems. Further studies aimed at analyzing the regional, cellular and molecular activities are required and planned. Taken together, our results demonstrate that early administration of PG leads to a significant reduction of neurodegenerative changes in the model of the focal cerebral ischemia. Importantly, in comparison with literary data, administration of PG shows minimal neurotoxicity in the immature brain. The present results suggest that use-dependent NMDA receptor antagonists, possessing neuroprotective properties and having minimal side effects can be promising drugs for future therapies.
Conclusions We demonstrated the neuroprotective effect of PG in ischemic immature brain. Well-timed systemic administration of PG significantly reduced range of the neuronal damage in ET-1 induced model of the focal cerebral ischemia. Importantly, as a potential therapeutic agent, this drug does not show direct neurotoxic effect in the immature brain. In the future we would like to focus on the importance of PG and other synthetic neuroactive steroids in the ischemic damage of the brain and in other pathophysiological processes influenced by glutamate excitotoxicity in adult and as well as immature rats.
Conflict of interest statement There is no conflict of interests.
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Acknowledgments We gratefully acknowledge the expert technical help by Mrs. Blanka Cejkova and Mrs. Michaela Fialova. We are appreciative to Dr. Ladislav Vyklicky, Dr. Hana Chodounska and Dr. Eva Kudova for scientific inspiration and for steroid synthesis. We would also like to express our gratitude to Dr. Jaroslava Folbergrova for critical reading of the manuscript and her inspiring comments. Last but not least, we want to express thank to Ms. Thuy Hua for language check. This study was supported by GACR grants P304/11/P386 P304/14/20613S P304/12/G069, P303/12/1464 TACR-TE01020028 and GAUK 604412, institutional support RVO: 67985823. References [1] Y. Ben-Ari, Effects of anoxia and aglycemia on the adult and immature hippocampus, Biology of the Neonate 62 (1992) 225–230. [2] H.S. Chen, J.W. Pellegrini, S.K. Aggarwal, S.Z. Lei, S. Warach, F.E. Jensen, S.A. Lipton, Open-channel block of N-methyl-d-aspartate (NMDA) responses by memantine: therapeutic advantage against NMDA receptor-mediated neurotoxicity, Journal of Neuroscience: Official Journal of the Society for Neuroscience 12 (1992) 4427–4436. [3] M.R. Del Bigio, L.E. Becker, Microglial aggregation in the dentate gyrus: a marker of mild hypoxic-ischaemic brain insult in human infants, Neuropathology and Applied Neurobiology 20 (1994) 144–151. [4] S.S. Deshpande, C.D. Smith, M.G. Filbert, Assessment of primary neuronal culture as a model for soman-induced neurotoxicity and effectiveness of memantine as a neuroprotective drug, Archives of Toxicology 69 (1995) 384–390. [5] R. Dingledine, K. Borges, D. Bowie, S.F. Traynelis, The glutamate receptor ion channels, Pharmacological Reviews 51 (1999) 7–61. [6] R. Druga, P. Mares, J. Otahal, H. Kubova, Degenerative neuronal changes in the rat thalamus induced by status epilepticus at different developmental stages, Epilepsy Research 63 (2005) 43–65. [7] K. Fuxe, B. Bjelke, B. Andbjer, H. Grahn, R. Rimondini, L.F. Agnati, Endothelin-1 induced lesions of the frontoparietal cortex of the rat. A possible model of focal cortical ischemia, NeuroReport 8 (1997) 2623–2629. [8] G. Gilmour, S.D. Iversen, M.F. O’Neill, D.M. Bannerman, The effects of intracortical endothelin-1 injections on skilled forelimb use: implications for modelling recovery of function after stroke, Behavioural Brain Research 150 (2004) 171–183. [9] M. Giroud, M. Lemesle, J.B. Gouyon, J.L. Nivelon, C. Milan, R. Dumas, Cerebrovascular disease in children under 16 years of age in the city of Dijon, France: a study of incidence and clinical features from 1985 to 1993, Journal of Clinical Epidemiology 48 (1995) 1343–1348. [10] I. Grivas, H. Michaloudi, C. Batzios, M. Chiotelli, C. Papatheodoropoulos, G. Kostopoulos, G.C. Papadopoulos, Vascular network of the rat hippocampus is not homogeneous along the septotemporal axis, Brain Research 971 (2003) 245–249. [11] C. Ikonomidou, F. Bosch, M. Miksa, P. Bittigau, J. Vockler, K. Dikranian, T.I. Tenkova, V. Stefovska, L. Turski, J.W. Olney, Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain, Science 283 (1999) 70–74. [12] M.V. Johnston, Excitotoxicity in perinatal brain injury, Brain Pathology 15 (2005) 234–240. [13] H. Kocaeli, E. Korfali, H. Ozturk, N. Kahveci, S. Yilmazlar, MK-801 improves neurological and histological outcomes after spinal cord ischemia induced by transient aortic cross-clipping in rats, Surgical Neurology 64 (Suppl 2) (2005) S22–S26, discussion S27. [14] M. Korinek, V. Kapras, V. Vyklicky, E. Adamusova, J. Borovska, K. Vales, A. Stuchlik, M. Horak, H. Chodounska, L. Vyklicky Jr., Neurosteroid modulation of N-methyl-d-aspartate receptors: molecular mechanism and behavioral effects, Steroids 76 (2011) 1409–1418. [15] P.A. Lapchak, 3alpha-OL-5-beta-pregnan-20-one hemisuccinate, a steroidal low-affinity NMDA receptor antagonist improves clinical rating scores in a rabbit multiple infarct ischemia model: synergism with tissue plasminogen activator, Experimental Neurology 197 (2006) 531–537.
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