Neuroprotective effects of recombinant human erythropoietin in the developing brain of rat after lithium-pilocarpine induced status epilepticus

Brain & Development 34 (2012) 189–195 www.elsevier.com/locate/braindev

Original article

Neuroprotective effects of recombinant human erythropoietin in the developing brain of rat after lithium-pilocarpine induced status epilepticus S ¸ ag˘layan So¨zmen a, Semra Hız Kurul b, Ulucß Yisß c,⇑, Kazım Tug˘yan d, ß ule C Basßak Baykara d, Osman Yılmaz e a _ Department of Pediatrics, School of Medicine, Dokuz Eylu¨l University, Izmir, Turkey _ Department of Pediatric Neurology, School of Medicine, Dokuz Eylu¨l University, Izmir, Turkey c Department of Pediatric Neurology, Gu¨lhane Military Medical School, Ankara, Turkey d _ Department of Histology and Embryology, School of Medicine, Dokuz Eylu¨l University, Izmir, Turkey e _ Animal Research Center, School of Medicine, Dokuz Eylu¨l University, Izmir, Turkey b

Received 28 March 2011; received in revised form 1 May 2011; accepted 2 May 2011

Abstract Status epilepticus triggers a mixture of apoptotic and necrotic cell death within the hippocampus. This neuronal loss may result in the development of epilepsy and cognitive deficits. Erythropoietin mediates a number of biological actions within the central nervous system and has been shown to be neuroprotective. In the present study, we investigated the effects of recombinant human erythropoietin on hippocampus of rat after lithium-pilocarpine induced status epilepticus. Twenty-one dam reared Wistar male rats, 21-day-old were divided into three groups: control group, lithium-pilocarpine induced status epilepticus and lithium-pilocarpine induced status epilepticus and erythropoietin treated group. Erythropoietin treated group received recombinant human erythropoietin 10 U/g intraperitoneally 40 min after pilocarpine injection for 5 days. Rats were sacrificed and brain tissues were collected at 5th day of experiment. Neuronal cell death and apoptosis were evaluated. Histopathological examination showed that erythropoietin significantly decreased neuronal cell death in CA1, CA2, CA3 and dentate gyrus regions of hippocampus. It also diminished apoptosis in the CA1 and dentate gyrus regions of hippocampus. In conclusion, erythropoietin may preserve the number of neurons and decrease apoptosis in model of status epilepticus induced by lithium-pilocarpine. This experimental study suggests that erythropoietin administration may be neuroprotective in status epilepticus. Ó 2011 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

1. Introduction Status epilepticus (SE) is a neurological emergency defined as recurrent seizures, lasting for more than 30 min without interictal resumption of baseline central nervous system function [1]. About 70% of episodes of status epilepticus are the initial seizure and up to 27% of children with epilepsy will present with one or more ⇑ Corresponding author. Tel.: +90 312 3041920.

E-mail address: [email protected] (U. Yisß).

episodes of SE [2]. SE is likely to induce changes in the brain, some of which may be harmful in both adults and children [3]. SE triggers both necrotic and apoptotic cell death that contribute to neuronal damage in hippocampus [4]. A wide range of neuropsychological deficits may follow the SE, which typically include learning and memory dysfunction and other cognitive deficits [5,6]. Symptomatic epilepsies typically develop in three phases. A brain-damaging insult occurs (e.g., SE) leading to epileptogenesis (latency period, during which there are no seizures), followed by recurrent seizures

0387-7604/$ - see front matter Ó 2011 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.braindev.2011.05.002

190

S ß .C¸ag˘layan So¨zmen et al. / Brain & Development 34 (2012) 189–195

or epilepsy [7]. Thus, the latent period after SE provides an opportunity for applying effective intervention strategies that are capable of preventing the progression of initial seizure induced neurodegeneration into chronic epilepsy, characterized by spontaneous recurrent motor seizures and learning and memory deficits. Furthermore, early intervention after the initial insult may modify the progression of disease considerably [8]. Recent studies in animal/human clarify the domain of anti-epileptogenic and neuroprotective strategies for salvaging, protecting and repairing neurons in post-SE condition. Most of the studies on neuroprotection are based on animal experimental models of neurodegeneration [9]. Erythropoietin (Epo) was first identified as a hematopoietic cytokine acting as a survival and differentiation factor [10]. However, several lines of evidences suggest that Epo and erythropoietin receptor (EpoR) are expressed by other tissues, including the nervous system. Different cell types including neurons, glial cells and endothelial cells in the nervous system produce Epo and express EpoR [11]. Recombinant human Epo (r-Hu-Epo) administered peripherally crosses the blood–brain barrier [12]. Multiple models of nervous system injury (mouse, rat, gerbil and rabbit) have been used to demonstrate the effectiveness of Epo as a neuroprotective agent, including focal and global cerebral ischemia, experimental traumatic brain injury, subarachnoid hemorrhage, spinal cord injury and retinal ischemia [13–17]. Our previous study results showed that Epo significantly diminished apoptosis in the CA1 region and dentate gyrus of hippocampus and parietal cortex after hyperoxia-induced neurodegeneration in the developing brain [18]. In the present study, we examined the effect of r-Hu-Epo treatment on neuronal death and apoptosis in the hippocampus of prepubertal rats after experimental model of SE. Although multiple regions of the brain are affected, the hippocampal region was chosen because of its highly plastic nature and increased susceptibility to seizure induced damage [19]. 2. Material and methods This study was performed in accordance with the guidelines provided by the Experimental Animal Laboratory and approved by the Local Ethic and Use Committee of the Dokuz Eylul University School of Medicine. A total of twenty-one dam reared Wistar male rats, 21-day-old were used. Each group was a litter. The study animals were housed in a controlled environment (constant temperature, 22–25 °C; humidity 50–60%; 12/12 light/dark cycle with lights on at 7 AM). Animals had free access to standart laboratory food and water. The animals were divided into three groups: control group saline treated (n = 7), SE + saline-treated group (n = 7) and SE + Epo-treated group (n = 7). In the

SE + saline treated group and SE + Epo treated group, all rats received lithium chloride (3 mEq/kg i.p., Sigma–Aldrich) 19–24 h prior to the administration of pilocarpine. Furthermore, rats were pretreated with atrophine (10 mg/kg i.p., 30 min prior to pilocarpine) to counteract the peripheral cholinomimetic effects of pilocarpine [20,21]. SE was induced by injecting pilocarpine hydrochloride (15 mg/kg i.p., Sigma–Aldrich) [20]. All pilocarpine-induced behavioral alterations were observed. Seizures were scored in each rat by Racine’s scale considering only stage 3 and more were convulsive seizures [22]. SE + Epo-treated group received r-HuEpo (Eprex 4000 IU/ml) 10 U/g i.p. 40 min after pilocarpine hydrochloride injection to evaluate effects of Epo [23]. Injections continued for 5 days. At sacrifice, animals were anesthetized with ether and brain tissues were collected at 5th day of experiment. Control animals and SE + saline treated groups were treated intraperitoneally with saline and did not receive any treatment. All histomorphological analyses described below were performed by an investigator with no prior knowledge of the treatment groups. 2.1. Light microscope and cresyl violet staining The brains were sectioned coronally into sequential 5 lm slices using a rat brain slicer. Brain sections were taken from each subject according to the Paxinos and Watson coordinates at the coronal plate [24]. For each brain, samples were taken from areas far from bregma 2.2, 2.84 and 3.43 mm that were seen at hippocampus CA1, CA2, CA3 and GD regions. The blocks were cut into 6 mm sections at multiple levels and stained with cresyl violet. 2.2. Estimation of hippocampus neuron density Each sample was subjected to the estimation of hippocampus CA1, CA2, CA3 and dentate gyrus neuron density. Three sections on average from each brain were selected according to the regions spanning from bregma 2.2, 2.84 and 3.43 mm that were seen at hippocampus CA1, CA2, CA3 and GD regions. The images were analyzed by using a computerassisted image analyzer system consisting of a microscope (Olympus BH-2 Tokyo, Japan) equipped with high-resolution video camera (JVC TK 890E, Japan). The numbers of CA1, CA2,CA3 and dentate gyrus were counted by the help of a 15,000 lm2 counting frame viewed through a 20 Nikon lens at the monitor. The counting frame was placed randomly five times on the image analyzer system monitor, and the neuron numbers of CA1, CA2, CA3 and dentate gyrus regions of hippocampus were counted (UTHSCA Image Tool for Windows version 3.0 software) and the average was taken. All counting and measurement procedures were performed blindly. The

S ß .C¸ag˘layan So¨zmen et al. / Brain & Development 34 (2012) 189–195

neuron numbers of CA1, CA2 and CA3 and gyrus dentatus regions of the hippocampus were calculated separately for the right and left hemispheres. 2.3. TUNEL staining To detect DNA fragmentation in cell nuclei, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) reaction was applied. TUNEL staining was performed using an In Situ Cell Death Detection KitÒ (Roche, Mannheim, Germany) according to the manufacturer’s protocol. Briefly, the sections were deparaffinized, hydrated by successive series of alcohol, washed in distilled water followed by phosphate-buffered saline (PBS) and deproteinized by proteinase K (20 lg/ml) for 30 min at 37 °C. Then the sections were rinsed and incubated in the TUNEL reaction mixture. The sections were rinsed and visualized using converter-POD with 0.02% 3,30 -diaminobenzidine (DAB). The sections were counter-stained with Harris hematoxylin. For quantitative analysis of TUNEL-positive cells in hippocampus, cells exhibiting apoptotic features (condensed cytoplasm and chromatin, intense TUNEL reactivity and a rounded cell body) were counted in CA1and dentate gyrus regions using a Olympus BH-2 Tokyo microscope at 200 magnification connected to a 14-in. monitor. For establishing apoptotic index, 1000 cells were counted in CA1and dentate gyrus regions of hippocampus. Cells showing apoptotic morphology were given as percentile. 2.4. Caspase-3 ımmune staining method For quantitative analysis of caspase-3 positive cells in hippocampus, cells exhibiting apoptotic features were counted in CA1 and dentate gyrus regions using a Olympus BH-2 Tokyo microscope at 200 magnification connected to a 14-in. monitor. For establishing apoptotic index, 1000 cells were counted in CA1 and dentate gyrus regions of hippocampus. Cells showing apoptotic morphology were given as percentile.

191

stage 3 or more according to Racine scale 30 min after pilocarpine injection. 3.2. Epo treatment suppressed neuronal death in hippocampus The number of neurons of CA1, CA2, CA3 and dentate gyrus of hippocampus were significantly less in the SE + saline group compared with the control group (p = 0.001, p = 0.001, p = 0.001 and p = 0.001). Treatment with erythropoietin significantly preserved the number of neurons in dentate gyrus, CA1, CA2 and CA3 regions of hippocampus when compared with SE + saline treated group (p = 0.001, p = 0.001, p = 0.001 and p = 0.001) (Table 1 and Fig. 1). 3.3. Epo treatment decreased the number of TUNNEL– positive cells TUNEL positive cells showed the typical morphological features of apoptosis such as the chromatin condensation, cytoplasmic budding and apoptotic bodies. When compared with the control group, the number of TUNEL + neurons in the CA1 and dentate gyrus of hippocampus increased in the SE + saline-treated group (p = 0.001 and p = 0.001). Epo significantly decreased the number of TUNEL + neurons in the CA1 and dentate gyrus of hippocampus when compared to SE + saline-treated group(p = 0.001 and p = 0.001) (Table 2 and Fig. 2). 3.4. Epo treatment decreased the ratio of caspase positive cells Apoptosis was also evaluated with caspase-3 immune staining method. One sample was randomly chosen from each group. Although it was not evaluated statistically, treatment with erythropoietin decreased the ratio of caspase positive cells of CA1 and dentate gyrus of hippocampus (Table 3 and Fig. 3).

2.5. Statistical analysis

4. Discussion

Values are presented as mean ± SD. One-way analysis of variance (ANOVA) along with Bonferroni post hoc analysis was used for statistical analysis. p < 0.05 is accepted as statistically significant.

The brain injury resulting from seizures is a dynamic process that comprises multiple factors contributing to neuronal cell death. Some of these dynamic neurochemical changes persist also in the chronically epileptic state or may be altered or substituted by other changes. They are accompanied by progressing rearrangement of neuronal circuitries, characterized by continuing neurodegeneration and by axonal outgrowth [25]. Based on these data, the use of neuroprotectants is considered one of the most appealing candidates for therapeutic approaches to prevent epileptogenesis without compromising recovery after brain damaging insult [26].

3. Results 3.1. Seizure activity after pilocarpine administration Animals in the SE + saline treated group and SE + Epo treated group showed behavioral changes of

S ß .C¸ag˘layan So¨zmen et al. / Brain & Development 34 (2012) 189–195

192

Table 1 The effect of erythropoietin treatment on neuronal density of the CA1,CA2, CA3 regions and dentate gyrus of hippocampus. Groups

Neuronaldensity CA1

CA2

CA3

GD

1-Control 2-SE + saline 3-SE + Epo

37.2 ± 1.19 25.15 ± 1.51 32.11 ± 0.65

41.31 ± 0.68 26.64 ± 1.67 34.15 ± 0.91

25.18 ± 1.06 15.51 ± 1.34 18.64 ± 0.47

70.27 ± 2.05 49.21 ± 2.33 55.15 ± 2.03

p values 1 vs.2 2 vs.3

0.001 0.001

0.001 0.001

0.001 0.001

0.001 0.001

vs., versus. The values are presented as mean ± SD.

Fig. 1. Effect of erythropoietin treatment on the neuron density in the CA1 region and dentate gyrus of hippocampus. Representative pictures are obtained by cresyl violet staining. When compared with the control group (A and B) the number of neurons of CA1, CA2, CA3 and dentate gyrus region of the hippocampus decreased in SE + saline treated group (C and D). Erythropoietin (E and F) preserved the number of neurons of CA1 and dentate gyrus regions of the hippocampus as compared with the SE + saline-treated group.

Further, if the development of epilepsy cannot be completely prevented, neuroprotective treatment might Table 2 The effect of erythropoietin treatment on TUNEL positive cells of the CA1 region and dentate gyrus of hippocampus. Groups

CA1

GD

Control SE + saline SE + Epo

3.53 ± 1.25 13.58 ± 0.62 9 ± 0.25

4.91 ± 1.18 17.58 ± 0.87 12.41 ± 0.52

p values 1 vs. 2 2 vs. 3

0.001 0.001

0.001 0.001

vs., versus. The values are presented as percentage of theapoptotic cells.

at least result in a milder and more easily treatable disease and less severe cognitive decline [27]. Research during the past years has clearly demonstrated that Epo is a potent promoter of neuronal survival [28]. Neuroprotection by Epo has been shown to associate with neuroregeneration, anti-inflammation and anti-apoptosis [29]. Previous studies showed preconditioning with Epo before SE exerts anti-apoptotic effects and prevents cognitive impairments in adult rats [22,30]. r-Hu-Epo also reduces the expression of neuron-specific enolase, S-100b and myelin basic protein and provides an early protective effect against epileptic brain injury [31]. Epo receptor was also found to be increased in the hippocampus after status epilepticus. During the latent period following SE, administered

S ß .C¸ag˘layan So¨zmen et al. / Brain & Development 34 (2012) 189–195

193

Fig. 2. Effect of erythropoietin treatment on the TUNEL immunoreactivity in the dentate gyrus of hippocampus. When compared with control group (A) the number of TUNEL + neurons (showed with arrows, dark cells) in hippocampus increased in SE-saline treated group (B and B1). Erythropoietin (C and C1) significantly reduced number of TUNEL + neurons of dentate gyrus as compared with SE + saline-treated group. Apoptotic cells at magnified (100 lm) images (B1 and C1).

Table 3 The effect of erythropoietin treatment on caspase-3positivecells of the CA1 regionanddentategyrus of hippocampus. Groups

CA1

DG

Control SE + saline SE + Epo

0.5 2.25 1.25

0.75 2.5 1.75

The values are presented as percentage of caspase-3 positive cells.

Epo prevented blood–brain barrier leakage, neuronal death and microglia activation in the dentate hilus, CA1, and CA3 and inhibited the generation of ectopic granule cells in the hilus and new glia in CA1. Moreover, Epo reduced the risk of spontaneous recurrent seizure development [32]. In our study, we showed that Epo significantly preserved number of neurons in CA1, CA2, CA3 regions and dentate gyrus of hippocampus. Apoptosis is a morphologically distinct form of cell death characterized by cytoplasmic condensation, preservation and packaging of intracellular organelles, DNA fragmentation, dispersal and phagocytosis of the cell as apoptotic bodies. In our study the extent of nuclear DNA fragmentation in injured cells in hippocampus was examined by using TUNEL staining. Epo treatment significantly reduced TUNEL-positive cells in CA1 and dentate gyrus of hippocampus. Apoptosis may be triggered by two main pathways. In the extrinsic pathway, activation of cell-surfaceexpressed death receptors of the TNF (tumor necrosis factor) superfamily leads to formation of an intracellu-

lar complex known as the DISC (death-inducing signaling complex) containing intracellular molecular adaptors such as FADD (Fas-associated death domain) and caspase 8 or 10. Caspases, cysteine proteases that cleave proteins at aspartate residues, play a prominent role in programmed cell death. Their targets include cytoskeleton proteins, DNA repair proteins and inhibitors of endonucleases. Caspase-mediated cleavage of intracellular proteins causes morphological changes of the cell such as shrinkage, chromatin condensation, DNA fragmentation and plasma membrane blebbing [33]. Caspase-3 is especially important because it is the central executioner caspase that is downstream of the intrinsic (mitochondrial) caspase-9 and extrinsic receptor-activated (Fas) caspase-8 pathways. It has been suggested that caspase-3 is activated and is responsible for seizure-induced neuronal death [8,29,34]. The intrinsic pathway is triggered following disruption to intracellular organelle homoeostasis or DNA damage. In our study, we aimed to support our findings on apoptosis with caspase immune staining to show exact mechanism of neuronal death. One criticizable point of the study is that we could not show the anti-apoptotic effects of Epo via caspase-3 statistically. But the ratio of caspase-3 positive cells reduced in the hippocampus of SE + Epo treated group when compared with the SE + saline treated group in the randomly selected slide. On the other hand, administration of r-Hu-Epo activates the PI3K/Akt signaling pathway in SE rats and increases the expression of p-Akt protein that regulate the expression of caspase-9, a regulatory factor of the

194

S ß .C¸ag˘layan So¨zmen et al. / Brain & Development 34 (2012) 189–195

Fig. 3. Effect of erythropoietin treatment on the caspase-3 immunoreactivity in dentate gyrus of hippocampus. When compared with control group (A) the number of caspase-3 + neurons (showed with arrows) in hippocampus increased in SE-saline treated group (B and B1). Erythropoietin (C and C1) significantly reduced number of caspase + neurons of dentate gyrus when compared with SE + saline-treated group. Apoptotic cells at magnified (100 lm) images (B1 and C1).

mitochondrial-dependent apoptotic pathway, and therefore provides anti-apoptotic and neuroprotective effects [35].Children who experience complex febrile seizures are at a higher risk of subsequent epileptic seizures. In an experimental study, molecular changes in the rat brain after febrile seizures were examined throughout the latent period and erythropoietin was administered as a potentially antiepileptic intervention. Erythropoietin treatment reduced the early inflammatory responses and modulated the molecular alterations after febrile seizures, thereby reducing the risk of subsequent spontaneous seizures [36]. In conclusion, Epo treatment may provide neuroprotection in the acute phase of seizure induced cell injury by suppressing apoptotic neuronal cell death. We suggest that Epo supplementation may promote enhanced neuronal survival following SE and may be used as a neuroprotective agent. References [1] Blume WT, Lu¨ders HO, Mizrahi E, Tassinari C, van Emde Boas W, Engel Jr J. Glossary of descriptive terminology for ictal semiology: report of the ILAE task force on classification and terminology. Epilepsia 2001;42:1212–8. [2] Sillanpa¨a¨ M, Shinnar S. Status epilepticus in a population-based cohort with childhood-onset epilepsy in Finland. Ann Neurol 2002;52:303–10. [3] Scantlebury MH, Heida JG, Hasson HJ, Velı´sˇkova´ J, Velı´sˇek L, Galanopoulou AS, et al. Age-dependent consequences of status epilepticus: animal models. Epilepsia 2007;48:75–82.

[4] Araki T, Simon RP, Taki W, Lan JQ, Henshall DC. Characterization of neuronal death induced by focally evoked limbic seizures in the C57BL/6 mouse. J Neurosci Res 2002;69:614–21. [5] Holmes MD, Silbergeld DL, Drouhard D, Wilensky AJ, Ojemann LM. Effect of vagus nerve stimulation on adults with pharmacoresistant generalized epilepsy syndromes. Seizure 2004;13:340–5. [6] Holmes GL. Clinical evidence that epilepsy is a progressive disorder with special emphasis on epilepsy syndromes that do progress. Adv Neurol 2006;97:323–31. [7] Pitka¨nen A, Kharatishvili I, Karhunen H, Lukasiuk K, Immonen R, Nairisma¨gi J, et al. Epileptogenesis in experimental models. Epilepsia 2007;48:13–20. [8] Naegele JR. Neuroprotective strategies to avert seizure-induced neurodegeneration in epilepsy. Epilepsia 2007;48:107–17. [9] Pitka¨nen A. Drug-mediated neuroprotection and antiepileptogenesis. Anim Data Neurol 2002;59:27–33. [10] Jelkmann W. Erythropoietin: structure, control of production, and function. Physiol Rev 1992;72:449–89. [11] Chin K, Yu X, Beleslin-Cokic B, Liu C, Shen K, Mohrenweiser HW, et al. Production and processing of erythropoietin receptor transcripts in brain. Brain Res Mol Brain Res 2000;81:29–42. [12] Brines ML, Ghezzi P, Keenan S, Agnello D, de Lanerolle NC, Cerami C, et al. Erythropoietin crosses the blood–brain barrier to protect against experimental brain injury. Proc Natl Acad Sci USA 2000;97:10526–31. [13] Bernaudin M, Marti HH, Roussel S, Divoux D, Nouvelot A, MacKenzie ET, et al. A potential role for erythropoietin in focal permanent cerebral ischemia in mice. J Cereb Blood Flow Metab 1999;19:643–51. [14] Sakanaka M, Wen TC, Matsuda S, Masuda S, Morishita E, Nagao M, et al. In vivo evidence that erythropoietin protects neurons from ischemic damage. Proc Natl Acad Sci USA 1998;95:4635–40. [15] Grasso G, Buemi M, Alafaci C, Sfacteria A, Passalacqua M, Sturiale A, et al. Beneficial effects of systemic administration of recombinant human erythropoietin in rabbits subjected to

S ß .C¸ag˘layan So¨zmen et al. / Brain & Development 34 (2012) 189–195

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24] [25]

subarachnoid hemorrhage. Proc Natl Acad Sci USA 2002;99:5627–31. Gorio A, Gokmen N, Erbayraktar S, Yilmaz O, Madaschi L, Cichetti C, et al. Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma. Proc Natl Acad Sci USA 2002;99:9450–5. Junk AK, Mammis A, Savitz SI, Singh M, Roth S, Malhotra S, et al. Erythropoietin administration protects retinal neurons from acute ischemia-reperfusion injury. Proc Natl Acad Sci USA 2002;99:10659–64. Yisß U, Kurul SH, Kumral A, Tug˘yan K, Cilaker S, Yılmaz O, et al. Effect of erythropoietin on oxygen-induced brain injury in the newborn rat. Neurosci Lett 2008;448:245–9. Acharya MM, Hattiangady B, Shetty AK. Progress in neuroprotective strategies for preventing epilepsy. Prog Neurobiol 2008;84:363–404. Glien M, Brandt C, Potschka H, Voigt H, Ebert U, Lo¨scher W. Repeated low- dose treatment of rats with pilocarpine: low mortality but high proportion of rats developing epilepsy. Epilepsy Res 2001;46:111–9. Suchomelova L, Baldwin RA, Kubova H, Thompson KW, Sankar R, Wasterlain CG. Treatment of experimental status epilepticus in immature rats: dissociation between anticonvulsant and antiepileptogenic effects. Pediatr Res 2006;59:237–43. Racine R. Modification of seizure activity by electrical stimulation: II. Motor seizure. Electroencephalogr Clin Neurophysiol 1972;32:281–94. Yang J, Huang YG, Yu X, Sun H, Li YN, Deng YC. Erythropoietin preconditioning suppresses neuronal death following status epilepticus in rats. Acta Neurobiol Exp 2007;67:141–8. Paxinos G, Watson G. The rat brain in stereotaxic coordinates. 2nd ed. New York: Academic Press; 1986. Houser CR, Miyashiro JE, Swartz BE, Walsh GO, Rich JR, Delgado-Escueta AV. Altered patterns of dynorphin immunoreactivity suggest mossy fiber reorganization in human hippocampal epilepsy. J Neurosci 1990;10:267–82.

195

[26] Clark S, Wilson WA. Mechanisms of epileptogenesis. Adv Neurol 1999;79:607–30. [27] Narkilahti S, Nissinen J, Pitka¨nen A. Administration of caspase 3 inhibitor during and after status epilepticus in rat: effect on neuronal damage and epileptogenesis. Neuropharmacology 2003;44:1068–88. [28] Bartesaghi S, Marinovich M, Corsini E, Galli CL, Viviani B. Erythropoietin: a novel neuroprotective cytokine. Neurotoxicology 2005;26:923–8. [29] Sola A, Wen TC, Hamrick SEG, Ferriero DM. Potential for protection and repair following injury to the developing brain: a role for erythropoietin? Pediatr Res 2005;57:7R–10R. [30] Jun Y, JiangTao X, YuanGui H, YongBin S, Jun Z, XiaoJun M, et al. Erythropoietin pre-treatment prevents cognitive impairments following status epilepticus in rats. Brain Res 2009;1282:57–66. [31] Jiang CM, DU JM, Liu ZL, Chen LQ, Feng M, Yang YH, et al. Effects of recombinant human erythropoietin on serum levels of neuron-specific enolase, S-100b protein and myelin basic protein in rats following status epilepticus. Zhongguo Dang Dai Er Ke Za Zhi (Chin J Contemp Pediatr). 2011;13:50–2, [in Chinese]. [32] Chu K, Jung KH, Lee ST, Kim JH, Kang KM, Kim HK, et al. Erythropoietin reduces epileptogenic processes following status epilepticus. Epilepsia 2008;49:1723–32. [33] Henshall DC. Apoptosis signalling pathways in seizure-induced neuronal death and epilepsy. Biochem Soc Trans 2007;35:421–3. [34] Kondratyev A, Gale K. Intracerebral injection of caspase-3 inhibitor prevents neuronal apoptosis after kainic acid-evoked status epilepticus. Brain Res Mol Brain Res 2000;75:216–24. [35] Wang WP, Shi ZQ, Yu JH, Guo L, Wang L, Han DL, et al. Effect of recombinant human erythropoietin on hippocampal p-Akt and caspase-9 expressions in rats with status epilepticus and the mechanism. Nan Fang Yi Ke Da Xue Xue Bao 2010;30:64–9, [in Chinese]. [36] Jung KH, Chu K, Lee ST, Park KI, Kim JH, Kang KM, et al. Molecular alterations underlying epileptogenesis after prolonged febrile seizure and modulation by erythropoietin. Epilepsia 2011;52:541–50.