- Email: [email protected]
Activity increase in EpoR and Epo expression by intranasal recombinant human erythropoietin (rhEpo) administration in ischemic hippocampi of adult rats
Neuroscience Letters 583 (2014) 16–20
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Activity increase in EpoR and Epo expression by intranasal recombinant human erythropoietin (rhEpo) administration in ischemic hippocampi of adult rats ˜ R. Castaneda-Arellano, A.I. Feria-Velasco, M.C. Rivera-Cervantes ∗ Laboratorio de Neurobiología Celular, Department of Cellular and Molecular Biology, CUCBA, Universidad de Guadalajara Zapopan Jal., Mexico
h i g h l i g h t s • Cell survival by rhEpo-intranasal administration in ischemic hippocampus. • Modified expression of Epo and EpoR by ischemia and after rhEpo-intranasal. • Increased phosphorilation of the EpoR by rhEpo administration.
a r t i c l e
i n f o
Article history: Received 8 July 2014 Received in revised form 26 August 2014 Accepted 4 September 2014 Available online 16 September 2014 Keywords: Erythropoietin Intranasal delivery EpoR phosphorylation Ischemia Neuroprotection
a b s t r a c t Erythropoietin in the nervous system is a potential neuroprotective factor for cerebral ischemic damage due to specific-binding to the erythropoietin receptor, which is associated with survival mechanisms. However, the role of its receptor is unclear. Thus, this work assessed whether a low dose (500 UI/Kg) of intranasal recombinant human erythropoietin administered 3 h after ischemia induced changes in the activation of its receptor at the Tyr456-phosphorylated site in ischemic hippocampi in rats. The results showed that recombinant human erythropoietin after injury maintained cell survival and was associated with an increase in receptor phosphorylation at the Tyr456 site as an initial signaling step, which correlated with a neuroprotective effect. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Ischemia produces a sequence of pathophysiological phenomena in the brain that damages neurons, glia and endothelial cells [1]. Animal models have determined that erythropoietin (Epo) promotes neuroprotection through the Epo receptor (EpoR) under conditions of brain damage [2], such as an ischemic event [3]. A deficiency in tissue oxygenation results in the production and secretion of Epo from the hippocampus and an increase in EpoR expression in astrocytes and neurons [4,5]. Epo binding changes the conformation of the EpoR, activates phosphorylation activity
∗ Corresponding author at: Laboratorio de Neurobiología Celular Dept. of Cellular and Molecular Biology, CUCBA, Universidad de Guadalajara, Km 15.5 Carretera a Nogales, Camino Ing, Ramón, Padilla Sánchez Km 2, Zapopan, Jalisco 45221, Mexico. Tel.: +52 33 37771191/37771150. E-mail addresses: [email protected], [email protected] (M.C. Rivera-Cervantes). http://dx.doi.org/10.1016/j.neulet.2014.09.013 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
of the Janus-tyrosine kinase 2 (Jak2) and this phosphorylates to the EpoR at multiple sites, such as the Tyr-456 motif, which is the preferred binding site for the transduction of intracellular signaling to foster neuroprotection [6–8]. A lower concentration of Epo induces erythropoiesis through EpoR as long as Epo is present. A typical dose of recombinant human erythropoietin (rhEpo) to 100 IU/kg provides serum Epo levels that trigger EpoR for approximately 24 h [9]. EpoR in the adult nervous system is not highly expressed under normal conditions, instead it is up-regulated following injury, and a high concentration of Epo is necessary for neuroprotection. Doses of rhEpo from 500 to 5000 IU/kg intravenously establishes tissue protection [10], but only a brief exposure of Epo triggers a sustained response and exerts multiple effects on the brain [11]. Intranasal delivery bypassed the blood–brain barrier and achieves potentially therapeutic levels of drugs in the CNS compared to systemic treatment due to the direct transport of absorbed drug into the systemic circulation, which bypasses the first-pass effect of the liver [12]. The intranasal administration of Epo reaches the damaged brain 10 times quicker
R. Casta˜ neda-Arellano et al. / Neuroscience Letters 583 (2014) 16–20
than the intravenous route [13]. The efficacy of this application has been demonstrated in models of ischemia in gerbils and rats treated with NeuroEpo and rhEpo, respectively [14–16]. The present study assessed whether a low dose of rhEpo (500 IU/Kg) induced cytoprotection and altered the expression levels of Epo and EpoR in hippocampi of ischemic rats. 2. Materials and methods 2.1. Animals All experimental animals in this study were used in accordance with NIH guidelines (NIH Guide for the Care and Use of Laboratory Animals). Adult male Wistar rats, weighing 250–300 g at the beginning of experiments were provided with food and water ad libitum in a room maintained at 22 ± 2 ◦ C under a 12 h light/dark cycle. Five study groups were used: intact group, intact animals without pharmacological treatment or surgical procedure; sham group, animals that underwent the surgical procedure, but without carotid occlusion; sham + rhEpo group, animals that underwent the surgical procedure, but without carotid occlusion and with administration of rhEpo (500 IU/kg, intranasally); ischemia, animals that underwent surgery by carotid occlusion, but without rhEpo; ischemia + rhEpo, the animals that underwent surgery by carotid occlusion and which were also treated with rhEpo (500 IU/kg, intranasally). 2.2. Model of 2-vessel occlusion Rats were anesthetized intraperitoneally with ketamine (90 mg/kg) and xylazine (10 mg/kg), and body temperature was continually monitored and maintained at 37–37.5 ◦ C using a pad and heat lamp. Occlusion of the common carotid arteries was performed as described previously [17] via 20 min of ligation. Sham-surgery animals were treated in the same manner except that the common carotid arteries were not clamped. All animals were killed by decapitation 24 h after surgery. 2.3. rhEpo intranasal treatment The dose of recombinant human erythropoietin was 500 IU/kg of body weight (Exetin-A, Pisa Pharmaceutical, Mexico). The animals were placed in supine position covering the mouth allowed the animal to breathe through the nose and absorb the drug, with a syringe pump (780010 KD Scientific) at a flow rate of 33 L/min for 3 h post-surgery. 2.4. Viability assay The hippocampus was extracted from the brains of the different study groups and dissected rapidly under cold conditions. Hippocampi remained in 1 mL of Hank’s solution with Mg2+ /Ca2+ , and they were washed and treated for cell dissociation with 100 L DNase (20 g/mL) via the addition of 100 L of trypsin solution 1:10 (100 L of trypsin in 900 L Hank’s solution Mg2+ /Ca2+ -free). Hippocampi were incubated at 37 ◦ C for 10 min and 100 L of a trypsin inhibitor was added. The cells were mechanically resuspended (15–20 steps) and centrifuged for 10 min at 200 × g at room temperature. A solution of fluorescein diacetate/propidium iodide (FDA-PI), 0.1 mL of FDA (5 mg/mL in acetone) and 0.03 mL (0.6 g) of PI was added directly to resuspended cells for staining, and 10 L of the cell suspension was removed and placed in a Neubauer chamber for cell counting under a fluorescence microscope (Olympus BX51 with Alexa absorption filter of 488–594 nm).
17
2.5. Semi-quantitative RT-PCR The expression of Epo and EpoR mRNA in the hippocampus was evaluated using reverse transcriptase-polymerase chain reaction (RT-PCR). RNA was isolated from hippocampus tissue (TRIzol® Reagent – Invitrogen, Carlsbad, CA), and first-strand cDNA was prepared (First Strand® , M-MLV RT, Invitrogen Cat# 28025-021). cDNA samples were probed with nucleotide primer sets Epo: forward 5 -GCT CCA ATC TTT GTG GCA TC-3 and reverse 5 -TGG CTT CGT GAC CCT CTG T-3 ; EpoR: forward 5 -CCG GGA TGG GCT TCA ACT AC-3 and reverse 5 -TCCAGTGGCACAAAACTCGAC-3 ; -actin: forward 5 -CACCACAGCTGAGAGGGAAATCGTGCGTGA-3 and reverse 5 -ATTTGCGGTGCACGATGGAGGGGCCGGACT-3 . The expression levels of mRNA transcripts were normalized to -actin for different testing sample-volumes for different PCR-reactions to obtain the same pattern of expression for each sample evaluated. The expression level of each target gene was also normalized to the expression of the -actin gene. 2.6. Western blotting The expression of Epo and EpoR proteins in the hippocampus was determined using Western blotting. Briefly, the hippocampi were rapidly harvested, homogenized on ice in 920 L lysis buffer containing 50 mM Tris–HCl, 150 mM NaCl, 25 mM NaF, 1% Nonidet P-40, and 1 mM Na3 VO4 plus 80 L of a protease inhibitor cocktail (sc-29130, Santa Cruz Biotechnology) and centrifuged at 14,000 rpm for 30 min. Equal amounts of protein (50 g) were loaded into each lane of a 12% SDS-PAGE gel, subjected to electrophoresis and transferred onto a nitrocellulose membrane. The membranes were blocked in 1× Animal-Free Blocker (SP-5030, VECTOR) for 2 h and incubated with 1:1000 dilutions of the following antibodies: rat anti-Epo (N-19, Santa Cruz Biotechnology), rat anti-EpoR recognizes non-phosphorylated form (M-20, Santa Cruz Biotechnology), rat anti-EpoR recognizes phosphorylated form in Tyr456 site (sc-20236-R, Santa Cruz Biotechnology) and 1:6000 of rat anti- actin (ab6276, Abcam) overnight at 4 ◦ C. The membranes were incubated with a biotinylated secondary antibody for 2 h followed by incubation with an avidin–biotin complex (ABC standard kit, VECTOR) for 45 min. The samples were washed twice with Tris–NaCl Buffer + Tween 20 (TBST), and the membranes were developed using diaminobenzidine (D-5905, Sigma). Band analysis was performed in Kodak Digital Science 1D Image Analysis, and relative band densities were normalized to the loading control -actin bands. 2.7. Data analysis All results are expressed as means ± standard error (SEM) and were analyzed using a one-way analysis of variance (ANOVA). Differences between groups were tested using Tukey’s post hoc test. In all cases, p < 0.05 was considered statistically significant. 3. Results 3.1. Cell survival after rhEpo-treatment under ischemic damage The intact, sham and sham + rhEpo groups were not significantly difference from each other in cell viabillity of the hippocampus (Fig. 1A and B). While ischemic injury (ischemia group) induced changes in cell viability decreasing 84% living cells and increasing 77% non-viable cells when compared to the intact group. On the other hand, treatment with intranasal rhEpo after injury (ischemia + rhEpo) significantly increased cell survival 74%, and fewer non-viable cells (66%) were observed versus the ischemic
18
R. Casta˜ neda-Arellano et al. / Neuroscience Letters 583 (2014) 16–20
Fig. 1. Effect of intranasal rhEpo on cell viability in rat hippocampus 24 h after reperfusion as determined using FDA and PI. (A) The number of viable cells and (B) the number of non-viable cells are plotted. The data represent the means ± SEM of 4 animals, conducted in duplicate samples: p < 0.05, compared to intact, + p < 0.05, compared to ischemia. The value of p < 0.05 was considered as statistically significant, ANOVA followed by Tukey’s post hoc test.
damage induced 2-vessel occlusion and evaluated using the FDA/IP assay (Fig. 1A and B). 3.2. Changes in the expression levels of Epo and EpoR mRNA following delivery of low-dose intranasal rhEpo to ischemic rats The analysis of mRNA expression of Epo and EpoR revealed no significant differences in the intact, sham or sham + rhEpo groups (Fig. 2C and D). However, Epo-mRNA levels in the ischemic injury (ischemia group) decreased to 33% and exhibited a 200% decrease in EpoR mRNA expression when compared to the intact group. While delivery of rhEpo intranasally following ischemic damage (ischemia + rhEpo group) increased Epo-expression levels to 43% and caused an increase of 410% in EpoR mRNA expression levels compared to ischemia (Fig. 2C and D).
(Fig. 3B). However, an approximately 28% greater expression was observed in the ischemia and ischemia + rhEpo groups compared to the intact group (Fig. 3B). Moreover, non-phospho EpoR showed no differences in the intact and sham groups, but an increase of 26% was observed in the ischemic brains (ischemia group). In the sham + rhEpo and ischemia + rhEpo groups, lower expression was detected in non-phospho EpoR of 68% compared to the ischemia group (Fig. 3C and D). The ratio between phospho-EpoR relative to non-phospho EpoR expression was analyzed. The levels of phospho-EpoR were not significantly different (p < 0.05) in intact and sham groups (Fig. 4A and B), but the level of phospho-EpoR in the sham + rhEpo group was twice as high as that in the intact group, and one fold increase was observed in the 2-vessel occlusion group. A 2.5-fold increase was observed after intranasal rhEpo delivery after ischemia (ischemia + rhEpo) when compared to the intact group (Fig. 4A and B).
3.3. Protein expression of Epo and EpoR after rhEpo treatment in cerebral ischemic rats
4. Discussion
Western blotting showed no significant differences in Epo expression between the intact, sham and sham + rhEpo groups
The Epo protein induces a neuroprotective effect in different ways, including the inhibition of neuronal apoptosis and
Fig. 2. Semiquantitative analysis of Epo and EpoR mRNA expression. Representative electrophoresis are shown for Epo-mRNA (A) and EpoR-mRNA (B). -Actin served as the gel-loading control. The data represent the means ± SEM of 4 animals, conducted in duplicate samples (C and D): p < 0.05, compared to intact, + p < 0.05, compared to ischemia. The value of p < 0.05 was considered as statistically significant, ANOVA followed by Tukey’s post hoc test.
R. Casta˜ neda-Arellano et al. / Neuroscience Letters 583 (2014) 16–20
19
Fig. 3. Levels of Epo and EpoR protein expression after induced ischemia (2VO) in the rat hippocampus. Representative Western blots are shown for Epo protein (A) and EpoR protein (B); -actin served as the gel-loading control. The data represent the means ± SEM of 4 animals, conducted in duplicate samples (C and D): p < 0.05, compared to intact, + p < 0.05, compared to ischemia. The value of p < 0.05 was considered as statistically significant ANOVA followed by Tukey’s post hoc test.
Fig. 4. Ratio of phosphorylated EpoR (EpoRp(Tyr456 )/non-phosphorilated EpoR in the hippocampus of ischemic rats. (A) Representative photograph of a membrane obtained by Western blotting for the EpoRp(Tyr456 and EpoR proteins. (B) The plotted data represent the means ± SEM of 4 animals, conducted in duplicate samples of ratio EpoRp(Tyr456) /EpoRtotal with respect to different study groups. p < 0.05, compared to intact, + p < 0.05, compared to ischemia. The value of p < 0.05 was considered as statistically significant, ANOVA followed by Tukey’s post hoc test.
cerebral inflammation, blockade of glutamatergic transmission and new vessel formation [18,19]. rhEpo at doses of 500 to 5000 IU/kg reduces infarct volume via the promotion of cell-survival [10,20]. However, these doses are quite large and significantly increase blood volume resultant from erythropoietic activity [21]. The present study used a modified 2-vessel occlusion that is a widely used experimental model of ischemia [17]. This procedure induces changes in cell viability in the hippocampus, which was evaluated by IP-incorporation into the cell due to drastically increase the plasma membrane permeability. This leads to cell death, contributing to tissue damage [22], according Jones and Senft [23] technique to evaluate cell viability. Furthermore, our data show that delayed (3 h) treatment with rhEpo administered intranasally at a dose of 500 IU/Kg decreased cell damage induced by ischemia. The neuroprotective effects of Epo have been demonstrated in several models
of stroke [10,14,19] and traumatic brain injury [24], which require the application of higher doses intravenously. Epo is a hormone involved in the regulation of neuroprotective functions, and its receptor is expressed on many neurons throughout the CNS, particularly hippocampus [20]. Epo and EpoR expression has been proposed as a mechanism of the tissueprotective response to injury in the brain [25,26]. The important issue to be addressed is whether the neuroprotective effects of rhEpo are initially triggered by its receptor. We showed that ischemic insult induced a decrease in the transcript level of Epo and its receptor, but delivery of rhEpo 3 h after damage increased the mRNA expression of both molecules 24 h post-damage. Repeated hypoxic exposures induce an increase in Epo and EpoR gene expression 3 h after re-oxygenation [2]. However, Spandou et al. [25] did not detect any significant alteration in EpoR expression 24 h after focal ischemic insult, which is similar to our results. Epo induces GATA-3 activity in neural cells, which is required for the active transcription of EpoR in brain development [14]. Therefore, this transcription factor may be regulated by rhEpo after ischemic damage, which leads to the substantial increase in expression level in response to injury. Furthermore, Epo protein levels increased 24 h after ischemic damage and by intranasal-rhEpo; however protein levels of nonphospho EpoR was lower detected in the groups receiving rhEpo compared to these untreated with rhEpo. As well as ischemic damage induced an increase in phospho-EpoR levels and even more intranasal rhEpo administration in the sham and ischemic groups, at same time. These data indicates rhEpo induced that an increase in EpoR-expression level, although in this time evaluated the receptor was detected higher in phosphorylated form. Before, it has only been reported that the rhEpo induced a significant increase in non-phospho EpoR expression, when is intravenous-administered and using a specific antibody [2,27,28] and the expression of the receptor is required to activate EpoR and generate a neuroprotective effect [2,29]. Besides, the EpoR-phosphorylation increased at Tyr456, which is the binding site associated with janus tyrosine kinase 2 (JAK-2) which in turn, leads to phosphorylation of the receptor [6,29]. This phosphorylation may be the initial step of Epo signaling in the injured brain [30,31]. A possible explanation for our results is that the nasal administration of rhEpo,
20
R. Casta˜ neda-Arellano et al. / Neuroscience Letters 583 (2014) 16–20
proved to be 10 times quicker than the intravenous route used for reaching the damaged brain [15], which could promote faster non-phospho EpoR-expression, 24 h earlier. Our results are in accordance with the fact that the neuroprotective effect requires the receptor induction and activation [32,33]. The intracellular signal pathways implicate protein phosphorylation that could affect the gene regulation involved in cell survival, which would not only be activated by both receptor activation and expression. A time course analysis of the intranasal administration of rhEpo to determine the effect on the changes in genetic and protein expression in ischemic conditions is required. 5. Conclusion In this work, a dose of 500 UI/kg rhEpo intranasally administered modifies the Epo and EpoR levels expression at 24 h after administratione. Additionally, rhEpo in these conditions promotes phosphorylation of EpoR at Tyr456, which would trigger the initial signaling steps correlated with a neuroprotective effect in which cell survival is included. Acknowledgments Grants from Consejo Nacional de Ciencia y Tecnología (CONACYT) grant 381861/253971 and Universidad de Guadalajara, Mexico P3E-221023 supported this work. References [1] S.L. Metha, N. Manhas, R. Raghubir, Molecular targets in cerebral ischemia for developing novel therapeutics, Brain Res. Rev. 54 (2007) 34–66. [2] P.E. Sanchez, R.P. Fares, J.J. Risso, C. Bonnet, S. Bouvard, M. Le-Cavorsin, B. Georges, C. Moulin, A. Belmeguenai, J. Bodennec, A. Morales, J.M. Pequignot, E.E. Baulieu, R.A. Levine, L. Bezin, Optimal neuroprotection by erythropoietin requires elevated expression of its receptor in neurons, PNAS 106 (2009) 9848–9853. [3] F. Zhang, A.P. Signore, Z. Zhou, S. Wang, G. Cao, J. Chen, Erythropoietin protects CA1 neurons against global cerebral ischemia in rat: potential signaling mechanisms, J. Neurosci. Res. 83 (2006) 1241–1251. [4] C. Dame, C.E. Juul, R.D. Christensen, The biology of erythropoietin in the central nervous system and its neurotrophic and neuroprotective potential, Biol. Neonate. 79 (2001) 228–235. [5] S. Genc, T.F. Koroglu, K. Genc, Erythropoietin as a novel neuroprotectant, Restor. Neurol. Neurosci. 22 (2004) 105–119. [6] O. Miura, J.L. Cleveland, J.N. Ihle, Inactivation of erythropoietin receptor function by point mutations in a region having homology with other cytokine receptors, Mol. Cell. Biol. 13 (1993) 1788–1795. [7] L. Mulcahy, The erythropoietin receptor, Semin. Oncol. 28 (2001) 19–23. [8] K. Maiese, F. Li, Z.Z. Chong, New avenues of exploration for erythropoietin, JAMA 293 (2005) 90–95. [9] M. Brines, A. Cerami, Erythropoietin-mediated tissue protection: reducing collateral damage from the primary injury response, J. Intern. Med. 264 (2008) 405–432. [10] Y. Wang, Z.G. Zhang, K. Rhodes, M. Renzi, R.L. Zhang, A. Kapke, M. Lu, C. Pool, G. Heavner, M. Chopp, Br. J. Pharmacol. 151 (2007) 1377–1384. [11] F. Zhang, J. Xing, A.K. Liou, S. Wang, Y. Gan, Y. Luo, X. Ji, R.A. Stetler, J. Chen, G. Cao, Enhanced delivery of erythropoietin across the blood–brain barrier for neuroprotection against ischemic neuronal injury, Transl. Stroke Res. 1 (2010) 113–121. [12] S.R. Alcalá-Barraza, M.S. Lee, L.R. Hanson, A.A. McDonald, W.H. Frey 2nd, L.K. McLoon, Intranasal delivery of neurotrophic factors BDNF, CNTF, EPO, and NT-4 to the CNS, J. Drug Target 18 (2010) 179–190.
[13] I.S. Teste, Y.M. Tamos, Y.R. Cruz, A.M. Cernada, J.C. Rodríguez, N.S. Martínez, R.M. Antich, A. González-Quevedo, J.C. Rodríguez, Dose effect evaluation and therapeutic window of the neuro-EPO nasal application for the treatment of the focal ischemia model in the Mongolian gerbil, Sci. World J. 2012 (607498) (2012) 12. [14] Y.P. Yu, Q.Q. Xu, Q. Zhang, W.P. Zhang, L.H. Zhang, E.Q. Wei, Intranasal recombinant human erythropoietin protects rats against focal cerebral ischemia, Neurosci. Lett. 387 (2005) 5–10. [15] I. Sosa, J.C. García Rodríguez, J. Santana, N. Subiros, C. González, Y. Rodríguez, J. Rodríguez, Intranasal administration of recombinant human erythropoietin exerts neuroprotective effects on post-ischemic brain injury in Mongolian gerbils, Pharmacologyonline 1 (2006) 100–112. [16] L. Fletcher, S. Kohli, S.M. Sprague, R.A. Scranton, S.A. Lipton, A. Parra, D.F. Jimenez, M. Digicaylioglu, Intranasal delivery of erythropoietin plus insulin-like growth factor-I for acute neuroprotection in stroke, J. Neurosurg. 111 (2009) 164–170. [17] M.L. Smith, G. Bendek, N. Dahlgren, I. Rosen, T. Wieloch, B.K. Siesjo, Models for studying long-term recovery following forebrain ischemia in the rat. 2. A 2-vessel occlusion model, Acta Neurol. Scand. 69 (1984) 385–401. [18] M. Leist, P. Ghezzi, G. Grasso, R. Bianchi, P. Villa, M. Fratelli, C. Savino, M. Bianchi, J. Nielsen, J. Gerwien, P. Kallunki, A.K. Larsen, L. Helboe, S. Christensen, L.O. Pedersen, M. Nielsen, L. Torup, T. Sager, A. Sfacteria, S. Erbayraktar, Z. Erbayraktar, N. Gokmen, O. Yilmaz, C. Cerami-Hand, Q.W. Xie, T. Coleman, C. Cerami, M. Brines, Derivatives of erythropoietin that are tissue protective but not erythropoietic, Science 305 (2004) 239–242. [19] P. Villa, J. van Beek, A.K. Larsen, J. Gerwien, S. Christensen, A. Cerami, M. Brines, M. Leist, P. Ghezzi, L. Torup, Reduced functional deficits, neuroinflammation, and secondary tissue damage after treatment of stroke by non-erythropoietic erythropoietin derivatives, J. Cereb. Blood Flow. Metab. 27 (2007) 552–563. [20] M.L. Brines, P. Ghezzi, S. Keenan, Erythropoietin cross the blood brain barrier to protect against experimental brain injury, PNAS 97 (2000) 10526–10531. [21] D. Agnello, P. Bigini, P. Villa, T. Mennini, A. Cerami, M.L. Brines, P. Ghezzi, Erythropoietin exerts an anti-inflammatory effect on the CNS in a model of experimental autoimmune encephalomyelitis, Brain Res. 952 (2002) 128–134. [22] M.A. Moskowitz, E.H. Lo, C. Iadecola, The science of stroke: mechanisms in search of treatments, Neuron 67 (2010) 181–198. [23] K. Jones, J.A. Senft, An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide, J. Histochem. Cytochem. 33 (1985) 77–79. [24] Y. Meng, Y. Xiong, A. Mahmood, Y. Zhang, C. Qu, M. Chopp, Dose-dependent neurorestorative effects of delayed treatment of traumatic brain injury with recombinant human erythropoietin in rats, J. Neurosurg. 115 (2011) 550–560. [25] E. Spandou, S. Papoutsopoulou, V. Soubasi, G. Karkavelas, C. Simeonidou, G. Kremenopoulos, O. Guiba-Tziampiri, Hypoxia-ischemia affects erythropoietin and erythropoietin receptor expression pattern in the neonatal rat brain, Brain Res. 1021 (2004) 167–172. [26] Z.Y. Chen, P. Asavaritikrai, J.T. Prchal, C.T. Noguchi, Endogenous erythropoietin signaling is required for normal neural progenitor cell proliferation, J. Biol. Chem. 282 (2007) 25875–25883. [27] A. Sola, M. Rogido, B.H. Lee, T. Genetta, T.C. Wen, Erythropoietin after focal cerebral ischemia activates the Janus kinase-signal transducer and activator of transcription signaling pathway and improves brain injury in postnatal day 7 rats, Pediatr. Res. 57 (2005) 481–487. [28] W.M. Campana, R.R. Myers, Exogenous erythropoietin protects against dorsal root ganglion apoptosis and pain following peripheral nerve injury, Eur. J. Neurosci. 18 (2003) 1497–1506. [29] S.C. Hellewell, E.B. Ya, D.S. Alwis, N. Bye, M.C. Morganti-Kossmann, Erythropoietin improves motor and cognitive deficit, axonal pathology, and neuroinflammation in a combined model of diffuse traumatic brain injury and hypoxia, in association with upregulation of the erythropoietin receptor, J. Neuroinflamm. 18 (2013) 10–156. [30] M. Kawakami, M. Sekiguchi, K. Sato, S. Kozaki, M. Takahashi, Erythropoietin receptor-mediated inhibition of exocytotic glutamate release confers neuroprotection during chemical ischemia, J. Biol. Chem. 276 (2001) 39469–39475. [31] M. Digicaylioglu, G. Garden, S. Timberlake, L. Fletcher, S.A. Lipton, Acute neuroprotective synergy of erythropoietin and insulin-like growth factor I, PNAS 101 (2004) 9855–9860. [32] L. Velly, L. Pellegrini, B. Guillet, N. Bruder, P. Pisano, Erythropoietin 2nd cerebral protection after acute injuries: a double-edged sword? Pharmacol. Ther. 128 (2010) 445–459. [33] S.A. Lipton, Pathologically activated therapeutics for neuroprotection, Nat. Rev. Neurosci. 8 (2007) 803–808.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Activity increase in EpoR and Epo expression by intranasal recombinant human erythropoietin (rhEpo) administration in ischemic hippocampi of adult rats ˜ R. Castaneda-Arellano, A.I. Feria-Velasco, M.C. Rivera-Cervantes ∗ Laboratorio de Neurobiología Celular, Department of Cellular and Molecular Biology, CUCBA, Universidad de Guadalajara Zapopan Jal., Mexico
h i g h l i g h t s • Cell survival by rhEpo-intranasal administration in ischemic hippocampus. • Modified expression of Epo and EpoR by ischemia and after rhEpo-intranasal. • Increased phosphorilation of the EpoR by rhEpo administration.
a r t i c l e
i n f o
Article history: Received 8 July 2014 Received in revised form 26 August 2014 Accepted 4 September 2014 Available online 16 September 2014 Keywords: Erythropoietin Intranasal delivery EpoR phosphorylation Ischemia Neuroprotection
a b s t r a c t Erythropoietin in the nervous system is a potential neuroprotective factor for cerebral ischemic damage due to specific-binding to the erythropoietin receptor, which is associated with survival mechanisms. However, the role of its receptor is unclear. Thus, this work assessed whether a low dose (500 UI/Kg) of intranasal recombinant human erythropoietin administered 3 h after ischemia induced changes in the activation of its receptor at the Tyr456-phosphorylated site in ischemic hippocampi in rats. The results showed that recombinant human erythropoietin after injury maintained cell survival and was associated with an increase in receptor phosphorylation at the Tyr456 site as an initial signaling step, which correlated with a neuroprotective effect. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Ischemia produces a sequence of pathophysiological phenomena in the brain that damages neurons, glia and endothelial cells [1]. Animal models have determined that erythropoietin (Epo) promotes neuroprotection through the Epo receptor (EpoR) under conditions of brain damage [2], such as an ischemic event [3]. A deficiency in tissue oxygenation results in the production and secretion of Epo from the hippocampus and an increase in EpoR expression in astrocytes and neurons [4,5]. Epo binding changes the conformation of the EpoR, activates phosphorylation activity
∗ Corresponding author at: Laboratorio de Neurobiología Celular Dept. of Cellular and Molecular Biology, CUCBA, Universidad de Guadalajara, Km 15.5 Carretera a Nogales, Camino Ing, Ramón, Padilla Sánchez Km 2, Zapopan, Jalisco 45221, Mexico. Tel.: +52 33 37771191/37771150. E-mail addresses: [email protected], [email protected] (M.C. Rivera-Cervantes). http://dx.doi.org/10.1016/j.neulet.2014.09.013 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
of the Janus-tyrosine kinase 2 (Jak2) and this phosphorylates to the EpoR at multiple sites, such as the Tyr-456 motif, which is the preferred binding site for the transduction of intracellular signaling to foster neuroprotection [6–8]. A lower concentration of Epo induces erythropoiesis through EpoR as long as Epo is present. A typical dose of recombinant human erythropoietin (rhEpo) to 100 IU/kg provides serum Epo levels that trigger EpoR for approximately 24 h [9]. EpoR in the adult nervous system is not highly expressed under normal conditions, instead it is up-regulated following injury, and a high concentration of Epo is necessary for neuroprotection. Doses of rhEpo from 500 to 5000 IU/kg intravenously establishes tissue protection [10], but only a brief exposure of Epo triggers a sustained response and exerts multiple effects on the brain [11]. Intranasal delivery bypassed the blood–brain barrier and achieves potentially therapeutic levels of drugs in the CNS compared to systemic treatment due to the direct transport of absorbed drug into the systemic circulation, which bypasses the first-pass effect of the liver [12]. The intranasal administration of Epo reaches the damaged brain 10 times quicker
R. Casta˜ neda-Arellano et al. / Neuroscience Letters 583 (2014) 16–20
than the intravenous route [13]. The efficacy of this application has been demonstrated in models of ischemia in gerbils and rats treated with NeuroEpo and rhEpo, respectively [14–16]. The present study assessed whether a low dose of rhEpo (500 IU/Kg) induced cytoprotection and altered the expression levels of Epo and EpoR in hippocampi of ischemic rats. 2. Materials and methods 2.1. Animals All experimental animals in this study were used in accordance with NIH guidelines (NIH Guide for the Care and Use of Laboratory Animals). Adult male Wistar rats, weighing 250–300 g at the beginning of experiments were provided with food and water ad libitum in a room maintained at 22 ± 2 ◦ C under a 12 h light/dark cycle. Five study groups were used: intact group, intact animals without pharmacological treatment or surgical procedure; sham group, animals that underwent the surgical procedure, but without carotid occlusion; sham + rhEpo group, animals that underwent the surgical procedure, but without carotid occlusion and with administration of rhEpo (500 IU/kg, intranasally); ischemia, animals that underwent surgery by carotid occlusion, but without rhEpo; ischemia + rhEpo, the animals that underwent surgery by carotid occlusion and which were also treated with rhEpo (500 IU/kg, intranasally). 2.2. Model of 2-vessel occlusion Rats were anesthetized intraperitoneally with ketamine (90 mg/kg) and xylazine (10 mg/kg), and body temperature was continually monitored and maintained at 37–37.5 ◦ C using a pad and heat lamp. Occlusion of the common carotid arteries was performed as described previously [17] via 20 min of ligation. Sham-surgery animals were treated in the same manner except that the common carotid arteries were not clamped. All animals were killed by decapitation 24 h after surgery. 2.3. rhEpo intranasal treatment The dose of recombinant human erythropoietin was 500 IU/kg of body weight (Exetin-A, Pisa Pharmaceutical, Mexico). The animals were placed in supine position covering the mouth allowed the animal to breathe through the nose and absorb the drug, with a syringe pump (780010 KD Scientific) at a flow rate of 33 L/min for 3 h post-surgery. 2.4. Viability assay The hippocampus was extracted from the brains of the different study groups and dissected rapidly under cold conditions. Hippocampi remained in 1 mL of Hank’s solution with Mg2+ /Ca2+ , and they were washed and treated for cell dissociation with 100 L DNase (20 g/mL) via the addition of 100 L of trypsin solution 1:10 (100 L of trypsin in 900 L Hank’s solution Mg2+ /Ca2+ -free). Hippocampi were incubated at 37 ◦ C for 10 min and 100 L of a trypsin inhibitor was added. The cells were mechanically resuspended (15–20 steps) and centrifuged for 10 min at 200 × g at room temperature. A solution of fluorescein diacetate/propidium iodide (FDA-PI), 0.1 mL of FDA (5 mg/mL in acetone) and 0.03 mL (0.6 g) of PI was added directly to resuspended cells for staining, and 10 L of the cell suspension was removed and placed in a Neubauer chamber for cell counting under a fluorescence microscope (Olympus BX51 with Alexa absorption filter of 488–594 nm).
17
2.5. Semi-quantitative RT-PCR The expression of Epo and EpoR mRNA in the hippocampus was evaluated using reverse transcriptase-polymerase chain reaction (RT-PCR). RNA was isolated from hippocampus tissue (TRIzol® Reagent – Invitrogen, Carlsbad, CA), and first-strand cDNA was prepared (First Strand® , M-MLV RT, Invitrogen Cat# 28025-021). cDNA samples were probed with nucleotide primer sets Epo: forward 5 -GCT CCA ATC TTT GTG GCA TC-3 and reverse 5 -TGG CTT CGT GAC CCT CTG T-3 ; EpoR: forward 5 -CCG GGA TGG GCT TCA ACT AC-3 and reverse 5 -TCCAGTGGCACAAAACTCGAC-3 ; -actin: forward 5 -CACCACAGCTGAGAGGGAAATCGTGCGTGA-3 and reverse 5 -ATTTGCGGTGCACGATGGAGGGGCCGGACT-3 . The expression levels of mRNA transcripts were normalized to -actin for different testing sample-volumes for different PCR-reactions to obtain the same pattern of expression for each sample evaluated. The expression level of each target gene was also normalized to the expression of the -actin gene. 2.6. Western blotting The expression of Epo and EpoR proteins in the hippocampus was determined using Western blotting. Briefly, the hippocampi were rapidly harvested, homogenized on ice in 920 L lysis buffer containing 50 mM Tris–HCl, 150 mM NaCl, 25 mM NaF, 1% Nonidet P-40, and 1 mM Na3 VO4 plus 80 L of a protease inhibitor cocktail (sc-29130, Santa Cruz Biotechnology) and centrifuged at 14,000 rpm for 30 min. Equal amounts of protein (50 g) were loaded into each lane of a 12% SDS-PAGE gel, subjected to electrophoresis and transferred onto a nitrocellulose membrane. The membranes were blocked in 1× Animal-Free Blocker (SP-5030, VECTOR) for 2 h and incubated with 1:1000 dilutions of the following antibodies: rat anti-Epo (N-19, Santa Cruz Biotechnology), rat anti-EpoR recognizes non-phosphorylated form (M-20, Santa Cruz Biotechnology), rat anti-EpoR recognizes phosphorylated form in Tyr456 site (sc-20236-R, Santa Cruz Biotechnology) and 1:6000 of rat anti- actin (ab6276, Abcam) overnight at 4 ◦ C. The membranes were incubated with a biotinylated secondary antibody for 2 h followed by incubation with an avidin–biotin complex (ABC standard kit, VECTOR) for 45 min. The samples were washed twice with Tris–NaCl Buffer + Tween 20 (TBST), and the membranes were developed using diaminobenzidine (D-5905, Sigma). Band analysis was performed in Kodak Digital Science 1D Image Analysis, and relative band densities were normalized to the loading control -actin bands. 2.7. Data analysis All results are expressed as means ± standard error (SEM) and were analyzed using a one-way analysis of variance (ANOVA). Differences between groups were tested using Tukey’s post hoc test. In all cases, p < 0.05 was considered statistically significant. 3. Results 3.1. Cell survival after rhEpo-treatment under ischemic damage The intact, sham and sham + rhEpo groups were not significantly difference from each other in cell viabillity of the hippocampus (Fig. 1A and B). While ischemic injury (ischemia group) induced changes in cell viability decreasing 84% living cells and increasing 77% non-viable cells when compared to the intact group. On the other hand, treatment with intranasal rhEpo after injury (ischemia + rhEpo) significantly increased cell survival 74%, and fewer non-viable cells (66%) were observed versus the ischemic
18
R. Casta˜ neda-Arellano et al. / Neuroscience Letters 583 (2014) 16–20
Fig. 1. Effect of intranasal rhEpo on cell viability in rat hippocampus 24 h after reperfusion as determined using FDA and PI. (A) The number of viable cells and (B) the number of non-viable cells are plotted. The data represent the means ± SEM of 4 animals, conducted in duplicate samples: p < 0.05, compared to intact, + p < 0.05, compared to ischemia. The value of p < 0.05 was considered as statistically significant, ANOVA followed by Tukey’s post hoc test.
damage induced 2-vessel occlusion and evaluated using the FDA/IP assay (Fig. 1A and B). 3.2. Changes in the expression levels of Epo and EpoR mRNA following delivery of low-dose intranasal rhEpo to ischemic rats The analysis of mRNA expression of Epo and EpoR revealed no significant differences in the intact, sham or sham + rhEpo groups (Fig. 2C and D). However, Epo-mRNA levels in the ischemic injury (ischemia group) decreased to 33% and exhibited a 200% decrease in EpoR mRNA expression when compared to the intact group. While delivery of rhEpo intranasally following ischemic damage (ischemia + rhEpo group) increased Epo-expression levels to 43% and caused an increase of 410% in EpoR mRNA expression levels compared to ischemia (Fig. 2C and D).
(Fig. 3B). However, an approximately 28% greater expression was observed in the ischemia and ischemia + rhEpo groups compared to the intact group (Fig. 3B). Moreover, non-phospho EpoR showed no differences in the intact and sham groups, but an increase of 26% was observed in the ischemic brains (ischemia group). In the sham + rhEpo and ischemia + rhEpo groups, lower expression was detected in non-phospho EpoR of 68% compared to the ischemia group (Fig. 3C and D). The ratio between phospho-EpoR relative to non-phospho EpoR expression was analyzed. The levels of phospho-EpoR were not significantly different (p < 0.05) in intact and sham groups (Fig. 4A and B), but the level of phospho-EpoR in the sham + rhEpo group was twice as high as that in the intact group, and one fold increase was observed in the 2-vessel occlusion group. A 2.5-fold increase was observed after intranasal rhEpo delivery after ischemia (ischemia + rhEpo) when compared to the intact group (Fig. 4A and B).
3.3. Protein expression of Epo and EpoR after rhEpo treatment in cerebral ischemic rats
4. Discussion
Western blotting showed no significant differences in Epo expression between the intact, sham and sham + rhEpo groups
The Epo protein induces a neuroprotective effect in different ways, including the inhibition of neuronal apoptosis and
Fig. 2. Semiquantitative analysis of Epo and EpoR mRNA expression. Representative electrophoresis are shown for Epo-mRNA (A) and EpoR-mRNA (B). -Actin served as the gel-loading control. The data represent the means ± SEM of 4 animals, conducted in duplicate samples (C and D): p < 0.05, compared to intact, + p < 0.05, compared to ischemia. The value of p < 0.05 was considered as statistically significant, ANOVA followed by Tukey’s post hoc test.
R. Casta˜ neda-Arellano et al. / Neuroscience Letters 583 (2014) 16–20
19
Fig. 3. Levels of Epo and EpoR protein expression after induced ischemia (2VO) in the rat hippocampus. Representative Western blots are shown for Epo protein (A) and EpoR protein (B); -actin served as the gel-loading control. The data represent the means ± SEM of 4 animals, conducted in duplicate samples (C and D): p < 0.05, compared to intact, + p < 0.05, compared to ischemia. The value of p < 0.05 was considered as statistically significant ANOVA followed by Tukey’s post hoc test.
Fig. 4. Ratio of phosphorylated EpoR (EpoRp(Tyr456 )/non-phosphorilated EpoR in the hippocampus of ischemic rats. (A) Representative photograph of a membrane obtained by Western blotting for the EpoRp(Tyr456 and EpoR proteins. (B) The plotted data represent the means ± SEM of 4 animals, conducted in duplicate samples of ratio EpoRp(Tyr456) /EpoRtotal with respect to different study groups. p < 0.05, compared to intact, + p < 0.05, compared to ischemia. The value of p < 0.05 was considered as statistically significant, ANOVA followed by Tukey’s post hoc test.
cerebral inflammation, blockade of glutamatergic transmission and new vessel formation [18,19]. rhEpo at doses of 500 to 5000 IU/kg reduces infarct volume via the promotion of cell-survival [10,20]. However, these doses are quite large and significantly increase blood volume resultant from erythropoietic activity [21]. The present study used a modified 2-vessel occlusion that is a widely used experimental model of ischemia [17]. This procedure induces changes in cell viability in the hippocampus, which was evaluated by IP-incorporation into the cell due to drastically increase the plasma membrane permeability. This leads to cell death, contributing to tissue damage [22], according Jones and Senft [23] technique to evaluate cell viability. Furthermore, our data show that delayed (3 h) treatment with rhEpo administered intranasally at a dose of 500 IU/Kg decreased cell damage induced by ischemia. The neuroprotective effects of Epo have been demonstrated in several models
of stroke [10,14,19] and traumatic brain injury [24], which require the application of higher doses intravenously. Epo is a hormone involved in the regulation of neuroprotective functions, and its receptor is expressed on many neurons throughout the CNS, particularly hippocampus [20]. Epo and EpoR expression has been proposed as a mechanism of the tissueprotective response to injury in the brain [25,26]. The important issue to be addressed is whether the neuroprotective effects of rhEpo are initially triggered by its receptor. We showed that ischemic insult induced a decrease in the transcript level of Epo and its receptor, but delivery of rhEpo 3 h after damage increased the mRNA expression of both molecules 24 h post-damage. Repeated hypoxic exposures induce an increase in Epo and EpoR gene expression 3 h after re-oxygenation [2]. However, Spandou et al. [25] did not detect any significant alteration in EpoR expression 24 h after focal ischemic insult, which is similar to our results. Epo induces GATA-3 activity in neural cells, which is required for the active transcription of EpoR in brain development [14]. Therefore, this transcription factor may be regulated by rhEpo after ischemic damage, which leads to the substantial increase in expression level in response to injury. Furthermore, Epo protein levels increased 24 h after ischemic damage and by intranasal-rhEpo; however protein levels of nonphospho EpoR was lower detected in the groups receiving rhEpo compared to these untreated with rhEpo. As well as ischemic damage induced an increase in phospho-EpoR levels and even more intranasal rhEpo administration in the sham and ischemic groups, at same time. These data indicates rhEpo induced that an increase in EpoR-expression level, although in this time evaluated the receptor was detected higher in phosphorylated form. Before, it has only been reported that the rhEpo induced a significant increase in non-phospho EpoR expression, when is intravenous-administered and using a specific antibody [2,27,28] and the expression of the receptor is required to activate EpoR and generate a neuroprotective effect [2,29]. Besides, the EpoR-phosphorylation increased at Tyr456, which is the binding site associated with janus tyrosine kinase 2 (JAK-2) which in turn, leads to phosphorylation of the receptor [6,29]. This phosphorylation may be the initial step of Epo signaling in the injured brain [30,31]. A possible explanation for our results is that the nasal administration of rhEpo,
20
R. Casta˜ neda-Arellano et al. / Neuroscience Letters 583 (2014) 16–20
proved to be 10 times quicker than the intravenous route used for reaching the damaged brain [15], which could promote faster non-phospho EpoR-expression, 24 h earlier. Our results are in accordance with the fact that the neuroprotective effect requires the receptor induction and activation [32,33]. The intracellular signal pathways implicate protein phosphorylation that could affect the gene regulation involved in cell survival, which would not only be activated by both receptor activation and expression. A time course analysis of the intranasal administration of rhEpo to determine the effect on the changes in genetic and protein expression in ischemic conditions is required. 5. Conclusion In this work, a dose of 500 UI/kg rhEpo intranasally administered modifies the Epo and EpoR levels expression at 24 h after administratione. Additionally, rhEpo in these conditions promotes phosphorylation of EpoR at Tyr456, which would trigger the initial signaling steps correlated with a neuroprotective effect in which cell survival is included. Acknowledgments Grants from Consejo Nacional de Ciencia y Tecnología (CONACYT) grant 381861/253971 and Universidad de Guadalajara, Mexico P3E-221023 supported this work. References [1] S.L. Metha, N. Manhas, R. Raghubir, Molecular targets in cerebral ischemia for developing novel therapeutics, Brain Res. Rev. 54 (2007) 34–66. [2] P.E. Sanchez, R.P. Fares, J.J. Risso, C. Bonnet, S. Bouvard, M. Le-Cavorsin, B. Georges, C. Moulin, A. Belmeguenai, J. Bodennec, A. Morales, J.M. Pequignot, E.E. Baulieu, R.A. Levine, L. Bezin, Optimal neuroprotection by erythropoietin requires elevated expression of its receptor in neurons, PNAS 106 (2009) 9848–9853. [3] F. Zhang, A.P. Signore, Z. Zhou, S. Wang, G. Cao, J. Chen, Erythropoietin protects CA1 neurons against global cerebral ischemia in rat: potential signaling mechanisms, J. Neurosci. Res. 83 (2006) 1241–1251. [4] C. Dame, C.E. Juul, R.D. Christensen, The biology of erythropoietin in the central nervous system and its neurotrophic and neuroprotective potential, Biol. Neonate. 79 (2001) 228–235. [5] S. Genc, T.F. Koroglu, K. Genc, Erythropoietin as a novel neuroprotectant, Restor. Neurol. Neurosci. 22 (2004) 105–119. [6] O. Miura, J.L. Cleveland, J.N. Ihle, Inactivation of erythropoietin receptor function by point mutations in a region having homology with other cytokine receptors, Mol. Cell. Biol. 13 (1993) 1788–1795. [7] L. Mulcahy, The erythropoietin receptor, Semin. Oncol. 28 (2001) 19–23. [8] K. Maiese, F. Li, Z.Z. Chong, New avenues of exploration for erythropoietin, JAMA 293 (2005) 90–95. [9] M. Brines, A. Cerami, Erythropoietin-mediated tissue protection: reducing collateral damage from the primary injury response, J. Intern. Med. 264 (2008) 405–432. [10] Y. Wang, Z.G. Zhang, K. Rhodes, M. Renzi, R.L. Zhang, A. Kapke, M. Lu, C. Pool, G. Heavner, M. Chopp, Br. J. Pharmacol. 151 (2007) 1377–1384. [11] F. Zhang, J. Xing, A.K. Liou, S. Wang, Y. Gan, Y. Luo, X. Ji, R.A. Stetler, J. Chen, G. Cao, Enhanced delivery of erythropoietin across the blood–brain barrier for neuroprotection against ischemic neuronal injury, Transl. Stroke Res. 1 (2010) 113–121. [12] S.R. Alcalá-Barraza, M.S. Lee, L.R. Hanson, A.A. McDonald, W.H. Frey 2nd, L.K. McLoon, Intranasal delivery of neurotrophic factors BDNF, CNTF, EPO, and NT-4 to the CNS, J. Drug Target 18 (2010) 179–190.
[13] I.S. Teste, Y.M. Tamos, Y.R. Cruz, A.M. Cernada, J.C. Rodríguez, N.S. Martínez, R.M. Antich, A. González-Quevedo, J.C. Rodríguez, Dose effect evaluation and therapeutic window of the neuro-EPO nasal application for the treatment of the focal ischemia model in the Mongolian gerbil, Sci. World J. 2012 (607498) (2012) 12. [14] Y.P. Yu, Q.Q. Xu, Q. Zhang, W.P. Zhang, L.H. Zhang, E.Q. Wei, Intranasal recombinant human erythropoietin protects rats against focal cerebral ischemia, Neurosci. Lett. 387 (2005) 5–10. [15] I. Sosa, J.C. García Rodríguez, J. Santana, N. Subiros, C. González, Y. Rodríguez, J. Rodríguez, Intranasal administration of recombinant human erythropoietin exerts neuroprotective effects on post-ischemic brain injury in Mongolian gerbils, Pharmacologyonline 1 (2006) 100–112. [16] L. Fletcher, S. Kohli, S.M. Sprague, R.A. Scranton, S.A. Lipton, A. Parra, D.F. Jimenez, M. Digicaylioglu, Intranasal delivery of erythropoietin plus insulin-like growth factor-I for acute neuroprotection in stroke, J. Neurosurg. 111 (2009) 164–170. [17] M.L. Smith, G. Bendek, N. Dahlgren, I. Rosen, T. Wieloch, B.K. Siesjo, Models for studying long-term recovery following forebrain ischemia in the rat. 2. A 2-vessel occlusion model, Acta Neurol. Scand. 69 (1984) 385–401. [18] M. Leist, P. Ghezzi, G. Grasso, R. Bianchi, P. Villa, M. Fratelli, C. Savino, M. Bianchi, J. Nielsen, J. Gerwien, P. Kallunki, A.K. Larsen, L. Helboe, S. Christensen, L.O. Pedersen, M. Nielsen, L. Torup, T. Sager, A. Sfacteria, S. Erbayraktar, Z. Erbayraktar, N. Gokmen, O. Yilmaz, C. Cerami-Hand, Q.W. Xie, T. Coleman, C. Cerami, M. Brines, Derivatives of erythropoietin that are tissue protective but not erythropoietic, Science 305 (2004) 239–242. [19] P. Villa, J. van Beek, A.K. Larsen, J. Gerwien, S. Christensen, A. Cerami, M. Brines, M. Leist, P. Ghezzi, L. Torup, Reduced functional deficits, neuroinflammation, and secondary tissue damage after treatment of stroke by non-erythropoietic erythropoietin derivatives, J. Cereb. Blood Flow. Metab. 27 (2007) 552–563. [20] M.L. Brines, P. Ghezzi, S. Keenan, Erythropoietin cross the blood brain barrier to protect against experimental brain injury, PNAS 97 (2000) 10526–10531. [21] D. Agnello, P. Bigini, P. Villa, T. Mennini, A. Cerami, M.L. Brines, P. Ghezzi, Erythropoietin exerts an anti-inflammatory effect on the CNS in a model of experimental autoimmune encephalomyelitis, Brain Res. 952 (2002) 128–134. [22] M.A. Moskowitz, E.H. Lo, C. Iadecola, The science of stroke: mechanisms in search of treatments, Neuron 67 (2010) 181–198. [23] K. Jones, J.A. Senft, An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide, J. Histochem. Cytochem. 33 (1985) 77–79. [24] Y. Meng, Y. Xiong, A. Mahmood, Y. Zhang, C. Qu, M. Chopp, Dose-dependent neurorestorative effects of delayed treatment of traumatic brain injury with recombinant human erythropoietin in rats, J. Neurosurg. 115 (2011) 550–560. [25] E. Spandou, S. Papoutsopoulou, V. Soubasi, G. Karkavelas, C. Simeonidou, G. Kremenopoulos, O. Guiba-Tziampiri, Hypoxia-ischemia affects erythropoietin and erythropoietin receptor expression pattern in the neonatal rat brain, Brain Res. 1021 (2004) 167–172. [26] Z.Y. Chen, P. Asavaritikrai, J.T. Prchal, C.T. Noguchi, Endogenous erythropoietin signaling is required for normal neural progenitor cell proliferation, J. Biol. Chem. 282 (2007) 25875–25883. [27] A. Sola, M. Rogido, B.H. Lee, T. Genetta, T.C. Wen, Erythropoietin after focal cerebral ischemia activates the Janus kinase-signal transducer and activator of transcription signaling pathway and improves brain injury in postnatal day 7 rats, Pediatr. Res. 57 (2005) 481–487. [28] W.M. Campana, R.R. Myers, Exogenous erythropoietin protects against dorsal root ganglion apoptosis and pain following peripheral nerve injury, Eur. J. Neurosci. 18 (2003) 1497–1506. [29] S.C. Hellewell, E.B. Ya, D.S. Alwis, N. Bye, M.C. Morganti-Kossmann, Erythropoietin improves motor and cognitive deficit, axonal pathology, and neuroinflammation in a combined model of diffuse traumatic brain injury and hypoxia, in association with upregulation of the erythropoietin receptor, J. Neuroinflamm. 18 (2013) 10–156. [30] M. Kawakami, M. Sekiguchi, K. Sato, S. Kozaki, M. Takahashi, Erythropoietin receptor-mediated inhibition of exocytotic glutamate release confers neuroprotection during chemical ischemia, J. Biol. Chem. 276 (2001) 39469–39475. [31] M. Digicaylioglu, G. Garden, S. Timberlake, L. Fletcher, S.A. Lipton, Acute neuroprotective synergy of erythropoietin and insulin-like growth factor I, PNAS 101 (2004) 9855–9860. [32] L. Velly, L. Pellegrini, B. Guillet, N. Bruder, P. Pisano, Erythropoietin 2nd cerebral protection after acute injuries: a double-edged sword? Pharmacol. Ther. 128 (2010) 445–459. [33] S.A. Lipton, Pathologically activated therapeutics for neuroprotection, Nat. Rev. Neurosci. 8 (2007) 803–808.