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Erythropoietin improves synaptic transmission during and following ischemia in rat hippocampal slice cultures
Brain Research 958 (2002) 305–311 www.elsevier.com / locate / brainres
Research report
Erythropoietin improves synaptic transmission during and following ischemia in rat hippocampal slice cultures Astrid Weber a , *, Rolf F. Maier a ,1 , Ulrike Hoffmann b , Martin Grips a , Marc Hoppenz a , Ayse G. Aktas a , Uwe Heinemann b , Michael Obladen a , Sebastian Schuchmann b a
Department of Neonatology, Charite´ , Humboldt University Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, D-13353 Berlin, Germany b ¨ Johannes Muller Institute of Physiology, Charite´ , Humboldt University Berlin, Tucholskystrasse 2, D-10117 Berlin, Germany Accepted 5 September 2002
Abstract Erythropoietin (EPO) prevents neuronal damage following ischemic, metabolic, and excitotoxic stress. In this study evoked extracellular field potentials (FP) were used to investigate the effect of EPO on synaptic transmission in hippocampal slice cultures. EPO treated cultured slices (40 units / ml for 48 h) showed significantly increased FP during and following oxygen and glucose deprivation compared with untreated control slices. The addition of the Jak2 inhibitor AG490 (50 mM for 48 h) blocked the EPO effect. These data suggest that EPO improves synaptic transmission during and following ischemia in hippocampal slice cultures. 2002 Elsevier Science B.V. All rights reserved. Theme: Excitable membranes and synaptic transmission Topic: Presynaptic mechanisms Keywords: Erythropoietin; Synaptic transmission; Ischemia; Neuroprotection; Hippocampal slice culture
1. Introduction The cytokine erythropoietin (EPO), well known for its crucial role in erythropoiesis, has been shown to possess neuroprotective characteristics following ischemic, metabolic and excitotoxic stress [4,13,16,17]. Expression of EPO and EPO receptor (EPOR) can be induced by hypoxia in immature and mature brains [7,11]. Therefore, it has been suggested that EPO could contribute to neuroprotection against acute and apoptotic cell death and may be involved in hypoxic–ischemic preconditioning [4], which has been shown to increase neuronal tolerance against subsequent prolonged ischemia [3]. Abbreviations: AG490, Janus kinase 2 inhibitor; AO, acridine orange; EPO, erythropoietin; EPOR, erythropoietin receptor; FP, extracellular field potential; Jak2, Janus kinase 2; OGD, oxygen and glucose deprivation; PI, propidium iodide *Corresponding author. Tel.: 149-30-450-566122; fax: 149-30-450566922. E-mail address: [email protected] (A. Weber). 1 Present address: Dept. of Neonatology, Philipps-University, Marburg, Deutschhausstr. 12, D-35033, Marburg, Germany.
The stimulation of EPOR activates different intracellular signaling pathways by activation of EPOR-associated Janus kinase 2 (Jak2, tyrosine kinase) in non-neuronal and neuronal cells [4,15]. Therefore a specific Jak2 tyrosine kinase inhibitor can be used to block EPO induced intracellular signaling [2]. In addition to the neuroprotective effect, little is known about the relevance of the EPO and EPOR signaling pathways on neuronal function. The present study was designed to investigate the effect of EPO preconditioning on the neuronal synaptic transmission under normoxic and ischemic conditions in cultured rat hippocampal brain slices.
2. Materials and methods
2.1. Preparation and culture of slices Combined entorhinal cortico-hippocampal slice cultures were prepared from 6–8-day-old Wistar rats and maintained as described before [19]. Briefly, after decapitation, the hippocampi with the entorhinal cortex attached were
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03604-1
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dissected and 400-mm slices were cut (McIlwain tissue chopper, Mickle Labaratories, UK) under sterile conditions in gassed (95% O 2 / 5%CO 2 ) ice-cold minimal essential medium (MEM; Gibco, Karlsruhe, Germany) at pH 7.35. Three slices were placed on each culture membrane (0.4 mm Minicell culture plate inserts; Millipore, Eschborn, Germany) and incubated in a medium containing 50% MEM, 25% Hank’s balanced salt solution (HBSS; Sigma, Deisenhofen, Germany), 25% horse serum (Gibco), 2 mM L-glutamine, pH 7.35 with 5% CO 2 at 36.5 8C. The antibiotic-free culture medium was completely replaced after day 2 and thereafter twice a week.
2.2. Preincubation For a number of experiments, slices were preincubated with EPO (Neorecormon, Roche, UK) or / and the Jak2 inhibitor AG490 (Calbiochem, Bad Soden, Germany) for 48 h. EPO was used at 40 units / ml (332 ng / ml) culture medium. The Jak2 inhibitor was dissolved at 100 mM in DMSO, stored at 220 8C and used in a final concentration of 50 mM. In n56 slice cultures we tested the direct effect of the Jak2 inhibitor AG490 and found no increased toxicity compared to control slices. Slices were divided into three groups: control (14 slices, five preparations; no preincubation), EPO (14 slices, five preparations; 40 units / ml EPO for 48 h), EPO plus Jak2 inhibitor (16 slices, two preparations; 40 units / ml EPO and 50 mM Jak2 inhibitor for 48 h), and Jak2 inhibitor (six slices, one preparation; 50 mM Jak2 inhibitor for 48 h).
2.3. Oxygen and glucose deprivation ( OGD) For the experiments, slice cultures were transferred to an interface chamber after 7–9 days in vitro. Slices were perfused at 1.6 ml / min with prewarmed (34 8C), gassed (95% O 2 / 5% CO 2 or 95% N 2 / 5% CO 2 ) artificial cerebrospinal fluid (aCSF) containing (in mM): NaCl 129; KCl 3; NaHPO 4 1.25; MgSO 4 1.8; CaCl 2 1.6; NaHCO 3 21; glucose 10; at pH 7.4. During the oxygen and glucose deprivation (OGD) slices were maintained using glucosefree aCSF gassed with 95% N 2 and 5% CO 2 for 30 min. The OGD period was followed by a reoxygenation period (120 min).
2.4. Electrophysiology For recordings a microelectrode was positioned in area CA1. Synaptic responses were obtained by electrical stimulation (100 ms duration) at the hilar border of area CA3 using a bipolar wire stimulation electrode. The stimulus strength was adjusted to twice the intensity needed to evoke a maximal extracellular field potential (FP) response. Only slice cultures in which orthodromic stimulation evoked a FP response of more than 1 mV were chosen for further investigations. Five evoked FP re-
sponses at intervals of 20 s were averaged and recorded every 5–10 min. The change in FP responses in CA1 following hilar stimulation were normalized to the FP response at the beginning of the experiment.
2.5. RNA isolation and reverse transcription–polymerase chain reaction ( RT–PCR) analysis Total RNA was prepared from hippocampal slices (7–9 days in vitro) by phenol / chloroform extraction using the RNAClean extraction kit (Hybaid, Heidelberg, Germany). Total RNA (250 ng) was reverse-transcribed using AMV reverse transcriptase (Promega, Madison, USA) and random hexamers (Promega). Four microliters of the resulting cDNAs were subjected to amplification in a total volume of 30 pl containing 103 Buffer, 0.5 mM of MgCl 2 , 0.13 mM each of dNTP, l unit Taq polymerase (Gibco, Karlsruhe, Germany) and a pair of specific primers (0.33 mM). Ten microliters of the PCR products were separated by 2.0% agarose gel electrophoresis and stained with ethidium bromide. RT products were 201 bp for EPO, 402 bp for EPOR, and 433 bp for b-actin.
2.6. Immunohistochemistry Hippocampal slice cultures (7–9 days in vitro) fixed over night in 5% neutral buffered formalin were paraffinembedded and sectioned. Rabbit polyclonal EPOR antibody (M20, Santa Cruz Biotechnology, Santa Cruz, USA), directed against the carboxy terminus of the EPO receptor of mouse origin, was applied at a dilution of 1:100 and a secondary antibody (biotinylated donkey anti-rabbit, Amersham Pharmacia Biotech, UK) was added at a dilution of 1:200. Sections were washed with PBS and incubated with Avidin–peroxidase (Sigma) at a dilution of 1:100. Slides were counterstained with hemalum solution, dehydrated and coverslipped for investigation under a light microscope. Absence of primary antibody was used as negative control.
2.7. Western blot Slice culture lysates were normalized for protein, subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose and stained with polyclonal anti-EPO-receptor antibody (M20, Santa Cruz Biotechnology), directed against the carboxy terminus of the EPOR of mouse origin at a dilution of 1:200. The secondary antibody was a donkey anti-rabbit antibody conjugated with horseradish peroxidase (Amersham Pharmacia Biotech) at a dilution of 1:10 000. Immunoreactive bands were visualized using chemiluminescence (ECLPlus, Amersham Pharmacia Biotech).
A. Weber et al. / Brain Research 958 (2002) 305–311
2.8. Fluorescence imaging In order to differentiate between living and dead cells in cultured slices, double staining with the intercalating dyes acridine orange (AO; 5 mM) and propidium iodide (PI; 5 mM) dissolved in aqueous solution was used. The membrane-impermeable PI binds to DNA only in dead cells (emission 605 nm, red), the membrane permeable AO interacts with RNA and DNA mainly in living cells (emission 515 nm, green). The two dyes (both from Molecular Probes, Leiden, the Netherlands) were simultaneously applied and the fluorescence emission signals were measured by exciting at 480 nm (acridine orange) and at 550 nm (propidium iodide) using an optical combination of a 565 nm dichroic mirror and a 590 nm long-pass filter.
2.9. Statistical evaluation Data are presented as mean6standard error of mean, statistical differences were tested using the two-sided student’s t-test for unpaired samples and the log rank test (Kaplan–Meier plot). Statistical significance was assumed if P was less than 0.05.
3. Results FP amplitudes were analyzed before, during and after an OGD period of 30 min duration. In the control group, FP amplitudes at the start of the experiment were 1.7660.1 mV. Following the OGD period, FP amplitudes decreased to 0.1360.08 mV (8.965.8% of control FP amplitude) and showed a restricted recovery during the reoxygenation period to 0.2960.13 mV (15.267.2% of control FP amplitude). In the EPO group, FP amplitudes were not significantly different at the start of the experiment compared to the control group (1.9960.37 mV; EPO vs. control, P50.540). Following the OGD period, FP amplitudes decreased to 0.4160.18 mV (26.2611.1% of control FP amplitude; EPO vs. control, P50.250) and recovered during the reoxygenation period to 1.4860.54 mV (70.6619.8% of control FP amplitude; EPO vs. control, P50.014). In n56 slice cultures we tested the effect of the Jak2 inhibitor AG490 on evoked FP during and following OGD. We found no deleterious effects compared with control slices. In the EPO plus Jak2 inhibitor group, FP amplitudes were significantly enlarged at the start of the experiment compared to the control group (2.8560.34 mV; EPO plus Jak2 inhibitor vs. control, P50.013). Following the OGD period, FP amplitudes dropped to 0.1760.17 mV (3.964.0% of control FP amplitude; EPO plus Jak2 inhibitor vs. control, P50.237; EPO plus Jak2 inhibitor vs. EPO, P50.056) and showed no recovery during the reoxygenation period (0.1160.06 mV, 10.965.2% of control FP amplitude; EPO plus Jak2 inhibitor vs. control, P50.620; EPO plus Jak2 inhibitor vs.
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EPO, P50.009). Fig. 1A,B summarizes the changes in FP amplitudes in the different groups before, during and after the OGD period. The results show a significantly improved recovery in FP amplitudes during the reoxygenation period in the EPO group compared to the control and the EPO plus Jak2 inhibitor group. After 120 min reoxygenation (95% O 2 , 5% CO 2 , 10 mM glucose) a FP amplitude .0.1 mV was recorded in 5 / 14 (35.7%) slices of the control group, in 12 / 14 slices (85.7%) of the EPO group and in 5 / 16 slices (31.2%) of the EPO plus Jak2 inhibitor group. Fig. 1C represents the outcome of the slices during the experiments as a Kaplan– Meier plot. The number of slices with FP amplitudes .0.1 mV was significantly higher in the EPO group compared to the control and the EPO plus Jak2 inhibitor group (control vs. EPO, P50.011; EPO vs. EPO plus Jak2 inhibitor, P50.012; control vs. EPO plus Jak2 inhibitor, P50.906). In order to check the presence of EPO and EPOR in cultured hippocampal slices, RT–PCR techniques were used to show EPO and EPOR mRNA after 7–9 days in vitro (Fig. 2A,B). Furthermore, immunohistochemistry techniques were used to show the expression of EPOR by hippocampal neurons from cultured hippocampal slices (Fig. 2C). Finally, fluorescence double staining with AO and PI was used to investigate semiquantitatively neuroprotective effects of EPO on hippocampal slice cultures following OGD periods (control 17 slices, EPO 15 slices, EPO plus Jak2 inhibitor eight slices). Fig. 2D shows typical cultured slices of the three groups immediately after OGD and a reoxygenation period of 120 min. An increased PI fluorescence signal in control and EPO plus Jak2 inhibitor slices compared to EPO slices indicates an enlarged number of dead cells in these slices. Furthermore, subtracting AO images and PI images accentuates the EPO neuroprotective effect after OGD and reoxygenation period in the EPO group vs. control group and EPO plus Jak2 inhibitor group. Additional staining of cultured slices (n5 11) treated with Jak2 inhibitor AG490 alone did not reveal any toxic effects compared with control slices.
4. Discussion The present study demonstrates for the first time that preconditioning of hippocampal slice cultures with EPO improves neuronal synaptic transmission during and following oxygen and glucose deprivation. In previous studies, pretreatment with EPO has been shown to prevent N-methyl-D-aspartate / glutamate, hypoxia and ischemia induced cell death in several in vitro and in vivo models [4,9,11,17]. The PI /AO double staining in the study presented here confirms in organotypic slice cultures the findings that EPO protects hippocampal neurons and that this effect is dependent on Jak2 associated EPOR. Following ischemic or glutamate induced lesions an increase of EPO and EPOR expression has been described in
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Fig. 1. Changes in extracellular field potential (FP) during and following ischemia (30 min OGD and 120 min reoxygenation) in slice cultures from control (no preincubation), EPO (40 units / ml EPO for 48 h) and EPO plus Jak2 inhibitor (40 units / ml EPO and 50 mM Jak2 inhibitor AG 490 for 48 h) group. (A) Characteristic traces of evoked extracellular field potential before, during and after OGD in the three experimental groups. Asterisks indicate the stimulation artifacts. (B) Changes in evoked extracellular field potential normalized to the FP amplitude under normoxic conditions in the control (s; n514 slices), the EPO (j; n514 slices), and the EPO plus Jak2 inhibitor (h; n516 slices) group. EPO-treated slice cultures show a significantly increased FP amplitude during and particularly following OGD compared with slice cultures from the control and the EPO plus Jak2 inhibitor group (EPO vs. control: *P,0.05; EPO vs. EPO plus Jak2 inhibitor: [ P,0.05). (C) In order to analyze the outcome of slice cultures, the normalized number of slices showing FP amplitudes .0.1 mV were summarized in a Kaplan–Meier plot. A significantly increased number of EPO-treated slice cultures (full line) show FP amplitudes .0.1 mV during and after OGD compared with slice cultures from control (cross-hatched line; P,0.05) and EPO plus Jak2 inhibitor (dotted line; P,0.05) group.
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Fig. 2. EPO and EPOR expression shown in slice cultures (DIV 7–9, no preincubation, no experiment performed) and EPO-induced neuroprotection in hippocampal slice cultures measured immediately after 30 min OGD and 120 min reoxygenation period. (A) Western analysis of protein lysates of three pooled untreated slice cultures (prior to electrophysiological investigation) shows specific EPOR immunoreactivity. Neonatal rat liver was used as positive control. (B) Detection of EPO and EPOR mRNA expression via RT–PCR in three pooled RNA extracts of slice cultures and corresponding beta-actin expression (prior to electrophysiological investigation). (C) Immunohistochemical detection of EPOR-positive neurons in a representative slice culture (prior to electrophysiological investigation). (D) Fluorescence imaging indicating dead (propidium iodide: red; left column) and living (acridine orange: green-yellow; middle column) cells immediately after 30 min OGD and 120 min reoxygenation in slice cultures from the control group, the EPO group and the EPO plus Jak2 inhibitor group. The right column shows a gray-scale differential image (propidium iodide image subtracted from acridine orange image), which indicates dead cells in dark-colored areas and living cells in light-colored areas. The anatomic structure of the slice cultures is displayed by the schematic plot.
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dissociated neuronal cultures, cultured cells with neuronal characteristics and in vivo [11,13,17]. The distinct expression of EPO and EPOR found in hippocampal slice cultures used in this study may partly result from the ischemic phase during the slice preparation. Which intracellular signaling cascade(s) initiated by EPO / EPOR interactions may lead to improvement of synaptic transmission during and following ischemia shown in this study? Moreover, are these intracellular cascade(s) divergent from the EPO mediated neuroprotective pathway? Recently, it has been suggested that the neuroprotective effect of EPO is regulated by activating NF-kB via Jak2 [4]. Jak2 is known to play an important role for signaling of cytokine receptors and therefore may influence indirectly neuronal development and function [15]. Evidence is accumulating for an important role of NF-kB in neuronal function. Thus, studies on neuronal excitation indicated that ionotropic glutamate receptors can activate NF-kB and point to a role of NF-kB as a signal transducer during synaptic transmission [6]. An increased level of NF-kB activity in nuclei of neurons has been suggested to regulate cellular anti-oxidative defense and therefore, may explain partly the neuroprotective effect [14]. Although the mechanism for NF-kB activation via Jak2 is discussed controversially, the role of NF-kB as intracellular second messenger transmitting EPOR derived signals to the nucleus appears unambiguously [2]. EPO has been suggested to modulate neurotransmitter release through activation of EPOR linked to a Jak2 pathway. Activation of EPOR has been shown to activate L-type Ca 21 channels and to modulate Ca 21 -induced dopamine release in clonal rat pheochromocytoma PC12 cells [8,10,12]. Dopamine has been shown to suppress synaptic transmission and inhibition in several regions of the brain, most likely by presynaptic mechanisms [1,5,18]. In the present study, significantly increased evoked FP amplitudes after blocking intracellular Jak2 signaling pathway by pre-treatment of slice cultures with the Jak2inhibitor AG490 may indicate an altered dopamine release under control conditions. Recently, it was suggested that EPO induced activation of EPOR might reduce the Ca 21 dependent release of glutamate from neurons of hippocampus and cerebellum in a model of chemical ischemia [9]. Therefore, EPO-induced modulation of neurotransmitter release may be a regulatory mechanism of presynaptic function in the brain. The improved synaptic transmission during and following ischemia demonstrated in the present study may thus be based on modulation of presynaptic neurotransmitter release. Further studies are necessary to elucidate the functional role of EPO and EPOR for synaptic transmission.
Acknowledgements We wish to thank J. Graulich for his crucial initiation to the collaboration group. We gratefully acknowledge techni-
cal support from S. Gabriel, H.J. Gabriel, S. Latta, and H. Siegmund and help with the preparation of the manuscript ¨ from S. Schurmann. This study was supported by a grant ¨ of the Wilhelm Sander-Stiftung, Munchen (No. 2000.091.1) and a grant of the Sonnenfeld-Stiftung, Berlin.
References [1] J. Behr, T. Gloveli, D. Schmitz, U. Heinemann, Dopamine depresses polysynaptic inhibition in rat subicular neurons, Brain Res. 861 (2000) 160–164. ¨ [2] T. Bittorf, T. Buchse, T. Sasse, R. Jaster, J. Brock, Activation of the transcript factor NF-kB by the erythropoietin receptor. Structural requirements and biological significance, Cell. Signal. 13 (2001) 673–681. [3] U. Bruer, M.K. Weih, N.K. Isaev, A. Meisel, K. Ruscher, A. Bergk, G. Trendelenburg, F. Wiegand, I.V. Victorov, U. Dirnagl, Induction of tolerance in rat cortical neurons: hypoxic preconditioning, FEBS Lett. 414 (1997) 117–121. [4] M. Digicaylioglu, S.A. Lipton, Erythropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-kB signaling cascades, Nature 412 (2001) 641–647. [5] C. Gonzalez-Islas, J.J. Hablitz, Dopamine inhibition of evoked IPSCs in rat prefrontal cortex, J. Neurophysiol. 86 (2001) 2911– 2918. [6] L. Guerrini, F. Blasi, S. Denis-Donini, Synaptic activation of NF-kB by glutamate in cerebellar granule neurons in vitro, Proc. Natl. Acad. Sci. USA 92 (1995) 9077–9081. [7] S.E. Juul, A.T. Yachnis, A.M. Rojiani, R.D. jj Christensen, Immunohistochemical localization of erythropoietin and its receptor in the developing human brain, Pediatr. Dev. Pathol. 2 (1999) 148–158. [8] M. Kawakami, S. Iwasaki, K. Sato, M. Takahashi, Erythropoietin inhibits calcium-induced neurotransmitter release from clonal neuronal cells, Biochem. Biophys. Res. Commun. 279 (2000) 293–297. [9] 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. [10] K. Koshimura, Y. Murakami, M. Sohmiya, J. Tanaka, Y. Kato, Effects of erythropoietin on neuronal activity, J. Neurochem. 72 (1999) 2565–2572. [11] P. Lewczuk, M. Hasselblatt, H. Kamrowski-Kruck, A. Heyer, C. ´ H. Ehrenreich, Survival of hippocampal Unzicker, A.-L. Siren, neurons in culture upon hypoxia: effect of erythropoietin, Neuroreport 11 (2000) 3485–3488. [12] S. Masuda, M. Nagao, K. Takahata, Y. Konishi, F. Gallyas, T. Tabira, R. Sasaki, Functional erythropoietin receptor of the cells with neural characteristics. Comparison with receptor properties of erythroid cells, J. Biol. Chem. 268 (1993) 11208–11216. [13] E. Morishita, S. Masuda, M. Nagao, Y. Yasuda, R. Sasaki, Erythropoietin receptor is expressed in rat hippocampal and cerebral cortical neurons, and erythropoietin prevents in vitro glutamate-induced neuronal cell death, Neuroscience 76 (1997) 105–116. [14] L.A. O’Neill, C. Kaltschmidt, NF-kappa B: a crucial transcription factor for glial and neuronal cell function, Trends Neurosci. 20 (1997) 252–258. [15] E. Parganas, D. Wang, D. Stravopodis, D.J. Topham, J.-C. Marine, S. Teglund, E.F. Vanin, S. Bodner, O.R. Colamonici, J.M. van Deursen, G. Grosveld, J.N. Ihle, Jak2 is essential for signalling through a variety of cytokine receptors, Cell 93 (1998) 385–395. [16] M. Sakanaka, T.C. Wen, S. Matsuda, S. Masuda, E. Morishita, M. Nagao, R. Sasaki, In vivo evidence that erythropoietin protects neurons from ischemic damage, Proc. Natl. Acad. Sci. USA 95 (1998) 4635–4640. ´ M. Fratelli, M. Brines, C. Goemans, S. Casagrande, P. [17] A.-L. Siren,
A. Weber et al. / Brain Research 958 (2002) 305–311 Lewczuk, S. Keenan, C. Gleiter, C. Pasquali, A. Capobianco, T. Mennini, R. Heumann, A. Cerami, H. Ehrenreich, P. Ghezzi, Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress, Proc. Natl. Acad. Sci. USA 98 (2001) 4044– 4049. [18] K. Stenkamp, U. Heinemann, D. Schmitz, Dopamine suppresses
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Research report
Erythropoietin improves synaptic transmission during and following ischemia in rat hippocampal slice cultures Astrid Weber a , *, Rolf F. Maier a ,1 , Ulrike Hoffmann b , Martin Grips a , Marc Hoppenz a , Ayse G. Aktas a , Uwe Heinemann b , Michael Obladen a , Sebastian Schuchmann b a
Department of Neonatology, Charite´ , Humboldt University Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, D-13353 Berlin, Germany b ¨ Johannes Muller Institute of Physiology, Charite´ , Humboldt University Berlin, Tucholskystrasse 2, D-10117 Berlin, Germany Accepted 5 September 2002
Abstract Erythropoietin (EPO) prevents neuronal damage following ischemic, metabolic, and excitotoxic stress. In this study evoked extracellular field potentials (FP) were used to investigate the effect of EPO on synaptic transmission in hippocampal slice cultures. EPO treated cultured slices (40 units / ml for 48 h) showed significantly increased FP during and following oxygen and glucose deprivation compared with untreated control slices. The addition of the Jak2 inhibitor AG490 (50 mM for 48 h) blocked the EPO effect. These data suggest that EPO improves synaptic transmission during and following ischemia in hippocampal slice cultures. 2002 Elsevier Science B.V. All rights reserved. Theme: Excitable membranes and synaptic transmission Topic: Presynaptic mechanisms Keywords: Erythropoietin; Synaptic transmission; Ischemia; Neuroprotection; Hippocampal slice culture
1. Introduction The cytokine erythropoietin (EPO), well known for its crucial role in erythropoiesis, has been shown to possess neuroprotective characteristics following ischemic, metabolic and excitotoxic stress [4,13,16,17]. Expression of EPO and EPO receptor (EPOR) can be induced by hypoxia in immature and mature brains [7,11]. Therefore, it has been suggested that EPO could contribute to neuroprotection against acute and apoptotic cell death and may be involved in hypoxic–ischemic preconditioning [4], which has been shown to increase neuronal tolerance against subsequent prolonged ischemia [3]. Abbreviations: AG490, Janus kinase 2 inhibitor; AO, acridine orange; EPO, erythropoietin; EPOR, erythropoietin receptor; FP, extracellular field potential; Jak2, Janus kinase 2; OGD, oxygen and glucose deprivation; PI, propidium iodide *Corresponding author. Tel.: 149-30-450-566122; fax: 149-30-450566922. E-mail address: [email protected] (A. Weber). 1 Present address: Dept. of Neonatology, Philipps-University, Marburg, Deutschhausstr. 12, D-35033, Marburg, Germany.
The stimulation of EPOR activates different intracellular signaling pathways by activation of EPOR-associated Janus kinase 2 (Jak2, tyrosine kinase) in non-neuronal and neuronal cells [4,15]. Therefore a specific Jak2 tyrosine kinase inhibitor can be used to block EPO induced intracellular signaling [2]. In addition to the neuroprotective effect, little is known about the relevance of the EPO and EPOR signaling pathways on neuronal function. The present study was designed to investigate the effect of EPO preconditioning on the neuronal synaptic transmission under normoxic and ischemic conditions in cultured rat hippocampal brain slices.
2. Materials and methods
2.1. Preparation and culture of slices Combined entorhinal cortico-hippocampal slice cultures were prepared from 6–8-day-old Wistar rats and maintained as described before [19]. Briefly, after decapitation, the hippocampi with the entorhinal cortex attached were
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03604-1
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A. Weber et al. / Brain Research 958 (2002) 305–311
dissected and 400-mm slices were cut (McIlwain tissue chopper, Mickle Labaratories, UK) under sterile conditions in gassed (95% O 2 / 5%CO 2 ) ice-cold minimal essential medium (MEM; Gibco, Karlsruhe, Germany) at pH 7.35. Three slices were placed on each culture membrane (0.4 mm Minicell culture plate inserts; Millipore, Eschborn, Germany) and incubated in a medium containing 50% MEM, 25% Hank’s balanced salt solution (HBSS; Sigma, Deisenhofen, Germany), 25% horse serum (Gibco), 2 mM L-glutamine, pH 7.35 with 5% CO 2 at 36.5 8C. The antibiotic-free culture medium was completely replaced after day 2 and thereafter twice a week.
2.2. Preincubation For a number of experiments, slices were preincubated with EPO (Neorecormon, Roche, UK) or / and the Jak2 inhibitor AG490 (Calbiochem, Bad Soden, Germany) for 48 h. EPO was used at 40 units / ml (332 ng / ml) culture medium. The Jak2 inhibitor was dissolved at 100 mM in DMSO, stored at 220 8C and used in a final concentration of 50 mM. In n56 slice cultures we tested the direct effect of the Jak2 inhibitor AG490 and found no increased toxicity compared to control slices. Slices were divided into three groups: control (14 slices, five preparations; no preincubation), EPO (14 slices, five preparations; 40 units / ml EPO for 48 h), EPO plus Jak2 inhibitor (16 slices, two preparations; 40 units / ml EPO and 50 mM Jak2 inhibitor for 48 h), and Jak2 inhibitor (six slices, one preparation; 50 mM Jak2 inhibitor for 48 h).
2.3. Oxygen and glucose deprivation ( OGD) For the experiments, slice cultures were transferred to an interface chamber after 7–9 days in vitro. Slices were perfused at 1.6 ml / min with prewarmed (34 8C), gassed (95% O 2 / 5% CO 2 or 95% N 2 / 5% CO 2 ) artificial cerebrospinal fluid (aCSF) containing (in mM): NaCl 129; KCl 3; NaHPO 4 1.25; MgSO 4 1.8; CaCl 2 1.6; NaHCO 3 21; glucose 10; at pH 7.4. During the oxygen and glucose deprivation (OGD) slices were maintained using glucosefree aCSF gassed with 95% N 2 and 5% CO 2 for 30 min. The OGD period was followed by a reoxygenation period (120 min).
2.4. Electrophysiology For recordings a microelectrode was positioned in area CA1. Synaptic responses were obtained by electrical stimulation (100 ms duration) at the hilar border of area CA3 using a bipolar wire stimulation electrode. The stimulus strength was adjusted to twice the intensity needed to evoke a maximal extracellular field potential (FP) response. Only slice cultures in which orthodromic stimulation evoked a FP response of more than 1 mV were chosen for further investigations. Five evoked FP re-
sponses at intervals of 20 s were averaged and recorded every 5–10 min. The change in FP responses in CA1 following hilar stimulation were normalized to the FP response at the beginning of the experiment.
2.5. RNA isolation and reverse transcription–polymerase chain reaction ( RT–PCR) analysis Total RNA was prepared from hippocampal slices (7–9 days in vitro) by phenol / chloroform extraction using the RNAClean extraction kit (Hybaid, Heidelberg, Germany). Total RNA (250 ng) was reverse-transcribed using AMV reverse transcriptase (Promega, Madison, USA) and random hexamers (Promega). Four microliters of the resulting cDNAs were subjected to amplification in a total volume of 30 pl containing 103 Buffer, 0.5 mM of MgCl 2 , 0.13 mM each of dNTP, l unit Taq polymerase (Gibco, Karlsruhe, Germany) and a pair of specific primers (0.33 mM). Ten microliters of the PCR products were separated by 2.0% agarose gel electrophoresis and stained with ethidium bromide. RT products were 201 bp for EPO, 402 bp for EPOR, and 433 bp for b-actin.
2.6. Immunohistochemistry Hippocampal slice cultures (7–9 days in vitro) fixed over night in 5% neutral buffered formalin were paraffinembedded and sectioned. Rabbit polyclonal EPOR antibody (M20, Santa Cruz Biotechnology, Santa Cruz, USA), directed against the carboxy terminus of the EPO receptor of mouse origin, was applied at a dilution of 1:100 and a secondary antibody (biotinylated donkey anti-rabbit, Amersham Pharmacia Biotech, UK) was added at a dilution of 1:200. Sections were washed with PBS and incubated with Avidin–peroxidase (Sigma) at a dilution of 1:100. Slides were counterstained with hemalum solution, dehydrated and coverslipped for investigation under a light microscope. Absence of primary antibody was used as negative control.
2.7. Western blot Slice culture lysates were normalized for protein, subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose and stained with polyclonal anti-EPO-receptor antibody (M20, Santa Cruz Biotechnology), directed against the carboxy terminus of the EPOR of mouse origin at a dilution of 1:200. The secondary antibody was a donkey anti-rabbit antibody conjugated with horseradish peroxidase (Amersham Pharmacia Biotech) at a dilution of 1:10 000. Immunoreactive bands were visualized using chemiluminescence (ECLPlus, Amersham Pharmacia Biotech).
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2.8. Fluorescence imaging In order to differentiate between living and dead cells in cultured slices, double staining with the intercalating dyes acridine orange (AO; 5 mM) and propidium iodide (PI; 5 mM) dissolved in aqueous solution was used. The membrane-impermeable PI binds to DNA only in dead cells (emission 605 nm, red), the membrane permeable AO interacts with RNA and DNA mainly in living cells (emission 515 nm, green). The two dyes (both from Molecular Probes, Leiden, the Netherlands) were simultaneously applied and the fluorescence emission signals were measured by exciting at 480 nm (acridine orange) and at 550 nm (propidium iodide) using an optical combination of a 565 nm dichroic mirror and a 590 nm long-pass filter.
2.9. Statistical evaluation Data are presented as mean6standard error of mean, statistical differences were tested using the two-sided student’s t-test for unpaired samples and the log rank test (Kaplan–Meier plot). Statistical significance was assumed if P was less than 0.05.
3. Results FP amplitudes were analyzed before, during and after an OGD period of 30 min duration. In the control group, FP amplitudes at the start of the experiment were 1.7660.1 mV. Following the OGD period, FP amplitudes decreased to 0.1360.08 mV (8.965.8% of control FP amplitude) and showed a restricted recovery during the reoxygenation period to 0.2960.13 mV (15.267.2% of control FP amplitude). In the EPO group, FP amplitudes were not significantly different at the start of the experiment compared to the control group (1.9960.37 mV; EPO vs. control, P50.540). Following the OGD period, FP amplitudes decreased to 0.4160.18 mV (26.2611.1% of control FP amplitude; EPO vs. control, P50.250) and recovered during the reoxygenation period to 1.4860.54 mV (70.6619.8% of control FP amplitude; EPO vs. control, P50.014). In n56 slice cultures we tested the effect of the Jak2 inhibitor AG490 on evoked FP during and following OGD. We found no deleterious effects compared with control slices. In the EPO plus Jak2 inhibitor group, FP amplitudes were significantly enlarged at the start of the experiment compared to the control group (2.8560.34 mV; EPO plus Jak2 inhibitor vs. control, P50.013). Following the OGD period, FP amplitudes dropped to 0.1760.17 mV (3.964.0% of control FP amplitude; EPO plus Jak2 inhibitor vs. control, P50.237; EPO plus Jak2 inhibitor vs. EPO, P50.056) and showed no recovery during the reoxygenation period (0.1160.06 mV, 10.965.2% of control FP amplitude; EPO plus Jak2 inhibitor vs. control, P50.620; EPO plus Jak2 inhibitor vs.
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EPO, P50.009). Fig. 1A,B summarizes the changes in FP amplitudes in the different groups before, during and after the OGD period. The results show a significantly improved recovery in FP amplitudes during the reoxygenation period in the EPO group compared to the control and the EPO plus Jak2 inhibitor group. After 120 min reoxygenation (95% O 2 , 5% CO 2 , 10 mM glucose) a FP amplitude .0.1 mV was recorded in 5 / 14 (35.7%) slices of the control group, in 12 / 14 slices (85.7%) of the EPO group and in 5 / 16 slices (31.2%) of the EPO plus Jak2 inhibitor group. Fig. 1C represents the outcome of the slices during the experiments as a Kaplan– Meier plot. The number of slices with FP amplitudes .0.1 mV was significantly higher in the EPO group compared to the control and the EPO plus Jak2 inhibitor group (control vs. EPO, P50.011; EPO vs. EPO plus Jak2 inhibitor, P50.012; control vs. EPO plus Jak2 inhibitor, P50.906). In order to check the presence of EPO and EPOR in cultured hippocampal slices, RT–PCR techniques were used to show EPO and EPOR mRNA after 7–9 days in vitro (Fig. 2A,B). Furthermore, immunohistochemistry techniques were used to show the expression of EPOR by hippocampal neurons from cultured hippocampal slices (Fig. 2C). Finally, fluorescence double staining with AO and PI was used to investigate semiquantitatively neuroprotective effects of EPO on hippocampal slice cultures following OGD periods (control 17 slices, EPO 15 slices, EPO plus Jak2 inhibitor eight slices). Fig. 2D shows typical cultured slices of the three groups immediately after OGD and a reoxygenation period of 120 min. An increased PI fluorescence signal in control and EPO plus Jak2 inhibitor slices compared to EPO slices indicates an enlarged number of dead cells in these slices. Furthermore, subtracting AO images and PI images accentuates the EPO neuroprotective effect after OGD and reoxygenation period in the EPO group vs. control group and EPO plus Jak2 inhibitor group. Additional staining of cultured slices (n5 11) treated with Jak2 inhibitor AG490 alone did not reveal any toxic effects compared with control slices.
4. Discussion The present study demonstrates for the first time that preconditioning of hippocampal slice cultures with EPO improves neuronal synaptic transmission during and following oxygen and glucose deprivation. In previous studies, pretreatment with EPO has been shown to prevent N-methyl-D-aspartate / glutamate, hypoxia and ischemia induced cell death in several in vitro and in vivo models [4,9,11,17]. The PI /AO double staining in the study presented here confirms in organotypic slice cultures the findings that EPO protects hippocampal neurons and that this effect is dependent on Jak2 associated EPOR. Following ischemic or glutamate induced lesions an increase of EPO and EPOR expression has been described in
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Fig. 1. Changes in extracellular field potential (FP) during and following ischemia (30 min OGD and 120 min reoxygenation) in slice cultures from control (no preincubation), EPO (40 units / ml EPO for 48 h) and EPO plus Jak2 inhibitor (40 units / ml EPO and 50 mM Jak2 inhibitor AG 490 for 48 h) group. (A) Characteristic traces of evoked extracellular field potential before, during and after OGD in the three experimental groups. Asterisks indicate the stimulation artifacts. (B) Changes in evoked extracellular field potential normalized to the FP amplitude under normoxic conditions in the control (s; n514 slices), the EPO (j; n514 slices), and the EPO plus Jak2 inhibitor (h; n516 slices) group. EPO-treated slice cultures show a significantly increased FP amplitude during and particularly following OGD compared with slice cultures from the control and the EPO plus Jak2 inhibitor group (EPO vs. control: *P,0.05; EPO vs. EPO plus Jak2 inhibitor: [ P,0.05). (C) In order to analyze the outcome of slice cultures, the normalized number of slices showing FP amplitudes .0.1 mV were summarized in a Kaplan–Meier plot. A significantly increased number of EPO-treated slice cultures (full line) show FP amplitudes .0.1 mV during and after OGD compared with slice cultures from control (cross-hatched line; P,0.05) and EPO plus Jak2 inhibitor (dotted line; P,0.05) group.
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Fig. 2. EPO and EPOR expression shown in slice cultures (DIV 7–9, no preincubation, no experiment performed) and EPO-induced neuroprotection in hippocampal slice cultures measured immediately after 30 min OGD and 120 min reoxygenation period. (A) Western analysis of protein lysates of three pooled untreated slice cultures (prior to electrophysiological investigation) shows specific EPOR immunoreactivity. Neonatal rat liver was used as positive control. (B) Detection of EPO and EPOR mRNA expression via RT–PCR in three pooled RNA extracts of slice cultures and corresponding beta-actin expression (prior to electrophysiological investigation). (C) Immunohistochemical detection of EPOR-positive neurons in a representative slice culture (prior to electrophysiological investigation). (D) Fluorescence imaging indicating dead (propidium iodide: red; left column) and living (acridine orange: green-yellow; middle column) cells immediately after 30 min OGD and 120 min reoxygenation in slice cultures from the control group, the EPO group and the EPO plus Jak2 inhibitor group. The right column shows a gray-scale differential image (propidium iodide image subtracted from acridine orange image), which indicates dead cells in dark-colored areas and living cells in light-colored areas. The anatomic structure of the slice cultures is displayed by the schematic plot.
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dissociated neuronal cultures, cultured cells with neuronal characteristics and in vivo [11,13,17]. The distinct expression of EPO and EPOR found in hippocampal slice cultures used in this study may partly result from the ischemic phase during the slice preparation. Which intracellular signaling cascade(s) initiated by EPO / EPOR interactions may lead to improvement of synaptic transmission during and following ischemia shown in this study? Moreover, are these intracellular cascade(s) divergent from the EPO mediated neuroprotective pathway? Recently, it has been suggested that the neuroprotective effect of EPO is regulated by activating NF-kB via Jak2 [4]. Jak2 is known to play an important role for signaling of cytokine receptors and therefore may influence indirectly neuronal development and function [15]. Evidence is accumulating for an important role of NF-kB in neuronal function. Thus, studies on neuronal excitation indicated that ionotropic glutamate receptors can activate NF-kB and point to a role of NF-kB as a signal transducer during synaptic transmission [6]. An increased level of NF-kB activity in nuclei of neurons has been suggested to regulate cellular anti-oxidative defense and therefore, may explain partly the neuroprotective effect [14]. Although the mechanism for NF-kB activation via Jak2 is discussed controversially, the role of NF-kB as intracellular second messenger transmitting EPOR derived signals to the nucleus appears unambiguously [2]. EPO has been suggested to modulate neurotransmitter release through activation of EPOR linked to a Jak2 pathway. Activation of EPOR has been shown to activate L-type Ca 21 channels and to modulate Ca 21 -induced dopamine release in clonal rat pheochromocytoma PC12 cells [8,10,12]. Dopamine has been shown to suppress synaptic transmission and inhibition in several regions of the brain, most likely by presynaptic mechanisms [1,5,18]. In the present study, significantly increased evoked FP amplitudes after blocking intracellular Jak2 signaling pathway by pre-treatment of slice cultures with the Jak2inhibitor AG490 may indicate an altered dopamine release under control conditions. Recently, it was suggested that EPO induced activation of EPOR might reduce the Ca 21 dependent release of glutamate from neurons of hippocampus and cerebellum in a model of chemical ischemia [9]. Therefore, EPO-induced modulation of neurotransmitter release may be a regulatory mechanism of presynaptic function in the brain. The improved synaptic transmission during and following ischemia demonstrated in the present study may thus be based on modulation of presynaptic neurotransmitter release. Further studies are necessary to elucidate the functional role of EPO and EPOR for synaptic transmission.
Acknowledgements We wish to thank J. Graulich for his crucial initiation to the collaboration group. We gratefully acknowledge techni-
cal support from S. Gabriel, H.J. Gabriel, S. Latta, and H. Siegmund and help with the preparation of the manuscript ¨ from S. Schurmann. This study was supported by a grant ¨ of the Wilhelm Sander-Stiftung, Munchen (No. 2000.091.1) and a grant of the Sonnenfeld-Stiftung, Berlin.
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